The development of air-filled respiratory organs is crucial for survival at birth. A combination of live imaging and genetic analysis was used to dissect respiratory organ maturation in the embryonic Drosophila trachea. Tracheal tube maturation was found to entail three precise epithelial transitions. Initially, a secretion burst deposits proteins into the lumen. Solid luminal material is then rapidly cleared from the tubes, and shortly thereafter liquid is removed. To elucidate the cellular mechanisms behind these transitions, gas-filling-deficient mutants were identified showing narrow or protein-clogged tubes. These mutations either disrupt endoplasmatic reticulum-to-Golgi vesicle transport or endocytosis. First, Sar1 was shown to be required for protein secretion, luminal matrix assembly, and diametric tube expansion. sar1 encodes a small GTPase that regulates COPII vesicle budding from the endoplasmic reticulum (ER) to the Golgi apparatus. Subsequently, a sharp pulse of Rab5-dependent endocytic activity rapidly internalizes and clears luminal contents. The coordination of luminal matrix secretion and endocytosis may be a general mechanism in tubular organ morphogenesis and maturation (Tsarouhas, 2007).

Branched tubular organs are essential for oxygen and nutrient transport. Such organs include the blood circulatory system, the lung and kidney in mammals, and the tracheal respiratory system in insects. The optimal flow of transported fluids depends on the uniform length and diameter of the constituting tubes in the network. Alterations in the distinct tube shapes and sizes cause pronounced defects in animal physiology and lead to serious pathological conditions. For example, tube overgrowth and cyst formation in the collecting duct are intimately linked to the pathology of Autosomal Dominant Polycystic Kidney Disease. Conversely, stenoses, the abnormal narrowing of blood vessels and other tubular organs, are associated with ischemias and organ obstructions (Tsarouhas, 2007 and references therein).

While the early steps of differentiation, lumen formation, and branch patterning begin to be elucidated in several tubular organs, only scarce glimpses into the cellular events of lumen expansion and tubular organ maturation are available. De novo lumen formation can be induced in three-dimensional cultures of MDCK cells. Recent studies in this system revealed that PTEN activation, apical cell membrane polarization, and Cdc42 activation are key events in lumen formation in vitro. In zebrafish embryos and cultured human endothelial cells, capillary vessels form through the coalescence and growth of intracellular pinocytic vesicles. These tubular vacuoles then fuse with the plasma membranes to form a continuous extracellular lumen. Salivary gland extension in Drosophila requires the transcriptional upregulation of the apical membrane determinant Crumbs (Crb), but the cellular mechanism leading to gland expansion remains unclear (Tsarouhas, 2007 and references therein).

The epithelial cells of the Drosophila tracheal network form tubes of different sizes and cellular architecture, and they provide a genetically amenable system for the investigation of branched tubular organ morphogenesis. Tracheal development begins during the second half of embryogenesis when 20 metameric placodes invaginate from the epidermis. Through a series of stereotypic branching and fusion events, the tracheal epithelial cells generate a tubular network extending branches to all embryonic tissues. In contrast to the wealth of knowledge about tracheal patterning and branching, the later events of morphogenesis and tube maturation into functional airways have yet to be elucidated. As the nascent, liquid-filled tracheal network develops, the epithelial cells deposit an apical chitinous matrix into the lumen. The assembly of this intraluminal polysaccharide cable coordinates uniform tube growth. Two luminal, putative chitin deacetylases, Vermiform (Verm) and Serpentine (Serp), are selectively required for termination of branch elongation. The analysis of verm and serp mutants indicates that modifications in the rigidity of the matrix are sensed by the surrounding epithelium to restrict tube length. What drives the diametric expansion of the emerging narrow branches to their final size? How are the matrix- and liquid-filled tracheal tubes cleared at the end of embryogenesis (Tsarouhas, 2007)?

This study used live imaging of secreted GFP-tagged proteins to identify the cellular mechanisms transforming the tracheal tubes into a functional respiratory organ. The precise sequence and cellular dynamics were characterized of a secretory and an endocytic pulse that precede the rapid liquid clearance and gas filling of the network. Analysis of mutants with defects in gas filling reveals three distinct but functionally connected steps of airway maturation. Sar1-mediated luminal deposition of secreted proteins is tightly coupled with the expansion of the intraluminal matrix and tube diameter. Subsequently, a Rab5-dependent endocytotic wave frees the lumen of solid material within 30 min. The precise coordination of secretory and endocytotic activities first direct tube diameter growth and then ensure lumen clearance to generate functional airways (Tsarouhas, 2007).

Two strong, hypomorphic sar1 alleles were identified in screens for mutants with tracheal tube defects. In wild-type embryos, the bulk of luminal markers 2A12, Verm, and Gasp has been deposited inside the DT lumen by stage 15. However, in zygotic sar1^P1 mutants (hereafter referred to as sar1), luminal secretion of 2A12, Verm, and Gasp was incomplete. The tracheal cells outlined by GFP-CAAX partially retained those markers in the cytoplasm. sar1 zygotic mutant embryos normally deposited the early luminal marker Piopio by stage 13. Luminal chitin was also detected in sar1 mutants at stage 15. However, the luminal cable was narrow, more dense, and distorted compared to wild-type. To test if the sar1 secretory phenotype in the trachea is cell autonomous, Sar1 was reexpressed specifically in the trachea of sar1 mutants by using btl-GAL4. Such embryos showed largely restored secretion of 2A12, Verm, and Gasp. Thus, it is concluded that tracheal sar1 is required for the efficient secretion of luminal markers, which are predicted to associate with the growing intraluminal chitin matrix (Tsarouhas, 2007).

sar1 mRNA has been reported to be abundantly maternally contributed. At later stages, zygotic expression of sar1 mRNA is initiated in multiple epithelial tissues. To monitor Sar1 zygotic expression in the trachea, a Sar1-GFP protein trap line was used. Embryos carrying only paternally derived Sar1-GFP show a strong zygotic expression of GFP in the trachea. An anti-Sar1 antibody was used to analyze Sar1 expression in the trachea of wild-type, zygotic sar1^P1, and sar1^EP3575Δ28 null mutant embryos were generated. Both zygotic mutants showed a clear reduction, but not complete elimination, of Sar1 expression in the trachea. To test the effects of a more complete inactivation of Sar1, transgenic flies were generated expressing a dominant-negative sar1^T38N form in the trachea. In btl > sar1^T38N-expressing embryos, early defects were observed in tracheal branching and epithelial integrity as well as a complete block in Verm secretion. In contrast to btl > sar1^T38N-expressing embryos, zygotic sar1^P1 mutant embryos show normal early tracheogenesis with no defects in branching morphogenesis and epithelial integrity (Tsarouhas, 2007).

In summary, tracheal expression of Sar1 is markedly reduced in zygotic sar1 mutant embryos. While maternally supplied Sar1 is sufficient to support early tracheal development, zygotic Sar1 is required for efficient luminal secretion (Tsarouhas, 2007).

Given the conserved role of Sar1 in vesicle budding from the ER, its subcellular localization in the trachea was determined by using anti-Sar1 antibodies. Sar1 localizes predominantly to the ER (marked by the PDI-GFP trap). Continuous COPII-mediated transport from the ER is required to maintain the Golgi apparatus and ER structure. To test if zygotic loss of Sar1 compromises the integrity of the ER and Golgi in tracheal cells, sar1 mutant embryos were stained with antibodies against KDEL (marking the ER lumen) and gp120 (highlighting Golgi structures). In sar1 mutant embryos, a strongly disrupted ER structure and loss of Golgi staining was observed in dorsal trunk (DT) cells at stage 14. Additionally, TEM of stage-16 wild-type and sar1 mutant embryos showed a grossly bloated rough ER structure in DT tracheal cells. Consistent with its functions in yeast and vertebrates, Drosophila Sar1 localizes to the ER and is not only required for efficient luminal protein secretion, but also for the integrity of the early secretory apparatus (Tsarouhas, 2007).

To analyze tracheal maturation defects in sar1 mutant embryos, sar1 strains were generated and imaged that carry either btl > ANF-GFP btl-mRFP-moe or btl > Gasp-GFP. In sar1 mutants, luminal deposition of both ANF-GFP and Gasp-GFP is reduced. Like endogenous Gasp in the mutants, Gasp-GFP was clearly retained in the cytoplasm of sar1 embryos. ANF-GFP was also retained in the tracheal cells of sar1 mutants, but to a lesser extent. Strikingly, sar1 mutants failed to fully expand the luminal diameter of the DT outlined by the apical RFP-moe localization. This defect was quantified by measuring diametric growth rates in metamere 6 for wild-type and sar1 mutant embryos. While early lumen expansion commences in parallel in both genotypes, the later diametric growth of sar1 mutants falls significantly behind compared to wild-type embryos. The DT lumen in sar1 mutants reaches only an average of 70% of the wild-type diameter at early stage 16. Identical diametric growth defects were detected in fixed sar1 mutant embryos expressing btl > GFP-CAAX by analysis of confocal yz sections or TEM. Reexpression of sar1 in the trachea of sar1 mutant embryos not only rescued secretion, but also the lumen diameter phenotype at stage 16. In contrast to the diametric growth defects, DT tube elongation in sar1 embryos was indistinguishable from that in wild-type. This demonstrates distinct genetic requirements for tube diameter and length growth. It also reveals that the sar1 DT luminal volume reaches less than half of the wild-type volume. Prolonged live imaging showed that sar1 mutants are also completely deficient in luminal protein and liquid clearance. Up to 80% of the rescued embryos also completed luminal liquid clearance, suggesting that efficient tracheal secretion and the integrity of the secretory apparatus are prerequisites for later tube maturation steps. Taken together, the above-described results show that tracheal Sar1 is selectively required for tube diameter expansion. Additionally, subsequent luminal protein and liquid clearance fail to occur in sar1 mutants (Tsarouhas, 2007).

Do the tracheal defects of sar1 reflect a general requirement for the COPII complex in luminal secretion and diameter expansion? To test this, lethal P element insertion alleles were examined disrupting two additional COPII coat subunits, sec13 and sec23. Mutant sec13 and sec23 embryos were stained for luminal Gasp and for Crb and α-Spectrin to highlight tracheal cells. At stage 15, embryos of both mutants show a clear cellular retention of Gasp. Furthermore, stage-16 sec13 and sec23 embryos show significantly narrower DT tubes when compared to wild-type. The average diameter of the DT branches in metamere 6 was 4.8 μm and 4.4 μm in fixed sec13 and sec23 embryos, respectively, compared to 6.3 μm in wild-type. Therefore, sec13 and sec23 mutants phenocopy sar1. The phenotypic analysis of three independent mutations disrupting ER-to-Golgi transport thus provides a strong correlation between deficits in luminal protein secretion and tube diameter expansion (Tsarouhas, 2007).

The live-imaging approach defines the developmental dynamics of functional tracheal maturation. At the organ level, three sequential and rapid developmental transitions were identified: (1) the secretion burst, followed by massive luminal protein deposition and tube diameter expansion, (2) the clearance of solid luminal material, and (3) the replacement of luminal liquid by gas. Live imaging of each event additionally revealed insights into the startlingly dynamic activities of the tracheal cells. ANF-GFP-containing structures and apical GFP-FYVE-positive endosomes rapidly traffic in tracheal cells during the secretion burst and protein clearance. The direct live comparison between wild-type and mutant embryos further highlights the dynamic nature of epithelial activity during each pulse (Tsarouhas, 2007).

This study identified several mutations that selectively disrupt distinct cellular functions and concurrently interrupt the maturation process at specific steps. This clearly demonstrates the significance of phenotypic transitions in epithelial organ maturation and establishes that secretion is required for luminal diameter expansion and endocytosis for solid luminal material clearance (Tsarouhas, 2007).

The sudden initiation of an apical secretory burst tightly precedes diametric tube expansion. The completion of both events depends on components of the COPII complex, further suggesting that the massive luminal secretion is functionally linked to diametric growth. How does apical secretion provide a driving force in tube diameter expansion? In mammalian lung development, the distending internal pressure of the luminal liquid on the epithelium expands the lung volume and stimulates growth. Cl⁻ channels in the epithelium actively transport Cl⁻ ions into luminal liquid. The resulting osmotic differential then forces water to enter the lung lumen, driving its expansion. By analogy, the tracheal apical exocytic burst may insert protein regulators such as ion channels into the apical cell membrane or add additional membrane to the growing luminal surface. Since the ER is a crucial cellular compartment for intracellular traffic and lipogenesis, its disruption in sar1 mutants may disrupt the efficient transport of so far unknown specific regulators or essential apical membrane addition required for diametric expansion. Alternatively, secreted chitin-binding proteins (ChB) may direct an increase of intraluminal pressure and tube dilation. Overexpression of the chitin-binding proteins Serp-GFP or Gasp-GFP was insufficient to alter the diametric growth rate of the tubes, suggesting that lumen diameter expansion is insensitive to increased amounts of any of the known luminal proteins. In sar1 mutants, the secretion of at least two chitin-binding proteins, Gasp and Verm, is reduced. Chitin, however, is deposited in seemingly normal quantities, but assembles into an aberrantly narrow and dense chitinous cable. This phenotype suggests that the correct ratio between chitin and multiple interacting proteins may be required for the correct assembly of the luminal cable. Interestingly, sar1, sec13, and sec23 mutant embryos form a severely defective and weak epidermal cuticle. The luminal deposition of ChB proteins during the tracheal secretory burst may orchestrate the construction and swelling of a functional matrix, which, in turn, induces lumen diameter dilation. While this later hypothesis is favored, it cannot be excluded that other mechanisms, either separately or in combination with the dilating luminal cable, drive luminal expansion (Tsarouhas, 2007).

During tube expansion, massive amounts of luminal material, including the chitinous cable, fill the tracheal tubes. This study found that Dynamin, Clathrin, and the tracheal function of Rab5 are required to rapidly remove luminal contents, indicating that endocytosis is required for this process. Several lines of evidence argue that the tracheal epithelium activates Rab5-dependent endocytosis to directly internalize luminal material. First, the tracheal cells of rab5 mutants show defects in multiple endocytic compartments. These phenotypes of rab5 mutants become apparent during the developmental period matching the interval of luminal material clearance in wild-type embryos. Second, tracheal cells internalize two luminal markers, the endogenously encoded Gasp and the dextran reporter, exactly prior and during luminal protein clearance. The number of intracellular dextran puncta reaches its peak during the clearance process and ceases shortly thereafter. Lastly, intracellular puncta of both Gasp and dextran colocalize with defined endocytic markers inside tracheal cells. The colocalization of Gasp and dextran with GFP-Rab7 and of Gasp with GFP-LAMP1 suggests that the luminal material may be degraded inside tracheal cells. Taken together, these data show that the tracheal epithelium activates a massive wave of endocytosis to clear the tubes (Tsarouhas, 2007).

Endocytic routes are defined by the nature of the internalized cargoes and the engaged endocytic compartments. What may be the features of the endocytic mechanisms mediating the clearance of luminal material? The phenotype of chc mutants and the presence of intracellular Gasp in CCVs indicate that luminal clearance at least partly relies on Clathrin-mediated endocytosis (CME). In addition to CME, Dynamin and Rab5 have also been implicated in other routes of endocytosis, suggesting that multiple endocytic mechanisms may be operational in tracheal maturation. The nature of the endocytosed luminal material provides an additional perspective. While cognate uptake receptors may exist for specific cargos such as Gasp, Verm, and Serp, the heterologous ANF-GFP, degraded chitin, and the fluid-phase marker dextran may be cleared by either fluid-phase internalization or multifunctional scavenger receptors. Interestingly, Rab5 can regulate fluid-phase internalization in cultured cells by stimulating macro-pinocytosis and the activation of Rabankyrin-5. The defective tracheal internalization of dextran in rab5 mutants provides further loss-of-function evidence for Rab5 function in fluid-phase endocytosis in vivo. The above-described arguments lead to the speculation that additional Rab5-regulated endocytic mechanisms most likely cooperate with CME in the clearance of solid luminal material (Tsarouhas, 2007).

How is liquid cleared from the lumen? While very little is known about this fascinating process, some developmental and mechanistic arguments suggest that this last maturation step is mechanistically distinct. First, the interval of luminal liquid clearance is clearly distinct from the period of endocytic clearance of solids. Second, the dynamic internalization of dextran and the abundance of GFP-marked endocytic structures decline before liquid clearance. Finally, assessment of liquid clearance further suggests that it requires a distinct cellular mechanism (Tsarouhas, 2007).

Viewing the entire process of airway maturation in conjunction, some general conclusions may be drawn. First, the three epithelial pulses are highly defined by their sequence and exact timing, suggesting that they may be triggered by intrinsic or external cues. Second, the analysis of mutants that selectively reduce the amplitude of the secretory or endocyic pulses demonstrates the requirement for each epithelial transition in the completion of the entire maturation process. These pulses are induced in the background of basal secretory and endocytic activities that operate throughout development. Third, specific cellular activities exactly precede each morphological transition. Finally, the separate transitions are interdependent in a sequential manner. Efficient secretion is a prerequisite for the endocytic wave. Similarly, protein endocytosis is a condition for luminal liquid clearance. This suggests a hierarchical coupling of the initiation of each pulse to completion of the previous one in a strict developmental sequence (Tsarouhas, 2007).

This study provides a striking example of how pulses of epithelial activity drive distinct developmental events and mold the nascent tracheal lumen into an air delivery tube. These findings are likely to be relevant beyond the scope of tracheal development. The uniform growth of salivary gland tubes in flies and the excretory canal and amphid channel lumen in worms also require the assembly of a luminal matrix for uniform tube growth. Luminal material is also transiently present during early developmental stages in the distal nephric ducts of lamprey. Thus, the coordinated, timely deposition and removal of transient luminal matrices may represent a general mechanism in tubulogenesis (Tsarouhas, 2007).

UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy, endocytosis and DNA damage repair, but how its loss contributes to colorectal cancer is poorly understood. This study shows that UVRAG deficiency in Drosophila intestinal stem cells leads to uncontrolled proliferation and impaired differentiation without preventing autophagy. As a result, affected animals suffer from gut dysfunction and short lifespan. Dysplasia upon loss of UVRAG is characterized by the accumulation of endocytosed ligands and sustained activation of STAT and JNK signaling, and attenuation of these pathways suppresses stem cell hyperproliferation. Importantly, the inhibition of early (dynamin-dependent) or late (Rab7-dependent) steps of endocytosis in intestinal stem cells also induces hyperproliferation and dysplasia. These data raise the possibility that endocytic, but not autophagic, defects contribute to UVRAG-deficient colorectal cancer development in humans (Nagy, 2016).

UVRAG encodes a homolog of yeast Vps38 in metazoans. UVRAG/Vps38 and Atg14 are mutually exclusive subunits of two different Vps34 lipid kinase complexes, both of which contain Vps34, Vps15 and Atg6/Beclin 1. It is well established that Vps38 is required for endosome maturation and vacuolar and lysosomal protein sorting, whereas Atg14 is specific for autophagy in yeast. However, the function of UVRAG is much more controversial in mammalian cells. Although UVRAG was originally found to have dual roles in autophagy through promotion of autophagosome formation and fusion with lysosomes in various cultured cell lines based on, predominantly, overexpression experiments, recent reports have described that autophagosomes are normally generated and fused with lysosomes in the absence of UVRAG in cultured mammalian (HeLa) cells and in the Drosophila fat body (Nagy, 2016 and references therein).

The discoveries of UVRAG mutations in colorectal cancer cells, and that its overexpression increases autophagy and suppresses the proliferation of certain cancer cell lines, altogether suggest that this gene functions as an autophagic tumor suppressor. Such a role for UVRAG is thought to be related to its binding to Beclin 1, a haploinsufficient tumor suppressor gene required for autophagy. UVRAG appears to play roles similar to yeast Vps38 in the Drosophila fat body, and developing eye and wing: its loss leads to the accumulation of multiple endocytic receptors and ligands in an endosomal compartment, impaired trafficking of Lamp1 and Cathepsin L to the lysosome, and defects in the biogenesis of lysosome-related pigment granules. However, whether this gene is also required for the maintenance of intestinal homeostasis in Drosophila was unclear because the loss of UVRAG did not lead to uncontrolled cell proliferation in the developing eye or wing according to these reports. The current results showing that Uvrag deficiency causes intestinal dysplasia suggest that this gene is also important for the proper functioning of the adult gut in Drosophila (Nagy, 2016).

A surprising aspect of this work is that UVRAG appears to function independently of autophagy in the intestine. There are other lines of evidence that also support that UVRAG has a more important role in endocytic maturation than in autophagy. First, it has been shown that truncating mutations in UVRAG that are associated with microsatellite-unstable colon cancer cell lines do not disrupt autophagy. Second, UVRAG depletion in HeLa cells does not prevent the formation or fusion of autophagosomes with lysosomes, but it does interfere with Egfr degradation. Third, a very recent paper has shown that overexpression of the colorectal-cancer-associated truncated form of UVRAG promotes tumorigenesis independently of autophagy status, that is, both in control and Atg5-knockout cells. That paper, again, relied on the overexpression of full-length or truncated forms of UVRAG, rather than the analysis of cancer-related mutations of the endogenous locus. Fourth, the endocytic function of UVRAG has been found to be required for developmental axon pruning that is independent of autophagy in Drosophila (Nagy, 2016 and references therein).

The results of this study indicate that UVRAG loss is accompanied with the sustained activation of JNK and STAT signaling in ISCs and EBs, and that these pathways are required for dysplasia in this setting. Sustained activation of these signaling routes is likely to be connected to the disruption of endocytic flux in the absence of UVRAG, because inhibiting endocytic uptake or degradation through dominant-negative dynamin expression or RNAi of Rab7, respectively, also leads to intestinal dysplasia. It is worth noting that the effects of inhibiting Shibire/dynamin function led to a much more severe hyperproliferation of ISCs and early death of animals. In line with this, the loss of early endocytic regulators, such as Rab5, in the developing eye causes overproliferation of cells and lethality during metamorphosis. Although eye development is not perturbed by the loss of the late endocytic regulators UVRAG or Rab7, these proteins are clearly important for controlling ISC proliferation and differentiation (Nagy, 2016).

A recent paper shows that hundreds of RNAi lines cause the expansion of the esg-GFP compartment in 1-week-old animals, which might be due to an unspecific ISC stress response in some cases. However, several lines of evidence support that impaired UVRAG-dependent endocytic degradation is specifically required to prevent intestinal dysplasia. First of all, activation of JNK stress signaling in esg-GFP-positive cells induces short-term ISC proliferatio, and almost all stem cells are lost through apoptosis by the 2- to 3-week age, the time when the Uvrag-mutant phenotype becomes obvious. In fact, UVRAG loss resembles an early-onset age-associated dysplasia that is normally observed in old (30-60 days) flies and involves the simultaneous activation of both JNK and STAT signaling. Second, UVRAG RNAi in ISCs and EBs leads to paracrine activation of the cytokine Unpaired3 in enterocytes, one of the hallmarks of niche appropriation by Notch-negative tumors. However, autocrine expression of the Unpaired proteins and JNK activation is observed in Uvrag-knockdown cells, unlike in Notch-negative tumors, and EBs with active Notch signaling accumulate in the absence of UVRAG, so the two phenotypes are clearly different. Third, it is the loss of autophagy that could be expected to mimic a stress response and perhaps induce stem cell tumors, but this does not seem to be the case – ISCs with Atg5 or Atg14 RNAi proliferate less in 3-week-old animals and an overall decrease of the esg-GFP compartment is seen, as opposed to the Uvrag-deletion phenotype (Nagy, 2016).

Taken together, this work indicates that endocytic maturation and degradation serves to prevent early-onset intestinal dysplasia in Drosophila, and its deregulation could be relevant for the development of colorectal cancer in humans (Nagy, 2016).

The Drosophila nephrocyte is a critical component of the fly renal system and bears structural and functional homology to podocytes and proximal tubule cells of the mammalian kidney. Nephrocytes are highly active in endocytosis and vesicle trafficking. Rab GTPases regulate endocytosis and trafficking but specific functions of nephrocyte Rabs remain undefined. This study analyzed Rab GTPase expression and function in Drosophila nephrocytes and found that 11 out of 27 Drosophila Rabs were required for normal activity. Rabs 1, 5, 7, 11 and 35 were most important. Gene silencing of the nephrocyte-specific Rab5 eliminated all intracellular vesicles and the specialized plasma membrane structures essential for nephrocyte function. Rab7 silencing dramatically increased clear vacuoles and reduced lysosomes. Rab11 silencing increased lysosomes and reduced clear vacuoles. These results suggest that Rab5 mediates endocytosis that is essential for the maintenance of functionally critical nephrocyte plasma membrane structures and that Rabs 7 and 11 mediate alternative downstream vesicle trafficking pathways leading to protein degradation and membrane recycling, respectively. Elucidating molecular pathways underlying nephrocyte function has the potential to yield important insights into human kidney cell physiology and mechanisms of cell injury that lead to disease (Fu, 2017).

Epithelial cells are characterized by apical-basal polarity. Intrinsic factors underlying apical-basal polarity are crucial for tissue homeostasis and have often been identified to be tumor suppressors. Patterning and differentiation of epithelia are key processes of epithelial morphogenesis and are frequently regulated by highly conserved extrinsic factors. However, due to the complexity of morphogenesis, the mechanisms of precise interpretation of signal transduction as well as spatiotemporal control of extrinsic cues during dynamic morphogenesis remain poorly understood. Wing posterior crossvein (PCV) formation in Drosophila serves as a unique model to address how epithelial morphogenesis is regulated by secreted growth factors. Decapentaplegic (Dpp), a conserved bone morphogenetic protein (BMP)-type ligand, is directionally trafficked from longitudinal veins (LVs) into the PCV region for patterning and differentiation. These data reveal that the basolateral determinant Scribbled (Scrib) is required for PCV formation through optimizing BMP signaling. Scrib regulates BMP-type I receptor Thickveins (Tkv) localization at the basolateral region of PCV cells and subsequently facilitates Tkv internalization to Rab5 endosomes, where Tkv is active. BMP signaling also up-regulates scrib transcription in the pupal wing to form a positive feedback loop. These data reveal a unique mechanism in which intrinsic polarity genes and extrinsic cues are coupled to promote robust morphogenesis.

This study shows that the Scrib complex, a basolateral determinant, is a novel feedback component that optimizes BMP signaling in the PCV region of the Drosophila pupal wing (Gui, 2016).

During PCV development, limited amounts of Dpp ligands are provided by the Dpp trafficking mechanism. Furthermore, amounts of receptors appear to be limited since tkv transcription is down-regulated in the cells in which the BMP signal is positive, a mechanism that serves to facilitate ligand diffusion and sustain long-range signaling in the larval wing imaginal disc. To provide robust signal under conditions in which both ligands and receptors are limiting, additional molecular mechanisms are needed. Previous studies suggest that two molecules play such roles. Crossveinless-2 (Cv-2), which is highly expressed in the PCV region, serves to promote BMP signaling through facilitating receptor-ligand binding. Additionally, the RhoGAP protein Crossveinless-c (Cv-c) provides an optimal extracellular environment to maintain ligand trafficking from LVs into PCV through down-regulating integrin distribution at the basal side of epithelia. Importantly, both cv-2 and cv-c are transcriptionally regulated by BMP signaling to form a feedback or feed-forward loop for PCV formation (Gui, 2016).

Scrib, a third component, sustains BMP signaling in the PCV region in a different manner. First, to utilize Tkv efficiently, Scrib regulates Tkv localization at the basolateral region in the PCV cells, where ligand trafficking takes place. Regulation of receptor localization could be a means of spatiotemporal regulation of signaling molecules during the dynamic process of morphogenesis. Second, to optimize the signal transduction after receptor-ligand binding, Scrib facilitates Tkv localization in the Rab5 endosomes. Localization of internalized Tkv is abundant at Rab5 endosomes in the PCV region of wild-type, but not scrib RNAi cells. While the physical interaction between Scrib, Tkv and Rab5 in the pupal wing remains to be addressed, the data in S2 cells suggest that physical interactions between these proteins are key for preferential localization of Tkv at the Rab5 endosomes. Recently, Scrib has been implicated in regulating NMDA receptor localization through an internalization-recycling pathway to sustain neural activity. Therefore, Scrib may be involved in receptor trafficking in a context-specific manner, the molecular mechanisms of which, however, remain to be characterized. Third, BMP/Dpp signaling up-regulates scrib transcription in the pupal wing. Moreover, knockdown of scrib leads to loss of BMP signaling in PCV region but not loss of apical-basal polarity. These facts suggest that upregulation of Scrib is key for optimizing BMP signaling by forming a positive feedback loop (Gui, 2016).

Previous studies indicate that cell competition takes place between scrib clones and the surrounding wild-type tissues in the larval wing imaginal disc. Moreover, cell competition has been documented between loss-of-Dpp signal and the surrounding wild-type tissues. It is presumed that the mechanisms proposed in this study are independent of cell competition for the following reasons. First, scrib RNAi and AP-2μ RNAi data reveal that loss of BMP signal in the PCV region is produced without affecting cell polarity. Thus, cell competition is unlikely to occur in this context. Second, BMP signal is intact in scrib mutant clones of the wing imaginal disc, suggesting that cell competition caused by scrib clones is not a direct cause of loss of BMP signaling in scrib mutant cells (Gui, 2016).

Previous studies established that receptor trafficking plays crucial roles in signal transduction of conserved growth factors, including BMP signaling. Several co-factors have been identified as regulators of BMP receptor trafficking. Some of them down-regulate BMP signaling while others help maintain it. It is proposed that the Scrib-Rab5 system is a flexible module for receptor trafficking and can be utilized for optimizing a signal. During larval wing imaginal disc development, BMP ligands are trafficked through extracellular spaces to form a morphogen gradient. Although previous studies indicate that regulation of receptor trafficking impacts BMP signaling in wing imaginal discs, BMP signaling persists in scrib or dlg1 mutant cells in wing discs. Wing disc cells interpret signaling intensities of a morphogen gradient. In this developmental context, an optimizing mechanism might not be beneficial to the system. In contrast, cells in the PCV region use the system to ensure robust BMP signaling (Gui, 2016).

Taken together, these data reveal that a feedback loop through BMP and Scrib promotes Rab5 endosome-based BMP/Dpp signaling during PCV morphogenesis. Since the components (BMP signaling, the Scrib complex, and Rab5 endosomes) discussed in this work are highly conserved, similar mechanisms may be widely utilized throughout Animalia (Gui, 2016).

The hematopoietic system of Drosophila is a powerful genetic model for studying hematopoiesis, and vesicle trafficking is important for signal transduction during various developmental processes; however, its interaction with hematopoiesis is currently largely unknown. Three endosome markers, Rab5, Rab7, and Rab11, were selected for study that play a key role in membrane trafficking, and it was determined whether they participate in hematopoiesis. Inhibiting Rab5 or Rab11 in hemocytes or the cortical zone (CZ) significantly induced cell overproliferation and lamellocyte formation in circulating hemocytes and lymph glands and disrupted blood cell progenitor maintenance. Lamellocyte formation involves the JNK, Toll, and Ras/EGFR signaling pathways. Notably, lamellocyte formation was also associated with JNK-dependent autophagy. In conclusion, Rab5 and Rab11 were identified as novel regulators of hematopoiesis, and the results advance the understanding of the mechanisms underlying the maintenance of hematopoietic homeostasis as well as the pathology of blood disorders such as leukemia (Yu, 2021).

Mutations in the ER-associated VAPB/ALS8 protein cause amyotrophic lateral sclerosis and spinal muscular atrophy. Previous studies have argued that ER stress may underlie the demise of neurons. This study found that loss of VAP proteins (VAPs) leads to an accumulation of aberrant lysosomes and impairs lysosomal degradation. VAPs mediate ER to Golgi tethering and their loss may affect phosphatidylinositol-4-phosphate (PtdIns4P) transfer between these organelles. Loss of VAPs elevates PtdIns4P levels in the Golgi, leading to an expansion of the endosomal pool derived from the Golgi. Fusion of these endosomes with lysosomes leads to an increase in lysosomes with aberrant acidity, contents, and shape. Importantly, reducing PtdIns4P levels with a PtdIns4-kinase (PtdIns4K) inhibitor, or removing a single copy of Rab7, suppress macroautophagic/autophagic degradation defects as well as behavioral defects observed in Drosophila Vap33 mutant larvae. It is proposed that a failure to tether the ER to the Golgi when VAPs are lost leads to an increase in Golgi PtdIns4P levels, and an expansion of endosomes resulting in an accumulation of dysfunctional lysosomes and a failure in proper autophagic lysosomal degradation (Mao, 2019).

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive motor neuron degeneration and muscle weakness. More than 20 ALS associated genes have been identified and these genes affect distinct cellular pathways including RNA processing, nuclear protein transport, and the unfolded protein response (UPR). One of the key pathological findings is the presence of TARDBP-positive protein aggregates in the cytoplasm of neurons in the brains and spinal cords of patients. Accumulation of protein aggregates in the ER induces a UPR, which attenuates protein translation and promotes proteasome-mediated degradation as well as expression of numerous ER chaperones. Several ALS-causing genes, including VAPB, VCP and UBQLN2, have been documented to play an important role in the ER, and the loss of these proteins promotes the UPR. In addition, ER stress has also been documented in SOD1G93A heterozygous mice. Whether ER stress is toxic or protective is still a matter of debate as some data argue that ER stress may be beneficial whereas other data dispute this. If the observed ER stress is protective, other defects may accelerate the demise of neurons given that defects in proteostasis are tightly linked to ALS (Mao, 2019).

Two major pathways regulate protein clearance: proteasome and autophagy-lysosome mediated degradation. Basal autophagy is required to maintain neuronal function, as loss of autophagy has been shown to induce neurodegeneration. Emerging evidence indicates that 2 genes associated with ALS, including TARDBP and C9orf72, play a role in autophagy but how they achieve this is not well defined (Mao, 2019).

Various mutations (P56S, T46I, A145V, S160Δ, V234I) in the gene encoding the human VAPB protein cause ALS8 (OMIM: 608627), a form of ALS and spinal muscular atrophy. Interestingly, mRNAs of VAPB are decreased in sporadic patients and in neurons derived from ALS8 patients as well as in human SOD1G93A transgenic mice, suggesting that VAPB may play a role in many forms of ALS. The VAPs belong to the VAMP-associated protein family and are highly conserved across species. There are 2 VAP homologs in mammals: VAPA and B (VAPA/B). However, Drosophila has a single VAP, Vap33 which corresponds to VPR-1 in C. elegans. Studies in Drosophila, C. elegans as well as mammalian cells have shown that VAPs (Vap33, VPR-1, VAPA/B) affect multiple cellular processes. These include the size and shape of neuromuscular junctions (NMJ), the presence of a UPR, the transfer of lipids from the ER to the Golgi, mitochondrial calcium homeostasis and muscle mitochondrial dynamics. VAPA and B share an N-terminal major sperm protein (MSP) domain followed by a coiled-coil domain and a C-terminal transmembrane domain that targets the protein to the ER. Previous work documented that Drosophila Vap33 functions in a cell non-autonomous manner by releasing and secreting the MSP domain (Tsuda, 2008). The MSP domain of the human VAPB is also detected in human blood and cerebrospinal fluid (CSF) and the levels of MSP in the CSF is reduced in patients with sporadic ALS, indicating that loss of MSP secretion may be associated with different forms of ALS (Mao, 2019).

In addition to the cell non-autonomous function, VAPB also functions cell autonomously in non-vesicular lipid transfer. VAP proteins directly interact with lipid transport proteins, such as OSBP (oxysterol binding protein) and COL4A3BP/CERT through a FFAT motif (2 phenylalanines in an acidic tract) to facilitate lipid transfer. Both the OSBP and COL4A3BP/CERT proteins contain a pleckstrin homology (PH) domain that interacts with PtdIns4P on the Golgi to promote membrane tethering and lipid transfer from the ER to the Golgi. The VAP-FFAT interaction is abolished in VAPBP56S, the most prevalent variant form of VAPB in ALS8 patients. This P56S variant functions as a loss-of-function mutation in some phenotypic assays and as a dominant-negative mutation as it traps endogenous wild-type VAPA and VAPB proteins in aggregates, resulting in a partial loss of function of both VAPA and VAPB. The tethering of the ER to the Golgi facilitates the transfer of PtdIns4P from the Golgi to the ER for hydrolysis and loss of VAPs affects PtdIns4P levels, including a general increase in the cytoplasm, a decrease in the Golgi, and an increase in endosomes. However, little is known about the role of PtdIns4P in the autophagic-lysosomal degradation pathway (Mao, 2019).

This study provides both in vivo and in vitro evidence that loss of VAPs impairs endo-lysosomal degradation. It was found that loss of VAPs leads to an obvious upregulation of the PtdIns4P levels in the Golgi, and a dramatic increase in the number of endosomes, lysosomes and autophagic vesicles. These compartments are defective because they do not acidify properly. Reducing the PtdIns4P levels significantly suppresses the autophagic and lysosomal defects, suggesting that the VAPs regulate autophagy-lysosomal degradation through a PtdIns4P-mediated endosomal trafficking pathway. Impairing this pathway causes a severe defect in lysosomal degradation that may play a critical role in ALS8 and other forms of ALS (Mao, 2019).

Based on the current studies, a model is proposed. VAP proteins localize to the ER and interact with lipid transfer proteins such as OSBP and COL4A3BP/CERT through their FFAT motif. The PH domains of OSBP and COL4A3BP/CERT interact with PtdIns4P anchored on the Golgi and tether the ER to the Golgi, facilitating PtdIns4P transfer from the Golgi to the ER for its hydrolysis by SACM1L. It is argued that this leads to an accumulation of PtdIns4P in the Golgi and increased production of RAB5- and RAB7-positive endosomes. These endosomes mature into lysosomes leading to an increase in the number of lysosomes with aberrant pH. These defective lysosomes affect protein degradation, and upon fusion with autophagosomes also impair autophagic degradation, resulting in an accumulation of autophagic vesicles (Mao, 2019).

The data argue that the defects in autophagic and lysosomal degradation in VAP mutant cells are due to PtdIns4P imbalance. Indeed, by reducing the PtdIns4P to more normal levels or removing one copy of the endosome proteins Rab5 or Rab7, a significant suppression of endosome and autophagy-lysosomal defects was observed in the Vap33 mutant. Modulating the PtdIns4P and endosome pathway also rescues the locomotion deficit in Vap33 mutant animals, suggesting a strategy to modify the phenotype in patients. At the root of the elevated level of PtdIns4P is the loss of ER-Golgi tethering, as promoting ER-Golgi tethering by overexpression of an OSBP that does not require VAPs significantly suppresses the motor deficit and early lethality of mutant flies (Mao, 2019).

A recent study by Gomez-Suaga (2017) argues that the function of VAPB is to inhibit autophagy by promoting ER-mitochondria tethering. The authors argue that siRNA-mediated knockdown of VAPB in HeLa cells disrupts ER-mitochondria tethering through VAPB and its interaction with RMDN3/PTPIP51. Loss of this interaction promotes autophagy and does not impair degradation. Given that this study observed dysfunctional autophagy in flies and mammalian cells upon loss of the VAPs it is argued that VAPA and VAPB are redundant and that removing VAPB alone appears insufficient to impair lysosomal degradation, but seems sufficient to promote autophagy induction (Mao, 2019).

The combined loss of function of VAPA and B may be relevant to ALS8. Indeed, the most prevalent form of the VAPB mutation found in these patients is the P56S mutation, which functions as a dominant negative allele in some contexts and traps both VAPA and VAPB in aggregates. Hence, reducing VAPA and B may better mimic the conditions of patients with the VAPBP56S mutation. This interpretation is also consistent with the observed accumulation of SQSTM1 in aged heterozygous VAPBP56S knockin mice (Mao, 2019).

The accumulation of lumenal tagged LAMP1-GFP argues that there is a defect in lysosomal acidification upon loss of Vap33. This phenotype needs to be reconciled with the increased LysoTracker Red staining and increased Magic Red CtsB1 staining observed in Vap33 mutant cells. LysoTracker Red is activated at pH = 6.5, a higher pH than what is required to quench GFP, which is 4.5. The lysosomal pH typically ranges from 4.5 to 5.0. Hence, LysoTracker Red should label lysosomes with a pH between 4.5 ~ 6.5, including many that may not be fully functional when VAPs are lost. Similarly, CTSB has been shown to have high proteolytic activity at pH>5. Hence, LAMP1-GFP reveals non-acidified lysosomes, whereas the increased LysoTracker Red and Magic Red CtsB1 staining indicate an expansion of lysosomes that may include acidified as well as poorly acidified lysosomes. However, the current data cannot exclude the possibility that loss of VAPs impairs the trafficking of some lysosomal proteins that are trapped in non-acidic endosomes. This trafficking defect would also result in dysfunctional lysosomes, consistent with the model (Mao, 2019).

The data show that there is an increase in the acidified lysosomal pool when VAPs are lost. Interestingly, lysosomal expansion is also observed in lysosomal storage diseases due to defects in lysosomal degradation. Indeed, lysosomal degradation defects impair the processing of cargo as well as the renewal of the lysosomal compartment, leading to the accumulation of aberrant lysosomes. Furthermore, loss of VAPs results in a significant disruption of the balance of the various hydrolases per lysosome, and are consistent with the lysosomal phenotypes observed in lysosomal storage diseases (Mao, 2019).

The importance of autophagic and lysosomal function in ALS has only recently come into focus. Loss of TARDBP was recently reported to elevate the levels of TFEB and impair the fusion of autophagosomes with lysosomes. Conversely, C9orf72, the most prevalent ALS-causing gene, has been shown to decrease autophagic flux upon its loss, whereas others have argued that loss of C9orf72 promotes autophagy. However, these studies were not performed in cells that carry the G4C2 hexanucleotide expanded repeat, and the role of autophagy in C9orf72 ALS patients therefore remains to be established. The current data in flies and human cells as well as the phenotypes associated with the mice carrying a single P56S mutation argue that autophagic and lysosomal degradation may be impaired in ALS8 patients and that the primary defect is due to the upregulation of PtdIns4P upon loss of the VAP-mediated anchoring of ER to Golgi (Mao, 2019).

Phagophore-derived autophagosomes deliver cytoplasmic material to lysosomes for degradation and reuse. Autophagy mediated by the incompletely characterized actions of Atg proteins is involved in numerous physiological and pathological settings including stress resistance, immunity, aging, cancer, and neurodegenerative diseases. This study characterized tg17/FIP200A, the Drosophila ortholog of mammalian RB1CC1/FIP200, a proposed functional equivalent of yeast Atg17. Atg17 disruption inhibits basal, starvation-induced and developmental autophagy, and interferes with the programmed elimination of larval salivary glands and midgut during metamorphosis. Upon starvation, Atg17-positive structures appear at aggregates of the selective cargo Ref(2)P/p62 near lysosomes. This location may be similar to the perivacuolar PAS (phagophore assembly site) described in yeast. Drosophila Atg17 is a member of the Atg1 kinase complex as in mammals, and it binds to the other subunits including Atg1, Atg13 and Atg101 (C12orf44 in humans, 9430023L20Rik in mice and RGD1359310 in rats). Atg17 is required for the kinase activity of endogenous Atg1 in vivo, as loss of Atg17 prevents the Atg1-dependent shift of endogenous Atg13 to hyperphosphorylated forms, and also blocks punctate Atg1 localization during starvation. Finally, it was found that Atg1 overexpression induces autophagy and reduces cell size in Atg17-null mutant fat body cells, and that overexpression of Atg17 promotes endogenous Atg13 phosphorylation and enhances autophagy in an Atg1-dependent manner in the fat body. A model is proposed according to which the relative activity of Atg1, estimated by the ratio of hyper- to hypophosphorylated Atg13, contributes to setting low (basal) vs. high (starvation-induced) autophagy levels in Drosophila (Nagy, 2014).

Almost all animals contain mitochondria of maternal origin only, but the exact mechanisms underlying this phenomenon are still vague. This stuy investigated the fate of Drosophila paternal mitochondria after fertilization. The sperm mitochondrial derivative (MD) is rapidly eliminated in a stereotypical process dubbed paternal mitochondrial destruction (PMD). PMD is initiated by a network of vesicles resembling multivesicular bodies and displaying common features of the endocytic and autophagic pathways. These vesicles associate with the sperm tail and mediate the disintegration of its plasma membrane. Subsequently, the MD separates from the axoneme and breaks into smaller fragments, which are then sequestered by autophagosomes for degradation in lysosomes. Evidence is provided for the involvement of the ubiquitin pathway and the autophagy receptor p62 in this process. Finally, it was shown that the ubiquitin ligase Parkin is not involved in PMD, implying a divergence from the autophagic pathway of damaged mitochondria (Politi, 2014).

Fast axonal transport of neuropeptide-containing dense core vesicles (DCVs), endolysosomal organelles, and presynaptic components is critical for maintaining neuronal functionality. How the transport of DCVs is orchestrated remains an important unresolved question. The small GTPase Rab2 mediates DCV biogenesis and endosome-lysosome fusion. This study used Drosophila to demonstrate that Rab2 also plays a critical role in bidirectional axonal transport of DCVs, endosomes, and lysosomal organelles, most likely by controlling molecular motors. It was further shown that the lysosomal motility factor Arl8 is required as well for axonal transport of DCVs, but unlike Rab2, it is also critical for DCV exit from cell bodies into axons. Evidence is provided that the upstream regulators of Rab2 and Arl8, Ema and BORC, activate these GTPases during DCV transport. These results uncover the mechanisms underlying axonal transport of DCVs and reveal surprising parallels between the regulation of DCV and lysosomal motility (Lund, 2021).

Recent experimental observations have shown evidence of an unexpected sudden drop-off in the dense core vesicles (DCVs) content at the ends of certain types of axon endings. A mathematical model was developed that is based on the conservation of captured and transiting DCVs in boutons. The model consists of 77 ordinary differential equations and is solved using a standard Matlab solver. It was hypothesized that the drop in DCV content in distal boutons is due to an insufficient supply of anterogradely moving DCVs coming from the soma. This hypothesis was tested by modifying the flux of DCVs entering the terminal, and it was found that the number of most distal boutons left unfilled increases if the DCV flux entering the terminal is decreased. The number of anterogradely moving DCVs in the axon can be increased either by the release of a portion of captured DCVs into the anterograde component or by an increase of the anterograde DCV flux into the terminal. This increase could lead to having enough anterogradely moving DCVs such that they could reach the most distal bouton and then turn around by changing molecular motors that propel them. The model suggests that this could result in an increased concentration of resident DCVs in distal boutons beginning with bouton 2 (the most distal is bouton 1). This is because in distal boutons, DCVs have a larger chance to be captured from the transiting state as they pass the boutons moving anterogradely and then again as they pass the same boutons moving retrogradely (Kuznetsov, 2021).

Adult stem cells are critical for the maintenance of residential tissue homeostasis and functions. However, the roles of cellular protein homeostasis maintenance in stem cell proliferation and tissue homeostasis are not fully understood. This study found that Derlin-1 and TER94/VCP/p97, components of the ER-associated degradation (ERAD) pathway, restrain intestinal stem cell proliferation to maintain intestinal homeostasis in adult Drosophila. Depleting any of them results in increased stem cell proliferation and midgut homeostasis disruption. Derlin-1 is specifically expressed in the ER of progenitors and its C-terminus is required for its function. Interestingly, it was found that increased stem cell proliferation is resulted from elevated ROS levels and activated JNK signaling in Derlin-1- or TER94-deficient progenitors. Further removal of ROS or inhibition of JNK signaling almost completely suppressed increased stem cell proliferation. Together, these data demonstrate that the ERAD pathway is critical for stem cell proliferation and tissue homeostasis. Thus this study provides insights into understanding of the mechanisms underlying cellular protein homeostasis maintenance (ER protein quality control) in tissue homeostasis and tumor development (Liu, 2021).

Lysosomal degradation, the common destination of autophagy and endocytosis, is one of the most important elements of eukaryotic metabolism. The small GTPases Rab39A and B are potential new effectors of this pathway, as their malfunction is implicated in severe human diseases like cancer and neurodegeneration. In this study, the lysosomal regulatory role of the single Drosophila Rab39 ortholog was characterized, providing valuable insight into the potential cell biological mechanisms mediated by these proteins. Using a de novo CRISPR-generated rab39 mutant, no failure was found in the early steps of endocytosis and autophagy. On the contrary, Rab39 mutant nephrocytes internalize and degrade endocytic cargo at a higher rate compared to control cells. In addition, Rab39 mutant fat body cells contain small yet functional autolysosomes without lysosomal fusion defect. These data identify Drosophila Rab39 as a negative regulator of lysosomal clearance during both endocytosis and autophagy (Lakatos, 2021).

Chronic exercise is widely recognized as an important contributor to healthspan in humans and in diverse animal models. Sestrins, a family of evolutionarily conserved exercise-inducible proteins, are critical mediators of exercise benefits in flies and mice. Knockout of Sestrins prevents exercise adaptations to endurance and flight in Drosophila, and similarly prevents benefits to endurance and metabolism in exercising mice. In contrast, overexpression of dSestrin in muscle mimics several of the molecular and physiological adaptations characteristic of endurance exercise. This study extends those observations to examine the impact of dSestrin on preserving speed and increasing lysosomal activity. dSestrin was found to be a critical factor driving exercise adaptations to climbing speed, but is not absolutely required for exercise to increase lysosomal activity in Drosophila. The role of Sestrin in increasing speed during chronic exercise requires both the TORC2/AKT axis and the PGC1α homolog spargel, while dSestrin requires interactions with TORC1 to cell-autonomously increase lysosomal activity. These results highlight the conserved role of Sestrins as key factors that drive diverse physiological adaptations conferred by chronic exercise (Sujkowski, 2021).

Neuronal target recognition is performed by numerous cell-surface transmembrane proteins. Correct folding of these proteins occurs in the endoplasmic reticulum (ER) lumen of the neuronal cells before being transported to the plasma membrane of axons or dendrites. Disturbance in this protein folding process in the ER leads to dysfunction of neuronal cell surface molecules, resulting in abnormal neuronal targeting. This study reports that the ER-resident protein Meigo in Drosophila, governs the dendrite targeting of olfactory projection neurons (PNs) along the mediolateral axis of the antennal lobe by regulating Toll-6 localization. Loss of Meigo causes Toll-6 mislocalization in the PNs and mediolateral dendrite targeting defects, which are suppressed by Toll-6 overexpression. Furthermore, this study found that the ER-chaperone protein, Gp93, also regulates the mediolateral targeting of PN dendrites by localization of the Toll-6 protein. Gp93 overexpression in the PN homozygous for the meigo mutation, partially rescued the dendrite targeting defect, while meigo knockdown decreased Gp93 expression levels in cultured cells. These results indicate that the ER-proteins Meigo and Gp93 regulate dendrite targeting by attenuating the amount and localization of cell surface receptors, including Toll-6, implying the unexpected but active involvement of ER proteins in neural wiring (Kamemura, 2022).

Warburg micro syndrome (WMS) is a hereditary autosomal neuromuscular disorder in humans caused by mutations in Rab18, Rab3GAP1, or Rab3GAP2 genes. Rab3GAP1/2 forms a heterodimeric complex, which acts as a guanosine nucleotide exchange factor and activates Rab18. Although the genetic causes of WMS are known, it is still unclear whether loss of the Rab3GAP-Rab18 module affects neuronal or muscle cell physiology or both, and how. This work characterized a Rab3GAP2 mutant Drosophila line to establish a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. These mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compa a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. These mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compartments and perturbation of lysosomal biosynthetic transport. Importantly, overexpression of UVRAG or loss of Atg14, two alternative subunits of the Vps34/PI3K (vacuole protein sorting 34/phosphatidylinositol 3-kinase) complexes in fat cells, mimics the autophagic phenotype of Rab3GAP-Rab18 module loss. This study finds that GTP-bound Rab18 binds to p62 (refractory to sigma P) and Atg8a was not apparent in Rab3GAP2 mutant head lysates was surprising; but, it is possible that only a subset of neurons is defective for autophagy in these flies, which this study failed to detect. Although studies in mammalian WMS models are focusing on neuronal defects, the data suggest that it may be worth investigating other tissues of WMS patients as well (Takats, 2020).

For characterizing the role of the Rab3GAP-Rab18 module in autophagy, the genetically mosaic fat tissue of L3 larvae, a well-established system for autophagy analysis in Drosophila, was used. By analyzing Rab3GAP2 mutants and multiple independent RNAi lines, this study shows that the loss-of-function cells are less effective in autophagosome–lysosome fusion and are defective in autolysosome morphology and maturation. Additionally, it was found that the lack of Rab3GAP2 function causes striking perturbation of Rab7-positive late endosomes, autophagosomes, and (auto)lysosomes, but it does not affect Rab5-positive early endosomes. Thus, the data suggest that the Rab3GAP-Rab18 module can be considered as a general regulator of lysosome maturation. These results fit well with the findings of a recently published paper showing that Rab18 acts in concert with Rab7 during the lysosomal fusion events of autophagy and in the axonal transport of lysosomes. On the other hand, previous studies in C. elegans and cultured human cells suggested that the Rab3GAP subunits and Rab18 are rather involved in early steps of autophagy; however, it is important to note that these latter studies focused only on the amount and localization patterns of the autophagy markers Atg8 and p62, with no particular emphasis on ultrastructural analysis of the integrity of the lysosomal system. Electron microscopy observations concerning that numerous autophagosomes and autophagosome clusters are present in the cytoplasm of Rab3GAP2 mutant fat cells further suggest that loss of the Rab3GAP-Rab18 module causes major defects in (auto)lysosome function rather than in autophagosome formation. Of course, the possibility that the discrepancies between these studies arise due to a tissue-specific role of the Rab3GAP-Rab18 module in autophagy cannot be ruled out. The finding that Rab3GAP2 mutant adults obviously accumulate much more autophagy cargos in muscles than in their brain further corroborates this notion (Takats, 2020).

Since this study demonstrated that loss of Vps34 Complex I function results in phenotypes similar to those of inhibition of the Rab3GAP-Rab18 module and, furthermore, a physical interaction between the permanent Vps34 complex subunit Atg6 and the GTP-bound Rab18 was proven, it is proposed that Complex I is likely a novel Rab18 effector (Takats, 2020).

Vps34 Complex I is one of the most important regulators of autophagosome formation, and it also has a role in autophagosome maturation and fusion [(Diao, 2015). As matured, intact autophagosomes in Rab3GAP2 mutant cells were detected, it seems likely that Rab18 is critical for Vps34 Complex I activity following autophagosome formation. Localization of Rab3GAP subunits and Rab18 to autophagosomes also suggests that this module plays a role in later steps of autophagosome maturation: It facilitates their fusion with lysosomes and further enhances the maturation of the newly formed autolysosomes into enlarged degradative compartments (Takats, 2020).

The question of how could the autophagosome-localized Rab3GAP-Rab18 module affect vesicle maturation is yet to be answered. Based on the current results showing that Rab7 becomes dispersed in cells lacking the Rab3GAP-Rab18 module, it is suggested that the most important role of this module is to stabilize the Rab7-containing compartment. During their maturation, (auto)lysosomes undergo a series of membrane fusion events with endosomes, Golgi-derived vesicles, and autophagosomes. All these steps are mediated by Rab7 and its effectors such as the tethering factor HOPS complex or the adaptor protein PLEKHM1. As matured autophagosomes are also positive for Rab7, the autophagy-derived Rab7 proteins can be an important source for the lysosomal Rab7 pool. This scenario is even more likely in cell types such as fat cells, which show relatively low endocytic but high autophagic activity. Still, it cannot be ruled out that the Rab3GAP-Rab18 complex is also present on maturing endosomes or Golgi-derived transport vesicles that may also act as important Rab7 sources for maturing lysosomes. The precise contribution of these membrane transport pathways to maintaining the lysosomal Rab7 pool needs to be further investigated in the future (Takats, 2020).

This research highlights that the Rab3GAP-Rab18 module, in concert with the activity of the Vps34 Complex I, maintains the integrity of the Rab7-positive late endosomal/lysosomal compartment. Additionally, these findings that loss of Rab3GAP-Rab18 function perturbs autolysosome maturation and autophagic degradation shed light on a new possible cause of WMS development and open up potential novel therapeutic perspectives for this disease (Takats, 2020).

Autophagy is mediated by membrane-bound organelles and it is an intrinsic catabolic and recycling process of the cell, which is very important for the health of organisms. The biogenesis of autophagic membranes is still incompletely understood. In vitro studies suggest that Atg2 protein transports lipids presumably from the ER to the expanding autophagic structures. Autophagy research has focused heavily on proteins and very little is known about the lipid composition of autophagic membranes. This study describes a method for immunopurification of autophagic structures from Drosophila melanogaster (an excellent model to study autophagy in a complete organism) for subsequent lipidomic analysis. Western blots of several organelle markers indicate the high purity of the isolated autophagic vesicles, visualized by various microscopy techniques. Mass spectrometry results show that phosphatidylethanolamine (PE) is the dominant lipid class in wild type (control) membranes. In Atg2 mutants (Atg2^-), phosphatidylinositol (PI), negatively charged phosphatidylserine (PS), and phosphatidic acid (PA) with longer fatty acyl chains accumulate on stalled, negatively charged phagophores. Tandem mass spectrometry analysis of lipid species composing the lipid classes reveal the enrichment of unsaturated PE and phosphatidylcholine (PC) in controls versus PI, PS and PA species in Atg2^-. Significant differences in the lipid profiles of control and Atg2^- flies suggest that the lipid composition of autophagic membranes dynamically changes during their maturation. These lipidomic results also point to the in vivo lipid transport function of the Atg2 protein, pointing to its specific role in the transport of short fatty acyl chain PE species (Laczko-Dobos, 2021).

Macroautophagy (autophagy hereafter) is a 'self-eating' process of eukaryotic cells, during which damaged or obsolete cytoplasmic components (organelles, proteins, lipids, sugars etc.) are removed and degraded in lysosomes\. Misregulation of autophagy contributes to neurodegenarative diseases, cancer, accelerated aging, etc. This fundamental catabolic process relies on the biogenesis of unique, very dynamic membranes and membrane-bound autophagic vesicles. The pathway initiates with the nucleation of a double-membrane structure called phagophore (formerly also known as isolation membrane), which expands and engulfs a portion of the cytoplasm. After closure and sealing, it will form the autophagosome, a double membrane vesicle. In the last step, autophagosomes will fuse with lysosomes to form autolysosomes, where the sequestered cytoplasmic material will be degraded and recycled back to the cytosol (Laczko-Dobos, 2021).

Fluorescence and electron microscopy are widely used to study these specific organelles. Autophagic structures can be isolated for example from human cell lines, yeast, mouse and rat tissues by applying subcellular fractionation, immunoprecipitation or combination of these two methods. Fruit flies are an excellent model organism to study autophagy in a complete animal as they can be genetically manipulated very easily, and about 75% of their genes show homology with disease associated human genes, so they can serve as models for various human diseases (Laczko-Dobos, 2021).

Autophagosomes are unique organelles regarding their lipid and protein composition, morphology and biogenesis. Autophagosome formation occurs very rapidly upon induction of autophagy, which requires a tremendous amount of membrane source(s). De novo synthesis of autophagic membranes is the most enigmatic field of autophagy; almost every compartment of the endomembrane system has been implicated in this process. Interestingly, also the contact sites between organelles such as ER and mitochondria may play an important role in this biogenesis event. During phagophore expansion, the synthesis or delivery of lipids must be distinctly controlled. Formation of these specific membranes relies on a collaborative work between proteins encoded by autophagy-related (Atg) genes and membrane lipids (Laczko-Dobos, 2021).

It has been shown recently that Atg2 protein is not only a potential tether between ER and phagophores, but it may also transport lipids from the ER to promote autophagosome biogenesis (Osawa, 2019a; Osawa, 2019b). Several in vitro studies on yeast (and human) Atg2 (Atg2A and B) showed the lipid transport activity of this special protein, which is able to transport several lipids at once using its hydrophobic cavity (Osawa, 2019b; Valverde, 2019; Maeda, 2019; Osawa, 2020). A very recent discovery is that the fast expansion of autophagic membranes after autophagy induction relies on localized 'on-demand' de novo phospholipid synthesis, by the aid of Acyl-CoA synthetase Faa1 enzymes identified on nucleated phagophores in yeast, and the Atg2-Atg18 protein complex may also be involved in this process. The P-element induced Drosophila mutant (Atg2EP3697), bearing the transposon insertion at the 5' non-translated region of the Atg2 gene that resulted in a strong hypomorphic allele, showed an autophagy defect similar to mammalian and C. elegans Atg2 mutants [26,27]. Microscopy and biochemical investigations support that loss of Atg2 protein function in Drosophila, worms, and also in mammalian cells causes a sealing defect of phagophores, leading to accumulation of enlarged phagophore-like structures. Other key players of autophagy are Atg8 proteins together with their lipid conjugation (including the E3-like Atg5-Atg12-Atg16 complex) and deconjugation machinery. They have important roles in the biogenesis of autophagic membranes, and they are reversebly conjugated to the PE head groups present in the membranes of autophagic structures. The two distinct forms of Atg8 proteins: the non-lipidated Atg8 (Atg8-I) and lipidated Atg8 (Atg8-II) are the most widely used autophagic markers, including Atg8a in Drosophila (Laczko-Dobos, 2021).

Lipids are largely unexplored players of the phagophore biogenesis machinery. The major structural lipids in eukaryotic membranes are glycerophospholipids, sphingolipids and sterols. The most abundant glycerophospholipid classes of Drosophila are: phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and less abundant lipid classes are phosphatidylglycerol (PG) and phosphatidic acid (PA). PC accounts for more than 50% of the phospholipids in most eukaryotic membranes. Interestingly, in Drosophila, PE is the most abundant structural lipid and also a component of the lipoproteins (like Atg8), in contrast with more PC-centric lipidome of mammalian cells. These main lipid classes differ in the chemical composition of their head groups and in length and saturation level of their fatty acyl chains. This determines the shape and physicochemical behavior of these lipid molecules, such as phase transition properties and thickness of the membranes. All these properties influence membrane curvature and fusion events (Laczko-Dobos, 2021).

Lipids are not only structural components of the membranes but they also play a regulatory role in cellular processes, such as autophagy. Inactivation of Desat1 (desaturase coding gene, responsible for double bond generation) in Drosophila resulted in an autophagic defect: autolysosomes could not form properly. Glycerolipids play important roles in the initiation of autophagy, elongation of phagophores, autophagosome maturation as well as in autophagosome-lysosome fusion. Different phosphorylated forms of PI including PI3P, PI4P, PI(4,5)P2, PI(3,5)P2 are also found in autophagic membranes, as different types of kinases present at these membranes are responsible for their generation. Although they are minor lipids (representing less than 1% of total lipids), together with PE they play important roles in autophagosome biogenesis by influencing the recruitment of specific proteins to the membrane. Interestingly, phosphatidic acid (PA) molecules may directly affect the physicochemical properties of lipid bilayers independently of protein effectors (Laczko-Dobos, 2021).

This study established a method for isolating autophagic structures from adult Drosophila melanogaster, which was optimized for subsequent lipidomic investigations. Deciphering the lipid composition of autophagic membranes is crucial to fully understand the mechanism of autophagy. Using powerful Drosophila genetics and the advantage of high-throughput mass spectrometry, this study points to the important in vivo lipid transport function of Atg2 protein (Laczko-Dobos, 2021).

Two related multisubunit tethering complexes promote endolysosomal trafficking in all eukaryotes: Rab5-binding class C core vacuole/endosome tethering (CORVET) that was suggested to transform into Rab7-binding homotypic fusion and vacuolar protein sorting (HOPS). Previous work has identified miniCORVET, containing Drosophila Vps8 and three shared core proteins that are required for endosome maturation upstream of HOPS in highly endocytic cells. This study shows that Vps8 overexpression inhibits HOPS-dependent trafficking routes including late endosome maturation, autophagosome-lysosome fusion, crinophagy and lysosome-related organelle formation. Mechanistically, Vps8 overexpression abolishes the late endosomal localization of HOPS-specific Vps41/Light and prevents HOPS assembly. Proper ratio of Vps8 to Vps41 is thus critical because Vps8 negatively regulates HOPS by outcompeting Vps41. Endosomal recruitment of miniCORVET- or HOPS-specific subunits requires proper complex assembly, and Vps8/miniCORVET is dispensable for autophagy, crinophagy and lysosomal biogenesis. These data together indicate the recruitment of these complexes to target membranes independent of each other in Drosophila, rather than their transformation during vesicle maturation (Lorincz, 2019).

Lysosomal degradation is essential for the survival and homeostasis of eukaryotic cells. The two main routes of lysosomal degradation are endocytosis and autophagy, and HOPS (homotypic fusion and vacuole protein sorting) tethering complex is a central player in both processes. HOPS was identified in yeast and is defined by two Ypt7 (Rab7 in higher eukaryotes) binding subunits Vps41 and Vps39 on its opposing ends. In metazoan cells including Drosophila, HOPS directly binds to Rab2 and Rab7-binding adaptors to ensure fusions of lysosomes with autophagosomes, late endosomes, secretory granules and Golgi derived vesicles (Lorincz, 2019 and references therein).

A closely related multisubunit complex termed CORVET (Class C core endosome vacuole tethering) mediates the tethering and fusions of Vps21 (Rab5 in higher eukaryotes) positive membranes. Both CORVET and HOPS share a common core of class C Vps proteins (Vps11, Vps16, Vps18 and Vps33), but in the former complex two Vps21 (Rab5 in higher eukaryotes) binding subunits: Vps8 and Vps3 are present instead of the Ypt7/Rab7 binding Vps41 and Vps39, respectively. Whilst HOPS is conserved across metazoans, higher eukaryotes lack Vps3, which is therefore yeast-specific. Mammalian CORVET contains Vps39-2 (also known as Tgfbrap1 or Trap1) in the place of Vps3, and Vps8 is conserved. Drosophila has a smaller CORVET variant termed miniCORVET, containing Vps8 and only three of the four class C Vps proteins (Dor/Vps18, Car/Vps33A and Vps16A). Thus, Vps11 is a HOPS specific protein in flies (Lorincz, 2016a; Lorincz, 2019).

Although CORVET and HOPS complexes share common subunits, the question whether these complexes assemble de novo or they can be converted into each other is still open. In yeast, a series of biochemical experiments on overexpressed complex specific subunits suggested the existence of intermediate complexes that contain one CORVET and one HOPS specific Vps protein. Moreover, overexpression of CORVET specific subunits can disturb endosome maturation and Vps3 can displace Vps39 from HOPS, potentially as a result of competition between complex specific subunits. These results raise the possibility that during assembly, complex-specific proteins may compete for class C proteins in yeast (Lorincz, 2019).

Previous work has shown that Drosophila is an excellent model to study miniCORVET and HOPS mediated vesicular trafficking processes, including endosome maturation in nephrocytes, autophagosome-lysosome fusion in fat cells, crinophagy in salivary glands and eye pigment granule biogenesis. This study aimed to answer the question whether the overexpression of CORVET-specific Vps8 or its HOPS-specific counterpart Vps41 could affect HOPS or CORVET dependent processes, respectively, and if so how (Lorincz, 2019).

Through a series of confocal and electron microscopy experiments, this study has shown that overexpression of Vps8 inhibits HOPS dependent trafficking, such as late endosome maturation in nephrocytes, autophagosome-lysosome fusion in fat cells, crinophagy in salivary glands and pigment granule biogenesis in eyes. Similar to the loss of HOPS, class C Vps core proteins or selected small GTPases, the late endosomal localization of Vps41 is lost in Vps8 overexpressing cells. Based on co-immunoprecipitation data, this study shows that the amount of HOPS decreases in Vps8 overexpressing animals, suggesting that Vps8 may negatively regulate HOPS by outcompeting Vps41. Since yeast Vps8 was suggested to be involved in autophagosome formation the possible function of miniCORVET was also examined, but this feature of CORVET is not conserved (Lorincz, 2019).

The complex functions of cellular membranes, and thus overall cell physiology, depend on the distribution of crucial lipid species. Sac1 is an essential, conserved, ER-localized phosphatase whose substrate, phosphatidylinositol 4-phosphate (PI4P), coordinates secretory trafficking and plasma membrane function. PI4P from multiple pools is delivered to Sac1 by oxysterol binding protein and related proteins in exchange for other lipids and sterols, which places Sac1 at the intersection of multiple lipid distribution pathways. However, much remains unknown about the roles of Sac1 in subcellular homeostasis and organismal development. Using a temperature-sensitive allele (Sac1^ts), this study shows that Sac1 is required for structural integrity of the Drosophila retinal floor. The βps-integrin Myospheroid, which is necessary for basal cell adhesion, is mislocalized in Sac1(ts) retinas. In addition, the adhesion proteins Roughest and Kirre, which coordinate apical retinal cell patterning at an earlier stage, accumulate within Sac1(ts) retinal cells due to impaired endo-lysosomal degradation. Moreover, Sac1 is required for ER homeostasis in Drosophila retinal cells. Together, these data illustrate the importance of Sac1 in regulating multiple aspects of cellular homeostasis during tissue development (Griffiths, 2020).

Although they comprise a minor fraction of total cellular phospholipid content, phosphoinositides, also known as phosphatidylinositol phosphates (PIPs), act as essential coordinators of membrane function and identity (Balla, 2013). PIPs are derived from the precursor phosphatidylinositol, whose inositol head group can be phosphorylated at any of three positions to yield seven unique PIP species that recruit distinct sets of effector proteins. Through the localized activity of PIP kinases and phosphatases, these species are interconverted to maintain enrichment in different membranes and to regulate numerous PIP effector-driven processes (Balla, 2013; Griffiths, 2020).

Sac1 is a conserved phosphatase whose substrate, phosphatidylinositol 4-phosphate (PI4P), coordinates multiple stages in secretory trafficking, participates in cellular signaling pathways, and acts as the precursor for PI(4,5)P2 at the plasma membrane (PM). PI4P is produced in the PM and Golgi, respectively, by two conserved type III PI 4-kinases (PI4Ks), PI4KIIIα, and PI4KIIIβ. In addition, a type II PI4K (PI4KIIα) produces PI4P in the trans-Golgi network (TGN) and on endosomes, where it is important for endosomal trafficking (Griffiths, 2020).

In contrast to the distribution of PI4Ks and PI4P, Sac1 localizes primarily to the ER, as well as the cis-Golgi under growth-limiting conditions. Although seemingly capable of acting in trans on PI4P in neighboring membranes in some scenarios, Sac1 appears to predominantly depend on delivery of PI4P to the ER via nonvesicular lipid transport at membrane contact sites (MCS). For instance, oxysterol-binding protein (OSBP), which localizes to ER-trans-Golgi MCS through interactions with the ER-resident vesicle-associated membrane protein-associated protein VAP as well as PI4P in the trans-Golgi, delivers PI4P from the trans-Golgi to the ER in exchange for sterols. Hydrolysis of incoming PI4P by Sac1 maintains a low concentration of PI4P in the ER that is necessary for sustained PI4P/sterol countertransport in vitro, although this relationship appears more nuanced in vivo. OSBP-related proteins (ORPs), which are encoded by 11 genes in humans and three in flies, function similarly to OSBP but differ in their localization and lipid-binding preferences. Despite its essential function, how Sac1 regulates different aspects of cellular homeostasis during animal development is not fully understood (Griffiths, 2020).

In Drosophila, null Sac1 mutants exhibit embryonic lethality due to defects in cell shape and ectopically activated JNK signaling that prevent dorsal closure. JNK signaling defects are also observed in Sac1 clones in larval imaginal discs. Moreover, Sac1 regulates Hedgehog signaling by inhibiting recruitment and activation of Smoothened at the PM in a PI4P-dependent manner (Yavari, 2010; Jiang, 2016). Sac1 is also required for axonal pathfinding in the embryonic central nervous system, as well as for axonal transport and synaptogenesis in larval neurons (Lee, 2011; Forrest, 2013; Griffiths, 2020).

In addition, loss of Sac1 causes severe tissue disorganization and degeneration during eye development. The Drosophila eye is composed of ~750 unit eyes called ommatidia. Presumptive ommatidia arise early in pupal development, where they initially comprise clusters of medial/basal photoreceptors and apical cone cells surrounded by a disordered pool of undifferentiated interommatidial cells (IOCs). During the first half of the ∼96-h pupal stage, two IOCs per ommatidium differentiate into primary pigment cells (1°pc), which encircle the cone cells. The remaining IOCs subsequently differentiate into a lattice of secondary and tertiary pc (2°/3°pc) and sensory bristles that separate neighboring ommatidia or are removed by apoptosis by 42 h after puparium formation (APF). Changes in IOC shape and position during this stage require the Irre cell recognition module (IRM) adhesion proteins Roughest (Rst) and Hibris (Hbs), as well as their paralogues Kirre and Sticks and stones (Sns). Rst/Kirre and Hbs/Sns are orthologues of mammalian Neph1 and nephrin, which are needed for formation of the renal slit diaphragm as well as during myoblast fusion. After IOC patterning, during late stages of pupal eye development (42-96 h APF), the retina elongates fivefold, and laminated corneal lenses with underlying gelatinous pseudocones are secreted, giving the eye its characteristic adult appearance (Griffiths, 2020).

Previously work has examined the role of Sac1 in the developing Drosophila eye using a hypomorphic Sac1 allele that is temperature sensitive (Sac1^ts). Sac1^ts flies develop morphologically normal eyes when reared at 18°C, but display a rough eye phenotype caused by defective IOC sorting when reared at or above 23.5°C. This study shows that Sac1^ts eyes exhibit structural defects at the retinal floor and mislocalization of the βps-integrin Myospheroid (Mys), which is required for retinal floor adhesion. This defect is not due to a loss of cell polarity, as apical adherens junctions are unaffected. However, a novel secondary defect was identified in the distribution of Rst and Kirre, which are apical transmembrane proteins. At 42 h APF, Sac1^ts 2°/3°pc contain an excess of intracellular Rst and Kirre due to impaired endo-lysosomal trafficking and degradation. Sac1^ts 2°/3°pc also accumulate PI4P and F-actin on enlarged, basal endosomes and exhibit ER stress. Thus, this study has identified novel roles for Sac1 in regulating cellular homeostasis during tissue morphogenesis (Griffiths, 2020).

The Drosophila pupal eye represents a powerful system to examine protein trafficking and turnover. Patterning of retinal cells requires spatially and temporally regulated expression as well as correct subcellular distribution of cell surface proteins that mediate cell-cell contacts and determine tissue architecture. Dysregulation of these processes can produce structural defects, which frequently persist in the adult eye. This study has taken advantage of these circumstances to demonstrate the importance of Sac1 in basal delivery of the βps-integrin Mys, which is required for retinal floor integrity, as well as endo-lysosomal regulation and turnover of the apical patterning determinants Rst and Kirre. The results also highlight the importance of Drosophila Sac1 in ER homeostasis, as had been reported in yeast. This could be due to deregulation of PI4P, phosphatidylserine, and sterol levels, which would be expected to disrupt ER membrane charge and lipid order (Griffiths, 2020).

Given the similarities between mys mutants and Sac1^ts, loss of Mys at the basal grommets in Sac1^ts likely causes the retinal floor defects observed in the adult eye. In addition, this phenotype resembles the basal retinal degeneration observed in an ALS-associated vap mutant, suggesting the underlying cause could be similar. However, it is unclear why basal distribution of Mys is perturbed while apical polarity is not. In the Drosophila follicular epithelium, Rab10 activity has been shown to be important for the distribution of basement membrane proteins independent of overall apical-basal polarity, in a manner dependent on PI(4,5)P2 at the apical PM. Previously study observed a decrease in apical PI(4,5)P2 abundance in Sac1^ts retinas at 24 h APF (Del Bel, 2018), which it is speculated could perturb basal trafficking. Alternatively, aberrant distribution of basal F-actin in Sac1^ts could inhibit localization of Mys to the grommets. Why some transmembrane proteins are sensitive to reduced Sac1 activity while others are not remains an open question. It is also unclear whether Mys mislocalization is linked to endosome dysfunction in Sac1^ts (Griffiths, 2020).

Whereas PI3P and PI(3,5)P2 are the canonical phosphoinositide regulators of endosomal progression (Wallroth, 2018), PI4P production has also emerged as an important factor in cargo delivery to lysosomes. In mammalian cells, PI4P is generated on late endosomes by type II PI4Ks (Baba, 2019). PI4KIIα is important for Golgi-to-lysosome trafficking of LIMP-2, as well as PM-to-lysosome trafficking of LAMP-1, and these proteins accumulate in enlarged endosomes when PI4KIIα levels are reduced. Furthermore, in macrophages, PI4KIIα-mediated PI4P enrichment on phagosomes occurs concurrently with Rab7 recruitment and is necessary for phagosome acidification and subsequent fusion with lysosomes (Griffiths, 2020).

This study has shown that Sac1-dependent depletion of PI4P is also important for endosomal trafficking and degradation of transmembrane proteins from the PM. This is consistent with a recent report by Mao and colleagues (Mao, 2019), who found that in multiple larval Drosophila tissues, loss of VAP, which recruits OSBP and a subset of ORPs to MCS, increases endosomal PI4P levels and inhibits autophagic degradation. Null vap mutants exhibit decreased lysosomal acidification, as well as an increase in the abundance of lysosomes, endosomes, autolysosomes, autophagosomes, and Ref(2)P (Mao, 2019). The authors propose that increased PI4P abundance up-regulates endosome formation and progression, which causes lysosomes to become oversaturated with incoming cargo. Indeed, loss of Ubiquilin, which contributes to lysosome acidification, also delays autophagy and causes Ref(2)P buildup. Notably, this study observed increased Ref(2)P abundance in Sac1^ts retinas, which suggests a similar delay in autophagy. It is a compelling notion that increased PI4P levels in Sac1^ts could promote excessive fusion of endosomes with lysosomes, which would replicate the effect described by Mao (2019). However, the accumulation of Rst and Kirre in Sac1^ts, which do not appear to be concentrated in lysosomes based on the lack of colocalization between Rst and Arl8, could also be caused by impaired endosomal progression or maturation, though this might stem from downstream lysosomal dysfunction. Indeed, the enlarged endosomes observed in Sac1^ts lacked both Vps16a and Arl8, suggesting they were not caused by excessive fusion with lysosomes. Further analysis of PI4P in endosomal dynamics and maturation is warranted to determine the precise role of Sac1 in late stages of protein degradation (Griffiths, 2020).

This study also found that reduced Sac1 function leads to basal accumulation of F-actin-positive enlarged endosomes. In mammalian cells, loss of both VAP isoforms has been shown to induce F-actin comet formation on endosomes via PI4P-dependent recruitment of the WASH-ARP2/3 complex. Notably, these do not resemble the more uniform F-actin coating on Sac1^ts endosomes. Rather, the structures that were observed appear more reminiscent of a phenomenon termed actin-flashing, wherein phagosomes become coated in F-actin by WASP-ARP2/3 to delay fusion with lysosomes. Endosomal phenotypes similar to those in Sac1^ts have also been observed when Arf6 activity is perturbed; increased Arf6 activity activates PIP5K, which has been shown to produce PI(4,5)P2 on endosomes and lead to F-actin polymerization via WASP, whereas loss of Arf6 increases endosomal PI4P levels and perturbs endosomal recycling. Intriguingly, in Caenorhabditis elegans, Sac1 inhibits Arf6 by sequestering the Arf6-GEF Bris-1. However, it is unknown whether this interaction is conserved or, more broadly, how Sac1 influences F-actin polymerization on endosomes (Griffiths, 2020).

It is noteworthy that enlarged endosomes were restricted to basal regions in Sac1^ts. Positioning of endosomes and lysosomes is mediated by bidirectional transport along microtubules, which influences their acidity and function. In mammalian cells, Rab7 recruits RILP, which activates endosomal dynein motors to promote minus end-directed transport toward perinuclear microtubule organizing centers. PI4P is also required for RILP recruitment, which implies that excess PI4P could lead to perinuclear endosome accumulation. Although the single Drosophila RILP orthologue has been shown to bind Arl8 rather than Rab7, it is possible that PI4P influences late endosome transport through analogous Rab7 effectors. Additionally, previous work has shown that Sac1^ts 2°/3°pc precursors contain unstable microtubules at 24 h APF (Del Bel, 2018), which could affect microtubule-based endosome positioning later in development (although this study was unable to detect microtubule defects by immunostaining at 42 h APF). However, it is also possible that enlarged endosomes accumulate basally for other reasons or are simply excluded from narrower apical-medial regions on the basis of size. It remains to be discerned whether Rst accumulation and the appearance of enlarged endosomes, which co-occurred between 24 and 42 h APF, share a causal basis or represent distinct, parallel phenotypes of reduced Sac1 activity (Griffiths, 2020).

Given the phenotypic similarities between Sac1^ts and vap mutants (Mao, 2019), it was surprising that osbp did not affect Mys distribution or cause severe Rst accumulation. However, this is reminiscent of previous results from Drosophila neurons, where loss of Vap but not OSBP caused protein accumulation and ER stress (Moustaqim-Barrette, 2014). It is possible that, as in yeast where the presence of one out of seven OSBP homologues is sufficient for viability, OSBP functions redundantly with one or more ORPs in regulating the endosomal pathway. Indeed, CG1513, which is synthetically lethal in combination with osbp (Moustaqim-Barrette, 2014), encodes an orthologue of mammalian ORP9, which functions similarly to OSBP in sterol-PI4P exchange at ER-Golgi MCS. Alternatively, CG3860 encodes an orthologue of mammalian ORP2, which localizes to late endosomes in HeLa cells and influences sterol levels in endosomes and the PM, although countertransport of PI4P has not been shown. Mammalian ORP2 also binds ORP1L, which acts at ER-endosome MCS and promotes endosome transport, though it is unclear whether such a role is conserved in Drosophila, which lack an ORP1L orthologue. Further characterization of the Drosophila ORPs is thus needed to clarify their respective contributions to lipid homeostasis and endosomal progression (Griffiths, 2020).

Recent years have seen a proliferation of research into Sac1's roles in lipid homeostasis and the importance of PI4P regulation, as well as the development of novel probes and methods for studying phosphoinositides in vivo. This study has provided new insights into Sac1's function in protein delivery and turnover in a developing tissue, which will serve as groundwork for further investigations into the significance of Sac1 in cell physiology, organismal development, and ultimately cellular homeostasis in human health and disease (Griffiths, 2020).

Plasma membranes fulfil many physiological functions. In polarized cells, different membrane compartments take on specialized roles, each being allocated correct amounts of membrane. The Drosophila tracheal system, an established tubulogenesis model, contains branched terminal cells with subcellular tubes formed by apical plasma membrane invagination. This study shows that apical endocytosis and late endosome-mediated trafficking are required for membrane allocation to the apical and basal membrane domains. Basal plasma membrane growth stops if endocytosis is blocked, whereas the apical membrane grows excessively. Plasma membrane is initially delivered apically and then continuously endocytosed, together with apical and basal cargo. An organelle is described carrying markers of late endosomes and multivesicular bodies (MVBs) that is abolished by inhibiting endocytosis and which is suggested to act as transit station for membrane destined to be redistributed both apically and basally. This is based on the observation that disrupting MVB formation prevents growth of both compartments (Mathew, 2020).

The Slit diaphragm (SD) is the key filtration structure in human glomerular kidney that is affected in many types of renal diseases. SD proteins are known to undergo endocytosis and recycling to maintain the integrity of the filtration structure. However, the key components of this pathway remain unclear. Using the Drosophila nephrocyte as a genetic screen platform, most genes involved in endocytosis and cell trafficking were screened, and the key components were identified of the cell trafficking pathway required for SD protein endocytosis and recycling. The SD protein endocytosis and recycling pathway was found to contain clathrin, dynamin, AP-2 complex, like-AP180 (Lap), auxilin and Hsc70-4 (the endocytosis part) followed by Rab11 and the exocyst complex (the recycling part). Disrupting any component in this pathway led to disrupted SD on the cell surface and intracellular accumulation of mislocalized SD proteins. This study provides the first in vivo evidence of trapped SD proteins in clathrin-coated pits at the plasma membrane when this pathway is disrupted. All genes in this SD protein endocytosis and recycling pathway, as well as SD proteins themselves, are highly conserved from flies to humans. Thus, these results suggest that the SD proteins in human kidney undergo the same endocytosis and recycling pathway to maintain the filtration structure, and mutations in any genes in this pathway could lead to abnormal SD and renal diseases (Wang, 2021).

Membrane trafficking plays many roles in morphogenesis, from bulk membrane provision to targeted delivery of proteins and other cargos. In tracheal terminal cells of the Drosophila respiratory system, transport through late endosomes balances membrane delivery between the basal plasma membrane and the apical membrane, which forms a subcellular tube, but it has been unclear how the direction of growth of the subcellular tube with the overall cell growth is coordinated. This study shows that endosomes also organize F-actin. Actin assembles around late endocytic vesicles in the growth cone of the cell, reaching from the tip of the subcellular tube to the leading filopodia of the basal membrane. Preventing nucleation of endosomal actin disturbs the directionality of tube growth, uncoupling it from the direction of cell elongation. Severing actin in this area affects tube integrity. These findings show a new role for late endosomes in directing morphogenesis by organizing actin, in addition to their known role in membrane and protein trafficking (Rios-Barrera, 2022).

The Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimer's disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. This study observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to human BIN1 isoform8 (BIN1iso8) and human BIN1 isoform9 (BIN1iso9), induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, thee data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation, which is an early pathophysiological hallmark of AD pathology (Lambert, 2022).

The amyloid precursor protein (APP) is a structurally and functionally conserved transmembrane protein whose physiological role in adult brain function and health is still unclear. Because mutations in APP cause familial Alzheimer's disease (fAD), most research focuses on this aspect of APP biology. This study investigated the physiological function of APP in the adult brain using the fruit fly Drosophila melanogaster, which harbors a single APP homologue called APP Like (APPL). Previous studies have provided evidence for the implication of APPL in neuronal wiring and axonal growth through the Wnt signaling pathway during development. However, like APP, APPL continues to be expressed in all neurons of the adult brain where its functions and their molecular and cellular underpinnings are unknown. This study reports that APPL loss of function (LOF) results in the dysregulation of endolysosomal function in neurons, with a notable enlargement of early endosomal compartments followed by neuronal cell death and the accumulation of dead neurons in the brain during a critical period at a young age. These defects can be rescued by reduction in the levels of the early endosomal regulator Rab5, indicating a causal role of endosomal function for cell death. Finally, this study shows that the secreted extracellular domain of APPL interacts with glia and regulates the size of their endosomes, the expression of the Draper engulfment receptor, and the clearance of neuronal debris in an axotomy model. It is proposes that APP proteins represent a novel family of neuroglial signaling factors required for adult brain homeostasis (Kessissoglou, 2020).

This study took advantage of D. melanogaster to investigate and unravel the physiological function of APPL, the single fly homologue of the human APP, in the adult brain. The key findings are as follows; (1) that APPL is required for neuronal survival during a critical period of early life; (2) that APPL regulates the size of endolysosomal vesicles in neurons and glia; and (3) that secreted APPL interacts with glial cells to enable the clearance of neuronal debris (Kessissoglou, 2020).

A homeostatic signaling system is composed of a set point, a feedback control, sensors, and an error signal. The error signal activates homeostatic effectors to drive compensatory alterations in the process being studied. This study proposes a model whereby the presence of APPL and its cleaved forms maintain the physiological flow of vesicular trafficking, either for degradation or for recycling, through the endolysosomal network in neurons. Simultaneously, in case of a system failure, a particular stress or an acute injury, there is an increased release of the secreted portion of APPL, SAPPL, the error signal, activating degradation in glial cells, the homeostatic effector, to reset the system to its baseline (Kessissoglou, 2020).

It has been observed that appl null flies have a shorter life span and develop large neurodegenerative vacuoles in their brain by 30 days old. This study demonstrated that the brain of appl null flies shows signs of dysfunctional homeostasis from a much younger age of 7 days old, resulting in a significantly increased number of apoptotic neurons and a significantly increased death rate from 20 days fibroblasts, AD mouse models, and recent studies using patients iPSCs have all shown evidence of a defective endolysosomal network]. In particular, neurons derived from AD patient iPSCs show that fAD mutations in APP or PSEN1 as well as knockout (KO) of APP, all cause alterations in the endolysosomal vesicle size and functionality. Some of the toxic effects on endolysosomal trafficking have been attributed not to amyloid accumulation but rather to the potential toxicity of the sAPPβ and/or APP β C-terminal fragment (APP&beta:CTF), while a wealth of literature suggests that full-length APP (flAPP) and sAPP&slpha; are neuroprotective (Kessissoglou, 2020).

Glial cells are the key immune responders of the brain that maintain neuronal homeostasis through neurotrophic mechanisms and by clearing degenerating neurons. The data show that neuronal expression of APPL is necessary and sufficient to activate glial clearance of neuronal debris and that glia take up neuronally released SAPPL. Moreover, this study showed that the function of APPL in response to an injury involves regulating the glial engulfment receptor, Draper, via an as of yet unknown mechanism. It has also previously been shown that acute injury of the adult brain elicited an increased expression of APPL at and near the site of injury [50]. Interestingly, a recent study using iPSCs derived astrocytes with APP KO and fAD mutations revealed that loss of flAPP impairs cholesterol metabolism and the ability of astrocytes to clear Aβ protein aggregates [51]. Moreover, up-regulation of APP expression in neurons and &slpha;-secretase expression in reactive astrocytes was observed after the denervation of the mouse dentate gyrus [44]. Together, these observations indicate that the expression and proteolytic processing of APP are part of a neuroglial signaling system responsible for monitoring brain health and activating glial responses to neuronal injury. Further future work will be needed to describe how exactly secreted APP fragments are taken up by glia and what cellular and molecular components they interact with and modify within glial cells to mediate appropriate levels of glial activation (Kessissoglou, 2020).

Our findings that the complete loss of the Drosophila APP homologue causes deficits in the endolysosomal pathway, in neuron-induced glial clearance of debris and in neuronal death and organismal life span strongly suggest that, in the adult brain, the physiological function of flAPP and the consequences of fAD mutations are mechanistically related to one another. Furthermore, the fact that neuronal death and defective neuronal endosomes are observed very early in life of appl mutant flies further supports the notion that significant deficits exist in the AD brain long before any clinical symptoms appear. This may suggest that examining the size and/or function of the early endosome may identify risk for future neurodegeneration and offer future treatment pathways (Kessissoglou, 2020).

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of many developmental pathways is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is critical for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila melanogaster, mutants were recovered that disrupt genes encoding serine palmitoyltransferase and Acetyl-CoA Carboxylase (ACC). Both mutants cause Notch, Wingless, the Epidermal Growth Factor Receptor (EGFR), and Patched to accumulate abnormally in endosomal compartments. In mosaic animals, mutant tissues exhibit an unusual non-cell-autonomous effect whereby mutant cells are functionally rescued by secreted activities emanating from adjacent wildtype tissue. Strikingly, both mutants display prominent tissue overgrowth phenotypes that are partially attributable to altered Notch and Wnt signaling. This analysis of the mutants demonstrates genetic links between abnormal lipid metabolism, perturbations in developmental signaling, and aberrant cell proliferation (Sasamura, 2013).

The importance of lipid metabolism for the formation and maintenance of cell membranes is well established. Both serine palmitoyltransferase (SPT) and acetyl-CoA carboxylase (ACC) are critical enzymes that control different steps of lipid metabolism, and are highly conserved in diverse animal species. Genetic elimination of ACC1 or the SPT subunits Sptlc1 or Sptlc2 cause early embryonic lethality in mice, although the cellular basis for this lethality is unknown. In D. melanogaster, RNA-interfering disruption of ACC activity in the fat body results in reduced triglyceride storage and increased glycogen accumulation, and in oenocytes leads to loss of watertightness of the tracheal spiracles causing fluid entry into the respiratory system. This study demonstrates that D. melanogaster mutants lacking functional SPT or ACC exhibit endosomal trafficking defects, causing Notch, Wingless, EGFR, and Patched to accumulate abnormally in endosomes and lysosomes. These effects are accompanied by significant alterations in Notch and Wingless signaling, as revealed by changes in downstream target gene activation for both pathways. However, the mutants do not fully inactivate these developmental signaling pathways, and instead display phenotypes consistent with more complex, pleiotropic effects on Notch, Wingless, and potentially additional pathways in different tissues. These findings reinforce the importance of lipid metabolism for the maintenance of proper developmental signaling, a concept that has also emerged from studies demonstrating that: D. melanogaster mutants for phosphocholine cytidylyltransferase alter endosomal trafficking and signaling of Notch and EGFR; mutants for alpha-1,4-N-acetylgalactosaminyltransferase-1 affect endocytosis and activity of the Notch ligands Delta and Serrate; mutants for the ceramide synthase gene shlank disrupt Wingless endocytic trafficking and signaling, and mutants for the glycosphingolipid metabolism genes egghead and brainiac modify the extracellular gradient of the EGFR ligand Gurken (Sasamura, 2013).

Most strikingly, lace and ACC mutants also display prominent tissue overgrowth phenotypes. These tissue overgrowth effects are linked to changes in Notch and Wingless signaling outputs, and they involve gamma-secretase, Su(H), and Armadillo activities, suggesting that the overgrowth reflects an interplay of Wingless inactivation and Notch hyperactivation. Consistent with the findings, both Notch and Wingless regulate cell proliferation and imaginal disc size in D. melanogaster. Moreover, several observations indicate that Notch and Wingless are jointly regulated by endocytosis, with opposing effects on their respective downstream pathway activities, a dynamic process that might be especially sensitive to perturbations in membrane lipid constituents. Wingless itself exerts opposing effects on disc size that might depend on the particular developmental stage or disc territory. For example, hyperactivation of Wingless or inactivation of its negative regulators cause overproliferation, but Wingless activity can also constrain wing disc growth. Similar spatiotemporal effects might underlie the variability detected in studies with lace and ACC mutant clones, in which both tissue overgrowth and developmentally arrested discs were observed. Although no obvious changes were detected in downstream signaling for several other cell growth pathways that were examined, the trafficking abnormalities seen for other membrane proteins aside from Notch, Delta, and Wingless, as well as the incomplete suppression of the overgrowth phenotypes by blockage of Notch and Wingless signaling, suggest that other pathways might also be dysregulated in lace and ACC mutants, possibly contributing to the observed tissue overgrowth (Sasamura, 2013).

Wingless is modified by lipid addition, and lipoprotein vesicles have been suggested to control Wingless diffusion. In D. melanogaster embryos, endocytosis of Wingless limits its diffusion and ability to act as a long-range morphogen. Endocytosis can also affect Wingless signaling in receiving cells, where endocytosis both promotes signal downregulation and positively facilitates signaling. The apparently normal diffusion ranges for overaccumulated Wingless in lace and ACC mutant clones, yet reduced downstream target gene expression, is consistent with the idea that SPT and ACC act by promoting endocytic trafficking of Wingless in receiving cells rather than influencing the secretion and/or diffusion of Wingless from signal-sending cells (Sasamura, 2013).

The finding that lace and ACC mutant overgrowth phenotypes are also partially Notch-dependent is reminiscent of similar overproliferation phenotypes seen in certain D. melanogaster endocytic mutants, such as vps25, and tsg101. The overproliferation of disc tissue in these mutants is attributable to Notch hyperactivation, reflecting the fact that non-ligand-bound Notch receptors that are normally targeted for recycling or degradation are instead retained and signal from endosomes. Analogous effects are likely to contribute to the lace and ACC mutant overgrowth, where significant Notch overaccumulation was observed throughout the endosomal-lysosomal routing pathway. Some ectopic Notch signaling might emanate from the lysosomal compartment, which is enlarged and accumulates particularly high levels of Notch in lace and ACC mutant clones. Analysis of D. melanogaster HOPS and AP-3 mutants, which affect protein delivery to lysosomes, has identified a lysosomal pool of Notch that is able to signal in a ligand-independent, gamma-secretase-dependent manner (Sasamura, 2013).

How do SPT and ACC contribute to endosomal trafficking of Notch and other proteins? In the yeast SPT mutant lcb1, an early step of endocytosis is impaired due to defective actin attachment to endosomes, a phenotype that is suppressed by addition of sphingoid base. However, the trafficking abnormalities seen in lace and ACC mutants do not resemble those in the yeast lcb1 mutant, perhaps because endocytic vesicle fission is primarily dependent upon dynamin in D. melanogaster and mammals, instead of actin as in yeast. Nevertheless, the requirement for SPT and ACC in D. melanogaster endosomal compartments might reflect possible functions in endosome-cytoskeleton interactions. Another possibility is that the defective endosomal trafficking seen in lace and ACC mutants is caused by the inability to synthesize specific phospholipids needed for normal membrane homeostasis. Finally, lace and ACC might be important for the formation and/or function of lipid rafts, specialized membrane microdomains that have been implicated in both signaling and protein trafficking (Sasamura, 2013).

A remarkable feature of the lace and ACC mutant phenotypes that suggests an underlying defect in lipid biogenesis is the non-autonomous effect in mutant tissue clones, wherein nearby wildtype cells generate a secreted activity that diffuses several cell diameters into the mutant tissue and rescues the trafficking and signaling defects. One possibility is that these secreted activities are diffusible lipid biosynthetic products of SPT and ACC, which enter the mutant cells and serve as precursors for further biosynthetic steps that do not require SPT or ACC. An intriguing alternative is that the SPT and ACC enzymes are themselves secreted and taken up by the mutant cells. A precedent for this mechanism has recently been demonstrated for D. melanogaster ceramidase, a sphingolipid metabolic enzyme that is secreted extracellularly, delivered to photoreceptors, and internalized by endocytosis to regulate photoreceptor cell membrane turnover (Sasamura, 2013).

Recent work has highlighted the importance of lipid metabolism for oncogenic transformation, and ACC has been advanced as a promising target for cancer drug development. ACC is upregulated in some cancers, possibly as a result of high demands for lipid biosynthesis during rapid cell divisions. Sphingolipids and their derivatives are also thought to influence the balance of apoptosis and cell proliferation during tissue growth, and thus have also garnered attention as potential cancer therapy targets. The current findings regarding the requirements of SPT and ACC for proper trafficking and signaling of key developmental cell-surface signaling molecules, including Notch and Wingless, provide insights into how lipid metabolic enzymes might influence cell proliferation and tissue patterning in multicellular animals. Complex lipid biosynthesis is essential for the creation of the elaborate, interconnected, and highly specialized membrane compartments in which developmental pathways operate, and perturbations in lipid biosynthesis that are tolerated by the cell might nevertheless exert significant pleiotropic effects on developmental patterning, cell proliferation, and other cellular processes. Exploration of lipid metabolic enzymes as pharmacological targets must therefore take into account potentially unfavorable effects on critical signaling pathways controlling development and organogenesis (Sasamura, 2013).

A striking variety of glycosylation occur in the Golgi complex in a protein-specific manner, but how this diversity and specificity are achieved remains unclear. This study shows that stacked fragments (units) of the Golgi complex dispersed in Drosophila imaginal disc cells are functionally diverse. The UDP-sugar transporter Fringe-connection (Frc) is localized to a subset of the Golgi units distinct from those harboring Sulfateless (Sfl), which modifies glucosaminoglycans (GAGs), and from those harboring the protease Rhomboid (Rho), which processes the glycoprotein Spitz (Spi). Whereas the glycosylation and function of Notch are affected in imaginal discs of frc mutants, those of Spi and of GAG core proteins are not, even though Frc transports a broad range of glycosylation substrates, suggesting that Golgi units containing Frc and those containing Sfl or Rho are functionally separable. Distinct Golgi units containing Frc and Rho in embryos could also be separated biochemically by immunoisolation techniques. Tn-antigen glycan is localized only in a subset of the Golgi units distributed basally in a polarized cell. It is proposed that the different localizations among distinct Golgi units of molecules involved in glycosylation underlie the diversity of glycan modification (Yano, 2005).

The pattern of glycosylation is extremely diverse, yet is highly specific to each protein. How can this specificity (and diversity) be achieved? There are >300 glycosylenzymes in humans and >100 in Drosophila, but is their enzymatic specificity sufficient to explain the precise modification of all substrates? One possible mechanism that might also contribute to the specific (and diverse) pattern of glycosylation would be the localization/compartmentalization of glycosylenzymes (Yano, 2005).

The Golgi complex, where protein glycosylation takes place, has been regarded as a single functional unit, consisting of cis-, medial-, and transcisternae in mammalian cells. However, the three-dimensional reconstruction of electron microscopic images of the mammalian Golgi structure has suggested the existence of more than one Golgi stack, with the individual stacks being connected into a ribbon by tubules bridging equivalent cisternae. Furthermore, during mitosis, the Golgi cisternae of mammalian cells become fragmented without their disassembly. In Drosophila, Golgi cisternae are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase, although there has been no evidence to date to indicate functional differences among the Golgi fragments (Yano, 2005).

The Golgi complex is a stack of cis-, medial-, and transcisternae in mammalian cells. In contrast, Golgi markers often do not overlap with each other in Saccharomyces cerevisiae, in which the Golgi cisternae are not stacked but disassembled. The Golgi cisternae of Drosophila are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase. To determine whether Drosophila imaginal disc cells have assembled or disassembled Golgi cisternae, the localizations were compared of the cis-cisternal marker dGM130, the transcisternal marker Syntaxin16 (Syx16), and the Golgi-tethered 120-kDa protein, which is commonly used to detect the Golgi complex in Drosophila. The 120-kDa protein was identified by immunoaffinity purification and protein sequencing as a Drosophila homolog of the vertebrate 160-kDa medial Golgi sialoglycoprotein (MG160), which resides uniformly in the medial-cisternae of the Golgi apparatus in vertebrate cells. An antibody specific for the 120-kDa protein also stained numerous Golgi fragments in imaginal disc cells. More than 80% of immunoreactivity for the 120-kDa protein was colocalized with both dGM130 and Syx16, suggesting that 120-kDa protein-positive fragments of the Golgi complex indeed comprise assembled cisternae; these fragments are referred to as 'Golgi units.' The distributions of the 120-kDa protein, dGM130, and peanut agglutinin (PNA), another transcisternal marker, also show that the markers are closely apposed but not identical, suggesting that the Golgi units are polarized. Interestingly, most of the PNA-positive transcisternae are oriented toward the basal side of the cell, within the Golgi complex, whereas most of the GM130-positive cis-cisternae are oriented toward the apical side of the cell. The cis-to-trans polarity of each Golgi unit thus appears to be correlated with the apico-basal polarity of the disc cells (Yano, 2005).

Drosophila mutant larvae defective in the UDP-sugar transporter Frc manifest a highly selective phenotype: the lack of Notch glycosylation in the presence of normal GAG synthesis (Goto, 2001). This limited phenotype is unexpected, given that Frc exhibits a broad specificity for UDP sugars used in the synthesis of various glycans including N-linked types, GAGs, and mucin types. However, given that the frc^R29 allele studied previously (Goto, 2001) is hypomorphic, whether the selective glycosylation defect might be a consequence of partial loss of Frc activity was examined. With the use of imprecise excision, a new allele was generated, frc^RY34, the presence of which results in the death of most larvae during the second-instar stage, much earlier than the death induced by frc^R29. Real-time PCR analysis revealed that the amount of frc transcripts in the second-instar larvae of frc^RY34 or frc^R29 mutants was 4.2% and 24.4% of that in the wild type, respectively. About 1 kb of the gene, including the transcription initiation site, was deleted in the frc^RY34 allele. Together, these observations suggest that frc^RY34 is essentially a null allele (Yano, 2005).

Clonal cells of the frc^RY34 mutant exhibited normal levels of GAGs, as detected by immunostaining with the 3G10 antibody, whereas the amount of GAGs was reduced in clones of tout-velu (ttv) mutant cells. Given that GAGs are required for signaling by Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp), the expression of corresponding target genes [patched (ptc) for Hh signaling and Dll for Wg and Dpp signaling] was examined in the wing discs of the frc^RY34 mutant. Expression of ptc and that of Dll in the ventral compartment of the wing discs were unaffected in the mutant clones, suggestive of normal GAG function (Yano, 2005).

Given that Notch glycosylation by Fringe (FNG), a fucose-specific beta1,3-N-acetylglucosaminyltransferase, requires Frc activity, Notch glycosylation was examined in the frc^RY34 mutant. The frc^RY34 mutant clones in the dorsal compartment, but not those in the ventral compartment, of the wing discs induced wg expression at their borders, suggesting that Notch glycosylation is impaired in the frc^RY34 mutant. The ectopic expression of Wg induced by the frc^RY34 mutant clones is likely responsible for the observed induction of Dll expression in the dorsal compartment (Yano, 2005).

To determine why the loss of a UDP-sugar transporter with a broad specificity selectively affects Notch glycosylation, the subcellular localization of Frc was investigated. Frc tagged with the Myc epitope was expressed in imaginal discs under the control of the arm-Gal4 driver. The Gal4-induced expression of Frc-Myc rescues the frc mutant phenotype, suggesting that Frc-Myc is functional and properly localized. Immunostaining of imaginal discs of wild-type larvae expressing Frc-Myc with antibodies to Myc and to the 120-kDa protein revealed that Frc is localized to only a small subset of Golgi units. This differential immunostaining of different Golgi units is not likely to be due to differential penetration of the antibodies or cripticity of the epitopes. The penetration of antibodies would not vary within the cell, because the Golgi units were distributed evenly throughout the cell, not in a biased manner. Moreover, it is unlikely that degradation of the epitopes during the immunostaining experiments due to contaminating proteases might alter the cripticity of the epitopes in different Golgi units, since the percentage of different Golgi units among the anti-120-kDa-positive Golgi units was statistically constant in several independent experiments. Thus, it is hypothesized that the Golgi units might be functionally heterogeneous, and that those containing Frc might modify some proteins, including Notch, but not others (Yano, 2005).

To test this hypothesis, the localizations of various molecules involved in protein modification in the Golgi complex were compared with that of Frc. It was found that Sfl is also restricted to a subset of Golgi units, but that its distribution does not overlap with that of Frc. This differential localization of Sfl and Frc might thus explain the observation that frc mutant clones in wing discs do not show any defect in GAG synthesis by Sfl (Yano, 2005).

The Spi-processing enzyme Rho is also localized to a subset of Golgi units distinct from those containing Frc, in addition to its presence in other compartments. This result indicates the existence of at least two types of Golgi units, those containing Rho and those containing Frc. To determine whether these two types of Golgi units differ functionally, the glycosylation state and function of Spi was examined in frc mutants (Yano, 2005).

Given that the extent of Notch glycosylation, as detected by wheat germ agglutinin (WGA), is markedly reduced in frc mutants compared with that in the wild-type background, whether the WGA-reactive glycan of Spi is also affected by frc mutation was also examined. Myc epitope-tagged Spi was expressed in the wild type or the frc^RY34 mutant. Spi-Myc was then precipitated from larval homogenates with antibodies to Myc and was examined for its glycosylation by SDS/PAGE and subsequent blot analysis with WGA. The reactivity of the Spi glycan with WGA was similar in the frc mutant and in the wild type. Whether the frc^RY34 mutation affects the Spi glycan was examined by mobility shift analysis. The electrophoretic mobility of glycosylated Spi from the wild type is similar to that from the frc mutant. Deglycosylation of Spi by neuraminidase, peptide-N-glycosidase (PNGase) F, and O-glycanases also increases its mobility to the same extent in wild-type and frc mutant larvae, suggesting that the core protein is not affected by the frc mutation. Together, these results indicate that the function of Frc is not necessary for formation of the Spi glycan. It is also concluded that the function of the Rho-Spi pathway is not affected by frc mutation (Yano, 2005).

To confirm that the Golgi units containing Frc and those containing Rho are distinct, whether these Golgi units could be selectively isolated by using antibodies to Myc (for Myc-tagged Frc) or HA (for HA-tagged Rho) was examined. Because it was very difficult to collect enough of the imaginal discs, the starting material was switched to embryos, and whether Frc and Rho localize to distinct Golgi units was examined in embryos. Frc-Myc and Rho-HA were coexpressed in the embryos by the arm-Gal4 driver, and immunostaining with antibodies to Myc and to HA revealed that the Golgi units containing Frc-Myc (45.4% of total Golgi units) and those containing Rho-HA (43.0% of total Golgi units) are largely distinct: only 11.6% of total Golgi units were positive for both Frc-Myc and Rho-HA. Immunoisolation was attempted from embryonic lysates by using either antibody to Myc or HA, and how much Frc-Myc and Rho-HA were coisolated in each immunoisolate was examined. When Frc-Myc was immunoisolated with an antibody to Myc, the recovery of Frc-Myc was 5.7 times greater than that of Rho-HA. Moreover, when Rho-HA was immunoisolated with an antibody to HA, the recovery of Rho-HA was 18.3 times greater than that of Frc-Myc. The immunoblot analysis of these immunoisolates with the anti-120-kDa antibody confirmed that the Golgi units were concentrated in these immunoisolates. These results support the notion that Frc-Myc-containing fraction is distinct and could be separated from Rho-HA-containing fraction (Yano, 2005). Whether these distinct Golgi units contain different constituents was examined. Fringe (Fng) is one of the candidate molecules that may be colocalized with Frc. Therefore, expression of ectopically expressed Fng was examined in Rho- and Frc-containing immunoisolates. It was found that expression of Fng in Frc-containing immunoisolates was 26 times greater than in Rho-containing immunoisolates, supporting the idea that Fng is localized in the Frc-positive Golgi units rather than the Rho-positive Golgi units. Immunostaining analysis confirmed that FNG was colocalized mostly with Frc (88.1% of the FNG-positive Golgi units), but not with Rho (16.6% of the FNG-positive Golgi units), by immunostaining analysis (Yano, 2005).

The data suggest that different Golgi units perform different functions, a notion that is also supported by the observation that Tn antigen (O-linked N-acetylgalactosamine) was detected in only a subset of Golgi units in imaginal eye disc cells. In addition, most of these Tn antigen-positive Golgi units were found to be distributed in the basal region of the disc cells, suggesting that the differential distribution of Golgi units might contribute to the apicobasal polarity of glycan distribution (Yano, 2005).

In contrast to the larval stage, Frc is required for GAG synthesis at the early embryonic stage. To determine why the Frc requirement for GAG synthesis differs between the embryonic and larval stages, embryos were stained expressing Frc-Myc with antibodies to Sfl and to Myc. Sfl was found to be colocalized with Frc, likely explaining the importance of Frc for GAG synthesis at the embryonic stage. In addition, this embryonic requirement of Frc for GAG synthesis excludes the possibility that the selective defects in Notch and not in GAG synthesis observed in frc mutant larvae are caused by the selective Frc-dependent transport of a subset of UDP-sugars used only for glycosylation of Notch but not for GAGs synthesis (Yano, 2005).

These results provide evidence for the existence of functionally distinct Golgi units in Drosophila cells. Such functional heterogeneity of Golgi units is likely responsible for the diversity of protein glycosylation. At least two types of Golgi units containing either Frc or Sfl were shown to be present in larval disc cells. Two distinct sets of proteins, exemplified by Notch and GAG core proteins, might thus be selectively transported to Frc- or Sfl-containing Golgi units, respectively, where they undergo glycosylation by different sets of molecules (Yano, 2005).

The variety of Golgi units might be established by separate transport of secretory proteins and glycosylenzymes from the endoplasmic reticulum (ER) to the distinct Golgi units. In yeast, glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in vesicles distinct from those containing other secretory protein. Given that the GAG core protein Dally in Drosophila is anchored to the membrane by GPI, it is possible that Dally and Notch are loaded into distinct vesicles as they exit the ER (Yano, 2005).

Combinations of glycosylenzymes and transporters, such as Sfl and Frc, contained in Golgi units of Drosophila differ not only between embryos and larval disc cells but also among cell types. For example, it was found that Frc is localized to all Golgi units in salivary gland cells at the larval stage. It has also been shown that all of the Golgi complexes dispersed in oocytes may have the ability to process the Gurken precursor protein, which is usually cleaved in a subset of the Golgi complexes residing in the dorso-anterior region. The Golgi units may thus be altered in a manner dependent on development, cell type, and signaling processes (Yano, 2005).

The functional diversity of Golgi units also might contribute to the polarized distribution of glycans along the apicobasal axis of cells. It was found that Tn antigen is synthesized in the basal Golgi units of larval disc cells. Furthermore, certain types of glycans are distributed along the apicobasal axis of pupal ommatidia. These glycans might thus be synthesized differentially in the Golgi units that are asymmetrically distributed along the apicobasal axis and then be secreted at either the apical or basal cell surface (Yano, 2005).

Whereas Golgi units are dispersed throughout Drosophila cells, the Golgi complex in mammalian cells is thought to be a single entity that is located in the pericentriolar region through its association with the microtubule-organizing center in interphase and which is fragmented at the onset of mitosis. The Golgi fragments apparent in mammalian cells during mitosis are highly similar to the Golgi units of Drosophila cells in both electron and confocal microscopic images. The mammalian Golgi complex during interphase may therefore be comprised of functionally distinct units that are associated with the microtubule-organizing center and connected with each other (Yano, 2005).

During the cell cycle, the Golgi, like other organelles, has to be duplicated in mass and number to ensure its correct segregation between the two daughter cells. It remains unclear, however, when and how this occurs. This study shows that in Drosophila S2 cells, the Golgi likely duplicates in mass to form a paired structure during G1/S phase and remains so until G2 when the two stacks separate, ready for entry into mitosis. Pairing requires an intact actin cytoskeleton which in turn depends on Abi/Scar but not WASP. This actin-dependent pairing is not limited to flies but also occurs in mammalian cells. It is further shown that preventing the Golgi stack separation at G2 blocks entry into mitosis, suggesting that this paired organization is part of the mitotic checkpoint, similar to what has been proposed in mammalian cells (Kondylis, 2007).

During the cell cycle, the Golgi, like other organelles, has to duplicate in mass and/or number to ensure its correct segregation between the two daughter cells. It remains unclear, however, when and how this occurs. The process of Golgi duplication and inheritance in mammalian cells is still debated, as different modes of Golgi biogenesis have been proposed. One reason why this issue is not yet settled could be due to the elaborate organization of the Golgi stacks, which are interconnected to form a single-copy organelle capping the nucleus, thus impeding clear visualization of organelle duplication and segregation. Therefore, this study has exploited the relatively small number of Golgi stacks in Drosophila tissue-cultured S2 cells to revisit this issue (Kondylis, 2007).

In S2 cells, the Golgi stacks are found in close proximity to transitional endoplasmic reticulum (tER) sites, forming tER-Golgi units (Kondylis, 2003; Herpers, 2004). Their number nearly doubles at G2 phase. In an effort to identify factors mediating this process, focus was placed on cytoskeletal elements that have been involved in the organization of the mammalian Golgi apparatus. Microtubules are involved in mammalian Golgi ribbon maintenance, as their depolymerization leads to its reorganization into individual Golgi stacks in close proximity to ER exit sites (Kondylis, 2007 and references therein).

F-actin has also been implicated in the organization of the mammalian Golgi apparatus; its depolymerization leads to a compact appearance of this organelle without disruption of cisternal stacking. A key regulator of actin polymerization is the Arp2/3 complex. Its F-actin nucleation activity is triggered both by Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous (WAVE/Scar) proteins, which are in turn regulated by Rho small GTPases. WASP exists in an autoinhibited state that is released by the cooperative action of Cdc42, PI(4,5)P, and other SH3-containing proteins. In contrast, WAVE/Scar proteins, together with Sra-1, Kette (Nap1), Abi, and HSPC300, form a stable complex, which is itself regulated by Rac (Kondylis, 2007 and references therein).

Rho GTPases have recently been implicated in maintaining Golgi architecture. Cdc42 has been localized on the Golgi membrane and shown to recruit the Arp2/3 complex around this organelle via ARHGAP10. Furthermore, suppression of the brain-specific Rho-binding protein Citron-N in neurons was shown to lead to fragmentation of the Golgi apparatus, and Rho1 was proposed to exert its local effect on F-actin by regulating ROCK and profilin activity (Kondylis, 2007 and references therin).

This study shows that drug-induced F-actin depolymerization in S2 cells leads to doubling of the number of tER-Golgi units independent of anterograde transport. Using live cell imaging, electron microscopy, and three-dimensional (3D) electron tomography, this study shows that each Golgi is organized as a pair of stacks held together by an actin-based mechanism, both in Drosophila and in human cells. In S2 cells, this is mediated by Abi and Scar, suggesting a novel role for the Rac signaling cascade in Golgi architecture. Last, it was shown that the Golgi stacks undergo separation at G2 through modulation of Abi and Scar, and that blocking this separation prevents cells from entering mitosis, supporting the existence of a G2/M checkpoint related to Golgi structural organization (Kondylis, 2007).

The two Golgi stacks could be physically linked without displaying membrane continuity or being interconnected, for instance through intercisternal tubular connections, either permanent or transient. Tubules connecting cisternae of adjacent stacks are involved in the formation of the Golgi ribbon in mammalian cells and, recently, GM130 and GRASP65 have been proposed to be required for their integrity. However, the putative tubules connecting the two stacks in the pair would have different molecular requirements, at least in Drosophila, since depletion of dGM130 or dGRASP does not lead to their separation (Kondylis, 2003; Kondylis, 2005; Kondylis, 2007 and references therein).

F-actin could provide a physical link holding the paired Golgi stacks together, or it could help in the formation/maintenance of intercisternal tubules. In addition, short actin filaments have been proposed to link spectrin mosaics leading to the formation of a skeleton that surrounds the Golgi complex. One of its functions could be to hold the two Golgi stacks close enough to allow the formation and fusion of the tubules. It could also surround the tubules themselves, thus providing membrane stability. The localization of Abi and Scar at the periphery of the tER-Golgi units and between the two stacks in a pair is consistent with both proposed functions. These tomography studies so far have not revealed clear membrane continuities between Golgi cisternae, though examples have been found of a tubular network which is shared by the paired stacks (Kondylis, 2007).

tER sites behave similarly to the Golgi, as they also separate at G2 and upon F-actin depolymerization. Because little is known about the mechanism regulating the biogenesis of tER sites, it is difficult to envisage how the two parts could be held together. The spectrin-actin mesh described above could be common to Golgi and tER sites, and Golgi and tER site scission could be achieved in a synchronized fashion. Alternatively, either of these organelles could split first and lead to the scission of the other, perhaps by providing positional information. Recently, the centrosome component centrin 2 that is also localized to tER sites in Trypanosoma has been shown to give such positioning information. A more in-depth study combining immunogold labeling and 3D tomography would be required to elucidate such fine details of tER-Golgi structural organization (Kondylis, 2007).

Drosophila Rho1 is unlikely to have a role in holding the two Golgi stacks together. The overexpression of the Rho1 constitutively inactive mutant or treatment of S2 cells with ROCK or myosin light chain inhibitors (Y27632 and blebbistatin) did not affect the Golgi number. Cdc42 is also unlikely to participate as the depletion of its downstream effector WASP did not lead to Golgi separation, although the overexpression of the Cdc42^T17N dominant negative did. However, this effect could be due to nonspecific sequestration of the guanine nucleotide exchange factor involved in maintaining the paired Golgi stacks and may be shared with other small GTPases (Kondylis, 2007).

Interestingly, the results are consistent with a role for Rac GTPases in Drosophila Golgi architecture. Expression of the constitutively inactive form of Rac1 led to a near-doubling in the Golgi number, and depletion of Scar/WAVE or Abi, which are regulated by Rac GTPases, led to a similar phenotype. The identical results obtained in Scar and Abi RNAi suggest that this well-established Scar/WAVE pentameric complex is involved in holding the paired Golgi stacks together by promoting F-actin polymerization. These data indicate that the Rac signaling pathway is involved. However, the Scar/Abi complex has recently been shown to also stimulate Arp2/3 and F-actin polymerization independently of Rac. This would need to be investigated further (Kondylis, 2007).

This study shows that the separation of the paired Golgi stacks occurs at G2, prior to mitosis. A similar phenomenon has already been reported during cell division in Toxoplasma gondii, where a single Golgi stack grows as a duplicated organelle and is separated as the cell divides. However, the mechanism underlying this separation is not known (Kondylis, 2007).

The Golgi doubling in number at G2 phase resembles many aspects of this observed upon F-actin depolymerization. In both cases, a similar increase in Golgi number and decrease in their size are observed. Furthermore, this study has shown that it is the modulation of the F-actin cytoskeleton and the activity of Abi/Scar at G2 that lead to Golgi stack separation. (1) It was found that both Scar and Abi localized to the Golgi, strongly arguing for having a role in actin remodeling around this organelle. (2) The Golgi stacks in G2 cells remain insensitive to F-actin depolymerization. (3) Cells depleted of Abi and Scar that exhibit separated Golgi stacks do not split them further at G2. (4) The overexpression of Abi prevents Golgi separation at G2. This strongly suggests that the F-actin/Abi/Scar-mediated link of the two stacks has been severed in a G2-specific manner, perhaps by kinases such as Polo (Kondylis, 2007).

Because tER sites and the Golgi apparatus ultimately disperse later in mitosis, both in mammalian and Drosophila S2 cells, the Golgi stack separation prior to dispersion might be part of the proposed Golgi G2/M checkpoint. Indeed, reagents that interfere with the GRASP65/55 phosphorylation by Polo and ERK/MEK, respectively, arrest or delay the cell cycle at the G2/M transition. This study shows that blocking Golgi separation at G2 by overexpressing Abi also prevents S2 cells from entering mitosis. This strengthens the relationship between Golgi organization and mitotic entry, although it cannot formally be excluded that the mitotic block observed is partly due to additional effects of Abi overexpression, for instance at the plasma membrane (Kondylis, 2007).

It is proposed that at G2, the paired stacks are separated along with the adjacent tER sites. As the cell enters mitosis, the Golgi membrane and the tER sites disperse, and are segregated into the two daughter cells, where the tER-Golgi units are rebuilt. The Golgi could be rebuilt as a very small paired stack in close association with Scar, Abi, and F-actin, or as a single stack that will duplicate by a mechanism that still needs to be unraveled. Since G1 cells are all sensitive to F-actin depolymerization, this suggests that the formation of the paired Golgi stack starts just after the exit from mitosis and persists until S phase, when the Golgi seems to grow significantly. A more detailed understanding will come from EM study of S and G2 cells (Kondylis, 2007).

One of the remaining questions regards the impact of the Abi/Scar role on Golgi organization during development. Using Scar/WAVE, Abi, Kette, and Sra-1 mutants, as well as transgenic flies carrying inducible RNAi constructs, it will be possible to assess whether any of the observed phenotypes (defects in oogenesis, cell and organ morphology, neuroanatomical malformations, and failure in cell migration) is in part due to defects in Golgi organization (Kondylis, 2007).

Microtubule nucleation is essential for proper establishment and maintenance of axons and dendrites. Centrosomes, the primary site of nucleation in most cells, lose their function as microtubule organizing centers during neuronal development. How neurons generate acentrosomal microtubules remains unclear. Drosophila dendritic arborization (da) neurons lack centrosomes and therefore provide a model system to study acentrosomal microtubule nucleation. This study investigated the origin of microtubules within the elaborate dendritic arbor of class IV da neurons. Using a combination of in vivo and in vitro techniques, it was found that Golgi outposts can directly nucleate microtubules throughout the arbor. This acentrosomal nucleation requires gamma-tubulin and CP309, the Drosophila homolog of AKAP450, and contributes to the complex microtubule organization within the arbor and dendrite branch growth and stability. Together, these results identify a direct mechanism for acentrosomal microtubule nucleation within neurons and reveal a function for Golgi outposts in this process (Ori-McKenney, 2012).

Microtubules are organized into dynamic arrays that serve as tracks for directed vesicular transport and are essential for the proper establishment and maintenance of neuronal architecture. The organization and nucleation of microtubules must be highly regulated in order to generate and maintain such complex arrays. Nucleating complexes, in particular, are necessary because spontaneous nucleation of new tubulin polymers is kinetically limiting both in vivo and in vitro. Gamma(Γ)-tubulin is a core component of microtubule organization centers and has a well-established role in nucleating spindle and cytoplasmic microtubules. Previous studies have proposed that in mammalian neurons, microtubules are nucleated by γ-tubulin at the centrosome, released by microtubule severing proteins, and then transported into developing neurites by motor protein. Indeed, injection of antibodies against γ-tubulin or severing proteins inhibited axon outgrowth in neurons cultured for one day in vitro (DIV1) (Ori-McKenney, 2012).

However, proper neuron development and maintenance may not rely entirely on centrosomal sites of microtubule nucleation. Although the centrosome is the primary site of microtubule nucleation at DIV2, it loses its function as a microtubule-organizing center during neuronal development. In mature cultured mammalian neurons (DIV 11-12), γ-tubulin is depleted from the centrosome, and the majority of microtubules emanate from acentrosomal sites. In Drosophila dsas-4 mutants that lack centrioles, organization of eye-disc neurons and axon outgrowth are normal in third-instar larvae. Within the Drosophila peripheral nervous system (PNS), although dendritic arborization neurons contain centrioles, they do not form functional centrosomes, and laser ablation of the centrioles does not perturb microtubule growth or orientation (Nguyen, 2011). These results indicate that acentrosomal generation of microtubules contributes to axon development and neuronal polarity. How and where acentrosomal microtubule nucleation may contribute to the formation and maintenance of the more complex dendrites, and what factors are involved in this nucleation is unknown. Dendritic arborization (da) neurons provide an excellent system for investigating these questions. They are a subtype of multipolar neurons in the PNS of Drosophila melanogaster which produce complex dendritic arrays and do not contain centrosomes. Based on their patterns of dendrite projections, the da neurons have been grouped into four classes (I-IV) with branch complexity and arbor size increasing with class number. Class IV da neurons are ideal for studying acentrosomal microtubule nucleation because they have the most elaborate and dynamic dendritic arbor, raising intriguing questions about the modes of nucleation for its growth and maintenance (Ori-McKenney, 2012).

One potential site of acentrosomal microtubule nucleation within these neurons is the Golgi complex. A number of studies have shown that the Golgi complex can nucleate microtubules in fibroblasts. Although, in these cell types, the Golgi is tightly coupled to the centrosome, it does not require the centrosome for nucleation. It does, however, require γ-tubulin, the centrosomal protein AKAP450, and the microtubule binding proteins CLASPs. When the Golgi is fragmented upon treatment with nocodazole, the dispersed Golgi ministacks can still promote microtubule nucleation, indicating that these individual ministacks contain the necessary machinery for nucleation (Ori-McKenney, 2012 and references therein).

In both mammalian and Drosophila neurons, the Golgi complex exists as Golgi stacks located within the soma and Golgi outposts located within the dendrites. In cultured mammalian hippocampal neurons, these Golgi outposts are predominantly localized in a subset of the primary branches; however, in Drosophila class IV da neurons, the Golgi outposts appear throughout the dendritic arbor, including within the terminal branches (Ye, 2007). The Golgi outposts may provide membrane for a growing dendrite branch, as the dynamics of smaller Golgi outposts are highly correlated with dendrite branching and extension. However, the majority of larger Golgi outposts remains stationary at dendrite branchpoints and could have additional roles beyond membrane supply. It is unknown whether Drosophila Golgi outposts contain nucleation machinery similar to mammalian Golgi stacks. Such machinery could conceivably support microtubule nucleation within the complex and dynamic dendritic arbor. This study identifies a direct mechanism for acentrosomal microtubule nucleation within the dendritic arbor and reveal a role for Golgi outposts in this process. Golgi outposts contain both γ-tubulin and CP309, the Drosophila homolog of AKAP450, both of which are necessary for Golgi outpost-mediated microtubule nucleation. This type of acentrosomal nucleation contributes not only to the generation of microtubules at remote terminal branches, but also to the complex organization of microtubules within all branches of the dendritic arbor. Golgi outposts are therefore important centers of acentrosomal microtubule nucleation, which is necessary to establish and maintain the complexity of the class IV da neuronal arbor (Ori-McKenney, 2012).

This study has addressed how microtubules are organized and nucleated within the complex arbor of class IV da neurons and how essential these processes are for dendrite growth and stability. Microtubule organization within different subsets of branches in da neurons must require many levels of regulation. This study has identified the first direct mechanism for acentrosomal microtubule nucleation within these complex neurons and has uncovered a role for Golgi outposts in this process. The data are consistent with the observation that pericentriolar material is redistributed to the dendrites in mammalian neurons (Ferreira, 1993) and that γ-tubulin is depleted from the centrosome in mature mammalian neurons (Stiess, 2010). This suggests that the Golgi outposts may be one structure involved in the transport of centriole proteins such as γ-tubulin and CP309. This study found that microtubule nucleation from these Golgi outposts correlates with the extension and stability of terminal branches, which is consistent with the observation that EB3 comet entry into dendritic spines accompanies spine enlargement in mammalian neurons (Jaworski, 2009). It is striking that microtubule organization in shorter branches, but not primary branches, mimics the organization in mammalian dendrites, with a mixed microtubule polarity in the secondary branches and a uniform plus end distal polarity in the terminal branches. Kinesin-2 and certain +TIPS are necessary for uniform minus end distal microtubule polarity in the primary dendrites of da neurons. Golgi outpost mediated microtubule nucleation could also contribute to establishing or maintaining this polarity both in the terminal branches and in the primary branches. It will be of interest to identify other factors that may be involved in organizing microtubules in different subsets of branches in the future (Ori-McKenney, 2012).

In vivo and in vitro data support a role for Golgi outposts in nucleating microtubules at specific sites within terminal and primary branches. However, it is noted that not all EB1 comets originate from Golgi outposts, indicating other possible mechanisms of generating microtubules. One potentially important source of microtubules is the severing of existing microtubules by such enzymes as katanin and spastin, both of which are necessary for proper neuronal development. It is likely that both microtubule nucleation and microtubule severing contribute to the formation of new microtubules within the dendritic arbor; however, the current studies suggest that Golgi-mediated nucleation is especially important for the growth and maintenance of the terminal arbor. In γ-tubulin and CP309 mutant neurons, the primary branches contain a similar number of EB1 comets, but only a small fraction of the terminal branches still contain EB1 comets. This result indicates that severing activity or other sources of nucleation may suffice for microtubule generation within the primary branches, but γ-tubulin mediated nucleation is crucial in the terminal branches. As a result, the terminal branch arbor is dramatically reduced by mutations compromising the γ-tubulin nucleation activity at Golgi outposts (Ori-McKenney, 2012).

It is important to note that Golgi outposts are present in the dendrites, but not in the axons of da neurons; thus, this mode of nucleation is dendrite specific and likely contributes to the difference in microtubule arrays in axons and dendrites. While the axon is one long primary branch with uniform microtubule polarity, the dendrite arbor is an intricate array of branches where microtubule polarity depends on branch length. Therefore, this more elaborate branched structure may have evolved a variety of nucleation mechanisms, including Golgi outpost nucleation and microtubule severing. Intriguingly, in da neurons lacking cytoplasmic dynein function, the Golgi outposts are mislocalized to the axon, which appears branched and contains microtubules of mixed polarity (Zheng, 2008). It is speculated that in these mutants, Golgi-mediated microtubule nucleation within the axon is contributing to the mixed microtubule orientation and formation of ectopic dendrite-like branches (Ori-McKenney, 2012).

Only a subpopulation of Golgi outposts could support microtubule nucleation both in vivo and in vitro. The results show that Golgi outpost mediated microtubule nucleation is restricted to stationary outposts and dependent upon γ-tubulin and CP309, but why some outposts contain these proteins while others do not is unknown. γ-tubulin and CP309 could be recruited to the Golgi outposts in the cell body and transported on the structure into the dendrites, or they could be recruited locally from soluble pools throughout the dendritic arbor. Golgi outposts are small enough to be trafficked into terminal branches that are 150-300 nm in diameter, and therefore may provide an excellent vehicle for transporting nucleation machinery to these remote areas of the arbor. It will be interesting to determine how these nucleation factors are recruited to the Golgi outposts (Ori-McKenney, 2012).

It has been previously shown that GM130 can recruit AKAP450 to the Golgi complex, but whether the first coiled-coil domain of the Drosophila AKAP450 homolog, CP309, can also bind GM130 is unknown. Interestingly, this study has observed that predominantly stationary Golgi outposts correlated with EB1 comet formation, indicating that this specific subpopulation may contain γ-tubulin and CP309. What other factors may be necessary to properly position the Golgi outposts at sites such as branchpoints, and how this is achieved will be a fascinating direction for future studies (Ori-McKenney, 2012).

Whether the acentrosomal microtubule nucleation uncovered in this study also occurs in the dendrites of mammalian neurons is a question of great interest. Golgi outpost distribution in cultured hippocampal neurons is significantly different than that in da neurons, and hippocampal neurons do not form as elaborate arbors as da neurons. However, other types of mammalian neurons form much more complex dendritic arbors and may conceivably require acentrosomal nucleation for the growth and perpetuation of the dendrite branches (Ori-McKenney, 2012).

This study provides the first evidence that Golgi outposts can nucleate microtubules at acentrosomal sites in neurons, shedding new light on the longstanding question about the origin of the microtubule polymer in elongated neuronal processes. This source of nucleation contributes to the complex organization of microtubules within all branches of the neuron, but is specifically necessary for terminal branch development. It is thus conclude that acentrosomal microtubule nucleation is essential for dendritic branch growth and overall arbor maintenance of class IV da neurons, and that Golgi outposts are important nucleation centers within the dendritic arbor (Ori-McKenney, 2012).

In this study, 546 lethal lines were screened for potential defects in Rh1 by examining the localization of Arr2::GFP in FLP/FRT-mediated mosaic retinas using two-color fluorescence imaging. A mutation was found in the Drosophila homolog of human PIG-U (Drosophila PIG-U), which encodes a subunit of GPI transamidase. Mutations in other genes of the GPI synthesis pathway but not in the GPI modification pathway gave rise to the same phenotype. Furthermore, the GPI-linked protein, Chp accumulates in the ER whereas the stalk membrane Crumbs protein and basolaterally localized Na⁺K⁺-ATPase were mis-sorted to the rhabdomere. Rh1 was found to be degraded before entering the post-Golgi vesicles, but Crb and Na⁺K⁺-ATPase are misrouted into vesicles destined for the rhabdomere in PIG mutant cells (Satoh, 2013).

There are two previous reports concerning GPI requirements for the transport of transmembrane proteins. In zebrafish, GPI transamidase has been found to be essential for the surface expression of voltage-gated sodium channels. In yeast, GPI synthesis is required for the surface expression of Tat2p tryptophan permease, which is associated with detergent-resistant membrane (DRM) in wild-type cells. In GPI-deficient yeast, Tat2p and Fur4p fail to associate with DRM and are retained in the ER. Although DRM forms in the ER in yeast, in mammalian cells, it is likely that DRM formation occurs only after Golgi entry. The reason for this is thought to be that GPI lipid remodeling occurs in different places: the ER in yeast and the Golgi body in mammalian cells. In mammalian cells, lipid rafts are postulated to concentrate some fractions of apically destined proteins owing to their affinity for the TGN or recycling endosomes (Satoh, 2013).

Along with the raft model, there are two possible explanations for the sorting phenotype of PIG mutant fly photoreceptors: (1) the polarized sorting of Rh1 depends on its affinity for the raft/DRM and the raft/DRM is deficient in PIG mutants; (2) unidentified GPI-anchored protein(s) play crucial roles in the polarized sorting of Rh1 and Na⁺K⁺-ATPase, and the raft/DRM provides a platform for the GPI-anchored protein(s). The first model predicts raft/DRM deficiency in PIG mutants, Rh1 association with lipid rafts and a stronger phenotype caused by mutations in the genes involved in raft formation. By contrast, the second model predicts that GPI deficiency produces a stronger phenotype than that caused by raft deficiency (Satoh, 2013).

Analysis of lipid raft deficiency does not support the first model in which the loss of polarized sorting of Rh1/Na⁺K⁺-ATPase in PIG mutants is a consequence of raft deficiency; instead, the current results support the second model in which unidentified GPI-anchored protein(s) concentrate Rh1 and exclude Na⁺K⁺-ATPase and Crb from post-Golgi vesicles destined for the rhabdomeres. Thus, loss of the GPI-anchored sorting protein(s) might cause most Rh1 to be directed into the endocytotic pathway and degraded by lysosomes while simultaneously allowing Na⁺K⁺-ATPase and Crb to be loaded into post-Golgi vesicles destined for the rhabdomeres. Chp is the only GPI-anchored protein known to be expressed in fly photoreceptors in the late-pupal stages. However, chp² mutants do not exhibit any mislocalization phenotype of Rh1 or Na⁺K⁺-ATPase. Identifying the GPI-anchored protein(s) responsible for the sorting in the TGN is an important step for understanding this mechanism of polarized transport (Satoh, 2013).

The biosynthetic pathway of GPI-anchored proteins has been well elucidated, but little was known to date about the phenotypic consequences of the loss of GPI synthesis in vivo. The present study demonstrates that GPI synthesis is essential for the sorting of non-GPI-anchored transmembrane proteins, including Rh1 and Na⁺K⁺-ATPase, without obvious defects in adherens junctions. Human PIGM or PIGV deficiency causes seizures or mental retardation. These neurological disorders might be also caused by the mis-sorting of some transmembrane proteins in addition to the defects in the formation of GPI-anchoring proteins. These findings aid the understanding of the pathology of diseases involving deficient GPI-anchoring protein synthesis (Satoh, 2013).

Neurons can maintain stable synaptic connections across adult life. However, the signals that regulate expression of synaptic proteins in the mature brain are incompletely understood. This study describes a transcriptional feedback loop between the biosynthesis and repertoire of specific phospholipids and the synaptic vesicle pool in adult Drosophila photoreceptors. Mutations that disrupt biosynthesis of a subset of phospholipids cause degeneration of the axon terminal and loss of synaptic vesicles. Although degeneration of the axon terminal is dependent on neural activity, activation of sterol regulatory element binding protein (SREBP) is both necessary and sufficient to cause synaptic vesicle loss. These studies demonstrate that SREBP regulates synaptic vesicle levels by interacting with tetraspanins, critical organizers of membranous organelles. SREBP is an evolutionarily conserved regulator of lipid biosynthesis in non-neuronal cells; these studies reveal a surprising role for this feedback loop in maintaining synaptic vesicle pools in the adult brain (Tsai, 2019).

These studies demonstrate that disrupting the biosynthesis of specific membrane phospholipids causes adult-onset degeneration of R cells and loss of synaptic vesicles. These two phenotypes arise via distinct molecular mechanisms that can be doubly dissociated using genetic and physiological manipulations. Degeneration of the axon terminal is an activity-dependent process that requires calcium-mediated vesicle fusion. Conversely, loss of synaptic vesicles is driven by activation of the transcription factor SREBP. Thus, in these cells, SREBP is activated by alterations in the levels of specific phospholipids. Here, SREBP affects the expression of a specific subset of genes that are largely not directly involved in lipid regulation, thus defining a previously unknown SREBP function. Rather, SREBP activation leads to reduced expression of four tetraspanins. Restoring expression of either of two of these tetraspanins suppresses the effects of SREBP activation, demonstrating that tetraspanins are functional effectors of SREBP in photoreceptors. Thus, a specialized feedback loop from the synaptic terminal to the nucleus links the levels of specific phospholipids to photoreceptor function and synaptic vesicle number. It is proposed that this feedback loop matches the vesicular demand for phospholipids to their production. As SREBP is evolutionarily conserved, and recent studies have linked SREBP to neuronal damage in several contexts, it is speculated that this feedback loop plays a central role in maintaining synaptic vesicle pools in the healthy aging brain (Tsai, 2019).

In Drosophila, mutations that disrupt phospholipid biosynthesis cause broad defects in brain function, including increased seizure activity and photoreceptor degeneration. However, these and other studies examining phospholipid composition in flies have either not quantified phospholipid levels or have not differentiated different phospholipid species. These studies using a high-resolution lipidomic approach demonstrate that biosynthesis of specific PE and PC species is required for maintaining synaptic vesicle pools and the axon terminal in adult photoreceptors. Moreover, the biosynthetic enzyme Pect is found at the axon terminal. It is speculated that the production of specific phospholipids can occur locally, coupling precise levels of phospholipids to the cellular processes that require them in the axonal compartment. Finally, recent work has demonstrated that derivatives of very long chain (VLC) PC species are neuroprotective in vertebrate photoreceptors and neurons. Although this study detected only one VLC PC precursor, PC c44:12, representing 0.01% of the total PC species in the fly retina, future studies will determine the extent to which derivatives of this or other PC or PE species play roles in maintaining adult photoreceptor axons and synapses in Drosophila. This work suggests the following model. The ultrastructural analysis of pect mutants reveals phenotypes in the axon terminal that are strongly reminiscent of those in endocytic mutants. Consistent with this, blocking exocytosis in pect mutants by either reducing light exposure or by genetic means suppresses axon terminal degeneration. It is therefore inferred that the inability to retrieve synaptic vesicles from the plasma membrane is sufficient to cause neuronal degeneration and that the availability of specific phospholipids can be rate limiting for endocytosis. These results are consistent with previous studies in C. elegans that demonstrated that a phospholipid desaturase causes defects in endocytosis through effects on synaptojanin, a critical component in endocytosis. At the same time, altering phospholipid production may also impair vesicle biogenesis, in which case blocking synaptic transmission could suppress neuronal degeneration by removing the demand for vesicle biogenesis via an as-yet-unknown mechanism (Tsai, 2019).

SREBP is a central regulator of genes involved in lipid biosynthesis in many cell types. The current data support the notion that SREBP plays an additional role in Drosophila photoreceptors. As the levels of only a few phospholipids are altered in pect mutants, SREBP activation appears linked to the detection of changes in levels in these PE and PC species. Moreover, although activation of SREBP does upregulate a small number of genes involved in lipid biosynthesis, it also downregulates many genes involved in phototransduction and synaptic function. Among these, genetic interaction studies demonstrate that tetraspanins are functionally critical SREBP effectors. Tetraspanins are transmembrane proteins that have been linked to synapse development, lysosomal function in R cells, and to outer segment structure and function in the vertebrate retina. Moreover, recent work has demonstrated that they can serve as cholesterol-binding proteins, further implicating this family in the regulation of membrane function. Although unraveling the specific molecular mechanisms that link tetraspanin function to synaptic vesicle pools remains a challenge for future studies, the current model for this role of SREBP represents an extension of SREBP's long-standing role in regulating lipid biosynthesis. In particular, a central role for phospholipids that is unique to neurons is as a critical component of synaptic vesicles. It is hypothesized that, when SREBP is activated and tetraspanin expression is reduced, either the biogenesis of synaptic vesicles is downregulated or their turnover and degradation is increased, shrinking the synaptic vesicle pool in an activity-independent manner. As a result, the cellular demand for the specific phospholipids found in synaptic vesicles is reduced. More broadly, these studies suggest that SREBP might complement its long-standing role in lipid biosynthesis with an additional role in controlling phospholipid utilization. Finally, by combining the high-resolution lipidomic approach this study developed to work with small populations of labeled cells with the powerful genetic tools available in this system, future work may shed further light on the regulation of SREBP activity and phospholipid levels (Tsai, 2019).

SREBP has been linked to both neurodegenerative disease and stroke. Recent studies in flies have demonstrated that reactive oxygen species can activate SREBP to cause lipid droplet formation in glia. However, the molecular mechanisms by which SREBP might act in these contexts are unknown. In addition, mutations in a human tetraspanins have been linked to intellectual disability. The demonstration that SREBP acts through tetraspanins to regulate synaptic vesicle pools and negatively regulates other genes required for synaptic function suggests a unifying mechanism for these seemingly disparate observations. Taken together, these studies argue that SREBP plays an evolutionarily conserved role in regulating neuronal and synaptic function, suggesting a link between the neuronal phospholipid repertoire and synapse maintenance in the adult brain (Tsai, 2019).

Protein trafficking and membrane addition in neurons need to be coordinated with the growth requirements of the axonal and dendritic plasma membranes, but the mechanisms that do so have been obscure. Abl pathway proteins associate with many of the ubiquitous guidance receptors that direct axon growth and guidance throughout phylogeny, including Netrin, Roundabout, the receptor tyrosine phosphatase DLAR, Notch and others. The data therefore suggest a potential link between the regulatory machinery that senses guidance information and the secretory machinery that helps execute those patterning choices. Indeed, preliminary experiments suggest that some of the axonal defects of Abl pathway mutants may arise from alterations in golgi function. Beyond this, Abl signaling is essential in neuronal migration, epithelial polarity and integrity, cell adhesion, hematopoiesis and oncogenesis, among other processes The data reported in this study now compel a reexamination of the many functions of Abl to ascertain whether some of these effects arise, at least in part, from regulation of secretory function (Kannan, 2014).

Cytokinesis requires a tight coordination between actomyosin ring constriction and new membrane addition along the ingressing cleavage furrow. However, the molecular mechanisms underlying vesicle trafficking to the equatorial site and how this process is coupled with the dynamics of the contractile apparatus are poorly defined. This study provides evidence for the requirement of Rab1 during cleavage furrow ingression in cytokinesis. The gene omelette (omt) was found encodes the Drosophila orthologue of human Rab1 and is required for successful cytokinesis in both mitotic and meiotic dividing cells of Drosophila melanogaster. Rab1 protein colocalizes with the conserved oligomeric Golgi (COG) complex Cog7 subunit and the phosphatidylinositol 4-phosphate effector GOLPH3 at the Golgi stacks. Analysis by transmission electron microscopy and 3D-SIM super-resolution microscopy reveals loss of normal Golgi architecture in omt mutant spermatocytes indicating a role for Rab1 in Golgi formation. In dividing cells, Rab1 enables stabilization and contraction of actomyosin rings. It was further demonstrated that GTP-bound Rab1 directly interacts with GOLPH3 and controls its localization at the Golgi and at the cleavage site. It is proposed that Rab1, by associating with GOLPH3, controls membrane trafficking and contractile ring constriction during cytokinesis (Sechi, 2017).

Cytokinesis represents the final act of cell division when a mother cell becomes fully partitioned into two daughter cells. Cytokinesis failures can contribute to several human diseases including blood disorders, age-related macular degeneration, Lowe syndrome, female infertility and cancer. In animal cells, cytokinesis relies upon constriction of a plasma membrane-anchored actomyosin ring, which leads to cleavage furrow ingression at the equatorial cortex. To fully separate each mother cell into two daughter cells, cytokinesis is also associated with a considerable expansion of cell plasma membrane. Insertion of new membrane during cytokinesis is achieved through shuttling of membrane vesicles to the ingressing cleavage furrow and involves both secretory and endocytic/recycling trafficking activities. Accumulating evidence also indicates that phosphoinositide lipids regulate both contractile ring dynamics and membrane trafficking during cytokinesis. Drosophila male meiosis provides an excellent cell system to dissect the vesicle trafficking pathways involved in cytokinesis. Indeed screens for mutants affecting spermatocyte cytokinesis have identified several components of the Golgi and endocytic/recycling machinery, comprising the conserved oligomeric Golgi complex (COG) subunits Cog5 and Cog7, the TRAPPII complex subunit Brunelleschi, the Dyntaxin 5 ER-to-Golgi vesicle-docking protein, the small GTPases Rab11 and Arf6, the COPI subunits and the exocyst complex proteins Sec8 and Exo84. Mutations affecting male meiotic cytokinesis have also revealed the requirement for proteins that regulate the phosphoinositide pathway including the Drosophila Phosphatidylinositol (PI) transfer protein (PITP), Giotto/Vibrator (Gio/Vib) and the PI 4-kinase III β Four wheel drive (Fwd). Both Fwd and Gio/Vib are required to localize Rab11 at the cleavage site. Fwd directly binds Rab11 at the Golgi and is required for synthesis of PI 4-phosphate (PI(4)P) on Golgi membranes and for localization of secretory organelles containing both PI(4)P and Rab11 at the cleavage site. Recent work has shown that the oncoprotein GOLPH3, described as a PI(4)P effector at the Golgi, accumulates at the cell equator of dividing cells and is required for cleavage furrow ingression in Drosophila. GOLPH3 function during cytokinesis is intimately connected to its ability to bind PI(4)P and regulates both the dynamics of the actomyosin ring and vesicle trafficking to the cleavage site (Sechi, 2017 and references therein).

This study provides the first comprehensive demonstration for Rab1 function in cytokinesis, in tissues of a multicellular organism. A possible involvement of Rab1 in mitotic cytokinesis was previously suggested by a genome-wide screen aimed at identifying genes required for cytokinesis in cultured Drosophila cells, reporting a slight increase of binucleate cells in RNAi-treated cells when compared with control. The current analysis reveals defects in early stages of cytokinesis of dividing spermatocytes, neuroblasts and S2 cells with reduced Rab1 protein expression, which result in incomplete contractile ring constriction. Although myosin II/anillin rings were observed in early telophases of omt/Df mutants, these structures failed to undergo full constriction during cytokinesis. A similar phenotype was found also in Drosophila S2 cells depleted of Rab1. Failure to assemble functional actomyosin rings is a commonly observed phenotype in male meiotic mutants of membrane-trafficking components including the COG subunits Cog5 and Cog7, the Arf6 and Rab11GTPases, the TRAPPII subunit Brunelleschi and GOLPH3. A model has been suggested whereby, during cytokinesis, assembly and dynamics of the contractile apparatus are intimately connected with vesicle trafficking and membrane remodelling at the cleavage furrow. In this context, membrane vesicles that fuse with the furrow membrane during cytokinesis might also transport structural components of the contractile ring or F-actin regulators. Indeed, visualization of actin and endocytic vesicles in cellularizing Drosophila embryos has suggested that F-actin and vesicles might be targeted as a unit to the furrow site (Sechi, 2017).

The finding that Rab1 localizes at the Golgi suggests a role for this protein in Golgi trafficking during cytokinesis. In agreement with this hypothesis, this analysis of Golgi in interphase spermatocytes by 3D-SIM super-resolution microscopy and TEM revealed a highly altered structure in Rab1 mutants, suggesting that defective trafficking through the Golgi may impair the flow of vesicle trafficking to the cleavage site and halt cytokinesis. Moreover, the characteristic organization of Drosophila Golgi into multiple discrete stacks, scattered throughout the cytoplasm, allowed has led to the uncovering of structural defects, caused by Rab1 mutations, that might not be identified in mammalian cells where the stacks are interconnected into a single ribbon-like Golgi. Indeed, mutations in Rab1 affected both the number and size of the Golgi stacks and disrupted the ultrastructure of Golgi cisternae. Golgi fragmentation is likely to result from defective COP I-mediated vesicle trafficking, which in turn depends on the GTPase Arf1 and its guanine nucleotide exchange factor GBF1. Consistent with this hypothesis, the current work demonstrates that Rab1 interacts with Garz, the Drosophila orthologue of GBF1, which is essential to recruit this protein to the Golgi. Golgi fragmentation and trafficking defects are also likely to result from decreased localization of the PI(4)P-binding protein GOLPH3. Remarkably, GOLPH3 is a key protein for maintaining Golgi architecture and vesicular release. A recent study has proposed that human Rab1B, in complex with PITPNC1, might control Golgi morphology by regulating Golgi PI(4)P levels and hence indirectly the abundance of the PI(4)P effector GOLPH3. In agreement with this work, the current data indicate that GOLPH3 requires wild-type function of Rab1 for its localization at the Golgi membranes during both interphase and telophase. Moreover, this study provides evidence that GOLPH3 protein directly interacts with Rab1-GTP and requires wild-type function of Rab1 for its recruitment to the cleavage site. Taken together these data suggest that Rab1 protein, by contributing to GOLPH3 recruitment, enables secretory vesicle trafficking and actomyosin constriction during cytokinesis. It cannot be excluded that the loss of Rab1 could have additional effects through other Golgi effectors in addition to GOLPH3 and that the cytokinesis defects might be the indirect consequences of altered secretory or endocytic pathways that are known to be important for cytokinesis. Indeed mutations in Rab1 do not affect Golgi localization of Cog7 but disrupt recruitment of the ArfGEF orthologue Garz, a known Golgi effector of Rab1. Nevertheless, these data indicate GOLPH3 is a major effector of Rab1 in mediating contractile ring constriction and cleavage furrow ingression during cytokinesis (Sechi, 2017).

In mammalian cells, a single molecular TRAPPII complex acts as a GDP-GTP exchange factor for Rab1 [60]. This complex appears to have a counterpart in Drosophila melanogaster where Bru, the fly orthologue of the TRAPPII subunit Trs120p, is also required for cleavage furrow ingression during male meiotic cytokinesis. In fission yeast and plant cells, this role appears to require both the TRAPPII and exocyst complexes that associate with vesicles in the cleavage furrows. These data suggest a possible conserved role for Rab1 together with the TRAPPII complex in guiding membrane addition to the cleavage furrow during cytokinesis that in animal cells may be played by a single complex. The investigation of such possibilities will be the topic of future work (Sechi, 2017).

Golgi phosphoprotein 3 (GOLPH3) is a highly conserved peripheral membrane protein localized to the Golgi apparatus and the cytosol. GOLPH3 binding to Golgi membranes depends on phosphatidylinositol 4-phosphate [PI(4)P] and regulates Golgi architecture and vesicle trafficking. GOLPH3 overexpression has been correlated with poor prognosis in several cancers, but the molecular mechanisms that link GOLPH3 to malignant transformation are poorly understood. It was recently shown that PI(4)P-GOLPH3 couples membrane trafficking with contractile ring assembly during cytokinesis in dividing Drosophila spermatocytes. This study used affinity purification coupled with mass spectrometry (AP-MS) to identify the protein-protein interaction network (interactome) of Drosophila GOLPH3 in testes. Analysis of the GOLPH3 interactome revealed enrichment for proteins involved in vesicle-mediated trafficking, cell proliferation and cytoskeleton dynamics. In particular, it was found that dGOLPH3 interacts with the Drosophila orthologs of Fragile X mental retardation protein and Ataxin-2, suggesting a potential role in the pathophysiology of disorders of the nervous system. These findings suggest novel molecular targets associated with GOLPH3 that might be relevant for therapeutic intervention in cancers and other human diseases (Sechi, 2021).

Dendrite aberration is a common feature of neurodegenerative diseases caused by protein toxicity, but the underlying mechanisms remain largely elusive. This study shows that nuclear polyglutamine (polyQ) toxicity resulted in defective terminal dendrite elongation accompanied by a loss of Golgi outposts (GOPs) and a decreased supply of plasma membrane (PM) in Drosophila class IV dendritic arborization (da) (C4 da) neurons. mRNA sequencing revealed that genes downregulated by polyQ proteins included many secretory pathway-related genes, including COPII genes regulating GOP synthesis. Transcription factor enrichment analysis identified CREB3L1/CrebA, which regulates COPII gene expression. CrebA overexpression in C4 da neurons restores the dysregulation of COPII genes, GOP synthesis, and PM supply. Chromatin immunoprecipitation (ChIP)-PCR revealed that CrebA expression is regulated by CREB-binding protein (CBP), which is sequestered by polyQ proteins. Furthermore, co-overexpression of CrebA and Rac1 synergistically restores the polyQ-induced dendrite pathology. Collectively, these results suggest that GOPs impaired by polyQ proteins contribute to dendrite pathology through the CBP-CrebA-COPII pathway (Chung, 2017).

Neurons have tremendously higher amounts of plasma membrane (PM) than other cell types, due to their highly elongated morphology. Recently, dendritic satellite organelles have been reported to be involved in the maintenance of integrity and dynamics of the PM in distal dendrites out of reach from somatic perinuclear organelles. The dendritic organelles have functional overlaps with the canonical perinuclear organelles. However, the restricted space appears to require the miniaturization of the satellite organelle systems, such as the endoplasmic reticulum (ER) and Golgi outposts (GOPs), compared to those localized around the nucleus, suggesting that the dendritic organelles may show different functional capacity from their perinuclear counterparts. Additionally, the distal areas of the dendrites may have quite different cellular environments, such as variations in the concentration of ions and proteins, compared to the perinuclear region. These differences may account for the vulnerability of dendrites over other neuronal domains to neuropathological insults, including protein toxicity conferred by the accumulation of mutant or misfolded proteins in neurons (Chung, 2017).

In Drosophila dendritic arborization (da) sensory neurons, GOPs were found to be located in dendrites, but not in axons, suggesting that GOPs may act as local stations to supply membranes to the PM in nearby dendrites (Ye, 2007). The synthesis of GOPs was reported to be regulated by secretory pathway-related genes, such as Sec31 and the transcription factor Cut (Iyer, 2013). Additionally, GOPs were shown to be transported toward or away from the soma by machinery consisting of a lava lamp protein (LVA), a golgin coiled-coil adaptor protein, and a motor dynein-dynactin protein complex, and their transport was found to be closely associated with the dynamics of dendrite growth. Moreover, GOPs have been implicated in the growth and maintenance of the dendrite arbor. For example, laser ablation of local GOPs decreased branch dynamics in da neurons (Ye, 2007), and the disruption of GOP trafficking blocked dendrite growth in developing hippocampal neurons, leading to the decreased length of dendrites (Chung, 2017).

Despite such functional roles of the GOPs in the growth and maintenance of the dendrite arbor under non-pathological conditions, it still remains unclear whether the GOPs can also contribute to dendrite aberration observed under pathological conditions induced by protein toxicity. Previously, nuclear proteotoxicity has been reported to cause severe dendrite pathology, but the mechanisms underlying dendrite pathology caused by protein toxicity remain largely elusive. Therefore this study investigated a mechanistic link of the GOPs to dendrite pathology caused by toxic nuclear polyQ proteins using Drosophila da sensory neurons that have been extensively utilized by various researchers as an in vivo cellular model for the study of the mammalian neuronal dendrite system with high fidelity dendrite pathology (Chung, 2017).

Toxic nuclear polyQ proteins cause terminal dendrite defects in C4 da neurons. This study examined morphological characteristics of dendrites of class IV (C4) da neurons expressing pathogenic Machado-Joseph's Disease (MJD) protein, MJDtr-78Q (78Q), known to induce nuclear protein toxicity. For this analysis, dendrite images of C4 da neurons expressing 78Q (78Q OE) and wild-type C4 da neuron controls were compared. A mechanistic model is proposed that links toxic nuclear polyQ proteins to the impaired elongation of terminal dendrites in 78Q OE through the CBP-CrebA-COPII-GOP pathway. Toxic nuclear polyQ proteins decreased the number of GOPs in the dendrites of C4 da neurons. mRNA sequencing revealed that the secretory pathway, including the COPII pathway, was a major pathway that was downregulated by toxic polyQ proteins. The disruption of the COPII pathway decreased GOP synthesis. TF enrichment analysis identified CrebA as a key regulator of the genes involved in the COPII pathway. CrebA overexpression restored the downregulation of the COPII pathway and the loss of GOPs caused by toxic polyQ proteins. It was further found that CBP regulated CrebA expression cooperatively with Cut and that toxic polyQ proteins interfered with CBP upstream of CrebA in the regulation of GOP synthesis. Finally, the co-overexpression of CrebA and Rac1 synergistically restored polyQ-induced defects in dendrite branching and elongation (Chung, 2017).

According to a mechanistic model, however, how the overexpression of Cut, unlike CBP, can potentially contribute to the restoration of GOPs is unclear, although it cooperatively regulates CrebA expression with CBP and interacts with CBP at the protein level. This can be explained by alterations in the stoichiometry between CBP and Cut for their complex formation under polyQ-expressed conditions. The results showed that polyQ proteins appeared to sequester CBP, but not Cut. The sequestration of CBP by polyQ proteins leads to a decreased amount of CBP available for the CBP-Cut complex formation. Accordingly, CBP overexpression can increase the amount of non-sequestered CBP under polyQ-expressed conditions, thereby restoring the CBP-Cut complex formation. By contrast, Cut overexpression does not contribute to an increase in complex formation because it has no effect on the depletion of CBP by polyQ proteins. Moreover, Rac1 was included as a component in the mechanistic model. Rac1 was connected to the polyQ-CBP-CrebA pathway based on the following results. First whether polyQ expression affected the expression of Rac1 was tested and a significant decrease in Rac1 expression was found in polyQ-expressing fly heads compared to w1118. Then TFs were sought that could be affected by polyQ expression and could regulate Rac1 expression. Previously, Cut was shown to regulate Rac1 expression. Thus, to examine the effect of polyQ proteins on Cut, whether polyQ proteins altered the intracellular distribution of Cut proteins was checked in polyQ-expressing da neurons, and no noticeable alteration was found in its distribution. As previously mentioned, Cut interacted with CBP in fly heads. Furthermore, Rac1 was shown to be epigenetically suppressed by H3K27me3, which can be antagonized by CBP-dependent H3K27ac. These data together provide a mechanistic connection of the CBP-Cut-Rac1 pathway to the polyQ-CBP-CrebA pathway through CBP (Chung, 2017).

This study proposed a polyQ-CBP-CrebA pathway for polyQ-induced terminal dendrite defects. However, an alternative possibility that dendrite perturbation may precede through CBP-CrebA-independent pathways, which then can result in altered gene expression, cannot be excluded. Consistent with such a possibility, nuclear polyQ proteins were shown to sequester nuclear pore complex proteins, potentially impairing the transport of proteins and RNA, independently of CBP or CrebA (Grima, 2017). Moreover, nuclear polyQ proteotoxicity induces the mis-localization of several TFs (e.g., TBP, Sp1, and p53) other than CBP, leading to the altered expression of their target genes. The perturbed functions of nuclear pore complexes and TFs may result in dendrite defects via CBP-CrebA-independent mechanisms and consequently alterations in gene expression. However, the restoration of GOP synthesis and PM protein supply via the overexpression of CBP or CrebA and the restoration of dendritic arborization defects via the co-overexpression of CrebA and Rac1 in polyQ-expressing neurons demonstrates that the CBP-CrebA pathway should be one of the important pathways that functionally regulates the polyQ-induced dendrite defects. Nevertheless, the relative importance of the CBP-CrebA pathway to the pathways that include other TFs affected by polyQ proteins can be questioned. However, this study found that the overexpression of the three key TFs (HLH106, gt, and Dref) did not restore the number of GOPs in polyQ-expressing neurons. Moreover, the effect of Sp1, which is known to be downregulated by polyQ proteins, on GOP synthesis was examined, and Sp1 knockdown did not significantly decrease the number of GOPs compared to the controls. Furthermore, it was also found that the overexpression of Cut and Scr, known upstream TFs of CrebA, failed to restore the loss of GOPs in polyQ-expressing neurons comparable to CrebA overexpression. Therefore, the data suggest that the mechanism by which polyQ toxicity induces dendrite pathology can be attributed to the polyQ-CBP-CrebA pathway proposed in this study (Chung, 2017).

The loss of GOPs was one of the most apparent dendrite defects caused by polyQ proteins. Thus, among many cellular processes downregulated by the polyQ proteins, this study focused on three cellular processes, (1) vesicle-mediated transport (VT) and (2) membrane organization (MO) and lipid biosynthetic process (LP), that were considered to be related to GOPs. However, these processes can also be related to other secretory-pathway-related processes in addition to GOP synthesis. For example, neuropeptide maturation and trafficking were previously shown to be modulated by the secretory pathway, suggesting the potential association of neuropeptide-containing vesicle trafficking with the three processes. Thus the effect of polyQ proteins on neuropeptide-containing vesicle trafficking was examined by measuring the amount of neuropeptide-containing vesicles in w1118 and polyQ-expressing neurons using a reporter. PolyQ expression was found to decrease the amount of ANF-EMD vesicles in dendrites and soma compared to controls, suggesting that polyQ perturbs the function of the secretory pathway related to neuropeptide-containing vesicle trafficking in addition to GOP synthesis. Additionally, the three processes can be associated with the synthesis of somatic Golgi. However, the data showed a larger loss of ManII-eGFP-positive puncta in the dendrites of C4 da neurons compared to the soma, suggesting that the three processes can be considered to be related more to GOP synthesis than to somatic Golgi synthesis. Thus, although the polyQ-induced downregulation of genes associated with the abovementioned three processes may impact the secretory pathway in general, the results appear to indicate that the synthesis of GOPs responds more to the downregulation of those genes compared to the synthesis of somatic Golgi (Chung, 2017).

In mRNA-sequencing analysis, fly heads were used to identify genes affected by toxic polyQ proteins. Considering that fly heads contain no C4 da neurons, alterations in gene expression obtained from mRNA sequencing in polyQ-expressing fly heads, compared to w1118 fly heads, should be defined by non-C4 da cells. Due to a small number (~50) of C4 da neurons per larva, however, it is challenging to isolate a sufficient number (>105 cells) of C4 da neurons for mRNA sequencing. Instead of C4 da neurons, fly heads were used for mRNA sequencing to obtain a clue for alteration of gene expression by polyQ proteins. Moreover, mRNA sequencing identified 5,385 differentially expressed genes between polyQ-expressed and control samples, of which 5,325 (98.9%) were downregulated. Such a large number and high percentage of downregulated genes suggests that there might be widespread cell death in polyQ-expressing fly brains. Thus, the extent of cell loss was checked in the polyQ-expressing adult fly brains on which mRNA sequencing was performed by measuring the number of cells that displayed cleaved caspase-3 through immunohistochemistry analysis. PolyQ-expressing brains showed minimal cell loss compared to controls in the condition where mRNA sequencing was performed. Additionally, no observable decrease was found in rRNA in polyQ-expressed adult fly heads compared to w1118 heads. These data suggest that the downregulation of the large number of genes is not likely to be caused by cell death. One alternative explanation can be the previously reported dysregulation of the following histone modifications caused by polyQ proteins: impaired acetylation of H3K27 and dysregulated tri-methylation of H3K9 and H3K27. These dysregulated histone modifications may result in the downregulation of 98.9% of DEGs through a global epigenetic silencing (Chung, 2017).

The normal length of the polyQ tract in humans has been reported to be less than 36 for the MJD protein. In flies, numerous studies have used the truncated MJD protein with only 27 polyQ repeats (MJDtr-27Q) as a control. However, in this study, w1118 rather than 27Q OE was used as the control for 78Q OE in most of the experiments because 27Q OE exhibited strong dendrite pruning defects compared to 78Q OE. A previous study also showed that Httex1p-Q93 caused strong pruning defects in C4 da neurons, whereas Httex1p-Q20 caused no such defects. Taken together, these data suggest that the pruning defects are likely not due to the context of the host proteins but to polyQ toxicity. However, there appears to be a threshold length (between 20Q and 27Q) of the polyQ tract to confer such polyQ toxicity. Nevertheless, further detailed studies should be performed to understand the mechanism underlying the protein toxicity of MJDtr-27Q to induce pruning defects (Chung, 2017).

Finally, this study identified three different nuclear polyQ toxicity models that exhibited decreased GOP synthesis and PM protein supply, indicating a potentially shared mode of nuclear proteotoxicity. Of note, the accumulation of misfolded proteins in the nucleus has been observed in other neuronal diseases or during aging. Thus, the proposed mechanistic model underlying terminal dendrite defects caused by the accumulation of nuclear toxic polyQ proteins can provide significant insights into other neuronal maladies linked to nuclear proteotoxicity and aging that can be further examined in detailed functional studies (Chung, 2017).

This study used highly elongated Drosophila bristle cells to dissect the role of dynein heavy chain (Dhc64C) in Golgi organization. Whereas in the bristle "somal" region Golgi units are composed of cis-, medial, and trans-Golgi compartments ("complete Golgi"), the bristle shaft contains Golgi satellites that lack the trans-Golgi compartment (hereafter referred to as "incomplete Golgi") and which are static and localized at the base area. However, in Dhc64C mutants, the entire bristle shaft was filled with complete Golgi units containing ectopic trans-Golgi components. To further understand Golgi bristle organization, the roles of microtubule (MT) polarity and the Dhc-opposing motor, kinesin heavy chain (Khc), were tested. Surprisingly, in Khc and Ik2Dominant-negative (DN) flies in which the polarized organization of MTs is affected, the bristle shaft was filled with complete Golgi, similarly to what is seen in Dhc64C flies. Thus, this study demonstrated that MTs and the motor proteins Dhc and Khc are required for bristle Golgi organization. However, the fact that both Dhc64C and Khc flies showed similar Golgi defects calls for an additional work to elucidate the molecular mechanism describing why these factors are required for bristle Golgi organization (Melkov, 2019).

Tango1 enables ER-to-Golgi trafficking of large proteins. Loss of Tango1, in addition to disrupting protein secretion and ER/Golgi morphology, causes ER stress and defects in cell shape. The previously observed dependence of smaller cargos on Tango1 is a secondary effect. If large cargos like Dumpy, which this study identifies as a Tango1 cargo, are removed from the cell, nonbulky proteins reenter the secretory pathway. Removal of blocking cargo also restores cell morphology and attenuates the ER-stress response. Thus, failures in the secretion of nonbulky proteins, ER stress, and defective cell morphology are secondary consequences of bulky cargo retention. By contrast, ER/Golgi defects in Tango1-depleted cells persist in the absence of bulky cargo, showing that they are due to a secretion-independent function of Tango1. Therefore, maintenance of ER/Golgi architecture and bulky cargo transport are the primary functions for Tango1 (Rios-Barrera, 2017).

The endoplasmic reticulum (ER) serves as a major factory for protein and lipid synthesis. Proteins and lipoproteins produced in the ER are packed into COPII-coated vesicles, which bud off at ER exit sites (ERES) and then move toward the Golgi complex where they are sorted to their final destinations. Regular COPII vesicles are 60-90 nm in size, which is sufficient to contain most membrane and secreted molecules. The loading of larger cargo requires specialized machinery that allows the formation of bigger vesicles to accommodate these bulky molecules. Tango1 (Transport and Golgi organization 1), a member of the MIA/cTAGE (melanoma inhibitory activity/cutaneous T cell lymphoma-associated antigen) family, is a key component in the loading of such large molecules into COPII-coated vesicles. Molecules like collagens and ApoB (apolipoprotein B)-containing chylomicrons are 250-450 nm long and rely on Tango1 for their transport out of the ER, by physically interacting with Tango1 or Tango1 mediators at the ERES (Rios-Barrera, 2017).

Tango1 is an ER transmembrane protein that orchestrates the loading of its cargo into vesicles by interacting with it in the ER lumen. The interaction of Tango1 with its cargo then promotes the recruitment of Sec23 and Sec24 coatomers on the cytoplasmic side, while it slows the binding of the outer layer coat proteins Sec13 and Sec31 to the budding vesicle. This delays the budding of the COPII carrier. Tango1 also recruits additional membrane material to the ERES from the Golgi intermediate compartment (ERGIC) pool, thereby allowing vesicles to grow larger. It also interacts directly with Sec16, which is proposed to enhance cargo secretion. A shorter isoform of mammalian Tango1 lacks the cargo recognition domain but nevertheless facilitates the formation of megacarrier vesicles (Rios-Barrera, 2017).

Apart from bulky proteins, some heterologous, smaller proteins like secreted horseradish peroxidase (ssHRP, 44 kDa) and secreted GFP (27 kDa) also depend on Tango1 for their secretion . Unlike for collagen or ApoB, there is no evidence for a direct interaction between Tango1 and ssHRP or secreted GFP. It is not clear why Tango1 would regulate the secretion of these molecules, but it has been proposed that in the absence of Tango1, the accumulation of nonbulky proteins at the ER might be due to abnormally accumulated Tango1 cargo blocking the ER; however, this has not been tested experimentally (Rios-Barrera, 2017).

Drosophila Tango1 is the only member of the MIA/cTAGE family found in the fruit fly, which simplifies functional studies. Like vertebrate Tango1, the Drosophila protein participates in the secretion of collagen. And as in vertebrates, ssHRP, secreted GFP, and other nonbulky molecules like Hedgehog-GFP also accumulate in the absence of Tango1. These results have led to the proposal that Tango1 participates in general secretion. However, most of the evidence for these conclusions comes from overexpression and heterologous systems that might not reflect the physiological situation (Rios-Barrera, 2017).

This study describes a tango1 mutant allele that was identified in a mutagenesis screen for genes affecting the structure and shape of terminal cells of the Drosophila tracheal system. Tracheal terminal cells form highly ramified structures with branches of more than 100 μm in length that transport oxygen through subcellular tubes formed by the apical plasma membrane. Their growth relies heavily on membrane and protein trafficking, making them a very suitable model to study subcellular transport. Terminal cells were used to study the function of Tango1, and loss of Tango1 was found to affect general protein secretion indirectly, and it also leads to defects in cell morphology and in the structure of the ER and Golgi. The defects in ER and Golgi organization of cells lacking Tango1 persist even in the absence of Tango1 cargo (Rios-Barrera, 2017).

These studies led to an explanation of why, in the absence of Tango1, nonbulky proteins accumulate in the ER despite not being direct Tango1 cargos. These cargos are retained in the ER as a consequence of nonsecreted bulky proteins interfering with their transport. However, the effect of loss of Tango1 on ER/Golgi morphology can be uncoupled from its role in bulky cargo secretion (Rios-Barrera, 2017).

This study has described a role of Tango1, which was initially identified through its function in tracheal terminal cells and other tissues in Drosophila embryos, larvae, and pupae. Due to their complex shapes and great size, terminal cells are a well-suited system to study polarized membrane and protein trafficking, with the easily scorable changes in branch number and maturation status providing a useful quantitative readout that serves as a proxy for functional membrane and protein trafficking machinery. Moreover, this analyses are conducted in the physiological context of different tissues in the intact organism (Rios-Barrera, 2017).

The loss-of-function allele tango1^2L3443 has a stop codon eight amino acids downstream of the PRD domain and eliminates the 89 C-terminal amino acids of the full-length protein. It is unlikely that the mutation leads to a complete loss of function. First, terminal cells expressing an RNAi construct against tango1 show stronger defects, with fewer branches per cell than homozygous tango1^2L3443 cells. Second, the mutant protein appears not to be destabilized nor degraded, but instead is present at apparently normal levels, albeit at inappropriate sites. Predictions of the deleted fragment of the protein suggest it is disorganized and that it contains an arginine-rich domain that has no known interaction partners and that is not present in human Tango1. In homozygous mutant terminal cells, the mutant tango1^2L3443 protein fails to localize at ERES. In mammalian Tango1, the Sec16-interacting region within the PRD domain is necessary for the localization of Tango1 to the ERES and for its interaction with Sec23 and Sec16, but since this domain is fully present in tango1^2L3443, the results mean that either the missing 89 C-terminal amino acids contain additional essential localization signals, or that the PRD domain is structurally affected by the truncation of the protein. The latter is considered less likely, as a truncation of eight amino acids downstream of the PRD domain is unlikely to destabilize the polyproline motifs, especially as the overall stability of the protein does not seem to be affected. Furthermore, this region shows a high density of phosphoserines (Ser-1345, Ser-1348, Ser-1390, and Ser-1392), suggesting it might serve as a docking site for adapter proteins or other interactors (Rios-Barrera, 2017).

Terminal cells lacking Tango1 have fewer branches than control cells and are often not properly filled with air. This loss-of-function phenotype is not due to a direct requirement for Tango1, as it is suppressed by the simultaneous removal of Dumpy (Dpy), an extracellular protein involved in epidermal-cuticle attachment, aposition of wing surfaces and trachea development. It also cannot be explained by the individual loss of crb, Piopio (Pio) a zona pellucida (ZP) domain protein that mediates the adhesion of the apical epithelial surface and the overlying apical extracellular matrix, or dpy, since knocking down any of these genes has no effect on cell morphology. Instead, it is proposed that the cell morphological defect is due at least in part to the activation of the ER stress response, since expression of Xbp1 is sufficient to recapitulate the phenotype. Xbp1 regulates the expression of genes involved in protein folding, glycosylation, trafficking, and lipid metabolism. It is possible that one or a small number of specific genes downstream of Xbp1 are responsible for defective branch formation or stability, but the phenotype could also be a secondary consequence of the physiological effects of the ER stress response itself, for example, a failure to deliver sufficient lipids and membrane from the ER to the apical plasma membrane (Rios-Barrera, 2017).

Collagen, with a length of 300 nm, and ApoB chylomicrons with a diameter of > 250 nm, have both been biochemically validated as Tango1 cargos. These molecules are not expressed in terminal cells, and therefore it was clear that Tango1 must have a different substrate in these cells. Given that Tango1 is known for the transport of bulky cargo, that Dpy is the largest Drosophila protein at 800 nm length, and that Dpy vesicles are associated with Tango1 rings in tracheal cells, it is proposed that Dpy is a further direct target of Tango1. Colocalization of Tango1 with its cargo has also been observed in other tissues: with collagen in Drosophila follicle cells and with ApoB in mammalian cell lines (Rios-Barrera, 2017).

No regions of sequence similarity that could represent Tango1-binding sites have been found in Tango1 cargos. There are several possible explanations for this. First, these proteins may contain binding motifs, but the motifs are purely conformational and not represented in a linear amino acid sequence. There is no evidence for or against this hypothesis, but it would be highly unusual, and there is support for alternative explanations. Thus, as a second possibility, all three proteins may require Tango1 for their secretion, but variable adapters could mediate the interactions. In vertebrates, Tango1 can indeed interact with its cargo through other molecules; for instance, its interaction with collagen is mediated by Hsp47. However, in Drosophila, there is no Hsp47 homolog. In the case of ApoB, it has been suggested that microsomal triglyceride transfer protein (MTP) and its binding partner, protein disulphide isomerase (PDI), might associate with Tango1 and TALI to promote ApoB chylomicrons loading into COPII vesicles. Evidence supporting this is that the lack of MTP leads to ApoB accumulation at the ER. It is not known if secretion of other Tango1 cargos like collagen or Dpy also depends on MTP and PDI, but PDI is known also to form a complex with the collagen-modifying enzyme prolyl 4-hydroxylase. Previous work has shown that terminal cells lacking MTP show air-filling defects and fail to secrete Pio and Uninflatable to the apical membrane, and that loss of MTP in fat body cells also affects lipoprotein secretion, as it does in vertebrates. Since cells lacking MTP or Tango1 have similar phenotypes, it is plausible that the MTP function might be connected to the activity of Tango1 (Rios-Barrera, 2017).

The data is interpreted to mean that in the absence of Tango1, primary cargo accumulates in the ER, and in addition, there are secondary, indirect effects that can be suppressed by reducing the Tango1 cargo that overloads the ER. The secondary effects include activation of the ER stress response and intracellular accumulation of other trafficked proteins like Crb, laminins, and overexpressed proteins and probably also the accumulation of heterologous proteins like secreted HRP or GFP in other systems (Rios-Barrera, 2017).

The data suggest that primary and secondary cargo reach the ERES but fail to be trafficked further along the secretory pathway. In this model, primary cargo, probably recruited by adaptors, would be competing with other secondary cargo for ERES/COPII availability, creating a bottleneck at the ERES. This is consistent with recent experiments that show that in tango1-knockdown HeLa cells, VSVG-GFP trafficking does not stop completely, but is delayed. Furthermore, in these experiments, VSVG-GFP is mostly seen in association with Sec16 and Sec31, supporting the clogging model (Rios-Barrera, 2017).

It is not immediately clear why cargo accumulation in terminal cells lacking Tango1 affects the secretion of Crb but not of βPS integrin. While steady states are looked at in this analyses, Maeda (2016) measured the dynamics of secretion and found that loss of Tango1 leads to a reduced rate of secretion of VSVG-GFP, an effect that would have been missed for any proteins the current study classified as not affected by loss of Tango1. Irrespective, a range of mechanisms can be thought of that might be responsible for this difference, including alternative secretion pathways and differences in protein recycling. Alternative independent secretory pathways have been reported in different contexts. For instance, while both αPS1 and βPS integrin chains depend on Sec16 for their transport, the αPS1 chain can bypass the Golgi apparatus and can instead use the dGRASP-dependent pathway for its transport. It would be possible then that in terminal cells, βPS integrin is also trafficked through an alternative pathway that is not affected by loss of Tango1. Similarly, tracheal cells lacking Sec24-CD accumulate Gasp, Vermiform, and Fasciclin III, but not Crb, supporting a role for alternative secretion pathways for different proteins, as has been already proposed. Following this logic, overexpressed βPS integrin would then also be trafficked through a different route from that of the endogenous βPS integrin, possibly because of higher expression levels or because of the presence of the Venus fused to the normal protein (Rios-Barrera, 2017).

Drosophila Tango1 was initially found to facilitate collagen secretion in the fat body. More recently, the accumulation of other nonbulky proteins at the ER in the absence of Tango1 has led to the proposal of two models to explain these results: one in which Tango1 regulates general secretion, and the second one where Tango1 is specialized on the secretion of ECM components, since loss of Tango1 leads to the accumulation of the ECM molecules SPARC and collagen. The current results suggest a third explanation, where cargo accumulation in the ER might not necessarily be a direct consequence of only the loss of Tango1. Instead, in addition to depending on Tango1, some proteins of the ECM appear also to depend on each other for their efficient secretion. This is the case for laminins LanB1 and LanB2, which require trimerization before exiting the ER, while LanA can be secreted as a monomer. Loss of collagen itself leads to the intracellular accumulation of ECM components in fat body cells, such as the laminins and SPARC. Conversely, SPARC is required for proper collagen and laminin secretion and assembly in the ECM. Furthermore, intricate biochemical interactions take place between ECM components. Hence, due to the complex genetic and biochemical interactions between ECM components, the dependence of any one of them on Tango1 is difficult to determine without further biochemical evidence. The concept of interdependent protein transport from the ER as such is not new, as it has also been observed in other systems, for instance in immune complexes. During the assembly of T-cell receptor complexes and IgM antibodies, subunits that are not assembled are retained in the ER and degraded (Rios-Barrera, 2017).

Nevertheless, these observations in glial cells, which express laminins but not collagen, allow at least these requirements to be partly separate. This study found that laminins are accumulated due to general ER clogging and not because they rely on Tango1 for their export. This is based on observations that once the protein causing the ER block is removed, laminin secretion can continue in the absence of Tango1. It is still unclear why glial cells can secrete laminins in the absence of collagen whereas fat body cells cannot, but presumably laminin secretion can be mediated by different, unidentified cargo receptors expressed in glial cells (Rios-Barrera, 2017).

This study found that Sec16 forms aberrant aggregates in cells lacking Tango1, as in mammalian cell lines, and that the number of Sec16 particles is reduced. Other studies have shown that Tango1 overexpression produces larger ERES, and that Tango1 and Sec16 depend on each other for localization to ERES. In addition, lack of Tango1 also affects the distribution of Golgi markers. Thus, Tango1 influences not only the trafficking of cargos, but also the morphology of the secretory system (Rios-Barrera, 2017).

It had been suggested that the disorganization of ER and Golgi apparatus in cells lacking Tango1 might be an indirect consequence of the accumulation of Tango1 cargo. The work of Maeda (2016) has provided a possible explanation for the molecular basis, and proposed that Tango1 makes general secretion more efficient, but it has not formally excluded the possibility that the primary cause for the observed defects is secretory protein overload. This study has now shown that this is not the case: In the absence of Tango1, an aberrant ER and Golgi morphology is still observed even after the main primary substrates of Tango1 were removed and, thereby, secretion of other molecules was restored and the ER stress response was prevented (Rios-Barrera, 2017).

ERGIC53 accumulates at the ERES in the absence of Tango1, and this can be partly reversed by removing dpy. This is in apparent contradiction to findings in mammalian cells where Tango1 was necessary for the recruitment of membranes containing ERGIC53 to the budding collagen megacarrier vesicle. However, ERGIC53 also has Tango1-independent means of reaching the ER. The current results indicate that its retrieval from the ER to the ERGIC compartment depends directly or indirectly partly on Tango1. As a cargo receptor for glycoproteins, ERGIC53 may be retained at the ERES as a consequence of the accumulation of its own cargo at these sites. This would mean that it cannot be delivered back to the ERGIC or the cis-Golgi apparatus for further rounds of retrograde transport, which may, in turn, be an explanation for the enlargement of the Golgi matrix protein 130 kD (GM130) compartment seen after Tango1 knockdown (Rios-Barrera, 2017).

The finding that Tango1-depleted cells have a functional secretory pathway despite the ER-Golgi disorganization was unexpected. Stress stimuli like amino acid starvation (but not ER stress response itself) lead to Sec16 translocation into Sec bodies and inhibition of protein secretion. However, uncoupling of ER-Golgi organization from functional secretion has also been observed in other contexts. Loss of Sec23 or Sec24-CD leads to a target peptide sequence KDEL appearing in aggregates of varying sizes and intensities similar to those observed for Sec16 and for KDEL-RFP in cells lacking tango1. Also, GM130 is reduced in Sec23 mutant embryos. Despite these structural problems, these embryos do not show generalized secretion defects and also do not affect the functionality of the Golgi apparatus, as determined by glycosylation status of membrane proteins (Rios-Barrera, 2017).

Thus, Tango1 appears to have an important structural function in coordinating the organization of the ER and the Golgi apparatus, and this, in turn, may enhance vesicle trafficking. This fits with the role of Tango1 in recruiting ERGIC membranes to the ERES, and also with the effects of loss of Tango1 in the distribution of ER and Golgi markers. It has been proposed that the ER and Golgi apparatus in insects, which unlike in mammalian cells is not centralized but spread throughout the cytoplasm, is less efficient for secretion of bulky cargo than mammalian cells that can accommodate and transport it more efficiently through the Golgi ribbon. This difference could explain why tango1 knockout mice seem to have only collagen secretion defects and die only as neonates. However, a complete blockage of the ER might also be prevented by the activity of other MIA3/cTAGE5 family homologs. In mammalian cell culture experiments, even if loss of tango1 affects secretion of HRP, the secretion of other overexpressed molecules like alkaline phosphatase is not affected. This could also be because of the presence of other MIA3/cTAGE5 family homologs. By contrast, because there are no other MIA3/cTAGE5 family proteins in Drosophila, loss of tango1 may lead to the accumulation of a wider range of overexpressed proteins and more overt mutant phenotypes than in mammals (Rios-Barrera, 2017).

Cell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. Here, this study shows that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. The exocyst component Sec15 was identfied as a RASSF8 interactor. Loss of RASSF8 elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, the depletion of which phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, these results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis (Chan, 2021).

The exocyst complex is an important regulator of intracellular trafficking and tethers secretory vesicles to the plasma membrane. Understanding of its role in neuron outgrowth remains incomplete, and previous studies have come to different conclusions about its importance for axon and dendrite growth, particularly in vivo. To investigate exocyst function in vivo Drosophila sensory neurons were used as a model system. To bypass early developmental requirements in other cell types, neuron-specific RNAi was used to target seven exocyst subunits. Initial neuronal development proceeded normally in these backgrounds, however, this was considered to be due to residual exocyst function. To probe neuronal growth capacity at later times after RNAi initiation, laser microsurgery was used to remove axons or dendrites and prompt regrowth. Exocyst subunit RNAi reduced axon regeneration, although new axons could be specified. In control neurons, a vesicle trafficking marker often concentrated in the new axon, but this pattern was disrupted in Sec6 RNAi neurons. Dendrite regeneration was also severely reduced by exocyst RNAi, even though the trafficking marker did not accumulate in a strongly polarized manner during normal dendrite regeneration. The requirement for the exocyst was not limited to injury contexts as exocyst subunit RNAi eliminated dendrite regrowth after developmental pruning. It is concluded that the exocyst is required for injury-induced and developmental neurite outgrowth, but that residual protein function can easily mask this requirement (Swope, 2022).

Exit of secretory cargo from the endoplasmic reticulum (ER) takes place at specialized domains called ER exit sites (ERESs). In mammals, loss of TANGO1 and other MIA/cTAGE (melanoma inhibitory activity/cutaneous T cell lymphoma-associated antigen) family proteins prevents ER exit of large cargoes such as collagen. This study shows that Drosophila melanogaster Tango1, the only MIA/cTAGE family member in fruit flies, is a critical organizer of the ERES-Golgi interface. Tango1 rings hold COPII (coat protein II; see Sec23) carriers and Golgi in close proximity at their center. Loss of Tango1, present at ERESs in all tissues, reduces ERES size and causes ERES-Golgi uncoupling, which impairs secretion of not only collagen, but also all other cargoes examined. Further supporting an organizing role of Tango1, its overexpression creates more and larger ERESs. These results suggest that spatial coordination of ERES, carrier, and Golgi elements through Tango1's multiple interactions increases secretory capacity in Drosophila and allows secretion of large cargo (Liu, 2017).

Secreted proteins reach the extracellular space through a controlled series of membrane traffic events ensuring fusion of cargo-containing secretory vesicles with the plasma membrane. After translocation into the ER, secretory cargo is collected at specialized cup-shaped regions of the ER and then loaded into membrane vesicles that transfer the cargo to the Golgi compartment. These specialized regions of the ER are known as ER exit sites (ERESs) or transitional ER, the latter emphasizing their dynamic relation with the Golgi. At the ERES, vesicles budding from the ER in the direction of the Golgi are generated by the coat protein II (COPII) complex, a set of proteins highly conserved in all eukaryotes. Structural studies have shown that budding of COPII vesicles from ERES is mediated by the assembly of a vesicle-enclosing cage of 60-90 nm in diameter, yet many secreted proteins exceed the dimensions of this cage and are efficiently secreted by cells, raising the question of how this happens. Examples of large secreted proteins include collagens, the main component of extracellular matrices in all animals, for which trimers assemble in the ER into long semirigid rods (Liu, 2017).

TANGO1, a protein belonging to the MIA/cTAGE family (melanoma inhibitory activity/cutaneous T cell lymphoma-associated antigen, has been shown to be involved in the transport of collagens from the ERES to Golgi. Tango1 was discovered in a screening for genes affecting secretion in Drosophila melanogaster S2 cells and confirmed in a second similar screening. It was later found that human TANGO1 was required for the secretion of collagen but not other secreted proteins. This was supported by a TANGO1 knockout mutant mouse which indeed showed defects in the deposition of multiple types of collagens (Wilson, 2011). TANGO1 is a transmembrane protein localized specifically at ERES. The luminal portion of TANGO1 contains an SH3-like domain at its N terminus that is capable of binding collagen at the ER lumen (Saito, 2009) through the chaperone Hsp49 (Ishikawa, 2016). The cytoplasmic portion contains a region with two presumed coiled coils and a Pro-rich region at its C terminus through which TANGO1 may interact with the COPII coat (Saito, 2009). It has been proposed that TANGO1 collects collagen at ERESs as a specific receptor while at the same time ensuring that a large enough vesicle is formed to package that cargo. Activities of TANGO1 in both retarding COPII coat assembly and recruiting ER-Golgi intermediate compartment (ERGIC) membranes to nascent vesicles have been proposed as mechanisms by which TANGO1 can mediate formation of such megacarrier vesicles (Liu, 2017).

Apart from TANGO1, the human genome contains additional TANGO1-like proteins of the MIA/cTAGE family. These include a short splice variant of TANGO1 (TANGO1S) and eight other members of the MIA/cTAGE family of proteins. Common to all these TANGO1-elike proteins is the presence of transmembrane, coiled-coil and Pro-rich regions highly similar to the cytoplasmic portion of TANGO1. In contrast to full-length TANGO1, however, they lack the SH3-like domain and extended intraluminal region. Nonetheless, a function in secretion has been shown for some of these proteins. TANGO1S, lacking the signal peptide and luminal domain of the full protein but preserving its transmembrane domain, is involved in collagen secretion (Maeda, 2016). Also involved in collagen secretion is cTAGE5. Finally, TALI, a chimeric protein resulting from fusion of MIA2 and cTAGE5 gene products, is required for the secretion of ApoB-containing large lipoparticles (Liu, 2017).

Besides TANGO1 and TANGO1-like proteins, loss of several factors potentially involved in general secretion have been shown to affect preferentially collagen secretion in mammalian cells. These include the TRAPP tethering complex component Sedlin, ubiquitination of Sec31 by the ubiquitin ligase KLHL12, Syntaxin 18, and the SNARE regulator Sly1. Notably, mutations in the Sec23A subunit of COPII led to craniofacial development defects attributable to aberrant collagen secretion. These studies suggest that secretion of collagen or large cargo, though using the same basic transport machinery as other cargoes, could be especially sensitive to impairments in that machinery (Liu, 2017).

The fruit fly Drosophila, in which Tango1 was first found, provides a very distinct advantage for studying the early secretory pathway in the form of limited gene redundancy compared with mammals. For instance, single Sar1 and Sec23 homologues are found in Drosophila. Similarly, only one Tango1 protein exists in Drosophila, in contrast to the presence of multiple TANGO1-like proteins with possible overlapping functions in humans. In addition, most proteins shown to play an essential role in secretory pathway function and organization have homologues encoded in the Drosophila genome as well, including Rab small GTPases, COPI and COPII coat components, SNAREs, Golgi matrix proteins, and Golgins. One of the main differences in secretory pathway organization between mammalian and Drosophila cells is that in mammals, ERES-derived vesicles fuse to form an ERGIC, where cargo transits en route to a single juxtanuclear Golgi ribbon. In flies, however, Golgi elements remain dispersed throughout the cytoplasm in close proximity to ERESs, forming ERES-Golgi units. Because this mode of organization is characteristic not just of flies, but probably of all nonmammalian animals and also plants, it is certain that ERES-ERGIC-Golgi secretory pathway organization in mammals is an elaboration on an ancestral, more simple theme represented by functionally independent ERES-Golgi units (Liu, 2017).

Besides its advantages for secretory pathway studies, the fruit fly Drosophila has strongly emerged in recent years as a convenient model to study the biology of collagen and the extracellular matrix. Compared with the 28 types of collagen found in mammals, Drosophila possesses a reduced complement of collagens, consisting of basement membrane Collagen IV and Multiplexin. Expression of Multiplexin, related to Collagens XV and XVIII, is restricted to the heart and central nervous system and is dispensable for viability. Collagen IV, in contrast, is abundantly present in all fly tissues. In Drosophila, as in all animals, Collagen IV is the main component of basement membranes, polymers of extracellular matrix proteins that underlie epithelia and surround organs and provide structural support to tissues. Drosophila Collagen IV is a heterotrimer composed of α chains encoded by Collagen at 25C (Cg25C; α1 chain) and viking (Vkg; α2 chain). The length of the Drosophila Collagen IV trimer is 450 nm, with a predicted molecular mass of 542.4 kD and increased flexibility caused by imperfections of the triple helix (Liu, 2017).

Having shown previously that Drosophila Tango1 is required for secretion of Collagen IV by fat body cells, their main source in the Drosophila larva (Pastor-Pareja, 2011), this study set out to characterize the expression of Tango1, loss-of-function phenotype, and specificity toward Collagen IV. In the course of this study, it was found that Tango1 is required to maintain the size and integrity of ERES-Golgi units, its loss of function impairing not only Collagen IV secretion, but also general secretion (Liu, 2017).

In this study, imaging of ERESs through super-resolution microscopy revealed close proximity of COPII carriers and cis-Golgi elements in the center of Tango1 rings (see also Raote, 2017). When the effects of Tango1 loss are examined, this study found that ERESs were reduced in size and frequently uncoupled from Golgi, indicating a requirement of Tango1 in the normal organization of ERES-Golgi units. Moreover, supporting an important role of Tango1 in the morphogenesis of Drosophila ERESs, overexpression of Tango1 created more and larger ERESs (Liu, 2017).

Overall, the results are consistent with a model in which the spatial organization of the ERES-Golgi interface provided by Tango1's multiple interactions helps build enlarged COPII carriers that canalize traffic in the center of ERESs. The proximity of ERESs and Golgi in Drosophila leads to an additional proposal that direct ERES-Golgi contact might be the way in which large cargo normally transfers from the ER to the Golgi in flies. Direct contact between ER and Golgi has been suggested as a mode of ER-to-Golgi transport in the yeast Saccharomyces cerevisiae and in plants, where ERESs and Golgi are, like in Drosophila, closely juxtaposed and possibly attached physically through a matrix. ERES-ERGIC contact also has been suggested as a transport mechanism in mammals. Careful electron tomography analysis and in vivo imaging could be used in the future to investigate in more detail the dynamics of cargo transfer among ERESs, COPII carriers, and Golgi at the center of Tango1 rings. Given the necessity to secrete not only Collagen IV or lipoprotein particles but also giant cuticular proteins like the 2,500-mol-wt protein Dumpy, ER-to-Golgi carriers in Drosophila must be necessarily large. Taking into account this and the narrow space in which Drosophila ERES-Golgi transport takes place, it is possible that such large carriers start fusion with the Golgi before having separated from the ERES, effectively creating intermittent tubular connections (Liu, 2017).

These experiments, importantly, revealed a wider role in secretion for Tango1, its knockdown causing intracellular retention of the multiple cargoes. Thus, large carriers or tubular connections built with the assistance of Tango1 may mediate not only the transport of large cargo, but also a significant portion of the total flow of general cargo. This is in contrast to the specific roles in secretion of collagens (TANGO1, TANGO1S, and cTAGE5) or lipoprotein particles (TANGO1 and TALI) proposed for mammalian members of the MIA/cTAGE family. Apart from Collagen IV (Pastor-Pareja, 2011), the ECM proteins Perlecan, Tiggrin, SPARC, and Laminin were previously observed to accumulate intracellularly in the absence of Drosophila Tango1, raising the possibility that these defects were secondary to Collagen IV retention or, alternatively, that Tango1 were required for secretion of large ECM proteins in general. The current results, however, show that small non-ECM cargoes like plain GFP were inefficiently secreted in the absence of Tango1 as well. Further supporting a general role of Drosophila Tango1 in secretion, Tango1 is expressed in all tissues of the larva, inconsistent with a relation with specific cargoes. The highest expression of Tango1 was found in the salivary gland, a dedicated secretory organ where genes encoding secretory pathway components are highly expressed as a group, including COPII and COPI genes. It would seem, therefore, that Tango1 expression correlates with secretory activity, but not with Collagen IV secretion because Collagen IV is not expressed in the salivary gland (Pastor-Pareja, 2011; Liu, 2017 and references therein).

Supporting both an organizing function of Tango1 at the ERES-Golgi interface and a wider role in secretion, the cytoplasmic part of Tango1 could rescue Tango1 loss in the fat body. The result of this rescue experiment additionally posits the question of what is the role of the intraluminal part of the Drosophila protein, through which mammalian TANGO1 is thought to interact with cargo. The intraluminal SH3-like domain of Tango1 is conserved among Drosophila and mammals, a sure sign of a biological role, and it is possible that this domain in Drosophila still has a role in binding cargoes, either directly or through several adaptors. Nonetheless, the results clearly show that Tango1 loss impairs general secretion and that the cytoplasmic part of the protein is by itself capable of enhancing Collagen IV secretion independent of the intraluminal part. Although it is conceivable that Drosophila Tango1 and mammalian TANGO1 have diverged in their function, the possibility that MIA/cTAGE5 family members are partially redundant in facilitating general secretion beyond any roles they may have as specific cargo adaptors is worth considering in light of these findings (Liu, 2017).

Recently, suppression of mammalian TFG expression has been shown to result in smaller ERESs that remain functional for the export of many secretory cargoes, but not collagen. TFG, a protein first characterized in the roundworm Caenorhabditis elegans, has been proposed to act in mammals by forming oligomeric assemblies that physically join ERESs and ERGIC (Johnson, 2015). Human and C. elegans TFG have no clear homologue in Drosophila. Conversely, C. elegans has no Tango1 homologue. This is despite the fact that C. elegans possess all four major basement membrane components, numerous collagens, and multiple other large ECM proteins. In this evolutionary context, work on Drosophila Tango1 shows that alternative mechanisms acting in ERES organization may exist in animal cells to increase capacity of ER-to-Golgi transport in terms of both cargo size and the amount of cargo to be secreted. Furthermore, because small COPII vesicles have seldom been observed in animal cells, it is possible that animals have largely abandoned these in favor of larger COPII-dependent carriers built with help from proteins like TFG and Tango1. Such proteins might have initially evolved to enable secretion of metazoan ECM and other large cargoes, creating in the process a mode of transport that increased efficiency of general ER export as well (Liu, 2017).

Secretory cargos are collected at endoplasmic reticulum (ER) exit sites (ERES) before transport to the Golgi apparatus. Decades of research have provided many details of the molecular events underlying ER-Golgi exchanges. Essential questions, however, remain about the organization of the ER-Golgi interface in cells and the type of membrane structures mediating traffic from ERES. To investigate these, transgenic tagging in Drosophila flies, 3D-structured illumination microscopy (SIM) and focused ion beam scanning electron microscopy (FIB-SEM) were used to characterize ERES-Golgi units in collagen-producing fat body, imaginal discs, and imaginal discs overexpressing ERES determinant Tango1. Facing ERES was fiybd a pre-cis-Golgi region, equivalent to the vertebrate ER-Golgi intermediate compartment (ERGIC), involved in both anterograde and retrograde transport. This pre-cis-Golgi is continuous with the rest of the Golgi, not a separate compartment or collection of large carriers, for which no evidence is found. This study observed, however, many vesicles, as well as pearled tubules connecting ERES and Golgi (Yang, 2021).

Regulated secretion is a conserved process occurring across diverse cells and tissues. Current models suggest that the conserved cargo receptor Tango1 mediates the packaging of collagen into large coat protein complex II (COPII) vesicles that move from the endoplasmic reticulum (ER) to the Golgi apparatus. However, how Tango1 regulates the formation of COPII carriers and influences the secretion of other cargo remains unknown. Through high-resolution imaging of Tango1, COPII, Golgi and secretory cargo (mucins) in Drosophila larval salivary glands, this study found that Tango1 forms ring-like structures that mediate the formation of COPII rings, rather than vesicles. These COPII rings act as docking sites for the cis-Golgi. Moreover, nascent secretory mucins were observed emerging from the Golgi side of these Tango1/COPII/Golgi complexes, suggesting that these structures represent functional docking sites/fusion points between the ER exit sites and the Golgi. Loss of Tango1 disrupted the formation of COPII rings, the association of COPII with the cis-Golgi, mucin O-glycosylation and secretory granule biosynthesis. Additionally, this study identified a Tango1 self-association domain that is essential for formation of this structure. These results provide evidence that Tango1 organizes an interaction site where secretory cargo is efficiently transferred from the ER to Golgi and then to secretory vesicles. These findings may explain how the loss of Tango1 can influence Golgi/ER morphology and affect the secretion of diverse proteins across many tissues (Reynolds, 2019).

Secretion of proteins is a highly conserved event occurring in all eukaryotic species and across many tissues. This process begins in the ER where proteins destined to be secreted are synthesized and transported to the cis-region of the Golgi apparatus in a COPII-dependent process. Appropriately modified and folded proteins are packaged into secretory vesicles emanating from the trans-Golgi network that then await appropriate signals before fusing with the plasma membrane to release their contents. However, the mechanisms whereby bulky cargo is efficiently packaged into small vesicular transport vehicles and moved between compartments of the secretory apparatus are largely unknown (Reynolds, 2019).

Recently studies have identified an essential cargo receptor (Tango1 or MIA Src homology 3 (SH3) domain ER export factor 3; MIA3) responsible for the efficient packaging and secretion of high-molecular-weight collagen. Tango1, a type I transmembrane protein located at the ER exit sites (ERES) was first identified in a screen for genes that affect general secretion and Golgi morphology in Drosophila cells (Bard, 2006). Subsequent studies demonstrated a role for Tango1 in collagen secretion, whereby it is thought to mediate the formation of large COPII megacarriers capable of transporting the large procollagen rods from the ER to the Golgi apparatus. Current models suggest that the luminal SH3 domain of Tango1 binds to the procollagen chaperone HSP47, directing procollagen to sites of COPII vesicle formation. Tango1 is thought to modulate the size of the COPII vesicles by recruiting factors that limit Sar1GTPase activity, thus allowing the vesicles to grow in size to accommodate this large cargo (Reynolds, 2019).

Many recent studies have suggested that Tango1 is important for the secretion of additional molecules other than collagen. In mammalian cells, Tango1 affects the export of bulky lipid particles such as pre-chylomicrons/very low-density lipoproteins. In Drosophila, loss of Tango1 results in defects in the secretion of mucins, laminins, perlecan, and other extracellular matrix proteins. Additional genetic studies suggest that Tango1 influences general secretion rather than the specific secretion of certain proteins. These studies also identified many additional Golgi proteins that interact with Tango1, either directly or indirectly, such as Grasp65 and GM130, and suggest that there may exist more direct contacts between the ER and Golgi that are mediated by Tango1. Other work suggests that although Tango1 is important for the secretion of bulky cargo, it has an additional role in ER-Golgi morphology. However, high-resolution visualization of Tango1 dynamics and COPII vesicle formation relative to endogenous cargo biosynthesis and packaging has been challenging given the small size of these structures and the resolution limits of light microscopy. Thus, the exact roles Tango1 plays in the packaging and secretion of diverse cargos, as well as ER-Golgi morphology, remain unclear (Reynolds, 2019).

This study used the Drosophila larval salivary gland (SG) to image the relationship between Tango1 and the synthesis and packaging of secretory cargo (mucins) in real time, taking advantage of the increased spatial resolution unique to this gland. The SG undergoes hormonally regulated secretory granule formation that results in secretory granules of 3-8 microns in diameter (~10-100x larger than those seen in mammalian systems) that are filled with highly O-glycosylated mucin proteins (19-23). Drosophila mucins are similar in structure to mammalian mucins (having serine/threonine-rich O-glycosylated regions) but are typically smaller in size. Fly lines expressing GFP-tagged versions of one secretory mucin (Sgs3-GFP) have allowed high-resolution, real-time imaging of secretory granule formation and secretion. The genetic tractability of Drosophila has also allowed the identification of factors that control secretory vesicle formation, morphology, and extrusion of bulky cargo, such as mucins. Through real-time imaging using this system, this study found that Tango1 undergoes regulated self-association and dynamic shape changes during hormonally induced secretion to form ring structures that mediate the formation of COPII rings rather than vesicles. These Tango1-COPII rings act as docking sites for the cis-Golgi. Moreover, nascent secretory mucins were imaged emerging from the Golgi side of these Tango1-COPII-Golgi complexes, suggesting that these structures represent functional docking sites/fusion points between the ER exit sites and the Golgi. Taken together, these data suggest that Tango1 acts as a scaffold for the formation of functional ER-Golgi junctions that allow the efficient synthesis, intraorganellar transport, and packaging of diverse secretory cargo (Reynolds, 2019).

Taking advantage of the high spatial resolution of secretory structures in the Drosophila larval SG, this study has demonstrated that Tango1 coordinates the formation of ER-Golgi interaction sites to mediate secretory vesicle formation. High resolution imaging of Tango1 relative to COPII, Golgi, and secretory cargo demonstrates that Tango1 self-associates to form rings that appear to orchestrate the formation of COPII rings. Moreover, cis-Golgi markers localize within these rings, forming a distinct Tango1-COPII-Golgi structure. In support of this Tango1-COPII-Golgi structure being a functional site of interaction between the ER and Golgi, secretory granules were found emanating from the trans-Golgi face of this structure. These results support a model where Tango1 mediates a functional interaction point between the ERES and Golgi to allow efficient transfer of cargo and the subsequent formation of secretory granules (Reynolds, 2019).

This unique structure and the formation of secretory vesicles are dependent on Tango1. This study found that loss of Tango1 resulted in the loss of the COPII rings, loss of the organized association of the cis-Golgi with COPII, and loss of secretory vesicles. Additionally, Tango1 overexpression in Drosophila cells was sufficient to drive COPII ring formation and Golgi association. Previous studies have demonstrated direct binding of Tango1 and COPII components, which likely orchestrates the overlapping ring formation. Likewise, COPII components are known to interact with various cis-Golgi proteins, which likely drives their association in this structure. The formation of this entire structure depends on Tango1 self-association via the CCD1 domain in the cytoplasmic region. This is similar to the domain responsible for Tango1 self-association in mammals, suggesting conserved aspects of Tango1 action between these species (Reynolds, 2019).

Interestingly, the COPII structures present in this system exist as rings rather than spherical vesicular structures. Whether the COPII ring represents a structure unique to Drosophila or whether these structures might also be present in mammals awaits further investigation. However, evidence exists for diverse COPII structures in different systems, including tubules and protruding saccules from the ER membrane. The flexibility of the COPII coat may allow unique adaptations, depending on the biological context. Indeed, one recent study in mammalian cells suggests that procollagen transport occurs via a 'short-loop pathway' from the ER to the Golgi in the absence of large COPII vesicular carriers. This study offers support for the possibility that similar COPII-dependent ER-Golgi interaction sites may exist in mammals (Reynolds, 2019).

Previous work in Drosophila also supports a model where Tango1 mediates a connection to the Golgi. In this study, the authors demonstrated that overexpression of the Tango1 cytoplasmic domain can increase the size and density of ERES and increase the number of Golgi units, strongly suggesting a role for Tango1 in organizing both ERES and the Golgi. Indeed, the authors propose that large COPII carriers may begin fusion with the Golgi before separating from the ERES. The current imaging clearly demonstrates that Tango1, COPII, and the Golgi lie in close proximity and that their spatial separation likely precludes the formation of a separate, large COPII vesicular carrier. Moreover, the finding that mucin cargo emerges from the trans-Golgi face of this structure strongly supports a model where Tango1 serves to organize ordered ERES/Golgi interactions sites through which cargo passes. The results and model would also explain previous Drosophila studies where loss of Tango1 affects Golgi structure as well as the secretion of diverse proteins. Tango1 was originally discovered in an RNAi screen in Drosophila cells for genes that affect both secretion and Golgi structure (Tango = transport and Golgi organization). Likewise, more recent studies have suggested that Tango1 plays a role in the interaction of the Golgi and ER that is independent of its role in trafficking bulky cargo proteins. Many of these studies also present evidence that loss of Tango1 affects constitutive secretion of all proteins, including small reporter proteins. If Tango1 functions to mediate docking sites between ERES and Golgi, then the loss of Tango1 would be expected to result in changes in Golgi structure. Indeed, evidence was seen of Golgi structural changes when Tango1 was deleted from this system. Likewise, if the rate of constitutive secretion also benefits from these contact sites, one would expect the loss of Tango1 to affect this as well. These results and model are therefore consistent with previous studies that suggest a role for Tango1 in Golgi structure and constitutive secretion (Reynolds, 2019).

Studies investigating the role of Tango1 in other systems have proposed diverse models for how Tango1 coordinates the secretion of specific cargo. In mammals, it is proposed that the SH3 region of Tango1 interacts with the HSP47 chaperone, which then binds collagen to mediate its entry into the nascent COPII vesicle. However, this model does not explain how diverse cargo and constitutive secretion can be affected by the loss of Tango1. Additionally, this model necessitates a second packaging event for bulky cargo on the trans-side of the Golgi that must take place. The data presented in this study suggest that tissues under a high secretory burden may use Tango1 to reduce the number of independent packaging steps required for bulky, highly glycosylated cargo (such as mucins) by forming direct connection points between the ER and Golgi. This may ensure the efficient production and packaging of large amounts of cargo into secretory vesicles over a short period of time (Reynolds, 2019).

The size of the secretory structures present in this genetically tractable system and its amenability to real-time imaging during the secretory process have led to key insights with regard to secretory granule biogenesis and secretion. Using this system, it was previously shown that clathrin and AP-1, which localize to the trans-Golgi network, are required for proper secretory granule formation. Subsequently, the activity of the phosphatidylinositol kinase PI4KII was shown to be essential for secretory granules to reach mature size, likely because of influences on homotypic fusion events. Previous work has identified a role for O-glycosylation in secretory granule morphology during granule maturation. Real-time imaging has outlined the steps involved in secretory granule fusion with the apical plasma membrane and identified factors required for proper secretion of the mucinous contents. The current results are consistent with these prior studies and shed light on how cargo moves efficiently from the ER to the Golgi through a unique secretory structure whose organization depends on Tango1. This structure may explain how Tango1 has diverse effects on both regulated and constitutive secretion across many cell and tissue types. Moving forward, this tractable genetic imaging system will be amenable to identifying additional factors responsible for the highly organized and incredibly robust secretory program of the SG. Moreover, understanding the mechanisms by which biological systems maximize secretory capacity and efficiency may provide insights into novel strategies to restore defective secretion in disease states (Reynolds, 2019).

Regulated exocytosis is an essential process whereby specific cargo proteins are secreted in a stimulus-dependent manner. Cargo-containing secretory granules are synthesized in the trans-Golgi Network (TGN); after budding from the TGN, granules undergo modifications, including an increase in size. These changes occur during a poorly understood process called secretory granule maturation. This study leveraged the Drosophila larval salivary glands as a model to characterize a novel role for Rab GTPases during granule maturation. Secretory granules were found to increase in size ~300-fold between biogenesis and release, and loss of Rab1 or Rab11 reduces granule size. Surprisingly, it was found that Rab1 and Rab11 localize to secretory granule membranes. Rab11 associates with granule membranes throughout maturation, and Rab11 recruits Rab1. In turn, Rab1 associates specifically with immature granules and drives granule growth. In addition to roles in granule growth, both Rab1 and Rab11 appear to have additional functions during exocytosis; Rab11 function is necessary for exocytosis, while the presence of Rab1 on immature granules may prevent precocious exocytosis. Overall, these results highlight a new role for Rab GTPases in secretory granule maturation (Neuman, 2021).

The Hippo pathway is an evolutionarily conserved developmental pathway that controls organ size by integrating diverse regulatory inputs, including actomyosin-mediated cytoskeletal tension. Despite established connections between the actomyosin cytoskeleton and the Hippo pathway, the upstream regulation of actomyosin in the Hippo pathway is less defined. This study identified the phosphoinositide-3-phosphatase Myotubularin (Mtm) as a novel upstream regulator of actomyosin that functions synergistically with the Hippo pathway during growth control. Mechanistically, Mtm regulates membrane phospholipid PI(3)P dynamics, which, in turn, modulates actomyosin activity through Rab11-mediated vesicular trafficking. PI(3)P dynamics were revealed to be a novel mode of upstream regulation of actomyosin and established Rab11-mediated vesicular trafficking as a functional link between membrane lipid dynamics and actomyosin activation in the context of growth control. This study also shows that MTMR2, the human counterpart of Drosophila Mtm, has conserved functions in regulating actomyosin activity and tissue growth, providing new insights into the molecular basis of MTMR2-related peripheral nerve myelination and human disorders (Hu, 2022).

During tissue morphogenesis, the changes in cell shape, resulting from cell-generated forces, often require active regulation of intracellular trafficking. How mechanical stimuli influence intracellular trafficking and how such regulation impacts tissue mechanics are not fully understood. This study identified an actomyosin-dependent mechanism involving Rab11-mediated trafficking in regulating apical constriction in the Drosophila embryo. During Drosophila mesoderm invagination, apical actin and Myosin II (actomyosin) contractility induces apical accumulation of Rab11-marked vesicle-like structures ("Rab11 vesicles") by promoting a directional bias in dynein-mediated vesicle transport. At the apical domain, Rab11 vesicles are enriched near the adherens junctions (AJs). The apical accumulation of Rab11 vesicles is essential to prevent fragmented apical AJs, breaks in the supracellular actomyosin network, and a reduction in the apical constriction rate. This Rab11 function is separate from its role in promoting apical Myosin II accumulation. These findings suggest a feedback mechanism between actomyosin activity and Rab11-mediated intracellular trafficking that regulates the force generation machinery during tissue folding (Chen, 2022).

Historically, the trans-Golgi network (TGN) has been recognized as a sorting center of newly synthesized proteins, whereas recycling endosome (RE) is a compartment where endocytosed materials transit before being recycled to the plasma membrane. However, recent findings revealed that both the TGN and RE connect endocytosis and exocytosis, and thus are functionally overlapping. This study reports, in both Drosophila and microtubule-disrupted HeLa cells, that REs are interconvertible between two distinct states, namely Golgi-associated REs and free REs. Detachment and reattachment of REs and Golgi stacks were often observed. These two types of REs were in the route of Glycosylphosphatidylinositol-anchored cargo protein released from the endoplasmic reticulum, but not in that of Vesicular stomatitis virus G protein. In plants, it has been established that there are two types of TGNs: the Golgi-associated TGN and Golgi-independent TGN. Dynamics of REs in both Drosophila and mammalian cells revealed strong similarity to plant TGNs. Together with the molecular-level similarity, these results indicate that fly/mammalian REs are equivalent organelles to TGNs in plants, and evoke reconsideration of identities and functional relationships between REs and TGNs (Fujii, 2020).

Rare genetic diseases preponderantly affect the nervous system causing neurodegeneration to neurodevelopmental disorders. This is the case for both Menkes and Wilson disease, arising from mutations in ATP7A and ATP7B, respectively. The ATP7A and ATP7B proteins localize to the Golgi and regulate copper homeostasis. Genetic and biochemical interactions were demonstrated between ATP7 paralogs with the Conserved Oligomeric Golgi complex, or COG complex, a Golgi apparatus vesicular tether. Disruption of Drosophila copper homeostasis by ATP7 tissue-specific transgenic expression caused alterations in epidermis, aminergic, sensory, and motor neurons. Prominent among neuronal phenotypes was a decreased mitochondrial content at synapses, a phenotype that paralleled with alterations of synaptic morphology, transmission, and plasticity. These neuronal and synaptic phenotypes caused by transgenic expression of ATP7 were rescued by downregulation of COG complex subunits. It is concluded that the integrity of Golgi-dependent copper homeostasis mechanisms, requiring ATP7 and COG, are necessary to maintain mitochondria functional integrity and localization to synapses (Hartwig, 2020).

Intracellular trafficking is a basic and essential cellular function required for delivery of proteins to the appropriate subcellular destination; this process is especially demanding in professional secretory cells, which synthesize and secrete massive quantities of cargo proteins via regulated exocytosis. The Drosophila larval salivary glands are professional secretory cells that synthesize and secrete mucin proteins at the onset of metamorphosis. Using the larval salivary glands as a model system, a role was identified for the highly conserved retromer complex in trafficking of secretory granule membrane proteins. This study demonstrates that retromer-dependent trafficking via endosomal tubules is induced at the onset of secretory granule biogenesis, and that recycling via endosomal tubules is required for delivery of essential secretory granule membrane proteins to nascent granules. Without retromer function, nascent granules do not contain the proper membrane proteins; as a result, cargo from these defective granules is mistargeted to Rab7-positive endosomes, where it progressively accumulates to generate dramatically enlarged endosomes. Retromer complex dysfunction is strongly associated with neurodegenerative diseases, including Alzheimer's disease, characterized by accumulation of amyloid β (Aβ). Ectopically expressed amyloid precursor protein (APP) undergoes regulated exocytosis in salivary glands and accumulates within enlarged endosomes in retromer-deficient cells. These results highlight recycling of secretory granule membrane proteins as a critical step during secretory granule maturation and provide new insights into our understanding of retromer complex function in secretory cells. These findings also suggest that missorting of secretory cargo, including APP, may contribute to the progressive nature of neurodegenerative disease (Neuman, 2020).

Plasma membrane protein trafficking is of fundamental importance for cell function and cell integrity of neurons and includes regulated protein recycling. This work reports a novel role of the endoplasmic reticulum (ER) for protein recycling as discovered in trafficking studies of the ion channel TRPL in photoreceptor cells of Drosophila. TRPL is located within the rhabdomeric membrane from where it is endocytosed upon light stimulation and stored in the cell body. Conventional immunohistochemistry as well as stimulated emission depletion super-resolution microscopy revealed TRPL storage at the ER after illumination, suggesting an unusual recycling route of TRPL. The results also imply that both phospholipase D (PLD) and retromer complex are required for correct recycling of TRPL to the rhabdomeric membrane. Loss of PLD activity in PLD(3.1) mutants results in enhanced degradation of TRPL. In the retromer mutant vps35^MH20, TRPL is trapped in a Rab5-positive compartment. Evidenced by epistatic analysis in the double mutant PLD^3.1 vps35^MH20, PLD activity precedes retromer function. A model is proposed in which PLD and retromer function play key roles in the transport of TRPL to an ER enriched compartment (Wagner, 2021).

Golgi stacks are the basic structural units of the Golgi. Golgi stacks are separated from each other and scattered in the cytoplasm of Drosophila cells. This study reports that the ARF-GEF inhibitor Brefeldin A (BFA) induces the formation of BFA bodies, which are aggregates of Golgi stacks, trans-Golgi networks and recycling endosomes. Recycling endosomes are located in the centers of BFA bodies, while Golgi stacks surround them on their trans sides. Live imaging of S2 cells revealed that Golgi stacks repeatedly merged and separated on their trans sides, and BFA caused successive merger by inhibiting separation, forming BFA bodies. S2 cells carrying genome-edited BFA-resistant mutant Sec71(M717L) did not form BFA bodies at high concentrations of BFA; S2 cells carrying genome-edited BFA-hypersensitive mutant Sec71(F713Y) produced BFA bodies at low concentrations of BFA. These results indicate that Sec71 is the sole BFA target for BFA body formation and controls Golgi stack separation. Finally, this study showed that impairment of Sec71 in fly photoreceptors induces BFA body formation, with accumulation of both apical and basolateral cargoes, resulting in inhibition of polarized transport (Fujii, 2020).

yata mutants of Drosophila melanogaster exhibit phenotypes including progressive brain shrinkage, developmental abnormalities and shortened lifespan, whereas in mammals, null mutations of the yata ortholog Scyl1 result in motor neuron degeneration. yata mutation also causes defects in the anterograde intracellular trafficking of a subset of proteins including APPL, which is the Drosophila ortholog of mammalian APP, a causative molecule in Alzheimer's disease. SCYL1 binds and regulates the function of coat protein complex I (COPI) in secretory vesicles. This study reveals a role for the Drosophila YATA protein in the proper localization of COPI. Immunohistochemical analyses performed using confocal microscopy and structured illumination microscopy showed that YATA colocalizes with COPI and GM130, a cis-Golgi marker. Analyses using transgenically expressed YATA with a modified N-terminal sequence revealed that the N-terminal portion of YATA is required for the proper subcellular localization of YATA. Analysis using transgenically expressed YATA proteins in which the C-terminal sequence was modified revealed a function for the C-terminal portion of YATA in the subcellular localization of COPI. Notably, when YATA was mislocalized, it also caused the mislocalization of COPI, indicating that YATA plays a role in directing COPI to the proper subcellular site. Moreover, when both YATA and COPI were mislocalized, the staining pattern of GM130 revealed Golgi with abnormal elongated shapes. Thus, these in vivo data indicate that YATA plays a role in the proper subcellular localization of COPI (Saito, 2021).

In eukaryotic cells, transmembrane proteins and secreted proteins are synthesized in the rough endoplasmic reticulum (ER) and then transported to their own destinations by intracellular vesicular trafficking. Transport vesicles are surrounded by coat proteins, whose type varies among distinct cellular locations. These coat proteins enable the efficient formation of transport carriers and incorporation of specific cargos into the vesicles. In the early steps of vesicular trafficking between the ER and the Golgi, vesicles are coated by coat protein complex I (COPI) or II (COPII) (Miller, 2013; Arakel, 2018; Bethune, 2018). COPI-coated vesicles transport proteins retrogradely from the Golgi to the ER, whereas COPII-coated vesicles transport proteins anterogradely from the ER to the Golgi. Proper regulation of vesicular trafficking between the ER and the Golgi is considered to be important in the retrieval of ER- and Golgi-resident proteins as well as appropriate quality control of synthesized proteins. Impairment of the early steps of vesicular trafficking has been suggested to cause some human genetic disorders (Saito, 2021).

A Drosophila yata mutant was previously identified that showed phenotypes in the compound eye, wing and brain. Homozygotes of the null allele of the yata gene, yata^KE2.1, show various phenotypes such as morphological abnormalities in the compound eye and wing, progressive reduction of brain volume and shortened lifespan. Electron microscopic examination of the internal morphology of the compound eye revealed that the yata mutation enhanced the formation of an abnormal cellular structure that formed as a bleb-like cellular protrusion and contained membranous organelles that had the appearance of lysosomes, autophagosomes and late endosomes (Arimoto, 2020). The yata gene has been suggested to be ubiquitously expressed. It encodes a protein that has no transmembrane domains and has a catalytic domain of a protein kinase. The kinase domain of YATA is predicted to be catalytically inactive because some of the amino acid residues that are essential for catalytic activity are not conserved. The yata mutant was originally identified as a locus that genetically interacted with the null allele of the Appl gene. The Appl gene encodes the orthologous protein of mammalian APP, which is a causative molecule of Alzheimer's disease. Double null mutants of yata and Appl showed exacerbated phenotypes in brain volume reduction and shortened lifespan. APPL proteins are synthesized in neuronal cell bodies and then transported to synaptic terminals by means of vesicular trafficking. Immunohistochemical analysis using an anti-APPL antibody revealed the aberrant accumulation of APPL in neuronal cell bodies in the pupal brain in yata mutants. This accumulation of APPL overlapped with a marker of the ER. In a Drosophila Alzheimer's disease model in which human mutant APP was ectopically expressed in larval motor neurons, RNAi-mediated knockdown of yata resulted in partial suppression of the anterograde transport of APP to synapses and of phenotypes caused by APP such as abnormal morphology of neuromuscular synapses and abnormal electrophysiological properties of neuromuscular synapses (Furotani, 2018). In addition to APPL, synaptic transport of another synaptic protein, Fasciclin II, which is a synaptic homophilic cell adhesion molecule orthologous to mammalian NCAM, is reduced in yata mutants. However, synaptic transport of Synaptotagmin I, which is a membrane-bound protein localized on synaptic vesicles, was not affected in the yata mutants, suggesting that yata mutation affects only a subset of proteins that are transported by vesicular trafficking (Saito, 2021).

yata orthologs occur in a variety of eukaryotes including yeast, plants, nematodes and mammals. Null mutation of the murine yata ortholog, Scyl1, causes motor neuron degeneration (Schmidt, 2007). Because neural-specific, but not skeletal muscle-specific, deletion of Scyl1 causes motor dysfunction, Scyl1 is thought to function autonomously in the nervous system (Pelletier, 2012). Because deletion of Scyl1 also caused the mislocalization and accumulation of the TAR DNA-binding protein of 43 kDa (TDP-43), mice with Scyl1 deletion are thought to share features with human neurodegenerative diseases, including amyotrophic lateral sclerosis. Mutation of human Scyl1 has been identified as a cause of a genetic disease that results in liver failure, peripheral neuropathy, cerebellar atrophy and ataxia (Schmidt, 2015; Lenz, 2018; Shohet, 2019). A previous mass spectrometry-based screen identified βCOP, a subunit of COPI, as the binding partner of SCYL1 (Burman, 2008). SCYL1 directly binds with COPI and colocalizes with COPI in the ER–Golgi intermediate compartment (ERGIC) and the cis-Golgi, which are the sites where buds of COPI-coated vesicles are formed. In cultured cells, RNAi-mediated knockdown of Scyl1 resulted in the inhibition of COPI-mediated retrograde trafficking of the KDEL receptor protein from the Golgi to the ER. Further study also identified class II Arfs, which are GTPases that are involved in the formation of COPI-coated vesicles, as another binding partner of SCYL1 (Hamlin, 2014). SCYL1 has been suggested to function as a scaffold protein for components of the COPI coat. In cultured cells, overexpression or knockdown of Scyl1 resulted in abnormal morphology of the Golgi and ERGIC, possibly due to loss of the scaffolding function of SCYL1 for COPI (Burman, 2010; Hamlin, 2014). In patients with a genetic disease caused by a mutation in Scyl1, enlargement of the Golgi has been observed in fibroblasts (Saito, 2021).

This study used immunostaining with anti-YATA and other marker antibodies to examine the subcellular localization of YATA in the Drosophila pupal brain. Observations obtained using confocal microscopy and structured illumination microscopy (SIM) revealed colocalization among YATA, COPI and a cis-Golgi marker. Thie further analysis using transgenically expressed YATA indicate that it has an in vivo role in the proper subcellular localization of COPI (Saito, 2021).

This study examined the subcellular localization of the YATA protein by immunostaining with an anti-YATA antibody and observation using confocal microscopy. Anti-YATA signals were observed to have a punctate pattern in the cytoplasm surrounding nuclei. These punctate signals colocalized with the signals for GM130, a marker of the cis-Golgi, and for COPI. Further analysis using SIM revealed that COPI and YATA were localized in a subset of regions within the GM130 punctae. YATA and COPI partially colocalized. These data suggest that the cis-Golgi, which is labeled by anti-GM130 antibody, contains several distinct subregions where YATA and COPI are localized. Subregions where GM130 colocalized with both YATA and COPI may be sites for the assembly and budding of COPI-coated secretory vesicles traveling from the Golgi to the ER. Consistent with this interpretation, when the expression of Arf4-GFP was induced, its localization showed mainly punctate signals that overlapped with the localization of YATA. A previous study has shown that Arf4-GFP is localized in punctae that seem to be the sites of formation of COPI-coated vesicles in Drosophila embryos, although it has not been proven that localization of Arf4-GFP completely overlaps with that of the endogenous Arf4 protein. On the other hand, when YFP-KDEL expression was induced, it was found to be localized in the regions that surrounded the nuclei and in some regions near the nuclei. The localization of YATA partially overlapped the YFP-KDEL signal, but the patterns and shapes presented by the signals were different. The C-terminal KDEL sequence is the ER retention motif, which is recognized by the KDEL receptor and functions in the retrieval of ER-resident proteins from the Golgi. In Drosophila neurons, staining with an anti-KDEL antibody and the localization of RFP-KDEL and Lysozyme-GFP-KDEL all reveal localization in the regions that surround the nuclei and in some regions near the nuclei. In addition, a previous immunoelectron microscopic study showed that a secreted protein, Hikaru genki, is localized in the region surrounding the nucleus in Drosophila neurons. These observations suggest that the regions found to be labeled by YFP-KDEL represent the ER. When the expression of Sar1-HA was induced, Sar1-HA showed a relatively strong punctate pattern of localization and a relatively weak pattern of perinuclear localization that seemed to correspond to the ER. A previous study has shown that Sar1-GFP is localized in the perinuclear region and that it also appears as relatively strongly stained punctae in Drosophila S2 cells. Costaining with dSec16 suggested that the relatively strongly stained punctae represent the ER exit sites at which COPII-coated vesicles are formed. Because the localization of Sar1-HA resembles that of Sar1-GFP, the punctate signals that arise from Sar1-HA seem to represent the ER exit sites, although it has not been proven that localization of Sar1-HA completely overlaps with that of endogenous Sar1 protein. YATA colocalized with the punctate Sar1-HA signal. Notably, the punctate signals arising from anti-YATA antibody labeling may not represent the entire areas of YATA localization. When YATAwt was overexpressed with no epitope tag, relatively weak staining was also observed in the perinuclear regions of the cytoplasm in addition to the relatively strong punctate signals that were found to colocalize with COPI and the cis-Golgi marker. Therefore, its COPI-related function may not be the only molecular function of YATA. In addition, the site of assembly of COPI-coated vesicles on the cis-Golgi and the site of assembly of COPII-coated vesicles on the ER may be spatially close to each other (Saito, 2021).

This study also assessed whether overexpression of YATA affects the localization of COPI. The results suggest that ectopic overexpression of YATAwt with no epitope tag increased the localization of COPI. This is consistent with the hypothesis that YATA directs COPI to the subregion of the cis-Golgi where COPI-coated vesicles are assembled, because an increased amount of YATA may be capable of directing an increased amount of COPI to this specific subregion of the cis-Golgi. However, in cultured cells, overexpression of SCYL1 conjugated with GFP caused an expanded morphology of the cis-Golgi, as revealed by labeling with anti-GM130 antibody (Hamlin, 2014). These differences may be due to species-specific differences, differences between cultured cells and living animals, an effect of the conjugated GFP, or differences in gene dosage (Saito, 2021).

Previous studies have shown that SCYL1 binds COPI at its C-terminal amino acid sequence (RKLD-COO-), which resembles the KKXX-COO- motif that functions as a COPI-binding motif in ER-resident proteins. Because the Drosophila YATA protein has an AKKL-COO- sequence at its C-terminus, whether mutating the C-terminal sequence of the YATA protein would influence the effect of YATA on COPI localization was tested. While overexpression of YATAwt increased the localization of COPI, the induction of YATAwt-HA, which has a 3×HA tag sequence at its C-terminus, did not affect COPI. These data suggest that the C-terminal sequence of YATA is required for its effect on COPI. The effects of HA-YATAwt and HA-YATAAAAL expression, both of which have a 3×HA tag at their N-terminus, were also tested. It was unexpectedly found that these N-terminally tagged YATA proteins did not exactly colocalize with the cis-Golgi marker but instead showed diffuse localization in the cytoplasm. These data suggest that the N-terminal sequence of YATA is required for its proper subcellular localization. Although the reason for the importance of the N-terminal sequence is unknown, mislocalization of N-terminally tagged YATA enabled examination of the effect of mistakenly localized YATA on the localization of COPI. The data show that the expression of mislocalized HA-YATAwt caused the mislocalization of COPI, while the expression of mislocalized HA-YATAAAAL did not. These data suggest that YATA functions to direct COPI to the proper subcellular site and that the C-terminal sequence of YATA is required for this function. The data regarding the localization of a cis-Golgi marker show that the Golgi exhibited an abnormal elongated shape when HA-YATAwt was expressed but that this effect was not observed when HA-YATAAAAL was expressed. These data suggest that the expression of mislocalized YATA protein that retains its original C-terminal sequence caused the cis-Golgi to acquire abnormal morphology. These observations are consistent with previous results showing that the overexpression or knockdown of Scyl1 resulted in an expanded morphology of the Golgi in cultured cells (Burman, 2010; Hamlin, 2014) and that the Golgi was enlarged in fibroblasts of patients with a genetic disease caused by mutation of Scyl1 (Schmidt, 2015; Saito, 2021 and references therein).

The data reveal a function of YATA, namely its involvement in the regulation of subcellular localization of COPI, and provide a basis for further in vivo genetic explorations of the mechanisms and physiological importance of COPI-mediated vesicular trafficking in Drosophila. However, the relevance of the COPI-regulating function of YATA to the phenotypes observed in yata null mutants is still unclear. yata mutants show impaired anterograde trafficking of a subset of proteins including APPL and Fasciclin II, although Synaptotagmin I trafficking is not affected (Sone, 2009; Furotani, 2018). In addition, the aberrant accumulation of Sec23, a component of COPII, was observed in yata mutants. One possibility is that the impairment of COPI-mediated retrograde trafficking from the Golgi to the ER disrupts the quality control system and metabolism of proteins synthesized in the ER, as previously suggested, because the retrograde trafficking system is necessary to recover some of the proteins that play roles in the ER and are transported from the ER to the Golgi. Alternatively, YATA could have another function in addition to the regulation of the subcellular localization of COPI in which it could affect the anterograde trafficking of some proteins. The important question of why the trafficking of only a subset of proteins is affected in yata mutants also remains unresolved. The fact that null mutants of yata are not lethal suggests that there are other molecules whose functions overlap with that of yata. Paralogs of yata that exist in the Drosophila genome and belong to the family of genes that encode SCYL family pseudokinases are candidates for such molecules. Further detailed analysis of the phenotypes observed in yata mutants and their relevance to the formation of COPI-coated vesicles and the regulation of intracellular vesicular trafficking will be necessary in the future (Saito, 2021).

Constructing the dendritic arbor of neurons requires dynamic movements of Golgi outposts (GOPs), the prominent component in the dendritic secretory pathway. GOPs move toward dendritic ends (anterograde) or cell bodies (retrograde), whereas most of them remain stationary. This study shows that Leucine-rich repeat kinase (Lrrk), the Drosophila melanogaster homologue of Parkinson's disease-associated Lrrk2, regulates GOP dynamics in dendrites. Lrrk localized at stationary GOPs in dendrites and suppressed GOP movement. In Lrrk loss-of-function mutants, anterograde movement of GOPs was enhanced, whereas Lrrk overexpression increased the pool size of stationary GOPs. Lrrk interacts with the golgin Lava lamp and inhibits the interaction between Lva and dynein heavy chain, thus disrupting the recruitment of dynein to Golgi membranes. Whereas overexpression of kinase-dead Lrrk causes dominant-negative effects on GOP dynamics, overexpression of the human LRRK2 mutant G2019S with augmented kinase activity promotes retrograde movement. This study reveals a pathogenic pathway for LRRK2 mutations causing dendrite degeneration (Lin, 2015).

Vps54 is an integral subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans-Golgi network (TGN). A destabilizing missense mutation in Vps54 causes the age-progressive motor neuron (MN) degeneration, muscle weakness, and muscle atrophy observed in the wobbler mouse, an established animal model for human MN disease. It is currently unclear how the disruption of Vps54, and thereby the GARP complex, leads to MN and muscle phenotypes. To develop a new tool to address this question, this study has created an analogous model in Drosophila by generating novel loss-of-function alleles of the fly Vps54 ortholog (scattered/scat). Null scat mutant adults are viable but have a significantly shortened lifespan. Like phenotypes observed in the wobbler mouse, this study shows that scat mutant adults are male sterile and have significantly reduced body size and muscle area. Moreover, this study demonstrates that scat mutant adults have significant age-progressive defects in locomotor function. Interestingly, sexually dimorphic effects are seen, with scat mutant adult females exhibiting significantly stronger phenotypes. Finally, it was shown that scat interacts genetically with rab11 in MNs to control age-progressive muscle atrophy in adults. Together, these data suggest that scat mutant flies share mutant phenotypes with the wobbler mouse and may serve as a new genetic model system to study the cellular and molecular mechanisms underlying MN disease (Wilkinson, 2021).

Ykt6 has emerged as a key protein involved in a wide array of trafficking events, and has also been implicated in a number of human pathologies, including the progression of several cancers. It is a complex protein that simultaneously exhibits a high degree of structural and functional homology, and yet adopts differing roles in different cellular contexts. Because Ykt6 has been implicated in a variety of vesicle fusion events, this study characterized the role of Ykt6 in oogenesis by observing the phenotype of Ykt6 germline clones. Immunofluorescence was used to visualize the expression of membrane proteins, organelles, and vesicular trafficking markers in mutant egg chambers. Ykt6 germline clones have morphological and actin defects affecting both the nurse cells and oocyte, consistent with a role in regulating membrane growth during mid-oogenesis. Additionally, these egg chambers exhibit defects in bicoid and oskar RNA localization, and in the trafficking of Gurken during mid-to-late oogenesis. Finally, it was shown that Ykt6 mutations result in defects in late endosomal pathways, including endo- and exocytosis. These findings suggest a role for Ykt6 in endosome maturation and in the movement of membranes to and from the cell surface (Pokrywka, 2021).

Membrane trafficking is essential for sculpting neuronal morphology. The GARP and EARP complexes are conserved tethers that regulate vesicle trafficking in the secretory and endolysosomal pathways, respectively. Both complexes contain the Vps51, Vps52, and Vps53 proteins, and a complex-specific protein: Vps54 in GARP and Vps50 in EARP. In Drosophila, both complexes were found to be required for dendrite morphogenesis during developmental remodeling of multidendritic class IV da (c4da) neurons. Having found that sterol accumulates at the trans-Golgi network (TGN) in Vps54KO/KO neurons, genes that regulate sterols and related lipids at the TGN were investigated. Overexpression of oxysterol binding protein (Osbp) or knockdown of the PI4K four wheel drive (fwd) exacerbates the Vps54KO/KO phenotype, whereas eliminating one allele of Osbp rescues it, suggesting that excess sterol accumulation at the TGN is, in part, responsible for inhibiting dendrite regrowth. These findings distinguish the GARP and EARP complexes in neurodevelopment and implicate vesicle trafficking and lipid transfer pathways in dendrite morphogenesis (O'Brien, 2023).

Zinc is a fundamental trace element essential for numerous biological processes, and zinc homeostasis is regulated by the Zrt-/Irt-like protein (ZIP) and zinc transporter (ZnT) families. ZnT7 is mainly localized in the Golgi apparatus and endoplasmic reticulum (ER) and transports zinc into these organelles. Although previous studies have reported the role of zinc in animal physiology, little is known about the importance of zinc in the Golgi apparatus and ER in animal development and neurodegenerative diseases. This study demonstrate that ZnT86D, a Drosophila ortholog of ZnT7, plays a pivotal role in the neurodevelopment and pathogenesis of Alzheimer disease (AD). When ZnT86D was silenced in neurons, the embryo-to-adult survival rate, locomotor activity, and lifespan were dramatically reduced. The toxic phenotypes were accompanied by abnormal neurogenesis and neuronal cell death. Furthermore, knockdown of ZnT86D in the neurons of a Drosophila AD model increased apoptosis and exacerbated neurodegeneration without significant changes in the deposition of amyloid beta plaques and susceptibility to oxidative stress. Taken together, these results suggest that an appropriate distribution of zinc in the Golgi apparatus and ER is important for neuronal development and neuroprotection and that ZnT7 is a potential protective factor against AD (Lee, 2022).

The Golgi apparatus (GA) is the hub of intracellular trafficking, but selectively targeting GA remains a challenge. This study shows an unconventional types of peptide thioesters, consisting of an aminoethyl thioester and acting as substrates of thioesterases, for instantly targeting the GA of cells. The peptide thioesters, above or below their critical micelle concentrations, enter cells mainly via caveolin-mediated endocytosis or macropinocytosis, respectively. After being hydrolyzed by GA-associated thioesterases, the resulting thiopeptides form dimers and accumulate in the GA. After saturating the GA, the thiopeptides are enriched in the endoplasmic reticulum (ER). Their buildup in ER and GA disrupts protein trafficking, thus leading to cell death via multiple pathways. The peptide thioesters target the GA of a wide variety of cells, including human, murine, and Drosophila cells. Changing d-diphenylalanine to l-diphenylalanine in the peptide maintains the GA-targeting ability. In addition, targeting GA redirects protein (e.g., NRAS) distribution. This work illustrates a thioesterase-responsive and redox-active molecular platform for targeting the GA and controlling cell fates (Tan, 2022).

The Golgi is the central sorting station in the secretory pathway. One set of proteins proposed to direct vesicle arrival at the Golgi are the golgins, long coiled-coil proteins localized to specific parts of the Golgi stack. In mammalian cells, three of the golgins, TMF, golgin-84, and GMAP-210, can capture intra-Golgi transport vesicles when placed in an ectopic location. However, the individual golgins are not required for cell viability, and mouse knockout mutants only have defects in specific tissues. To further illuminate this system, this study examined the Drosophila orthologs of these three intra-Golgi golgins. The study shows that ectopic forms can capture intra-Golgi transport vesicles, but strikingly, the cargo present in the vesicles captured by each golgin varies between tissues. Loss-of-function mutants show that the golgins are individually dispensable, although the loss of TMF recapitulates the male fertility defects observed in mice. However, the deletion of multiple golgins results in defects in glycosylation and loss of viability. Examining the vesicles captured by a particular golgin when another golgin is missing reveals that the vesicle content in one tissue changes to resemble that of a different tissue. This reveals a plasticity in Golgi organization between tissues, providing an explanation for why the Golgi is sufficiently robust to tolerate the loss of many of the individual components of its membrane traffic machinery (Park, 2022).

Selective autophagy receptors and adapters contain short linear motifs called LIR motifs (LC3-interacting region), which are required for the interaction with the Atg8-family proteins. LIR motifs bind to the hydrophobic pockets of the LIR motif docking site (LDS) of the respective Atg8-family proteins. The physiological significance of LDS docking sites has not been clarified in vivo. This study shows that Atg8a-LDS mutant Drosophila flies accumulate autophagy substrates and have reduced lifespan. Using quantitative proteomics to identify the proteins that accumulate in Atg8a-LDS mutants, this study identified the cis-Golgi protein GMAP (Golgi microtubule-associated protein) as a LIR motif-containing protein that interacts with Atg8a. GMAP LIR mutant flies exhibit accumulation of Golgi markers and elongated Golgi morphology. These data suggest that GMAP mediates the turnover of Golgi by selective autophagy to regulate its morphology and size via its LIR motif-mediated interaction with Atg8a (Rahman, 2022).

Consanguineous kindred presented with an autosomal recessive syndrome of intrauterine growth retardation, marked developmental delay, spastic quadriplegia with profound contractures, pseudobulbar palsy with recurrent aspirations, epilepsy, dysmorphism, neurosensory deafness and optic nerve atrophy with no eye fixation. This study aimed at elucidating the molecular basis of this disease. Genome-wide linkage analysis combined with whole exome sequencing were performed to identify disease-causing variants. Functional consequences were investigated in fruit flies null mutant for the Drosophila SEC31A orthologue. SEC31A knockout SH-SY5Y and HEK293T cell-lines were generated using CRISPR/Cas9 and studied through qRT-PCR, immunoblotting and viability assays. Through genetic studies, a disease-associated homozygous nonsense mutation in SEC31A was identified. SEC31A was shown to be ubiquitously expressed, and the mutation triggers nonsense-mediated decay of its transcript, comprising a practical null mutation. Similar to the human disease phenotype, knockdown SEC31A flies had defective brains and early lethality. Moreover, in line with SEC31A encoding one of the two coating layers comprising the Coat protein complex II (COP-II) complex, trafficking newly synthesised proteins from the endoplasmic reticulum (ER) to the Golgi, CRISPR/Cas9-mediated SEC31A null mutant cells demonstrated reduced viability through upregulation of ER-stress pathways. This study demonstrated through human and Drosophila genetic and in vitro molecular studies, that a severe neurological syndrome is caused by a null mutation in SEC31A, reducing cell viability through enhanced ER-stress response, in line with SEC31A's role in the COP-II complex (Halperin, 2018).

Other evidence strongly supports the idea that CD63-positive puncta secreted from SCs are exosomes and not vesicles shed from the plasma membrane. This includes the observation that CD63-positive puncta are found inside both acidic Rab7-positive MVB-like compartments as well as nonacidic Rab11-positive vacuoles and require the ESCRT and ESCRT-associated proteins Hrs and ALiX, as well as several Rabs linked to mammalian exosome secretion, to be formed and secreted. Secreted puncta counts have been used previously in flies to study genetic control of exosome secretion. A criticism of this approach is that reduced puncta numbers may merely reflect aggregation. However, the transfer of CD63-GFP to females was drastically reduced in mutant backgrounds, arguing against a simple aggregation model. Furthermore, because genetic manipulation of ESCRT function does not alter other secretory processes in SCs, this strongly implicates the endocytic pathway in secretion of tagged CD63 (Corrigan, 2014).

Studies of exosomes in Drosophila as well as mammals already suggest that multiple exosome subtypes exist and may be regulated differently, e.g., different roles for ALiX, Hrs, and Evi. If different exosome subtypes are made in SCs, these cells should offer an ideal system to study their differential regulation (Corrigan, 2014).

The remarkably large size of endosomal compartments in SCs provides new opportunities to study exosome biogenesis in vivo. To date, many studies of the intracellular exosome biogenesis machinery and endolysosomal trafficking in higher eukaryotes have relied on expressing an activated form of Rab5 or addition of the ionophore monensin in cell culture to artificially enlarge the endolysosomal compartments, disrupting normal trafficking events. Hence, this new SC in vivo model should allow reinvestigation of previously reported regulators of exosome biogenesis and identify functional differences in trafficking phenotypes, as has been seen for Hrs and ALiX (Corrigan, 2014).

This study has already revealed a surprisingly dynamic interaction between the secretory and endolysosomal systems in SCs. Communication between these compartments using vesicular transport and tubulation processes has been reported in other cell types in flies and mammals, but this study suggests that direct fusion can also be involved. Indeed, the data are also consistent with mMVBLs forming after fusion between SVs and iLEs, suggesting that fusion events may play a critical role in establishing distinct compartments within SCs. In light of this dynamic flux between compartments, it remains unclear whether CD63-GFP-labeled exosomes might be released by the classical route involving mMVBL fusion to the plasma membrane or via an intermediate secretory compartment (Corrigan, 2014).

Although most analysis of the fly AG has highlighted roles for MC products, such as SP, in reprogramming female postmating responses, several recent studies have also suggested a central but poorly defined function for SCs. A transcriptional program regulated by the Hox gene Abd-B controls vacuole formation in SCs (Gligorov, 2013). These findings now indicate that at least one of the effects mediated by SCs, altered receptivity to remating, requires exosome secretion (Corrigan, 2014).

It is difficult to accurately estimate the frequency of SC exosome-sperm fusion events in each female fly because they can probably only be visualized transiently, and many may involve fusion to the very long sperm tail. Sperm play an essential role as mediators of SP-dependent postmating effects in females, so it is plausible that exosome fusion to sperm may modulate specific SP functions. Another appealing hypothesis is that SC exosomes also interact with the female reproductive tract to influence female behavior. However, whatever the target tissues, the data clearly demonstrate a role for SC exosomes in female reprogramming. Furthermore, like human prostasomes, SC exosomes fuse with sperm, highlighting possible conserved roles for exosomes in male reproductive biology. In prostate cancer, prostasomes are inappropriately secreted into the bloodstream, so that other cells in the body may be subjected to these powerful reprogramming functions, potentially supporting tumor-stroma interactions and metastasis (Corrigan, 2014).

Reducing BMP signaling in SCs inhibits exosome secretion and leads to the formation of a novel mMVBL compartment that is filled with fluorescent CD63-GFP. A simple interpretation of this result is that MVBL compartments in these cells do not mature properly, blocking exosome secretion. Consistent with this, increasing BMP signaling in these cells produces a highly enlarged acidic compartment (Corrigan, 2014).

Previous studies have shown that blocking endosomal maturation by knockdown of the early ESCRT component Hrs increases the size of immature endosomal class E compartments lacking ILVs and also results in increased BMP signaling. The data demonstrate that elevated BMP signaling increases mMVBL size, suggesting that there is a complex bidirectional interaction between mMVBL maturation and size and the level of BMP signaling in SCs (Corrigan, 2014).

The findings are consistent with a model in which BMP signaling also controls SC growth by driving endolysosomal trafficking and maturation events. Late endosomes and lysosomes have previously been shown to house major nutrient sensors and cell growth machinery, including the mTORC1 complex, which is activated by intraluminal amino acids. Interestingly, the growth rate of knockdown cells with reduced ESCRT function appears to correlate with mMVBL size rather than exosome secretion rate. Whether growth in these cells is mTORC1 dependent needs to be tested (Corrigan, 2014).

Whatever the explanation for the growth defects in SCs, these data very clearly implicate BMP signaling in the regulation of endolysosomal trafficking and exosome secretion. It will now be important to test whether BMP signaling plays a similar role in mammalian glands that secrete exosomes, such as prostate and breast, and determine whether this role is affected in diseases such as cancer (Corrigan, 2014).

To identify coats that might function in granule biogenesis, the subcellular distribution of clathrin heavy chain was examined, as well as subunits of the clathrin adaptor protein complexes AP-1 and AP-3, which reside on intracellular organelles (note that Drosophila lacks AP-4). First clathrin, AP-1, and AP-3 were examined in salivary gland cells at stage 0, just prior to glue production. At this stage, Golgi bodies are easily visualized using antibodies directed against the golgin Lava lamp (Lva), which localizes to the cis-Golgi. Note that the cis-Golgi has a cup-shaped appearance. A monomeric red fluorescent protein fusion to clathrin heavy chain (RFP-Chc) predominantly localized to large puncta adjacent to the concave face of the cis-Golgi, consistent with a previous report showing localization of endogenous Chc to intracellular puncta in these cells (Wingen, 2009). Endogenous AP-1γ showed a similar distribution. A projection constructed from serial confocal sections revealed numerous Golgi units scattered throughout the cytoplasm. There was a one-to-one correspondence between AP-1γ- and Lva-positive structures, with the cis-Golgi cups surrounding AP-1γ in a manner consistent with AP-1 localizing to the TGN. Indeed AP-1γ and RFP-Chc colocalized with the trans-Golgi protein EpsinR (also called Liquid facets-Related or LqfR). In contrast, AP-1 showed only minimal overlap with the recycling endosome regulator Rab11. AP-1γ and RFP-Chc colocalized at the TGN, although AP-1γ distribution appeared slightly more diffuse in salivary gland cells expressing RFP-Chc than in nonexpressing cells. Localization of AP-1 to the TGN is adaptor-protein specific, because a functional monomeric cherry fluorescent protein (mCherry) fusion to AP-3δ (called Garnet in Drosophila) showed no overlap with a Venus fluorescent protein (VFP) fusion to AP-1μ (called AP-47 in Drosophila), but rather colocalized with the late endosome marker Rab7. Given the high degree of colocalization of clathrin and AP-1, it was asked whether AP-1 might be required to recruit clathrin to the TGN (Burgess, 2011).

To test whether AP-1 recruits clathrin to the TGN, use was made of a μ1-adaptin null allele, AP-47^SHE-11. To bypass late embryonic lethality caused by this allele, mosaic clones were generated in the salivary gland using FLP-FRT-based recombination. Briefly, the wild-type chromosome carries a copy of green fluorescent protein (GFP) such that homozygous mutant cells are marked by the absence of GFP expression and heterozygous and wild-type cells are marked by one or two copies of GFP, respectively. AP-47^SHE-11 clones were generated during embryogenesis and analyzed in third-instar larval salivary glands at stage 0, just prior to glue production. To determine whether other AP-1 subunits can localize to the TGN in the absence of AP-47, the distribution of AP-1γ was examined, and its punctate localization was found to be entirely lost in AP-47^SHE-11 mutant cells. Hence AP-47 is required for efficient recruitment or stability of AP-1γ, similar to what was previously observed in μ1-adaptin-deficient mouse embryonic fibroblasts. Not all trafficking markers were affected by the loss of AP-47, as the early endosome marker Rab5 was unperturbed (Burgess, 2011).

Strikingly, in AP-47^SHE-11 mutant cells, RFP-Chc localization to the Golgi was dramatically reduced. The effect on RFP-Chc distribution was also observed in salivary gland cells in which expression of a double-stranded RNA was used to knock down expression of AP-1γ by RNA interference (RNAi). Most cells depleted of AP-1γ exhibited strong delocalization of RFP-Chc, with only a few cells retaining weak RFP-Chc localization at the TGN. Hence the TGN is the major site of clathrin localization in these cells, and AP-1 plays a pivotal role in clathrin recruitment. Importantly, Golgi integrity per se (as assessed by distribution of Lva) was not affected by disruption of AP-1 (Burgess, 2011).

This study has provided compelling evidence of a previously unknown function for clathrin and AP-1 in the formation of mucin-type secretory granules. Clathrin and AP-1 were shown to localize to the TGN prior to synthesis of secretory cargo, colocalize with newly synthesized secretory cargo, and are required for secretory granule formation. Hence AP-1 and clathrin play a crucial role in early stages of secretory granule formation in salivary gland cells. Consistent with this idea, clathrin becomes delocalized upon AP-1 depletion, indicating that other adaptors cannot recruit clathrin in the absence of AP-1 at this stage of salivary gland development (Burgess, 2011).

The results suggest that formation of mucin-containing glue granules and Weibel-Palade bodies might be similar. Weibel-Palade bodies have an unusual cigar-shaped appearance and it was proposed that AP-1 and clathrin might participate in their formation at the TGN by allowing lumenal cargo to properly fold and aggregate or by preventing premature scission. Indeed, depletion of AP-1 in endothelial cells results in the formation of small, round von Willebrand factor-containing organelles lacking other Weibel-Palade body markers. The data demonstrate that the requirement for clathrin and AP-1 is not restricted to one specific type of granule. Depletion of clathrin or AP-1 in Drosophila salivary glands resulted in the accumulation of glue protein both at the TGN and in small organelles of aberrant morphology. This finding extends the role of AP-1 and clathrin to the formation of granules containing mucoprotein cargo and suggests a broader requirement for this coat complex in granule production (Burgess, 2011).

How might AP-1 participate in glue granule formation? One possibility is that AP-1 and clathrin are directly involved in packaging glue granule cargo at the TGN. In mammalian cells, several transmembrane proteins are targeted to regulated secretory granules, including peptidyl-α-amidating monooxygenase, muclin, and phogrin. Indeed, phogrin has been shown to bind to AP-1 and AP-2 through well-conserved tyrosine and dileucine sorting motifs present in its cytosolic tail. How AP-1, a cytosolic coat protein, might interact with lumenal glue proteins in salivary cells remains to be determined. Because none of the known granule proteins contains a predicted transmembrane domain, a yet-unidentified transmembrane receptor might mediate this interaction (Burgess, 2011).

A distinct possibility is that AP-1 might be required to maintain a steady-state distribution of proteins that shuttle between the TGN and endosomes such that they are available at the TGN during granule formation. For instance, the protein convertase furin recycles between the TGN and endosomes and is required to process numerous secreted proteins such as von Willebrand factor. Importantly, furin is no longer concentrated at the TGN in μ1A-deficient fibroblasts. Thus failure to recycle transmembrane enzymes that play a crucial role in processing secreted cargo could also contribute to defective granule formation (Burgess, 2011).

Reduced levels of AP-1 resulted in intermediate-sized granules, suggesting AP-1 might have an additional role during glue granule maturation. The development of Drosophila glue granules is characterized by an overall increase in size and decrease in number, consistent with homotypic fusion of smaller granules over time (Farkas, 1999). Whether small and large granules are equally capable of fusing and whether fusion events are temporally regulated is not known. AP-1 might regulate granule maturation by sorting or retrieving membrane proteins required for homotypic fusion and eventual exocytosis. Additionally, AP-1 might function directly on maturing granules to remove missorted proteins, such as lysosomal hydrolases, similar to what has been reported for other types of secretory granules. In support of this view, live imaging revealed a dynamic association of AP-1 with immature granules. Further studies are needed to resolve whether AP-1 functions in the addition and/or removal of proteins from maturing glue granules (Burgess, 2011).

On the basis of the small size of mutant cells, AP-1 likely participates in additional trafficking pathways. In mammalian cells, AP-1A is ubiquitously expressed and required for trafficking between TGN and endosomes, whereas AP-1B is present only in polarized epithelial cells and is required for basolateral sorting from recycling endosomes. The sole AP-1 complex in Drosophila might mediate both functions in a single cell type. Interestingly, depletion of AP-1γ in salivary glands after granule formation caused the basolateral protein Discs large to redistribute to the apical surface, suggesting that AP-1 is required for basolateral targeting of proteins in this tissue. However, an independent analysis of AP-1μ null cells in the dorsal thorax epithelium failed to reveal a similar polarity defect (Benhra, 2011). This discrepancy might be due to cell type-specific requirements for AP-1 or to differences in RNAi versus mutant clones (Burgess, 2011).

The observation that the abundance of Sgs3-DsRed protein and several Sgs mRNAs is reduced upon AP-1 knockdown suggests the existence of a negative-feedback loofp, whereby a block in anterograde secretory trafficking results in down-regulation of secretory genes. A block in secretion at the TGN could potentially induce the unfolded protein response, analogous to what happens upon depletion of the Arf1 GEF GBF1. However, GBF1 functions early in the secretory pathway, and knockdown of two Arf-GEFs that act on the TGN did not elicit a similar response. Alternatively, a block in anterograde trafficking might repress transcriptional activation of secretory genes by Drosophila CrebA and Forkhead (Fkh) by some as-yet-unknown mechanism (Burgess, 2011).

In addition to the AP-1 complex, the Drosophila genome encodes two other Golgi-localized clathrin adaptor proteins, EpsinR/LqfR and Golgi-localized, γ-ear-containing, ADP-ribosylation factor-binding (GGA) protein (Drosophila has only one GGA). LqfR partially colocalizes with AP-1 at the TGN in salivary gland cells and lqfR mutants exhibit small salivary glands, suggesting defects in granule biogenesis. It will be interesting to determine whether LqfR and GGA participate in glue granule biogenesis, especially since these clathrin adaptors might facilitate sorting of other types of cargo. For example, EpsinR has been shown to bind SNARE proteins and could function to provide vesicle identity to nascent glue-containing granules. SNAP-24 was previously identified as a glue granule-specific SNARE, although whether this SNARE mediates homotypic fusion of granules or functions during exocytosis of granules at the plasma membrane is unclear. Given the apparent similarities between glue granule and Weibel-Palade body biogenesis, as well as the high degree of conservation of TGN sorting machinery in Drosophila, the current findings suggest that Drosophila salivary glands are of great utility to further elucidate the mechanisms of biogenesis of regulated secretory granules (Burgess, 2011).

In summary, this work provides strong evidence that E is released from the PG by calcium-stimulated, vesicle-mediated exocytosis. Therefore, it is suggested that the prevailing 'free diffusion' model of steroid hormone secretion needs to be reconsidered. It also follows that if E uses an active export process, then the import of many hormones, in particular 20E, is also likely controlled by transporters. Given the diversity of physiological processes regulated by steroid hormones, additional characterization of the mechanisms responsible for their import and export from various cell types and tissues will have significant impact on both basic and clinical aspects of steroid hormone physiology (Yamanaka, 2015)

The small G protein Arf like 1 (Arl1) is found at the Golgi apparatus, and in the GTP-bound form it recruits to the Golgi several effectors including GRIP-domain containing coiled-coil proteins, and the Arf1 exchange factors Big1/2. To investigate the role of Arl1, this study has characterised a loss of function mutant of the Drosophila Arl1 orthologue. The gene is essential, and examination of clones of cells lacking Arl1 shows that it is required for recruitment of three of the four GRIP domain golgins to the Golgi, with dGCC185 being less dependent on Arl1. At a functional level, Arl1 is essential for formation of secretory granules in the larval salivary gland. When Arl1 is missing, the Golgi are still present but there is a dispersal of AP-1, a clathrin adaptor that requires Arf1 for its membrane recruitment and which is known to be required for secretory granule biogenesis. Arl1 does not appear to be required for AP-1 recruitment in all tissues, suggesting that it is critically required to enhance Arf1 activation at the trans-Golgi in particular tissues (Torres, 2014).

Localized signaling in neuronal dendrites requires tight spatial control of membrane composition. Upon initial synthesis, nascent secretory cargo in dendrites exits the endoplasmic reticulum (ER) from local zones of ER complexity that are spatially coupled to post-ER compartments. Although newly synthesized membrane proteins can be processed locally, the mechanisms that control the spatial range of secretory cargo transport in dendritic segments are unknown. This study, carried out in mammalian neuronal cell cultures, monitored the dynamics of nascent membrane proteins in dendritic post-ER compartments under regimes of low or increased neuronal activity. In response to activity blockade, post-ER carriers are highly mobile and are transported over long distances. Conversely, increasing synaptic activity dramatically restricts the spatial scale of post-ER trafficking along dendrites. This activity-induced confinement of secretory cargo requires site-specific phosphorylation of the kinesin motor Kif17 (see Drosophila KIF17) by Ca²⁺/calmodulin-dependent protein kinases (CaMK) (see for example Drosophila CaMKII). Thus, the length scales of early secretory trafficking in dendrites are tuned by activity-dependent regulation of microtubule-dependent transport (Hunus, 2014. PubMed ID: 24931613).

Transglutaminases (TGs) play essential intracellular and extracellular roles by covalently cross-linking many proteins. Drosophila TG is encoded by one gene and has two alternative splicing-derived isoforms, TG-A and TG-B, which contain distinct N-terminal 46- and 38-amino acid sequences, respectively. Immunocytochemistry revealed that TG-A localizes to multivesicular-like structures, whereas TG-B localizes to the cytosol. TG-A, but not TG-B, was found to be modified concomitantly by N-myristoylation and S-palmitoylation. Moreover, TG-A, but not TG-B, was secreted in response to calcium signaling induced by Ca2+ ionophores and uracil, a pathogenic bacteria-derived substance. Brefeldin A and monensin, inhibitors of the ER/Golgi-mediated conventional pathway, did not suppress TG-A secretion, whereas inhibition of S-palmitoylation by 2-bromopalmitate blocked TG-A secretion. TG-A was shown to be secreted via exosomes together with co-transfected mammalian CD63, an exosomal marker, and the secreted TG-A was taken up by other cells. The 8-residue N-terminal fragment of TG-A containing the fatty acylation sites was both necessary and sufficient for the exosome-dependent secretion of TG-A. In conclusion, TG-A is secreted through an unconventional ER/Golgi-independent pathway involving two types of fatty acylations and exosomes (Shibata, 2017).

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. This study found that the Galphao-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Galphao induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Galphao, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Galphao interactors, this study pinpoints several basic cellular activities, including vesicular trafficking, as being regulated by Galphao. It was further found small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Galphao. This KDELR --> Galphao --> Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues (Solis, 2017).

Intracellular signaling pathways currently emerge more as dynamic networks of protein interactions rather than linear cascades of activation/inactivation reactions. In this regard, thorough elucidation of the interaction targets of heterotrimeric G proteins (the immediate transducers of GPCRs) is of crucial importance to advance the understanding of this type of signal transduction. It is especially true for Gαo. Being the most abundant G protein in the nervous system and controlling multiple evolutionary conserved developmental, physiologic, and pathologic programs, it has been remarkably shy in revealing its signaling partners. This study discloses results of multiple overlapping screens, identifying > 250 interaction partners of Gαo. Each of the screens performed has its inherent advantages and limitations, and by complementation, it is thought that a near complete coverage was obtained of the Gαo interactome -- an endeavor rarely performed for a signaling protein. Cherry-picking of individual proteins from this network resulted in detailed descriptions of mechanisms of Gαo-controlled regulation of Wnt/Fz signaling, synapse formation, PCP, asymmetric cell divisions, endocytic regulation, etc., validating the interactome findings (Solis, 2017).

As opposed to characterizations of selected individual Gαo partners, this study aimed at identifying functional modules within the interactome. For this, bioinformatics analysis clustering was performed the individual components by their functions. This resulted in appearance of several major cellular activities, which now emerge to be regulated by Gαo-dependent GPCR signaling. One of them, vesicular trafficking, was selected for detailed investigation. Many important components of this cellular function, both endocytic and exocytic, are found among Gαo targets. A study previously characterized interaction of Gαo and the endocytic master regulator Rab5, important for GPCR internalization and signaling. This study now focuses more on the exocytic function of Gαo. In various cell types (neuronal, epithelial, mesenchymal) of different animal groups (insect and mammalian) this study now finds a dual localization of Gαo to Golgi and PM, and the coordinated action of the two pools is found in exocytosis and formation of various types of cellular protrusions. This study further uncovered the evolutionary conserved KDELR --> Gαo --> Rab1/Rab3 pathway at Golgi, required for stimulated material delivery to PM and the growing protrusions (Solis, 2017).

KDELR is a Golgi-residing GPCR-like receptor, activated by the cargo delivery from ER and regulating both anterograde and retrograde trafficking from Golgi. This study shows that from Drosophila to mammals, KDELR binds Gαo and activates it, potentiating Gαo-induced cellular responses. Intriguingly, this study shows that it is the βγ-free form of Gαo that is the binding and activation partner of KDELR (in a sharp contrast to the action of typical PM-localized GPCRs, which act on heterotrimeric Gαβγ complexes. It was further found that KDELR and Gαo form a multi-subunit complex, additionally containing Rab1/Rab3 GTPses and αGDI. Activation of KDELR results in the nucleotide exchange on Gαo and its dissociation from KDELR. Although recombinant Rabs interact stronger with the GTP-loaded Gαo in vitro in absence of αGDI, in cells it was found that activation of Gαo leads to dissociation of the Rab1/Rab3-αGDI complexes, ultimately resulting in activation of the small GTPases and stimulated anterograde material delivery, necessary for the growth and stabilization of cellular protrusions. Activation of KDELR is known to induce formation of multicomponent aggregates recruiting a number of additional proteins (Majoul, 2001); recruitment of Rab-GEFs to these complexes to mediate ultimate activation of Rab1/Rab3 is also conceivable but will require further investigation. Importantly, the Golgi pool of Gαo plays key roles in these processes, as the anterograde transport as well as KDELR-mediated Rab1 activation are inhibited upon depletion of Gαo (Solis, 2017).

Based on the data presented in this study, a model emerges whereas specific Gαo pools at PM and Golgi play different but cooperative roles during neuritogenesis and protrusion formation in general. At PM, Gαo initiates neurite formation regulating actin and microtubule cytoskeletons in response to activation by specific GPCRs. At Golgi, the atypical GPCR KDELR induces activation of βγ-free Gαo, which subsequently activates Rab1 and Rab3, and the combined action of these proteins potentiates the PM-directed trafficking required for elongation and stability of membrane protrusions. Being conserved from Drosophila to mammals, this molecular mechanism is of basic importance for the understanding of G protein functions in development, physiology, and disease (Solis, 2017).

Viruses are classically characterized as being either enveloped or nonenveloped depending on the presence or absence of a lipid bi-layer surrounding their proteinaceous capsid. In recent years, many studies have challenged this view by demonstrating that some nonenveloped viruses (e.g. hepatitis A virus) can acquire an envelope during infection by hijacking host cellular pathways. This study examined the role of exosome-like vesicles (ELVs) during infection of Drosophilia melanogaster S2 cells by Cricket paralysis virus (CrPV). Utilizing quantitative proteomics, it was demonstrated that ELVs can be isolated from both mock- and CrPV-infected S2 cells that contain distinct set of proteins compared to the cellular proteome. Moreover, 40 proteins increased in abundance in ELVs derived from CrPV-infected cells compared to mock, suggesting specific factors associate with ELVs during infection. Interestingly, peptides from CrPV capsid proteins (ORF2) and viral RNA were detected in ELVs from infected cells. Finally, ELVs from CrPV-infected cells are infectious suggesting that CrPV may hijack ELVs to acquire an envelope during infection of S2 cells. This study further demonstrates the diverse strategies of nonenveloped viruses from invertebrates to vertebrates to acquire an envelope in order to evade the host response or facilitate transmission (Kerr, 2018).

The cellular machinery required for the fusion of constitutive secretory vesicles with the plasma membrane in metazoans remains poorly defined. To address this problem a powerful, quantitative assay was developed for measuring secretion, and it was used in combination with combinatorial gene depletion studies in Drosophila cells. This has allowed identification of at least three SNARE complexes mediating Golgi to PM transport: (1). STX1, SNAP24/SNAP29 and Synaptobrevin; (2). STX1, SNAP24/29 and YKT6; and (3).STX4, SNAP24 and Synaptobrevin. RNAi mediated depletion of YKT6 and VAMP3 in mammalian cells also blocks constitutive secretion suggesting that YKT6 has an evolutionarily conserved role in this process. The unexpected role of YKT6 in plasma membrane fusion may in part explain why RNAi and gene disruption studies have failed to produce the expected phenotypes in higher eukaryotes (Gordon, 2017).

Constitutive secretion delivers newly synthesised proteins and lipids to the cell surface and is essential for cell growth and viability. This pathway is required for the exocytosis of molecules such as antibodies, cytokines and extracellular matrix components so has both significant physiological and commercial importance. The majority of constitutive secreted proteins are synthesised at the endoplasmic reticulum, pass through the Golgi, and are transported to the cell surface in small vesicles and tubules which fuse with the plasma membrane. Constitutive secretory vesicles are not stored within the cell and do not require a signal to trigger their fusion with the plasma membrane which is in contrast to dense core secretory granules or synaptic vesicles. In some cell types, such as MDCK cells and macrophages, there is evidence that constitutive secretory cargo passes through a endosomal intermediate on its way to the cell surface. However, in non-polarised cells endosomal intermediates do not appear to play a major role in this pathway (Gordon, 2017).

Vesicle fusion is driven by a family of molecules known as SNAREs. SNARE are generally small (14-42kDa), C-terminally anchored proteins that have a highly conserved region termed the SNARE motif that has the ability to interact with other SNAREs. For membrane fusion to occur, SNAREs on opposing membranes must come together and their SNARE motifs zipper up to form a SNARE complex. Detailed characterisation of the neuronal SNARE complex (syntaxin 1A/VAMP2/SNAP25) required for synaptic vesicle fusion has provided a mechanistic framework for understanding the function of SNAREs. There are 38 SNAREs encoded in the human genome and they can be classified as either R or Q-SNAREs depending on the presence of a conserved arginine or glutamine in their SNARE motif. Q-SNAREs can be further subdivided into Qa, Qb and Qc SNAREs based on their homology to syntaxin and SNAP25. A typical fusogenic SNARE complex will contain four SNARE motifs (Qa, Qb, Qc and R). Qbc-SNAREs such as SNAP23, 25, 29 and 47 contribute two SNARE motifs to the SNARE complex. R-SNAREs can also be further classified as either longin or brevin type SNAREs. Longin type R-SNAREs contain a longin type fold and are found in all eukaryotes and while brevin type SNAREs are less widely conserved across species (Gordon, 2017).

Over the past twenty years significant progress has been made defining the SNARE complexes required for the majority of intracellular transport steps within eukaryotic cells. In addition, there are an increasing number of examples where the SNARE complexes required for the secretion of specific cargo such as Wnt, TNF and IL-6 have been identified. However, these proteins are not delivered directly to the cell surface from the TGN but pass through an endosomal compartment. Many labs have attempted to identify the machinery which drive the fusion of constitutive secretory vesicles with the plasma membrane and on the whole very little progress has been made. This in part may be due to the fact that there are multiple routes to the cell surface from the Golgi and redundancy in the fusion machinery. If just the R-SNAREs are considered, the human genome encodes seven post-Golgi SNAREs and a typical mammalian cell line can express at least five R-SNAREs so disruption of just one R-SNARE is unlikely to block secretion if they are functionally redundant. To overcome this problem SNARE function was characterized in Drosophila cells , as they have a simpler genome with less redundancy. The Drosophila genome encodes 26 SNAREs with 16 of them predicted to be localised to post-Golgi membranes based on their homology to mammalian SNAREs. The complexity is reduced even further as Drosophila cell lines just express two post-Golgi R-SNAREs, Syb and VAMP7 (based on publically available microarray data generated by the modENCODE project) (Gordon, 2017).

This study has developed a novel, quantitative assay for measuring constitutive secretion based on a reporter cell line that can be effectively used to monitor secretion by flow cytometry, immunoblotting and fluorescence microscopy. Depletion of known components of the secretory pathway in Drosophila cells (STX5, SLH and ROP) causes robust blocks in ER to Golgi and Golgi to plasma membrane transport, therefore validating this approach. As predicted, there is redundancy in the post-Golgi SNAREs and multiple SNAREs must be depleted to obtain robust blocks in secretion. This study has detected strong negative genetic interactions between Drosophila STX1 and STX4, SNAP24 and SNAP29, STX1 and Syb, and SNAP24 and Syb. A novel and unexpected genetic interaction was detected between Syb and YKT6. Depletion of YKT6 and VAMP3 in mammalian cells also causes a robust block in secretion indicating that this negative genetic interaction is conserved across species and provides evidence that these two R-SNAREs function in the late secretory pathway (Gordon, 2017).

Using well characterised targets (STX5, SLY1 and ROP) this study has validated the system and the assay was shown to be capable of differentiating blocks in ER to Golgi and Golgi to plasma membrane transport based on proteolytic processing and accumulation of the secretory cargo in post-Golgi transport vesicles. The experimental data suggests that there are at least three fusion complexes operating at the Drosophila PM. The first complex consists of STX1, SNAP24/29 and Syb. The second complex consists of STX4, SNAP24/29 and Syb. The third complex consists of STX1, SNAP24 and YKT6. The reason the possibility of a STX4, SNAP24/29, YKT6 complex was excluded is because depletion of both STX1 and Syb led to a complete block in secretion. Indicating that STX4 and YKT6 are unable to form a SNARE complex that can substitute for the loss of STX1 and Syb. Genetic interaction data also suggests that SNAP29 is unable to substitute for the loss of SNAP24 under conditions when both SNAP24 and Syb are depleted. This data suggests that the third SNARE complex specifically consists of STX1, SNAP24 and YKT6. At present it is unclear whether these SNARE complexes define parallel pathways to the plasma membrane or simply reflect the ability of these SNAREs to substitute with each other (Gordon, 2017).

The most striking observation in this study is that an unexpected role for YKT6 in the fusion of secretory carriers with the plasma membrane was uncovered. Depletion of YKT6 and Syb/VAMP3 in combination causes a complete block in secretion and leads to an accumulation of post-Golgi transport vesicles within Drosophila cells. YKT6 is a lipid anchored R-SNARE that has been shown to function on many pathways including ER to Golgi transport, intra-Golgi transport, endosome-vacuole fusion, endosome to Golgi transport and exosome fusion with the plasma membrane. YKT6 actively cycles on and off membranes in a palmitoylation dependant manner so potentially it is well suited to function on a wide variety of intracellular pathways. Due to the promiscuous nature of YKT6 some caution must be taken when interpreting rhe functional data. It is possible that loss of YKT6 may be indirectly affecting post-Golgi transport and fusion at the plasma membrane. However, the simplest interpretation of this data is YKT6 is directly involved in this process as this study was able to biochemically detect an interaction between YKT6 and STX1 (Gordon, 2017).

Using the knowledge obtained from the Drosophila system, the role of R-SNAREs in constitutive secretion in mammalian cells was reexamined. As previously reported, depletion of VAMP3 and other post-Golgi R-SNAREs did not perturb secretion in HeLa cells. However, depletion of VAMP3 and YKT6 in combination caused a complete block in secretion. This data suggests that YKT6 and VAMP3 may be functioning in the fusion of secretory carriers with the plasma membrane in mammalian cells. Significant efforts were made to localise endogenous YKT6 and VAMP3 on post-Golgi secretory carriers. However, the attempts have been hampered by the fact the endogenus YKT6 is expressed at very low levels and over expressed YKT6 does not target correctly to membranes and remains cytoplasmic (Gordon, 2017).

As expected, there is redundancy in the Q-SNAREs required for the fusion of secretory carriers with the plasma membrane. However, it is clear that certain SNAREs have a more prominent role in this process. The main Q-SNAREs at the Drosophila plasma membrane are STX1 and STX4. Depletion of STX1 causes a partial block in secretion while depletion of STX4 does not. It is unclear why STX1 is the favoured Qa-SNARE. It could simply be that STX1 is more abundant than STX4 or has a higher affinity for the R-SNARE on the vesicle. It may also reflect the route by which the synthetic cargo traffics to the cell surface. This study also observed redundancy between the Qbc-SNAREs SNAP24 and SNAP29 (orthologues of Sec9). A complete block in secretion is detected when both are depleted. It has previously been shown that SNAP29 interacts with STX1. However, the complexes it forms are not SDS-resistant suggesting that they may not be fusogenic (Gordon, 2017).

A potential problem with gene disruption and RNAi mediated depletion studies is compensation by other genes in the same family. For example, VAMP2 and 3 are upregulated in certain tissues of the VAMP8 knockout mouse and VAMP3 is upregulated in VAMP2 deficient chromafin cells isolated from VAMP2 null mice. Based on immunoblotting data this study did not observe any compensation between R-SNAREs when they are depleted using RNAi in Drosophila cells. No evidence was seen of this in previous work performed in HeLa cells. It was initially thought that STX1 and STX4 were being upregulated in STX5 and Syb depleted cells based on immunoblotting. However, when the samples were directly prepared in Laemmli sample buffer, rather than a TX100 based extraction buffer, no difference in the levels of these SNAREs was observed. It is possible that the change in extractability may be caused by an alteration in the localisation of the Q-SNAREs from TX100 insoluble micro-domains at the plasma membrane. However, this hypothesis was not tested. To directly assess changes in gene expression during the RNAi experiments the mRNA levels were measured of several SNAREs using RT-PCR. Depletion of STX1 leads to an upregulation of STX4 and Syb. However, no significant change was observed in the protein level of these SNAREs by immunoblotting. Thus it is unclear how significant these changes are. In the future, it will be interesting to determine how the expression levels of SNAREs, which function on the same pathway, are co-ordinated and regulated (Gordon, 2017).

To validate the genetic interaction data a published S. cerevisiae proliferation-based genetic interaction map was have interrogated to determine if the yeast homologues share similar genetic interactions to those observed in Drosophila cells (under the assumption that constitutive secretion is essential for growth). Negative genetic interactions were observed between Drosophila STX1 and STX4, STX1 and Syb, Syb and SNAP24, SNAP24 and SNAP29, YKT6 and Sec22b and Syb and YKT6. Similar genetic interactions were also observed in S. cerevisiae indicating that the data generated from Drosophila cells is physiologically relevant and the genetic interactions are evolutionary conserved. Importantly the homologues of YKT6 and Syb/VAMP3 were also found to genetically interact in yeast (YKT6 and SNC2) (Gordon, 2017).

In summary, this study has identified the SNARE complexes required for the fusion of constitutive secretory vesicles with the plasma membrane in Drosophila cells. This study has uncovered a novel role for YKT6 in the fusion of secretory vesicles with the plasma membrane which is conserved from yeast to man. This observation may in part explain why RNAi and gene disruption studies in higher eukaryotes have failed to yield the expected phenotypes. In the future, it should be possible to use the secretion assay in combination with SNARE depletion as a tool to further dissect the post-Golgi pathways involved in secretion and generate post-Golgi secretory carriers for proteomic profiling (Gordon, 2017).

What would the expected consequences be of upregulating VMAT and neuropeptides in vesicles? Upregulating VMAT will increase the speed of vesicle loading when there is exocytosis-endocytosis cycling or kiss-and-run release. Hence, increased VMAT will affect release more when vesicle emptying by release is most marked. In Drosophila, the effect of increased VMAT on behavior has not been examined. However, the dopamine precursor L-DOPA increases vesicular monoamine content and ameliorates Parkinsonian symptoms in humans. Thus, by analogy, it is suggested that octopaminergic signaling would be boosted by increased vesicular octopamine packaging induced by synaptic VMAT upregulation. Of course, increased co-transmission by neuropeptides could further alter octopamine action. Therefore, it would be interesting to explore how Rptps in the brain affect octopamine-dependent fly behaviors such as feeding and egg laying (Tao, 2019).

Exocytosis is a fundamental process in physiology, communication between cells, organs and even organisms. Hormones, neuropeptides and antibodies, among other cargoes are packed in exocytic vesicles that need to reach and fuse with the plasma membrane to release their content to the extracellular milieu. Hundreds of proteins participate in this process and several others in its regulation. This study reports a novel component of the exocytic machinery, the Drosophila transmembrane immunophilin Zonda (Zda), previously found to participate in autophagy. Zda is highly expressed in secretory tissues, and regulates exocytosis in at least three of them: the ring gland, insulin-producing cells and the salivary gland. Using the salivary gland as a model system, Zda was found to be required at final steps of the exocytic process for fusion of secretory granules to the plasma membrane. In a genetic screen, the small GTPase RalA was identified as a crucial regulator of secretory granule exocytosis that is required, similarly to Zda, for fusion between the secretory granule and the plasma membrane (de la Riva Carrasco, 2020).

Dysregulation of collagen production and secretion contributes to aging and tissue fibrosis of major organs. How procollagen proteins in the endoplasmic reticulum (ER) route as specialized cargos for secretion remains to be fully elucidated. This study reports that TMEM39, an ER-localized transmembrane protein, regulates production and secretory cargo trafficking of procollagen. The C. elegans ortholog TMEM-39 was identified from an unbiased RNAi screen; deficiency of tmem-39 leads to striking defects in cuticle collagen production and constitutively high ER stress response. RNAi knockdown of the tmem-39 ortholog in Drosophila causes similar defects in collagen secretion from fat body cells. The cytosolic domain of human TMEM39A binds to Sec23A, a vesicle coat protein that drives collagen secretion and vesicular trafficking. TMEM-39 regulation of collagen secretion is independent of ER stress response and autophagy. It is proposed that the roles of TMEM-39 in collagen secretion and ER homeostasis are likely evolutionarily conserved (Zhang, 2021).

The duplication and 9-fold symmetry of the Drosophila centriole requires that the cartwheel molecule, Sas6, physically associates with The dimeric Golgi protein Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. This study used hydrogen-deuterium exchange mass spectrometry to define Gorab's interacting surfaces that mediate its sub-cellular localization. A core stabilization sequence within Gorab's C-terminal coiled-coil domain was identified that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer's coiled-coil domain undergoes an anti-parallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable hetero-trimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6's Gorab-binding domain generates a Sas6 variant with a 16-fold reduced binding affinity for Gorab that can not support centriole duplication. Thus Gorab dimers at the Golgi exist in equilibrium with Sas-6 associated monomers at the centriole to balance Gorab's dual role (Fatalska, 2021).

Centrioles are the ninefold symmetrical microtubule arrays found at the core of centrosomes, the bodies that organize cytoplasmic microtubules in interphase and mitosis. Centrioles also serve as the basal bodies of both non-motile and motile cilia, and flagellae. The core components of centrioles and the molecules that regulate their assembly are highly conserved. The initiation of centriole duplication first requires that the mother and daughter pair of centrioles at each spindle pole disengage at the end of mitosis. Plk4 then phosphorylates Ana2 (Drosophila)/STIL(human) at its N-terminal part, which promotes Ana2 recruitment to the site of procentriole formation, and at its C-terminal part, which enables Ana2 to bind and thereby recruit Sas6. The ensuing assembly of a ninefold symmetrical arrangement of Sas6 dimers provides the structural basis for the ninefold symmetrical cartwheel structure at the procentriole's core. Sas6 interacts with Cep135 and in turn with Sas4 (Drosophila)/CPAP (human), which provides the linkage to centriole microtubules (Fatalska, 2021).

An unexpected requirement has been identified for the protein, Gorab, to establish the ninefold symmetry of centrioles (Kovacs, 2018). Flies lacking Gorab are uncoordinated due to basal body defects in sensory cilia, which lose their ninefold symmetry, and also exhibit maternal effect lethality due to failure of centriole duplication in the syncytial embryo. Gorab is a trans-Golgi-associated protein. Its human counterpart is mutated in the wrinkly skin disease, gerodermia osteodysplastica. By copying a missense mutation in gerodermia patients that disrupts the association of Gorab with the Golgi, this study was able to create mutant Drosophila Gorab, which was also unable to localize to trans-Golgi. However, this mutant form of Gorab was still able to rescue the centriole and cilia defects of gorab null flies. It was also found that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype that can be overcome by a second mutation preventing Golgi targeting. Thus, centriole and Golgi functions of Drosophila Gorab are separable (Fatalska, 2021).

The Golgi apparatus both delivers and receives vesicles to and from multiple cellular destinations and is also responsible for modifying proteins and lipids. Gorab resembles a group of homodimeric rod-like proteins, the golgins, which function in vesicle tethering. The golgins associate through their C-termini with different Golgi domains, and their N-termini both capture vesicles and provide specificity to their tethering. There is known redundancy of golgin function, reflected by the overlapping specificity of the types of vesicles they capture. Gorab is rapidly displaced from the trans-side of the Golgi apparatus by Brefeldin A, suggesting that its peripheral membrane association requires ARF-GTPase activity (Fatalska, 2021).

Previous studies of human Gorab indicated its ability to form a homodimer in complex with Rab6 and identified its putative coiled-coil region as a requirement to localize at the trans-Golgi (Egerer, 2015; Witkos, 2019). Studies on its Drosophila counterpart supported Gorab's ability to interact with itself, potentially through the predicted coiled-coil motif. However, this region was also found to overlap with the region required for Gorab's interaction with Sas6 (Kovacs, 2018). These findings raised the questions of how Gorab's putative coiled-coil region could facilitate interactions with the Golgi, on the one hand, and its Sas6 partner, on the other. To address this, s hydrogen-deuterium exchange (HDX) was employed in conjunction with mass spectrometry (MS). HDX enables the identification of dynamic features of protein by monitoring the exchange of main chain amide protons to deuteria in solution. This study used HDX-MS to monitor the retarded exchange of amide protons localized between interacting regions of Gorab and Sas6 to identify the interacting surfaces within the Gorab-Sas6 complex. Together with other biophysical characterizations, this has revealed that Gorab is able to form a homo-dimer through its coiled-coil region but that it interacts as a monomer with the C-terminal coiled-coil of Sas6. Mutation of a critical amino acid in Sas6's Gorab-binding domain generates a variant of Sas6 with a sixteenfold reduced binding affinity for Gorab that is no longer able to support centriole duplication (Fatalska, 2021).

Together, these findings indicate that Gorab exists at the trans-Golgi network as a homodimer. Dimerization requires its coiled-coil motif (residues 200-315) within which is a core sequence (residues 270-287) that represents the most stable part of this dimerization region. Dimerization enables Gorab to interact with Rab6, and this in turn enables its association with the trans-Golgi. In contrast, Gorab interacts with Sas6 as a monomer. Gorab's binding to Sas6 occurs with a higher affinity than its homodimerization, enabling a Gorab monomer to associate with the Sas6 dimer. Thus, the relatively small number of Sas6 molecules at the centriole would more avidly bind the Gorab monomer, allowing greater excess of Gorab to accumulate as dimers at the trans-Golgi. Sas6 and Gorab interact through short interfaces within their coiled-coil regions. Disruption of this region of Sas6 through mutation of a single conserved leucine residue, L447, results in a failure of Gorab to bind to Sas6 and localize to the centriole. While the possibility cannot be formally excluded that the L447A mutation affects some other aspect of Sas6 function, the finding that expression of this mutant phenocopies a strong gorab hypomorph in its effects upon both co-ordination and centriole duplication suggests that failure to recruit Gorab is responsible for the Sas6-L447A defect. The finding of some residual apparent Gorab-like function in Sas6-L447A-expressing flies may reflect the overexpression of the protein due to the technical requirements of the experiment and the fact that Sas6-L447A still binds Gorab but with a sixteenfold reduced affinity compared to wild-type Sas6. Given that Sas6-L447A greatly diminishes the interaction with Gorab, whereas the mutation, M440A, in the adjoining 'a' position of the 'a-g' coiled-coil heptad repeat does not, leads to the conclusion that Gorab binds to a narrow region near the C-terminus of the coiled coil of Sas6 (Fatalska, 2021).

Gorab shows many of the properties typical of golgins, a family of tentacle-like proteins that protrude from the Golgi membranes to capture a variety of target vesicles. Redundancy between golgins in their ability to bind target vesicles could act as a functional safeguard and might explain why loss-of-function gorab mutants display no obvious Golgi phenotype, contrasting to the Golgi defects shown by the C-terminally tagged Gorab molecule (Kovacs, 2018). Gorab is similar to other golgins, which also associate with the Golgi membranes through their C-terminal parts in interactions that require Rab family member proteins to interact with the C-terminal part of the golgin dimer. The N-terminal parts of the golgins interact with their vesicle targets. Human GORAB's N-terminal part interacts with Scyl1 to promote the formation of COPI vesicles at the trans-Golgi (Witkos, 2019). However, its precise role in the transport of COPI vesicles is not clear, particularly why loss of human GORAB affects Golgi functions in just bone and skin when COPI function is required in multiple tissues. Drosophila Gorab also co-purifies and physically interacts with both Yata, counterpart of Scyl1, and COPI vesicle components, and its importance for transport of COPI vesicles in Drosophila is similarly unclear (Fatalska, 2021).

This study offers a perspective on how Gorab interacts with Sas6 at the centriole and suggests the possibilities for why this interaction is essential to establish the centriole's ninefold symmetry. The heterotrimeric structure formed by a Sas6 dimer and the Gorab monomer will together constitute a single spoke plus central hub unit of the centriole's cartwheel. The C-terminal part of Gorab would be expected to lie in a tight antiparallel association with the C-terminal part of Sas6's coiled-coil region. Gorab's N-terminus might thus be expected to extend towards the centriolar microtubules and their associated proteins. As the microtubules of Drosophila's somatic centrioles exist as doublets of A- and B-tubules, it is tempting to speculate that Gorab interacts with the centriole wall in a region occupied in other cell types by the C-tubule. This could account for the lack of any requirement for Sas6-Gorab interaction in the male germ-line, where centrioles have triplet microtubules and a C-tubule occupies this space. Gorab's partner proteins interacting with its N-terminal region are therefore of great interest at both the Golgi and in the centriole, and it will be key to understand the nature of these interactions in future studies (Fatalska, 2021).

Exocrine secretion commonly employs micron-scale vesicles that fuse to a limited apical surface, presenting an extreme challenge for maintaining membrane homeostasis. Using Drosophila melanogaster larval salivary glands, this study shows that the membranes of fused vesicles undergo actomyosin-mediated folding and retention, which prevents them from incorporating into the apical surface. In addition, the diffusion of proteins and lipids between the fused vesicle and the apical surface is limited. Actomyosin contraction and membrane crumpling are essential for recruiting clathrin-mediated endocytosis to clear the retained vesicular membrane. Finally, membrane crumpling was also observed in secretory vesicles of the mouse exocrine pancreas. It is concluded that membrane sequestration by crumpling followed by targeted endocytosis of the vesicular membrane, represents a general mechanism of exocytosis that maintains membrane homeostasis in exocrine tissues that employ large secretory vesicles (Kamalesh, 2021).

Maintaining synaptic structure and function over time is vital for overall nervous system function and survival. The processes that underly synaptic development are well understood. However, the mechanisms responsible for sustaining synapses throughout the lifespan of an organism are poorly understood. This study demonstrates that a previously uncharacterized gene, CG31475, regulates synaptic maintenance in adult Drosophila NMJs. CG31475 was named mayday, due to the progressive loss of flight ability and synapse architecture with age. Mayday is functionally homologous to the human protein Cab45 (SDF4 - stromal cell derived factor 4), which sorts secretory cargo from the Trans Golgi Network (TGN). Mayday was found to be required to maintain trans-synaptic BMP signaling at adult NMJs in order to sustain proper synaptic structure and function. Finally, mutations in mayday were shown to result in the loss of both presynaptic motor neurons as well as postsynaptic muscles, highlighting the importance of maintaining synaptic integrity for cell viability (Sidisky, 2021).

Among the most prominent neuromuscular synapses in adult Drosophila are those of the indirect flight muscles. One set of IFMs, the Dorsal Longitudinal Muscles (DLMs), are composed of six large muscle fibers innervated by five motor neurons on each side of the thorax. Once the DLM NMJs are established, these stable structures are present throughout the lifespan of the organism. These NMJs are part of the Giant Fiber (GF) pathway that propels flight behavior. Thus, the activity of DLMs can be monitored by assaying flight behavior as a readout of synaptic integrity. Additionally, the DLM NMJs form a tripartite synapse composed of a presynaptic motor neuron, postsynaptic muscle cell, and associated glial cell, that provide the ability to understand synaptic function at the cellular and molecular level. This model also allows for expression of transgenes in non-essential tissue, particularly the DLM motor neurons that are easily accessible. Together, it is possible assess the morphological and functional properties of adult DLM NMJs to elucidate the mechanisms responsible for sustaining synapses in aging adults, then apply this to understand how synapses deteriorate in neurodegenerative diseases (Sidisky, 2021).

Although the processes involved in maintaining synaptic structure and function may not be understood, there are a few key pathways that are crucial for regulating synaptic growth, organization and stability during synaptic development. Specifically, in Drosophila one key signaling cascade that involves coordination between the presynaptic motor neurons and postsynaptic muscle cells is the bone morphogenic protein (BMP) signaling cascade. The morphogen glass bottom boat (Gbb), the Drosophila ortholog to mammalian BMP7, is secreted in a retrograde manner from the postsynaptic muscle cell to the presynaptic motor neuron. Currently, it is not understood how this pathway could function past development. This suggests that this signaling cascade could play a role in maintaining synaptic integrity (Sidisky, 2021).

Gaining a better understanding of synaptic dysfunction should help to identify strategies involved in maintaining synaptic integrity with age. This study identified Mayday, a resident Golgi protein that is required to maintain trans-synaptic signaling across adult NMJs. Mutations in mayday impair retrograde BMP signaling, resulting in degradation of synaptic structure and function. Finally, this study demonstrates that this sustained trans-synaptic signaling is required to maintain the viability of both presynaptic motor neurons and postsynaptic muscles (Sidisky, 2021).

The current study describes mayday (myd), a previously uncharacterized gene that plays a role in maintaining synaptic integrity with age by promoting trans-synaptic signaling. We found that myd^3PM71 mutants have structural and functional deficits in adult DLM NMJs. Through tissue-specific RNAi and rescue experiments, it was determined that Myd is necessary in both postsynaptic muscle tissue and presynaptic motor neurons to maintain synaptic integrity. Myd localizes to the TGN and shares functional homology with human Cab45. Myd sustains retrograde BMP signaling in adult DLM NMJs through genetic interactions with gbb, tkv, wit, and mad mutants and staining of Gbb and pMad markers. Finally, myd sustains the viability of presynaptic motor neurons and postsynaptic muscles (Sidisky, 2021).

From developmental studies, it was learned that Gbb is a morphogen secreted in a retrograde manner trans-synapticaly from postsynaptic muscle tissue to the presynaptic motor neuron in larval NMJs to promote synaptic growth. However, relatively little is known regarding the roles of this pathway in fully developed organisms. Recent evidence demonstrates that sustained BMP signaling is required to maintain FMRFamide expression in a subset of neurons in the Drosophila brain. The current results here further demonstrate that retrograde BMP signaling that regulates NMJ development is required in adult NMJs to sustain synaptic integrity with age. It is also possible that several other signaling pathways crucial for organism development may be required throughout the life of the organism (Sidisky, 2021).

Knockdown and rescue experiments using myd demonstrate that it maintains synaptic integrity through roles in both pre- and postsynaptic tissue. While most studies involving BMP in synaptic growth report a retrograde signaling mechanism, recent evidence suggests that this pathway could also signal in an anterograde fashion. While genetic studies provide support for retrograde BMP signaling, it cannot be ruled out that anterograde BMP signaling also plays an important role in maintaining synaptic integrity. In particular, the levels of pMAD that were observed were present within both presynaptic motor neuron terminals as well as postsynaptic muscles. Further studies aimed at characterizing BMP signal activation within muscle cells should help with our understanding of the mechanisms responsible for synaptic maintenance (Sidisky, 2021).

While trans-synaptic BMP signaling plays a clear role in maintaining synapses, myd mutations likely impair other pathways associated with cargo trafficking. In addition to secretory cargo, Cab45 also has a role in trafficking lysosomal proteases. Given the functional homology shared between Cab45 and Myd, it is possible that the trafficking of these lysosomal hydrolases needed for autophagy could be disrupted. Defects in autophagy have been strongly linked with neurodegenerative diseases. Therefore, it is possible that myd mutants have disruptions in autophagy that lead to the loss of synaptic integrity. It will be interesting to investigate how Myd impacts these other processes that are associated with neuronal dysfunction (Sidisky, 2021).

This assessment of synaptic dysfunction in the current study includes flight performance as a readout of functional integrity, as well as morphological measurements of branch length, branch number, and bouton number using a presynaptic membrane marker. In future studies, it will be helpful to further evaluate synaptic integrity in mayday mutants. Additional functional assays may include electrophysiological measurements of activity across these NMJs, and more structural data could be obtained through the use of a wide array of synaptic markers, as well as ultrastructural analysis using Transmission Electron Microscopy. Together, these types of studies should allow for an even greater understanding of synaptic dysfunction and the mechanisms required to maintain these critical structures (Sidisky, 2021).

The ability of stem cells to switch between quiescent and proliferative states is crucial for maintaining tissue homeostasis and regeneration. In Drosophila, quiescent neural stem cells (qNSCs) extend a primary protrusion, a hallmark of qNSCs. This study found that qNSC protrusions can be regenerated upon injury. This regeneration process relies on the Golgi apparatus that acts as the major acentrosomal microtubule-organizing center in qNSCs. A Golgi-resident GTPase Arf1 and its guanine nucleotide exchange factor Sec71 promote NSC reactivation and regeneration via the regulation of microtubule growth. Arf1 physically associates with its new effector mini spindles (Msps)/XMAP215, a microtubule polymerase. Finally, Arf1 functions upstream of Msps to target the cell adhesion molecule E-cadherin to NSC-neuropil contact sites during NSC reactivation. These findings have established Drosophila qNSCs as a regeneration model and identified Arf1/Sec71-Msps pathway in the regulation of microtubule growth and NSC reactivation (Gujar, 2023).

This work has established Drosophila qNSC protrusion as a regeneration model. It shares two features with other regeneration models, including adult rat retinal ganglion cells (RGCs) and adult C. elegans motor neurons. First, Drosophila qNSCs exhibit age-dependent decline in regeneration capability. This is similar to age-dependent decline in axon regeneration capacity after axotomy in C. elegans mechanosensory neurons and in corticospinal and rubrospinal neurons in the adult mammalian CNS. Second, alterations in microtubule dynamics affect regeneration. The effects of microtubule dynamics on qNSC regeneration are in line with findings in the mammalian CNS neurons that microtubule manipulation promotes axon regeneration after injury (Gujar, 2023).

One interesting aspect of Drosophila qNSC regeneration is that the distal tip of the protrusion does not degenerate after severing but is capable of the regeneration similar to the proximal end. Likely, certain cues or signals from the neuropil to which the protrusion is attached play an important role in the regeneration. It would be worthwhile to understand how the damaged plasma membrane is repaired during qNSC regeneration. Interestingly, re-sealing of the plasma membrane during axon regeneration is dependent on actin and calcium (Gujar, 2023).

This study shows that Golgi functions as a MTOC in qNSCs. The centrosomes may take over Golgi as the MTOC when NSCs re-enter the cell cycle as the proximity between the immature centrosomes and the apical Golgi likely facilitates this transition. Golgi is required for the regeneration of qNSC protrusions upon injury, likely via microtubule growth. Similarly, moderately stable microtubules are required for efficient axon regeneration in central neurons following spinal cord injury. The anterograde transport of cargo, such as mitochondria and vesicles, is also important for their delivery to lesioned axonal tip for axon regeneration. Although it was previously thought that qNSCs retract their protrusions before cell-cycle re-entry, recent work has shown that qNSCs can retain their protrusions throughout the first post-reactivation division (Gujar, 2023).

We also observed that the primary protrusion of larval brain NSCs persists during reactivation and subsequently the first division, although it remains unknown if this applies to most of the reactivating NSCs. Observations also suggest that regeneration of the protrusion precedes NSC reactivation at the event of injury (Gujar, 2023).

This study has identified Arf1 and Sec71 as key regulators of acentrosomal microtubule nucleation, growth, and orientation in the primary protrusion of Drosophila qNSCs. The role of Arf1 in regulating microtubule growth is unexpected, as it is well established that Arf1-6 proteins are critical for membrane trafficking, while Arf-like (Arl) proteins such as Arl2 are important for microtubule functions. Interestingly, mammalian Arf1 recruits tubulin-binding cofactor E (TBCE), which is known to associate with the microtubule regulator Arl2 during microtubule polymerization, to the Golgi, and Arf1 overexpression increases tubulin abundance in motor neurons (Gujar, 2023).

It will be particularly interesting to investigate a similar microtubule regulatory role for Arf1 in other Drosophila cell types as well as in different organisms (Gujar, 2023).

This study reports that in Drosophila qNSC regeneration, the distal tip is capable of the regeneration similar to the proximal end, and focus was placed on the intrinsic role of Arf1 and Msps in regeneration. It remains to be tested whether they could also function in neuropil to facilitate the regeneration of the distal protrusion. EB1-GFP comets going into PIS Golgi and coming out from it in a few seconds were assigned as 'not from Golgi,' which may underestimate the number of Golgi-derived EB1-GFP comets. Furthermore, due to technical limitations, it was not possible to pinpoint the changes in microtubule dynamics or cargo transport in qNSCs after injury. Further studies are warranted to better understand how microtubule growth and dynamics enhance qNSC regeneration (Gujar, 2023).

The Pallidin protein is a central subunit of a multimeric complex called biogenesis of lysosome-related organelles complex 1 (BLOC1) that regulates specific endosomal functions and has been linked to schizophrenia. Downregulation of Pallidin and other members of BLOC1 in the surface glia, the Drosophila equivalent of the blood-brain barrier, reduces and delays nighttime sleep in a circadian-clock-dependent manner. In agreement with BLOC1 involvement in amino acid transport, downregulation of the large neutral amino acid transporter 1 (LAT1)-like transporters JhI-21 and mnd, as well as of TOR (target of rapamycin) amino acid signaling, phenocopy Pallidin knockdown. Furthermore, supplementing food with leucine normalizes the sleep/wake phenotypes of Pallidin downregulation, and this study identified a role for Pallidin in the subcellular trafficking of JhI-21. Finally, evidence is provided that Pallidin in surface glia is required for GABAergic neuronal activity. These data identify a BLOC1 function linking essential amino acid availability and GABAergic sleep/wake regulation (Li, 2023).

Biogenesis of lysosome-related organelles complex 1 (BLOC1) is an octameric complex linked to endosomal compartments and the cytoskeleton (Cheli, 2010). The genes coding for the 8 subunits in mice (Pallidin, dysbindin, BLOS1, BLOS2, BLOS3, cappuccino, muted, and snapin) are broadly expressed within the brain and in peripheral tissues. The complex regulates the trafficking of various receptors and transporters and appears to play a prominent role in the biogenesis of recycling endosomes. Mice bearing severe or complete loss-of-function mutations in BLOC1 genes are viable and fertile. They display common phenotypes, originating from defects in highly specialized lysosome-related organelles, such as reduced pigmentation due to impaired retinal and epidermal melanosomes or extended bleeding times resulting from the lack of dense granules in platelets. In humans, mutations in BLOC1 genes and other functionally related genes are found in the Hermansky-Pudlak syndrome (Li, 2023).

In addition, genetic studies have identified variants of the dysbindin gene and other BLOC1 genes as risk factors for developing schizophrenia. Although the latter results are debated, several postmortem studies have reported reduced levels of dysbindin mRNA and protein in the brain of schizophrenics. Furthermore, genome-wide association studies also reported a link between dysbindin genetic variants and cognitive abilities (Li, 2023).

These findings have led to investigations aiming at deciphering the role of BLOC1 in neuronal function using not only mouse models defective for individual BLOC1 gene function but also the Drosophila model, in which the complex is well conserved. These studies confirmed the involvement of BLOC1 in behavior and memory. They identified potential cellular and molecular mechanisms such as abnormal glutamatergic, GABAergic, and dopaminergic transmission. However, the vast majority of these studies have been carried out in animals with spontaneous or artificial mutations. This approach makes the interpretation of the resulting phenotypes challenging, given the broad expression of BLOC1 in many cell types. Furthermore, the complex is expressed during development, when it can be involved in neurodevelopmental diseases such as autism spectrum disorders (Li, 2023).

Apart from one recent report (Lee, 2018) the involvement of BLOC1 in sleep/wake regulation has not been investigated. Interestingly, it has been found that Pallidin is upregulated in a somnolent mouse model with defective histaminergic transmission (Seugnet, 2023). Thus, further investigation is required given the critical implication of sleep in brain function, and in particular in schizophrenia, in which sleep disruption could be in part a consequence of the pathology, or an aggravating factor as seen in human and rodent models. This study investigated the potential role of BLOC1 in sleep/wake regulation in Drosophila. Neurotransmission systems, ion channels, and glial functions in Drosophila and mammals are globally conserved (Li, 2023).

There are differences regarding sleep/wake in insects compared with mammals, such as the absence of slow wave and paradoxical sleep. Nevertheless, the evidence for conserved regulatory principles and functions is compelling. The flexibility and short generation time of the Drosophila model is an asset in molecular genetic studies, providing hypotheses that can be tested in rodents and ultimately used for therapy. A conditional knockdown strategy was used to target Pallidin, a major component of BLOC1. The pool of Pallidin in the cell is associated almost entirely with the complex, and this protein plays a central role through its interactions with dysbindin, BLOS1, and cappuccino (Cheli, 2010), with its loss leading to their degradation (Li, 2023).

This study focused on glia, as a previous results suggest a function for Pallidin in non-neuronal cells. Glial cells are increasingly proven to play very significant roles in the control of sleep/wake and circadian rhythms both in Drosophila and in rodent models. Glia-dependent neurotransmitter reuptake, neurotransmitter metabolism, and glial calcium transients critically influence neuronal networks that control sleep timing and sleep homeostasis. Neuroglia signaling pathways have been shown to modulate sleep-deprivation-induced learning impairments. Recent reports have demonstrated the prominent role of glial cells at the interface between the brain and the circulating fluids during the sleep/wake cycle (Li, 2023 and references therein).

This study provides evidence in Drosophila that Pallidin and other BLOC1 components regulate the initiation of sleep in the early part of the night, at the level of surface glial (SG) cells. In Drosophila, SG cells express tight junctions and molecular components in common with the mammalian BBB. As in the BBB this organization prevents the entry of macromolecules and tightly regulates the exchanges of solutes between the circulatory system (the hemolymph in Drosophila) and the brain, thus maintaining the particular interstitial fluid composition necessary for neuronal activity. This function relies on intense transporter activity. The data support a mechanism whereby BLOC1 regulates LAT1-like transporters subcellular trafficking in both perineurial and subperineurial cells, leading to adequate essential amino acid supply and GABAergic neuronal activity promoting sleep, in a circadian-clock-dependent manner. This study observed that downregulating Pallidin using a pan-neuronal driver also resulted in reduced night sleep, suggesting that SG is not the only cell type in the brain where Pallidin is required to regulate sleep (Li, 2023).

Blocking the cycling of the molecular clock by placing flies in a per⁰ mutant background completely abolishes the Pallidin knockdown sleep/wake phenotype, suggesting that the circadian clock is implicated. Accordingly, it was found that hyperpolarization of PDF-expressing clock neurons is sufficient to normalize the main Pallidin knockdown phenotype: increased sleep latency at night. This raises the possibility that Pallidin affects clock-dependent sleep/wake regulatory networks and in particular, those under the control of the large PDF-expressing neurons (lLNv). In lLNv neurons, GABAergic input is modulated in a circadian manner, resulting in higher inhibition in the early night, promoting sleep, while a disruption of this input results in delayed and fragmented sleep (Li, 2023).

The GABAergic neurons responsible for lLNv inhibition are so far unidentified. The delayed and reduced night sleep observed upon Pallidin knockdown in SG is also similar to the phenotype previously reported with pan-GABAergic neuron inhibition. Conversely, feeding flies the GABA agonist THIP65 or increasing GABAergic neuronal firing by expressing TrpA1 can potently induce sleep. The latter manipulation cannot promote sleep when Pallidin is downregulated in SG, suggesting that the gene is required to allow sustained GABAergic neuronal activity. Alternatively, Pallidin function could be regulated directly by the clock present in perineurial cells, which modulates the efflux properties of subperineurial cells in a circadian manner (Li, 2023).

Downregulation of several members of BLOC1 in SG phenocopies the knockdown of Pallidin, strongly suggesting that the whole complex is involved in this Pallidin-dependent sleep/wake regulation. This is consistent with the observation that the pool of Pallidin and dysbindin present in the cell is almost entirely associated with the complex and that the lack of one subunit leads to a destabilization of the others (Li, 2023).

The lack of sleep alteration with snapin manipulation may result from lower efficiency of the genetic tools used for downregulation. Alternatively, it may also reflect different gene dosage sensitivity and functionality for the different members of the complex, as previously reported, or the existence of multiple subunits within the complex2 or different complexes containing a subset of the subunits (Li, 2023).

In Drosophila larval neuromuscular junction, Snapin has, for example, been shown to regulate synaptic homeostasis (Dickman, 2012), while Pallidin does not seem to affect baseline neurotransmission and is required during sustained neuronal activity to replenish the pool of releasable synaptic vesicles (Chen, 2017). BLOC1 has been shown to be required for the biogenesis of recycling endosomes through its interactions with sorting endosomes and the cytoskeleton (Delevoye, 2016). These BLOC1 functions and the regulation of sleep by BLOC1 members reported in this study are in agreement with a recent report showing bidirectional interactions between sleep and endocytosis in SG (Lee, 2018). Intriguingly, the endocytosis in SG was reported to be the most intense in the early part of the night, when sleep is the deepest and the phenotype of Pallidin downregulation is the most pronounced. In addition, the activity of the recycling endosome associated small GTPase Rab11 appears to play a prominent role in this context. Consistent with these findings, extensive protein-protein interaction analyses identified rab11 as the best interacting partner for BLOC1 in both Drosophila and humans.5 Thus, BLOC1 may facilitate the high endocytic activity of SG during the early part of the night, notably the biogenesis of recycling endosomes, while having a less prominent role at other times during the day. In line with the results, Pallidin mutant mice display reduced sleep amount during the light phase of the day, the primary sleep period in rodents, and shorter average sleep-bout duration (Li, 2023).

However, BBB-specific and conditional knockout models in mice would be necessary to determine whether the model outlined in this study applies to mammals. In humans, the incidence of sleep/wake disorders in the rare Hermansky-Pudlak syndrome, bearing deficits in BLOC1 or other functionally related complexes, is unknown but deserves scrutiny. In contrast, sleep disruption is common among patients suffering from schizophrenia, another pathology associated with BLOC1 deficits. Interestingly, one of the prominent sleep abnormalities in schizophrenia patients is a reduction in sleep spindle density, which strongly relies on GABAergic activity, and the latter is also affected in this pathology. Accordingly, previous studies in dysbindin mutant mice have shown that BLOC1 can disrupt GABAergic activity (Li, 2023).

Thus, a potential role for amino acid transport at the BBB in those contexts would deserve investigation given these results. Early studies of Pallidin function pointed to a role in amino acid import into the brain transport, as suggested by lower sensitivity to intraperitoneal injections of the LAT1 substrates l-DOPA and tryptophan. The LAT1 transporter plays a major role in the import of these amino acids and in the import of large neutral essential amino acids such as leucine, isoleucine, and histidine (Li, 2023).

A conditional knockout of LAT1 in brain endothelial cells confirmed the crucial role of LAT1 in the regulation essential amino acid abundance within the brain, its impact on GABAergic transmission, and its potential implication in autism spectrum disorders. Intriguingly, the relative abundance of amino acids in the brain of Pallidin mutant mice resembles those found in mice with a BBB specific knockout LAT1 (Li, 2023).

This study provides evidence that Pallidin function in SG is linked to essential amino acid supply and to LAT1-like transporter activity. First, downregulation of the LAT1-like transporters and of TOR signaling phenocopy knock down, to a large extent or completely, the BLOC1 genes. Second, supplementing the food with leucine can normalize the JhI-21, Pallidin, and Blos2 phenotypes. Interestingly, the phenotype of the JhI-21 transporter knockdown could be rescued by valine and tryptophan supplementation, while Pallidin knockdown could be rescued only by leucine. This suggests that Pallidin does not solely modulate LAT1-like transporter function and has complex effect on multiple transport systems, as previously suggested (Li, 2023).

JhI-21 and minidisc are the Drosophila closest homologs of LAT1 light-chain. JhI-21 is expressed broadly in the Drosophila brain, with most of the signal in perinuclear punctae that colocalizes partially with the lysosomal marker ATG8. The subcellular trafficking of JhI-21 in SG is abnormal following Pallidin downregulation, with a substantial fraction of the transporter localized in the nucleus. This abnormal trafficking is likely to reduce the functionality of the transporter, explaining the similar sleep/wake phenotypes of JhI-21 and Pallidin downregulation in SG and their normalization by essential amino acid supplementation. A nuclear localization has already been reported in glioma cell lines for the solute carrier (SLC) Eaat1 and Eaat2 transporters, and for the K+ inwardly rectifying channel. In both cases, this unusual subcellular localization was associated with an overall reduced functionality of the protein in the cell. Aside transporters, several full-length transmembrane receptors have been repeatedly reported to be localized in the nucleus where some of them may act as a transcription factors (Li, 2023).

The mechanisms underlying this phenomenon are mostly unknown. In this study, it was not possible to detect obvious changes in autophagy, suggesting that Pallidin downregulation does not induce major changes in lysosomal activity and rather affects more specifically particular cargos, as previously suggested. Interestingly, in addition to their cytoplasmic localization, a nuclear localization has previously been reported for two BLOC1 subunits: BLOS2102 and, in particular, dysbindin. ATG8 is also found both in lysosomes and in the nucleus where it can regulate gene expression in association with transcription factors. Such lysosomal-nuclear connections open the possibility of trapping a diversity of unexpected proteins in the nucleus in normal as well as in abnormal conditions (Li, 2023).

In Drosophila, protein intake, threonine intake, and D-serine levels play a role in sleep/wake regulation. Although the changes in amino acid intake presumably change global free amino acid levels within the brain, the effect on sleep/wake regulation seems to originate from specific sleep/wake regulatory networks or neurotransmission systems. In humans, amino-acid-supplemented diets have been designed to improve sleep and wakefulness, on the basis of the principle that this will increase the synthesis of monoamines. The results point to a model independent from these previously identified mechanisms: higher recycling endosome activity in the early part of the night, mediated by BLOC1, would lead to high LAT1-like activity and essential amino acid import in the brain. The TOR signaling appears also to be required and may facilitate LAT1-like transporter function. This assessment suggests that Pallidin-dependent sleep regulation does not involve a modulation of monoamine levels, in contrast with previous reports. However, in these reports the conclusions were reached using complete knock out and not by downregulation in specific cell types, making comparison with the present results difficult. For example, Drosophila Dysbindin has been suggested to regulate global brain dopamine level by modulating its recycling through glial cells (Li, 2023).

In contrast, the current data suggest that essential amino acid import to the brain facilitated by Pallidin would enhance GABAergic transmission required for sleep. This study showed that a large number of GABAergic neurons in the adult fly brain are in direct cellular contacts with SG. Although the significance and the function of these contacts remain to be determined, these observations fit the idea that a significant subset of these neurons have particular metabolic needs (Li, 2023).

How could amino acid transport regulate GABAergic transmission? Amino acids are at the interplay among many processes, being involved in protein synthesis, energy metabolism, neurotransmitter synthesis, and degradation. Amino acid transporters may further intertwine these processes: as a prime example, LAT1 is an antiporter that can associate the import of leucine to the export of glutamine, two amino acids that are crucial in the glutamate/GABA/glutamine cycle in the brain (Li, 2023).

Pharmacological experiments have indeed suggested that LAT1 in the BBB regulates GABA homeostasis in the interstitial fluid. Branched-chain amino acids (BCAA) and leucine in particular are thought to be critical providers of nitrogen for glutamate synthesis through transamination, affecting glutamate/GABA/glutamine cycling. Enzymes for BCAA metabolism are conserved in Drosophila, suggesting similar metabolic regulation in this model organism. An interesting example is Drosophila mutants for GABA transaminase (GABAT), which sleep 2–3 h longer than their genetic controls because of impaired GABA degradation. For survival, these mutants require that food include glutamate or BCAAs (leucine and valine), whose transamination can provide glutamate in the cells. These data indicate that GABA metabolism and BCAAs as a source of glutamate play a substantial role in sleep/wake regulation and brain energy metabolism. Interestingly, this study found that the GABA/glutamate ratio tends to be decreased in Pallidin knockdown flies, while the glutamate/aspartate ratio is increased compared with controls. These results suggest that BLOC1 function in SG modulates neurotransmitter and brain amino acid metabolism on a global level and may control the inhibitory/excitatory balance (Li, 2023).

The data presented in this study emphasize the implication of circulating amino acids, in particular BCAA, in sleep/wake regulation, corroborating several recent metabolomics studies in mammalian models and in humans, in which branched-chain amino acid (BCAA) levels have been repeatedly shown to be modulated by the circadian clock and/or the sleep homeostat. For example, a recent study identified in insomniac patients prominent changes in the levels of several circulating BCAA, including increased levels of leucine during the night. Food supplemented with BCAA can correct sleep disorders in a mouse model with chronic sleep disruption. It is worth noting that genes involved in amino acid transport are among those commonly disrupted in autism and schizophrenia. In conclusion, this study provides potential mechanisms at the blood-brain interface that may be relevant to both sleep disruption and psychosis and emphasizes the possibility of diet-based therapies (Li, 2023).

One limitation of this study has been the inability to monitor Pallidin protein expression using immunofluorescence. A previously published antibody produced a non-specific signal in the adult brain, and attempts to generate a new antibody were unsuccessful. This precluded determining the subcellular localization of the protein and assessing the local efficiency of the knockdown constructs. The lack of appropriate drivers prevented more precise manipulation of the neuronal targets affected by Pallidin function: the GABAergic neurons presynaptic to lLNv neurons are unknown, and therefore there is no identified driver making it possible to monitor or control their activity, and in addition, there are no available LexA construct specifically expressed in lLNv neurons. For future investigations, it will be important to develop an efficient conditional system, independent of temperature, to limit the knockdown of Pallidin to the adult stage. Finally, further work is required to elucidate the involvement of the TOR signaling pathway in this context and the Pallidin-JhI-21-dependent interactions between SG and GABAergic neurons (Li, 2023).

This study demonstrated that a Drosophila Golgi protein, Gorab, is present not only in the trans-Golgi but also in the centriole cartwheel where, complexed to Sas6, it is required for centriole duplication. In addition to centriole defects, flies lacking Gorab are uncoordinated due to defects in sensory cilia, which lose their nine-fold symmetry. The separation of centriole and Golgi functions of Drosophila Gorab were demonstrated in two ways: first, Gorab variants were created that are unable to localize to trans-Golgi but can still rescue the centriole and cilia defects of gorab null flies; second, it was shown that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype overcome by mutations preventing Golgi targeting. These findings suggest that during animal evolution, a Golgi protein has arisen with a second, apparently independent, role in centriole duplication (Kovacs, 2018).

This study has identified a tissue specific role for Golgi-associated Gorab in centriole duplication in Drosophila. Gorab physically interacts with the centriole cartwheel component, Sas6, with which it co-localizes from the onset of procentriole formation. Centrosomes fail to duplicate in gorab-mutant-derived embryos and in diploid tissues of gorab-null Drosophila, which lose coordination through defects in their mechanosensory cilia. Such cilia have a single, mother centriole-derived basal body with six to ten sets of microtubules, and this abnormal symmetry extends into the ciliary axoneme (Kovacs, 2018).

Loss of nine-fold symmetry in gorab-mutant centrioles is reminiscent of Sas6 mutants. It suggests the Gorab–Sas6 partnership is required for both centriole duplication and symmetry. The formation of centrioles with correct symmetry can still be directed around Sas6 variants that are unable to establish nine-fold symmetry. This suggests other components of the centriole, in addition to Sas6, also contribute to its symmetry. Gorab could be one such contributing molecule, at least in part. However, this cannot be universally true, because centrioles and axonemes in the gonads of fully fertile gorab-null males have correct nine-fold symmetry. This could be either because maternal Gorab protein perdures sufficiently in male germ cells to permit centriole duplication or because Gorab is substituted by another molecule in spermatogenesis. These possibilities, either of which could reflect the distinctive morphology of Drosophila's spermatocyte centrioles, require further study (Kovacs, 2018).

The trans-Golgi localization of human GORAB is mirrored in multiple Drosophila tissues, including salivary glands, imaginal discs, the central nervous system, and in the male and female germ lines, but not in syncytial embryos, where Golgi has yet to form. Accordingly, Gorab's association with COPI coatomer components in cultured cells suggests involvement in retrograde vesicle transport from Golgi to ER consistent with its resemblance to a golgin. The rod-like golgins, which bind Rab, Arf, or ADP-ribosylation family GTPases, are tethered to Golgi membranes by their C termini and protrude outwards to capture vesicles at their N termini. Overlapping specificity in vesicle targeting provides redundancy of function. Thus, both golgins GMAP-210 and GM130 can capture ER-derived carriers; both GMAP-210 and Golgin-84 can capture cis-Golgi derived vesicles; and so on. Such redundancy might account for the lack of any Golgi phenotype in gorab-null mutants. However, Gorab's functional relevance at Drosophila Golgi is indicated by the cytokinesis defects in male meiosis caused by expression of C-terminally tagged Gorab, which are strikingly similar to those following disruption of COPI-mediated vesicle trafficking. This accords with Gorab's association with COPI proteins and reinforces suggestions that the integrity of ER and other membranous structures is interdependent with astral and spindle microtubule function in male meiosis (Kovacs, 2018).

By generating the counterpart of a gerodermia osteodysplastica missense mutant that prevents human GORAB from localizing to Golgi, Drosophila Gorab's Golgi and centriole functions can be separated. This p.Val266Pro mutation prevents Gorab from associating with Golgi but fully rescues centriole duplication defects of gorab-null mutants and restores their ciliary function. A proline residue at this site could strongly influence structure of the Golgi-interacting region because of its side chain's rigidity and ability to undergo cis–trans isomerization. The mutation did not, however, interfere with Gorab's ability to bind Sas6. Moreover, introducing p.Val266Pro into C-terminally tagged Gorab prevented its localization to Golgi and so relieved the cytokinesis defect. Thus the male sterility resulting from a C-terminal GFP tag is mediated through Gorab's Golgi association. Gorab's precise Golgi functions in Drosophila, most likely redundant with other golgins, must await further genetic and molecular studies (Kovacs, 2018).

Gorab is not required for centriole duplication or Golgi function in unicellular organisms such as ciliated eukaryotes. Its evolutionary appearance in animals may reflect increased proximity and functional interactions between the Golgi, centrosomes, and cilia. Such co-evolution could have facilitated the emergence of proteins with dual functions, allowing a component of one organelle to take on an additional function in its neighbor. However, Gorab is not present in all animal species; it is absent, for example, from C. elegans. This could possibly reflect the assembly of C. elegans SAS6 into a spiral rather than the ring-shaped oligomers characteristic of the centriole cartwheels in most species, which may obviate the need for interactions with a Gorab-like protein. Moreover, even within a single species, Gorab may be required for centriole duplication in some tissues and not others, as was found in Drosophila. Such tissue specificity might account for findings with a GORAB-mutant mouse, which has few primary cilia in dermal condensate cells responsible for Hedgehog signaling in hair follicles but does have primary cilia on keratinocytes. This could reflect tissue-specific failure of centriole duplication in the GORAB-null mouse, even though there is currently no evidence to support this notion (Kovacs, 2018).

Although the above defects in cilia development in the GORAB-null mouse require molecular analysis, they suggest a possibility of conserved roles for GORAB. Both fly and human proteins are not only found at the trans-Golgi but also at the centriole. GFP-tagged GORAB was expressed in U2OS cells, and it was found at both centrosomes and Golgi. The Golgi localization was abolished by the p.Ala220Pro mutation but centrosome association remained. This study also found that anti-GORAB antibodies could detect human GORAB at the centriole, albeit not always together with Sas6 as in Drosophila. This might reflect different requirements for GORAB and Sas6 at the centriole in the two organisms; Sas6 remains centriole-associated throughout the Drosophila duplication cycle, whereas it is first recruited and then is later absent from the lumen of the mother centriole for a substantial part of the human duplication cycle. It will be of future interest to track the precise behaviors of SAS6 and GORAB throughout the centriole duplication cycle in human cells (Kovacs, 2018).

Currently, however, it remains uncertain whether GORAB functions in centriole duplication in human cells as in insects. Because mammalian cells lacking centrosomes are prevented from cell cycle progression by a p53-dependent pathway, attempts were made to assess the consequences of GORAB depletion on centrosome number in a human osteosarcoma (U2OS) line expressing dominant-negative p53 (U2OS p53DD). It is found that some more-stable centriole proteins require more rounds of knockdown before a duplication phenotype can be observed and, unfortunately, GORAB RNAi led to cell death before depletion was complete. This was possibly due to compromised Golgi function, making it difficult to assess the effect upon centriole duplication. However, GORAB RNAi enhanced the centrosome loss seen after depletion of SASS6 alone, suggesting the possibility of a cooperative role between the two proteins. It was also found that depletion of human GORAB abolished the centrosome overduplication that occurs in U2OS cells held in S-phase following aphidicolin and hydroxyurea treatment. However, because Golgi function is also compromised by these treatments, it is not certain that human GORAB is required for centrosome duplication as in flies (Kovacs, 2018).

It is noted that the p.Ala220Pro mutation results in a disease phenotype comparable to null mutations8. As GORAB p.Ala220Pro can still associate with the centrosome, this suggests that the gerodermia osteodysplastica phenotype is likely to result predominantly from defective Golgi functioning. However, it would still be worthwhile to re-examine cells from different tissues of patients with GORAB null mutations for potential additional defects in centriole duplication and/or formation of primary cilia. It will also be important to examine Gorab−/− mice further to determine whether the reported loss of cilia could arise through failure of centriole duplication rather than as a secondary consequence of Golgi malfunction (Kovacs, 2018).

To conclude, these findings bring insight into the dual life of a protein with Golgi and centriole functions but also raise new future questions. An understanding of the precise role of Gorab at the Golgi awaits a greater knowledge of Gorab's Golgi partners and its redundancy with other golgins. Moreover, full understanding of Gorab's centriole duplication function in Drosophila awaits future studies of its precise structural interactions with Sas6 and other centriole proteins (Kovacs, 2018).

Previous work has revealed that alterations in components of endocytic compartments, such as Rab5 overexpression or mutation of tsg101/ept or vps25, disrupt epithelial cell polarity and upregulate Notch signaling. However, in these cases, it is unclear whether perturbation of endocytosis alters Notch signaling via cell polarity disruption or whether changes in endocytic compartments directly impact on Notch. This study shows that without cell polarity loss, lgl- tissue displays altered endocytic compartments and upregulates Notch signaling, indicating that changes in endocytosis alone are sufficient to upregulate Notch pathway activity. Moreover, this study shows that reducing aPKC-Baz-Par6 complex activity does not rescue Notch pathway upregulation in lgl- tissue, revealing that Lgl's roles in regulating cell polarity and endocytosis are separable. Interestingly, the Crb polarity protein also has separable roles in the regulation of cell polarity and Notch signaling and endocytosis, via different mechanisms to Lgl (Parsons, 2014).

The discovery that Lgl depletion increases Notch activation without cell polarity loss has implications for tumorigenesis. In the developing eye epithelium, increased Notch signaling results in upregulation of the cell cycle regulator, CycA, and the cell survival regulator, Rst, which in lgl- tissue, is expected to contribute to increased cell proliferation and survival, concomitant with impaired Hippo signaling. Because elevated Notch signaling is associated with various human cancers, the finding that Lgl regulates Notch signaling warrants investigation of whether elevated Notch signaling in human cancer is associated with Lgl depletion. Notably, Lgl1 knockout in the mouse brain induces hyperproliferation and decreased differentiation, associated with increased Notch signaling, and mutation of zebrafish Lgl disrupts retinal neurogenesis, dependent on increased Notch signaling. Moreover, the finding that Lgl plays a novel role in regulating endosomal acidification and the striking suppressive effect of chloroquine on the adult lgl- mosaic phenotype reveal the importance of acidification in tumor growth, perhaps by also modulating Hippo signaling. The data, together with evidence that many cancers show higher acidity due to increased V-ATPase activity, which contributes to tumorigenesis, posit the question of whether Lgl dysfunction might contribute to acidification defects in human cancer (Parsons, 2014).

Cell fate decision during asymmetric division is mediated by the biased partition of cell fate determinants during mitosis. In the case of the asymmetric division of the fly sensory organ precursor cells, directed Notch signaling from pIIb to the pIIa daughter endows pIIa with its distinct fate. Previous studies have shown that Notch/Delta molecules internalized in the mother cell traffic through Smad anchor for receptor activationSara endosomes and are directed to the pIIa daughter. This study shows that the receptor Notch itself is required during the asymmetric targeting of the Sara endosomes to pIIa. Notch binds Uninflatable, and both traffic together through Sara endosomes, which is essential to direct asymmetric endosomes motility and Notch-dependent cell fate assignation. The data uncover a part of the core machinery required for the asymmetric motility of a vesicular structure that is essential for the directed dispatch of Notch signaling molecules during asymmetric mitosis (Loubery, 2014).

The Notch signaling pathway plays multiple roles in organisms ranging from flies and worms to mammals. A powerful model system to elucidate the cell biology of Notch signaling is the Drosophila sensory organs. Each sensory organ precursor (SOP) cell divides asymmetrically to produce a pIIa cell and a pIIb daughter cell, which perform directed Notch signaling: pIIb signals to pIIa. Four independent endocytic mechanisms control asymmetric signaling in the SOP. These include asymmetric endocytic events mediated by the E3 ubiquitin ligase Neuralized, recycling endosomes, and the endocytic adaptors α- and γ-adaptin together with Numb (Loubery, 2014).

During SOP cytokinesis, a fourth mechanism involves a population of endosomes marked by the adaptor protein Sara. Sara endosomes contain as cargo a pool of endocytosed Notch and Delta molecules. Notch and Delta reach the Sara endosome 20 min after their endocytosis in the SOP; this pool is dispatched into pIIa during cytokinesis. In contrast, the pools of Notch in endosomal populations upstream (Rab5 early endosomes) or downstream (Rab7 late endosomes) of Sara endosomes are segregated symmetrically. The specific pool of Notch in Sara endosomes is relevant for signaling: it is cleaved in a ligand- and gamma-secretase-dependent manner to release the transcriptionally active Notch intracellular domain (NICD) in pIIa (Loubery, 2014).

A key question is what machineries control the asymmetric targeting of these endosomes. Is the cargo (the ligand Delta or its receptor Notch) playing a role on the specific targeting of these endosomes? To unravel the machinery regulating the behavior of Sara endosomes during SOP mitosis, candidate factors from previously reported proteomics approaches or genetic screens were tested for Notch signaling. Thus, Uninflatable was identified as a factor involved in the asymmetric dynamics of Sara endosomes (Loubery, 2014).

MARCM homozygous mutant clones were generated for a null allele of Uninflatable (Uif2B7) an the trafficking of Delta, Notch, and the Notch effector Sanpodo through Sara endosomes was monitored. To look at the motility of the endogenous population of Sara endosomes, the cohort of internalized Delta molecules 20 min after its endocytosis was followed in the SOP by means of a pulse-chase antibody uptake assay. Delta, Notch, and Sanpodo traffic normally through Sara endosomes in the absence of Uif, and these endosomes are targeted to the cleavage plane (the central spindle) in cytokinesis (Loubery, 2014).

In Uif mutants or RNAi knockdown conditions, iDl20'/Sara endosomes fail to be asymmetrically dispatched to pIIa after their targeting to the central spindle. These results indicate that Uif is not required to bring Notch to the Sara endosomes or to target the endosomes to the central spindle. However, once in the spindle, Uif is essential for the specific dispatch of Sara endosomes from the spindle into the pIIa cell (Loubery, 2014).

This function of Uninflatable is specific to the asymmetric segregation of Sara endosomes. To gain mechanistic insights into the mechanism of action of Uif, this study has analyzed the density of microtubules in the central spindle and has shown that Uninflatable does not regulate the organization of the microtubular cytoskeleton. In contrast, it was found that Uif controls the residence time of Sara endosomes on the central spindle: in control SOPs, Sara endosomes depart from the central spindle with a decay time of 103 ± 21 s, whereas upon Uif downregulation this decay time goes up to 175 ± 42 s. These data indicate that Uif is not involved in the organization of the spindle, but rather in the motility properties of the endosomes, particularly their last step of departing from the central spindle and end up in pIIa (Loubery, 2014).

Consistent with the role of Uif in the asymmetric targeting of Sara endosomes, Uif contributes to Notch-dependent cell fate assignation in the SOP lineage. To address this, the composition of SOP lineages was examined in homozygous Uif2B7 MARCM clones or upon Uif RNAi. In wild-type animals, the SOP lineage consists of four different cells: two external cells (the shaft and the socket) originating from pIIa and two internal cells (the sheath and the neuron) from pIIb, which can be identified by immunostaining. In Uif mutant clones, instead of a sheath and a neuron per SOP lineage, two sheath cells can be frequently observed in the notum, indicating a symmetric division in the pIIb lineage. Similarly, upon Uif downregulation in the postorbital SOPs, duplications of sockets were observed, which is diagnostic of symmetric divisions in the pIIa lineage. These data uncover a role for Uninflatable in Notch-dependent asymmetric cell fate assignation that is mediated by the asymmetric dispatch of the Sara endosomes (Loubery, 2014).

The Uif phenotype during asymmetric endosomal targeting and cell fate assignation prompted us to look whether Uif is a cargo of Sara endosomes. To detect the endogenous protein, anti-Uif antibodies were generated. To look at Uif trafficking in vivo, transgenic flies were generated expressing a Uif-GFP protein, which can provide activity to rescue the lethality of a Uif lethal mutation at least partly (Loubery, 2014).

Uif-GFP is strongly colocalized with both Sara-GFP and iDelta20'. Since a cargo of Sara endosomes is Notch itself (73% ± 2.7% of the vesicular population of Notch molecules is in Sara endosomes), the presence of Notch cargo was examined in Uif vesicles: 44% ± 4.7% of Uif-positive vesicular structures contain Notch. Therefore, a population of Uninflatable and Notch traffics through Sara endosomes during SOP asymmetric mitosis (Loubery, 2014).

The fact that Uninflatable controls the asymmetric dispatch of the Sara endosomes, which contain internalized Notch and Uninflatable, prompted a look at a possible molecular interaction between Uninflatable and Notch. Uif- and Notch-expressing plasmids were cotransfected in S2 cells and immunoprecipitation experiments were performed by using anti-Uif-coupled beads, followed by immunoblotting with a clean anti-Notch antibody that was purified from a hybridoma cell line (DSHB #C17.9C6). Uif was shown to immunoprecipitate Notch. This coimmunoprecipitation can be reproduced from lysates of S2 cells expressing Notch and Uif tagged with the PC peptide tag and anti-PC-coupled beads; as a control, other transmembrane proteins such as Tkv-GFP are not coimmunoprecipitated with Uif-PC. Together, these results indicate a specific molecular interaction between Notch and Uif (Loubery, 2014).

Uninflatable is a transmembrane protein that, like Notch, contains an array of epidermal growth factor (EGF) repeats. It has been shown that Notch is engaged in protein-protein interactions through its EGF repeats with other factors containing EGF repeats. These include its ligand Delta, but also a number of noncanonical Notch ligands, secreted or membrane proteins lacking the DSL domain characteristic of canonical Notch ligands (Dlk-1, Dlk-2, DNER, Trombospondin, LRP1, EGFL7, and Weary). Consistently, it has recently been reported that a synergistic genetic interaction between Uif and Notch depends on Notch EGF repeats. Therefore, studies were performed to discover which EGF repeats of Uif could be involved in the molecular interaction with Notch. A coimmunoprecipitation experiment was performed in S2 cells coexpressing Notch and an N-terminal, truncated form of Uif tagged with PC (UifΔCter-PC) that lacks the four EGF domains flanking the transmembrane domain but still contains the other 17 EGF repeats and other extracellular domains. While full-length Uif-PC coimmunoprecipitates Notch, UifΔCter-PC does not. This indicates that the interaction between Uif and Notch may be mediated by the four EGF domains of Uif flanking its transmembrane domain (Loubery, 2014).

Although Uif binds and colocalizes with Notch, it does not play a role in core Notch signaling: embryos deprived of maternal and zygotic Uif in germline clones do not show a Notch signaling phenotype, whereas they display loss of inflation of the trachea as previously reported. Consistently, loss of Uif in wing mosaics does not cause a defect of Notch-dependent expression of Wingless at the wing margin. This indicates that Uninflatable interaction with Notch is not essential during core Notch signaling, but rather during the asymmetric dispatch of Notch-containing Sara endosomes during asymmetric cell division. This prompted the possibility that Notch itself is required for the asymmetric motility of the endosomes (Loubery, 2014).

To study whether Notch plays a role during the asymmetric dispatch of Sara endosomes, the trafficking was studied of a Notch-GFP fusion expressed at endogenous levels. The idea was to confirm previous observations using a Notch antibody uptake assay to follow Notch expressed at endogenous levels. To achieve this, a reporter transgenic fly strain was set up in which Notch-GFP fusion is driven by the Notch endogenous promoter and is expressed at endogenous levels. In this fusion, GFP is inserted in the middle of the Notch-intra domain. Since in protein fusions GFP is frequently cleaved out, whether the fusion protein is intact was examined. This would be particularly important in this case, since a cleavage event would lead to a truncated Notch-intra peptide (Loubery, 2014).

In these transgenic Notch-GFP flies, GFP is very efficiently cleaved out (74% of total GFP is cleaved, leading to truncated Notch-intra peptides that can only partially support Notch function and thereby cause a highly penetrant mutant phenotype. This precludes the usage of this reagent as a bona fide marker for Notch. In particular, the cytosolic GFP signal cannot be used as a readout of signaling as previously reported: a nuclear accumulation of the GFP signal in these flies does not solely reflect the accumulation of Notch-intra-GFP, but rather the overall accumulation of different GFP-containing fragments (Loubery, 2014).

Whether, in these conditions, the pool of membrane associated GFP-Notch traffics through Sara endosomes and is asymmetrically dispatched to the pIIa cell was studied. Only 11% ± 1.3% of the total GFP signal in these flies is membrane associated (plasma membrane and intracellular vesicular structures). The rest, representing the vast majority (89%), corresponds to cytosolic and nuclear cleaved GFP (Loubery, 2014).

In Notch-GFP flies, 3.1% of the total GFP signal is associated with intracellular vesicular structures. These correspond to various intracellular vesicular compartments, including Notch in the secretory pathway, as well as in early endosomes, Sara endosomes, recycling endosomes, and late endosomes. To measure the size of the specific pool of Notch in Sara endosomes, a Notch antibody internalization assay was performed, and internalized Notch was chased 20 min after its endocytosis (iNotch20'). As previously established, 73% ± 2.7% of Notch-GFP vesicles are positive for iNotch20'. Of this iNotch20'-positive pool, 79% would be targeted to pIIa . This is consistent with only 65% ± 3.1% of the total pool of Notch-GFP being dispatched to pIIa (Loubery, 2014).

Whether Notch itself plays a role on the asymmetric targeting of Sara endosomes was addressed. Notch was depleated in the SOP by expressing a previously validated Notch dsRNA, and the behavior of Sara endosomes was examined. Upon Notch knockdown in the SOP, iDl20'/Sara endosomes are still targeted to the central spindle, but the subsequent directed dispatch to pIIa is defective. This indicates that Notch itself contributes to the endosomal recruitment of the machinery that endows the Sara endosomes with their asymmetric behavior (Loubery, 2014).

It has been shown that the targeting of Notch to Sara endosomes does not depend on Uninflatable; it was then determined whether the recruitment of Uninflatable on Sara endosomes depended on Notch. Interestingly, it was found that, conversely, the targeting of Uif to Sara endosomes is not controlled by Notch. This implies that these two molecules use different machineries to get to the endosome, where they can interact and are both required for the asymmetric motility of the endosome (Loubery, 2014).

Since the Notch receptor itself is required for the asymmetric targeting of Sara endosomes, it was asked whether Notch signaling plays a role in the process. Notch signaling was blocked by inactivating the ligand Delta through overexpression of Tom in the SOP cell; Tom overexpression leads to inactivation of the Ubiquitin ligase Neuralized and thereby blocks endocytosis-dependent activation of Delta. In the absence of Notch signaling, targeting of Sara endosomes to the central spindle and their asymmetric dispatch to the pIIa cell remains intact. This indicates that although the Notch receptor is essential for the asymmetric targeting of Sara endosomes, Notch signaling is not (Loubery, 2014).

This report has started to unravel the machinery that mediates asymmetric endosome motility during asymmetric cell division. Both Notch and Uninflatable were shown to play a key role in the last step of the asymmetric motility of endosomes: the final, specific stride of the Sara endosomes from the central spindle into the anterior pIIa cell. This is based on the following four key sets of observations (Loubery, 2014).

First, it was confirmed that a functional Notch-GFP fusion expressed at endogenous level does traffic through Sara endosomes, which are indeed dispatched asymmetrically during SOP mitosis. Second, Notch binds Uninflatable, and both colocalize in Sara endosomes. Third, neither Notch nor Uninflatable is essential for the targeting of Notch/Delta/Uif to the Sara endosomes or the targeting of those endosomes to the central spindle, but they are essential for the final dispatch from the central spindle into the pIIa cell. Although Notch is necessary for this process, Notch signaling is not. Fourth, Uninflatable is not an integral component of the Notch signaling pathway, but it plays a role during asymmetric Notch signaling in the SOP, and therefore mutant Uif conditions lead to a lineage identity phenotype. It remains to be elucidated what machineries downstream of Notch/Uninflatable implement the control of the final step toward pIIa and what is asymmetrical in the cytoskeleton so that this final step occurs toward pIIa and not pIIb (Loubery, 2014).

During asymmetric division, fate assignation in daughter cells is mediated by the partition of determinants from the mother. In the fly sensory organ precursor cell, Notch signalling partitions into the pIIa daughter. Notch and its ligand Delta are endocytosed into Sara endosomes in the mother cell and they are first targeted to the central spindle, where they get distributed asymmetrically to finally be dispatched to pIIa. While the processes of endosomal targeting and asymmetry are starting to be understood, the machineries implicated in the final dispatch to pIIa are unknown. This study shows that Sara binds the PP1c phosphatase and its regulator Sds22. Sara phosphorylation on three specific sites functions as a switch for the dispatch: if not phosphorylated, endosomes are targeted to the spindle and upon phosphorylation of Sara, endosomes detach from the spindle during pIIa targeting (Loubery, 2017).

Asymmetric cell division plays many roles in development. In particular, stem cells divide asymmetrically to self-renew while also forming differentiated cells. Asymmetric cell division involves the specific partitioning of cell fate determinants (RNA, proteins or organelles) in one of the two sibling daughter cells. The Sensory Organ Precursor cells (SOPs) of the Drosophila notum are a model system of choice to unravel the molecular mechanisms of asymmetric cell division (Loubery, 2017).

The division of each SOP gives rise to a pIIa and a pIIb daughter cell and, after two more rounds of asymmetric cell divisions, to the four cells of the sensory organ: the outer cells (shaft and socket) are progeny of the pIIa, while the pIIb forms the inner cells (sheath and neuron) and a glial cell that rapidly undergoes apoptosis. The Notch signalling pathway controls cell fate determination in this system: a signalling bias between the pIIa-pIIb sibling cells is essential to obtain a correct lineage (Loubery, 2017).

The asymmetric dispatch of cell fate determinants during SOP division is governed by the polarity of the dividing cell. The Par complex (composed by the aPKC, Par-3 and Par-6 proteins) is the master regulator of the establishment of this polarity. Downstream the Par complex, Notch signalling is regulated by endocytosis and endosomal trafficking through four independent mechanisms: (1) The E3 Ubiquitin ligase Neuralized is segregated to the pIIb cell, where it induces the endocytosis and thereby the activation of the Notch ligand Delta; (2) Recycling endosomes accumulate in the perinuclear region of the pIIb cell, in which they enhance the recycling and activation of Delta; (3) The endocytic proteins α-adaptin and Numb are segregated to the pIIb cell, where they inhibit the Notch activator Sanpodo; (4) During SOP mitosis, Sara endosomes transport a signalling pool of Notch and Delta to the pIIa cell, where Notch can be activated. Asymmetric Sara endosomes have also been shown to operate in the larval neural stem cells (Coumailleau, 2009) as well as in the adult intestinal stem cells in flies, where they also play a role during asymmetric Notch signalling. In fish, Sara endosomes mediate asymmetric cell fate assignation mediated by Notch during the mitosis of neural precursor of the spinal cord (Loubery, 2017).

Sara endosomes are a subpopulation of Rab5-positive early endosomes characterised by the presence of the endocytic protein Sara. Sara directly binds the lipid phosphatidyl-inositol-3-phosphate and both molecules are found at the surface of these endosomes. A pulse-chase antibody uptake assay has been established to monitor the trafficking of endogenous internalised Notch and Delta and showed that both Notch and Delta traffic through Sara endosomes. Furthermore, it was shown that Sara endosomes are specifically targeted to the pIIa cell during SOP division, mediating thus the transport of a pool of Notch and Delta that contribute to the activation of Notch in the pIIa. The Notch cargo and its Uninflatable binding partner are required for this asymmetric dispatch. Targeting of Sara endosomes to the central spindle is mediated by a plus-end-directed kinesin, Klp98A. The asymmetric distribution of endosomes at the central spindle results from a higher density of microtubules in pIIb with their plus ends pointed towards pIIa15 (Loubery, 2017).

This study shows that the Sara protein itself controls both the targeting and the final dispatch of Sara endosomes to the pIIa daughter cell. Sara binds and is a target of the PP1 phosphatase complex. The phosphorylation state of Sara functions as a switch that enables the targeting of Sara endosomes to the central spindle of the dividing SOP, and their subsequent detachment from the central spindle, which is necessary to allow their movement to the pIIa daughter cell (Loubery, 2017).

Previous work has shown that a subpopulation of Rab5 early endosomes positive for Sara are asymmetrically dispatched into the pIIa daughter cell during cytokinesis of the SOP. This was monitored by following in vivo either GFP-Sara or internalized Delta or Notch, which reach the Sara endosomes 20 min after their endocytosis in the mother cell. These vesicles were termed iDelta^20' endosomes. In contrast, the pools of Notch in endosomal populations upstream or downstream of the Sara endosomes (that is, the Rab5 early endosomes with low Sara levels and the Rab7 late endosomes, respectively) were segregated symmetrically. Rab5 endosomes show different levels of Sara signal: by a progressive targeting of Sara to the Rab5 endosomes, Rab5 early endosomes mature into Sara endosomes. This prompts the question whether the levels of Sara in endosomes correlate indeed with their asymmetric behaviour (Loubery, 2017).

To study the relationship between the levels of Sara in endosomes and their targeting to the spindle, Matlab codes were written to perform automatic 3D-tracking of the Sara endosomes. Sara endosomes were detected by monitoring a GFP-Sara fusion, which was overexpressed through the UAS/Gal4 system. This way, the position of the endosomes, their displacement towards and away from the central spindle was monitored as well as the levels of Sara. In addition, the position was detected automatically of the Pon cortical crescent, which forecasts the side of the cell that will become the pIIb cell (Loubery, 2017).

The localization of endosomes was studied with respect to a 2 μm-wide box centered in the central spindle during SOP mitosis. The enrichment was measured of endosomes in this central spindle as a function of time. Two phases were observed in the movement of the endosomes during mitosis: (1) targeting to the central spindle and (2) departure into the pIIa cell. The endosomes are progressively accumulating in the central spindle area from the end of metaphase (~450 s before abscission) through anaphase and during cytokinesis until they are enriched at the central spindle by about 10-fold at 250 s before abscission (Loubery, 2017).

Subsequently, the endosomes depart from the central spindle area into the pIIa cell. By fitting an exponential decay to the profile of abundance of the endosomes at the central spindle, the characteristic residence time of the endosomes at the central spindle was measured after the recruitment phase: after recruitment, endosomes remain at the central spindle 98±9.8 s before they depart into one of the daughter cells, preferentially the pIIa cell (Loubery, 2017).

To address a potential role of Sara on central spindle targeting and asymmetric segregation, the behaviour was tracked and quantified of the endosomes in a Sara loss of function mutant (Sara¹²) and in conditions of Sara overexpression in the SOP (Neur-Gal4; UAS-GFP-Sara). In Sara¹² SOPs, targeting of iDelta^20' endosomes to the cleavage plane is severely impaired. Consistent with the fact that the asymmetric dispatch of endosomes to pIIa requires first their targeting to the central spindle as previously shown, in Sara¹² SOPs the dispatch to the pIIa daughter is strongly affected. A slight bias (60% pIIa targeting) is, however, retained in the mutant, consistent with a previous report (Loubery, 2017).

Conversely, overexpression of Sara increases targeting to the central spindle. In these conditions, Sara is found not only in Rab5 endosomes, but also in Rab7 late endosomes as well as in the Rab4 recycling endosomes. Correlating with this, Rab4, Rab5 and Rab7 endosomes, which are not all recruited to the central spindle in wild-type conditions, are now targeted to the central spindle upon Sara overexpression and are asymmetrically targeted (Loubery, 2017).

Furthermore, consistent with the correlation that is observed between the levels of Sara at the endosomes and their displacement towards the cleavage plane, quantification of central spindle targeting of the Sara endosomes upon its overexpression shows that targeting of the endosomes to the cleavage plane is increased by a factor of 2.5 in these conditions. These observations indicate that Sara plays a crucial role on the targeting of the endosomes to the spindle and the subsequent dispatch of the Notch/Delta containing endosomes to pIIa. Does this play a role during Notch-dependent asymmetric cell fate assignation? (Loubery, 2017).

Sara function contributes to cell fate assignation through asymmetric Notch signalling, but this activity is redundantly covered by Neuralized. Neuralized E3 Ubiquitin ligase does play an essential role during the endocytosis and activation of the Notch ligand Delta. Therefore, during larval development, Neuralized is essential for Notch-mediated lateral inhibition in the proneural clusters, which leads to the singling-out of SOP cells from the proneural clusters. Later, during pupal development, Neuralized appears as a cortical crescent in the pIIb side of the dividing SOPs, thereby biasing Delta activation in the pIIb cell and asymmetric activation of Notch in pIIa6 (Loubery, 2017).

Consistently, a partial loss of function of Neuralized by RNAi interference in the centre of the notum (Pnr>Neur^RNAi Control) showed lateral inhibition defects in the proneural clusters, causing the appearance of supernumerary SOPs as well as asymmetric Notch signalling defects in the SOP lineage, leading to supernumerary neurons and loss of the external shaft/socket cells in the lineage. The remaining Neuralized activity in this partial loss of function condition allows many sensory organs (more than forty in the centre of the notum) to perform asymmetric cell fate assignation and to develop, as in wild type, into structures containing at least the two external cells (Loubery, 2017).

In Pnr>Neur^RNAi, Sara¹²/Df(2R)48 transheterozygote mutants, the number of supernumerary SOPs is increased by 35% with respect to the Pnr>Neur^RNAi controls (668±38 versus 498±52). This indicates that during lateral inhibition, Sara endosomes contributes to Notch signalling. This general role of Sara is uncovered when the Neuralized activity during Notch signalling is compromised (Loubery, 2017).

In the case of Neuralized, its localization to the anterior cortex biases Notch signalling to be elicited in the pIIa cell. This is the same in the case of Sara endosomes: asymmetric dispatch of Sara endosomes also biases Notch signalling to pIIa10. Indeed, in Pnr>Neur^RNAi, Sara¹²/Df(2R)48 transheterozygote mutants, the number of bristles (external shaft/socket cells) in the notum is strongly reduced at the expense of supernumerary neurons compared to the Pnr>Neur^RNAi controls. This indicates that Notch-dependent asymmetric cell fate assignation in the SOP lineage is synergistically affected in the Sara/Neuralized mutant. This implies that the SOP lineages which still could generate bristles with lower levels of Neuralized function in Pnr>Neur^RNAi need Sara function to perform asymmetric cell fate assignation: in Pnr>Neur^RNAi, Sara¹²/Df(2R)48 and Pnr>Neur^RNAi, Sara¹²/Sara1 transheterozygote mutants, these lineages failed to perform asymmetric signalling, causing the notum to be largely bald. Therefore, Sara contributes to Notch signalling and asymmetric cell fate assignation, as observed in conditions in which other redundant systems for asymmetric Notch signalling are compromised (Loubery, 2017).

Both Neuralized and Sara play general roles in Notch signalling: they are both involved in lateral inhibition at early stages and, at later stages, in asymmetric cell fate assignation. Indeed, both Neuralized and Sara mutants show early defects in lateral inhibition and, accordingly, they show supernumerary SOPs. In addition, Neuralized and Sara mutant conditions also show defective Notch signalling during cell fate assignation in the SOP lineage and therefore cause the transformation of the cells in the lineage into neurons. In this later step, Notch signalling is asymmetric. The possibility that both Sara and Neuralized play key roles in ensuring the asymmetric nature of this signalling event is only correlative: in the case of Neuralized, it is enriched in the anterior cortex of the cell, which will give rise to pIIb; in the case of Sara, (1) both Delta and Notch are cargo of these endosomes, (2) cleaved Notch is seen in the pIIa endosomes and (3) Sara endosomes are dispatched asymmetrically to pIIa10. It is tantalizing to conclude that the asymmetric localization of these two proteins mediate the asymmetric nature of Notch signalling in the SOP lineage, but further assays will be necessary to unambiguously address this issue. Clonal analysis is unfortunately a too slow assay to sort out the specific requirement of these cytosolic factors (Sara and Neuralized) in the pIIa versus the pIIb cell (Loubery, 2017).

Sara mediates the targeting of Notch/Delta containing endosomes to the central spindle and could contributes to Notch-mediated asymmetric signalling in the SOP lineage. What machinery controls in turn the Sara-dependent targeting of endosomes to the central spindle? Previous proteomic studies uncovered bona fide Sara-binding factors, including the Activin pathway R-Smad, Smox17 and the beta subunit of the PP1c serine-threonine phosphatase (PP1β(9C)). In an IP/Mass Spectrometry approach, those interactions were confirmed and in addition to PP1β(9C), two of the other three Drosophila isoforms of PP1c: PP1α(87B) and PP1α(96A) were found. Furthermore, the PP1c regulatory subunit Sds22 was found, suggesting that Sara binds the full serine-threonine PP1 phosphatase complex. The interaction with Sds22 was confirmed by immunoprecipitation of overexpressed Sds22-GFP and western blot detection of endogenous Sara in the immunoprecipitate (Loubery, 2017).

Prompted by these results, whether the PP1 complex plays a role in the asymmetric targeting of the Sara endosomes was explored by manipulating the activity of Sds22, the common regulatory unit in all the complexes containing the different PP1 isoforms. Sds22 was overexpressed specifically during SOP mitosis, by driving Sds22-GFP under the Neur-Gal4 driver with temporal control by the Gal80ts system. In SOPs where PP1-dependent dephosphorylation is enhanced by overexpressing Sds22, the Sara endosomes fail to be dispatched asymmetrically toward the pIIa daughter cell (Loubery, 2017).

The role of PP1-dependent dephosphorylation in the SOP was examined by knocking down Sds22 (through a validated Sds22-RNAi). Loss of function Sds22 did also affect the asymmetric targeting of endosomes. These data uncover a key role for phosphorylation and PP1-dependent dephosphorylation as a switch that contributes to the asymmetric targeting of Sara during asymmetric cell division (Loubery, 2017).

The observations raise the question of which is the step in the asymmetric dispatch of the endosomes that is controlled by the levels of phosphorylation: central spindle targeting, central spindle detachment or targeting to the pIIa cell? PP1/Sds22-dependent dephosphorylation controls a plethora of mitotic events, including mitotic spindle morphogenesis, cortical relaxation in anaphase, epithelial polarity and cell shape, Aurora B activity and kinetochore-microtubule interactions as well as metabolism, protein synthesis, ion pumps and channels. Therefore, to establish the specific event during the asymmetric dispatch of Sara endosomes that is controlled by PP1/Sds22 dephosphorylation, focus was placed on the phosphorylation state of Sara itself and its previously identified phosphorylation sites. This allowed specific interference with this phosphorylation event and thereby untangle it from other cellular events also affected by dephosphorylation (Loubery, 2017).

PP1/Sds22 was shown to bind Sara. It has previously been shown that mammalian Sara itself is phosphorylated at multiple sites and that the level of this Sara phosphorylation is independent on the level of TGF-beta signalling. Three phosphorylation sites have been identified at position S636, at position S709, and at position S774 in Sara protein and these sites were confirmed by Mass Spectrometry of larval tissue expressing GFP-Sara. Phosphorylation of Sara had been previously reported to be implicated in BMP signalling during wing development. However, the role of these three phosphorylation sites during asymmetric division are to date unknown (Loubery, 2017).

ProQ-Diamond phospho-staining of immunoprecipitated GFP-Sara confirmed that Sara is phosphorylated. To test whether PP1/Sds22 controls the phosphorylation state of Sara, ProQ-Diamond stainings of GFP-Sara were performed with and without down-regulation of Sds22. Downregulating Sds22 induced a 40%-increase in the normalized quantity of phosphorylated Sara, showing that PP1/Sds22 does control the phosphorylation state of Sara (Loubery, 2017).

To study the role of Sara phosphorylation during asymmetric targeting of the endosomes, the mitotic behaviour of the endosomes was analyzed in conditions of overexpression of mutant versions of Sara where (1) the three phosphorylated Serines (at position S636, S709, and S774) were substituted by Alanine (phosphorylation defective: GFP-Sara3A) or (2) the PP1 interaction was abolished by an F678A missense mutation in the PP1 binding domain (hyper-phosphorylated: GFP-SaraF678A). Neither mutation affects the general levels of abundance of the Sara protein in SOPs, the targeting of Sara itself to the endosomes, nor the residence time of Sara in endosomes as determined by FRAP experiments. Also, the targeting dynamics of internalized Delta to endosomes are not affected in these mutants (Loubery, 2017).

Upon overexpression of GFP-Sara3A in SOPs, the rate of targeting of the endosomes to the central spindle is greatly increased. In addition, GFP-Sara3A shows impaired departure from the spindle: while the residence time of Sara endosomes at the central spindle after their recruitment is around 100 s in wild type, GFP-Sara3A endosomes stay at the spindle significantly longer (151±21 s). In GFP-Sara3A endosomes, impaired departure leads to defective asymmetric targeting to the pIIa cell while, in wild type, departure from the central spindle occurs well before abscission, in the GFP-Sara3A condition, endosomes that did not depart are caught at the spindle while abscission occurs. These data indicate that the endosomal targeting to the central spindle is greatly favoured when these three sites in Sara are dephosphorylated and suggest that the departure from the microtubules of the central spindle requires that the endosomes are disengaged by phosphorylation of Sara (Loubery, 2017).

Loss of Sara phosphorylation in these sites impairs disengagement from the central spindle. Conversely, impairing Sara binding to the PP1 phosphatase results in defective targeting to the central spindle. Indeed, when binding of Sara to the PP1/Sds22 phosphatase is impaired in the GFP-SaraF678A overexpressing SOP mutants, Sara endosomes fail to be targeted to the spindle. Mistargeted away from the central spindle, the GFP-SaraF678A endosomes fail thereby to be asymmetrically targeted to the pIIa cell. Loss and gain of function phenotypes of the Phosphatase regulator Sds22 during endosomal spindle targeting support the role of Sara phosphorylation during targeting to the central spindle microtubules suggested by the GFP-Sara3A and GFP-SaraF678A experiments (Loubery, 2017).

What are the functional consequences on signalling of impaired phosphorylation/dephosphorylation in Sara mutants? The presence of Sara in endosomes is itself essential for Notch signalling. Sara loss of function mutants show a phenotype in SOP specification (supernumerary SOPs) as well as during fate determination within the SOP lineage (all cells in the lineage acquire a neural fate). In addition, this study showed that Sara is also essential for the targeting of endosomes to the spindle: in the absence of Sara, endosomes fail to move to the spindle in the SOP. They are therefore dispatched symmetrically, but those endosomes do not mediate Notch signalling. As a consequence, both daughters fail to perform Notch signalling in sensitized conditions in which Neuralized is compromised. The result is a Notch loss of function phenotype: the whole lineage differentiates into neurons (Loubery, 2017).

In both Sara3A and SaraF678A mutants, because of reasons that are different in the two cases (either they do not go to the spindle or their departure from the spindle is impaired), functional Sara endosomes are dispatched symmetrically (Fig. 6a,b,e). In contrast to the situation in the Sara loss of function mutant, those endosomes are functional Sara signalling endosomes, which can mediate Notch signalling in both cells. Therefore, these mutations are consistently shown to cause a gain of function Sara signalling phenotype: supernumerary sockets are seen in the lineages (88% of the lineages for Sara3A and 82% of the lineages for SaraF678A). A milder version of this phenotype can be also seen by overexpressing wild-type Sara (34% of the lineages) consistent again with some gain of function Notch signalling phenotype when Sara concentrations are elevated. In summary, this implies that the 3A and F678A mutations impair the phosphorylation state of Sara (with consequences in targeting), but not its function in Notch signalling (Loubery, 2017).

These results indicate that Sara itself plays a key, rate limiting role on the asymmetric targeting of the endosomes by controlling the targeting to the spindle and its departure. Maturation of the early endosomes by accumulating PI(3)P leads to accumulation of the PI(3)P-binding protein Sara to this vesicular compartment. At the endosome, the phosphorylation state of Sara indeed determines central spindle targeting and departure: in its default, dephosphorylated state, Sara is essential to engage the endosomes with the mitotic spindle. Phosphorylation of Sara disengages the endosomes from the central spindle allowing the asymmetric departure into the pIIa cell (Loubery, 2017).

Membrane trafficking is defined as the vesicular transport of proteins into, out of, and throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways result in impaired function and are linked to diseases. However, how these trafficking pathways regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo system, we investigated enterocyte-specific functions for the early endosomal machinery.This study focused on Rab21, which regulates specific steps in early endosomal trafficking. Depletion of Rab21 in enterocytes led to abnormalities in intestinal morphology, with deregulated cellular equilibrium associated with a gain in mitotic cells and increased cell death. Increases in apoptosis and Yorkie signaling were responsible for compensatory proliferation and tissue inflammation. Using an RNA interference screen, this study identified regulators of autophagy and membrane trafficking that phenocopied Rab21 knockdown. It was further shown that Rab21 knockdown-induced hyperplasia was rescued by inhibition of epidermal growth factor receptor signaling. Moreover, quantitative proteomics identified proteins affected by Rab21 depletion. Of these, changes were validated in apolipoprotein ApoLpp and the trehalose transporter Tret1-1, indicating roles for enterocyte Rab21 in lipid and carbohydrate homeostasis, respectively. These data shed light on an important role for early endosomal trafficking, and Rab21, in enterocyte-mediated intestinal epithelium maintenance (Nassari, 2022).

The Adaptor Protein (AP)-3 complex is a molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. A forward screening was performed for genetic modifiers of the eye pigmentation AP-3 (carmine) gene in Drosophila. One modifier was the Atg2 gene, encoding a conserved protein involved in autophagy. Loss of one copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, Biogenesis of lysosome-related organelles complex 1, subunit 1 (Blos1) and lightoid (Rab32), and even increased the eye pigment content of wild-type flies. A second modifier was the ArfGAP1 gene, encoding a conserved GTPase-activating protein. Loss of a single copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants. Strikingly, loss of the second copy of the gene elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 or lightoid. These results provide evidence for functional links connecting the machinery for biogenesis of LROs with autophagy and small GTPase regulation (Rodriguez-Fernandez, 2015).

Dynactin is a multi-subunit complex that functions as a regulator of the Dynein motor. A central component of this complex is Dynamitin/p50 (Dmn). Dmn is required for endosome motility in mammalian cell lines. However, the extent to which Dmn participates in the sorting of cargo via the endosomal system is unknown. This study examined the endocytic role of Dmn using the Drosophila melanogaster oocyte as a model. Yolk proteins are internalized into the oocyte via clathrin-mediated endocytosis, trafficked through the endocytic pathway, and stored in condensed yolk granules. Oocytes that were depleted of Dmn contained fewer yolk granules than controls. In addition, these oocytes accumulated numerous endocytic intermediate structures. Particularly prominent were enlarged endosomes that were relatively devoid of yolk proteins. Ultrastructural and genetic analyses indicate that the endocytic intermediates are produced downstream of Rab5. Similar phenotypes were observed upon depleting Dynein heavy chain (Dhc) or Lis1. Dhc is the motor subunit of the Dynein complex and Lis1 is a regulator of Dynein activity. It is therefore proposed that Dmn performs its function in endocytosis via the Dynein motor. Consistent with a role for Dynein in endocytosis, the motor co-localized with the endocytic machinery at the oocyte cortex in an endocytosis-dependent manner. These results suggest a model whereby endocytic activity recruits Dynein to the oocyte cortex. The motor along with its regulators, Dynactin and Lis1, functions to ensure efficient endocytic uptake and maturation (Liu, 2015).

How vesicle trafficking components actively contribute to regulation of paracrine signaling is unclear. This study has genetically uncovered a requirement for alpha-Soluble NSF Attachment Protein (alpha-Snap) in the activation of the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway during Drosophila egg development. alpha-Snap, a well-conserved vesicle trafficking regulator, mediates association of N-ethylmaleimide-Sensitive Factor (NSF) and SNAREs to promote vesicle fusion. Depletion of alpha-Snap or the SNARE family member Syntaxin1A in epithelia blocks polar cells maintenance and prevents specification of motile border cells. Blocking apoptosis rescues polar cell maintenance in alpha-Snap-depleted egg chambers, indicating that the lack of border cells in mutants is due to impaired signaling. Genetic experiments implicate alpha-Snap and NSF in secretion of a STAT-activating cytokine. Live imaging suggests that changes in intracellular calcium may be linked to this event. These data suggest a cell-type specific requirement for particular vesicle trafficking components in regulated exocytosis during development. Given the central role for STAT signaling in immunity, this work may shed light on regulation of cytokine release in humans (Saadin, 2018).

F-BAR proteins are prime candidates to regulate membrane curvature and dynamics during different developmental processes. This study analyzed nostrin (nost), a novel Drosophila F-BAR protein related to Cip4. Genetic analyses revealed a strong synergism between nost and cip4 functions. While single mutant flies are viable and fertile, combined loss of nost and cip4 results in reduced viability and fertility. Double mutant escaper flies show enhanced wing polarization defects and females exhibit strong egg chamber encapsulation defects. Live-imaging analysis suggests that the observed phenotypes are caused by an impaired E-cadherin membrane turnover. Simultaneous knock-down of Cip4 and Nostrin strongly increases the formation of tubular E-cadherin vesicles at adherens junctions. Cip4 and Nostrin localize at distinct membrane subdomains. Both proteins prefer similar membrane curvatures but seem to form different membrane coats and do not heterooligomerize. These data suggest an important synergistic function of both F-BAR proteins in membrane dynamics. A cooperative recruitment model is proposed in which first Cip4 promotes membrane invagination and early actin-based endosomal motility while Nostrin makes contact with microtubules through the kinesin Khc-73 for trafficking of recycling endosomes (Zobel, 2015).

Members of the Fes-CIP4 homology Bin-amphiphysin-Rvs161/167 (F-BAR) protein family form crescent-shaped dimers that are able to shape membranes into vesicles and tubules. F-BAR proteins have been grouped into six subfamilies, the Cdc42-interacting protein 4 (Cip4) subfamily, the Fes subfamily of non-receptor tyrosine kinases, the protein kinase C and casein kinase substrate in neurons protein (pacsin) subfamily, the Slit-Robo RhoGTPase-activating proteins (SrGAPs), the FCH-domain-only (FCHO) and the proline-serine-threonine phosphatase-interacting protein (PSTPIP) subfamilies. The phylogenetic subgrouping is mainly based on structural similarities of the N-terminal F-BAR module, and on the composition and architecture of C-terminal domains. Distinct differences of the intrinsic F-BAR domain curvature observed among the different F-BAR-domain proteins are thought to reflect characteristic preferences in sensing and/or inducing membrane invaginations of different curved geometry. Consistent with this idea, members of FCHO subfamily bind to very low membrane curvatures and are found to be essential for the initial step of membrane invagination in endocytosis. Other F-BAR proteins, such as members of the Cip4 subfamily, which includes the Cdc42-interacting protein 4 (Cip4), the transducer of Cdc42-dependent actin assembly (Toca-1) and the formin-binding protein 17 (FBP17), have a preference for higher membrane curvatures present in later steps during vesicle formation (Zobel, 2015 and references therein).

Unlike those of the FCHO subfamily, Cip4 subfamily proteins contain a C-terminal SH3 domain that binds dynamin and factors that promote actin filament formation. All three members of the Cip4 subfamily are able to activate N-WASP by promoting Arp2/3-mediated actin nucleation in vitro. Cip4 and Toca-1 also associate with Cdc42 through a central coiled-coil region. The current view is that Cip4-related proteins may stabilize plasma membrane invaginations and, subsequently, recruit dynamin and WASP proteins to the neck of endocytic pits that mediate the constriction and scission of vesicles. Recruitment of WASP proteins to newly formed vesicles also promotes the formation of actin comet tails that provide the driving force for endocytic vesicle movement. However, understanding of how F-BAR proteins function in vivo in a physiological context is still incomplete because loss-of-function studies in higher organisms are limited. Mice that lack Cip4 are viable and show only a weak endocytosis defect of the insulin-responsive glucose transporter Glut4. Mutant animals also display a reduced platelet production and defective integrin-dependent T-cell adhesion. Both defects are probably caused by decreased WASP-dependent actin polymerization, rather than impaired endocytosis (Chen, 2013). Given the mild phenotypes, the two other Cip4-like subfamily members Toca-1 and FBP17 might have redundant functions and could compensate for the loss of Cip4 function (Zobel, 2015).

Initial RNA interference (RNAi) studies in Drosophila melanogaster revealed that Cip4 regulates dynamin-dependent endocytosis of E-cadherin at adherens junctions. As in mammals, function of cip4 is not essential for fly development. cip4 mutants show duplicated wing hairs because of an impaired endocytosis. Further analyses revealed that Cip4 acts downstream of Cdc42 to activate the WASP-WAVE-Arp2/3 pathway in the notum and the wing epithelium. In addition, a postsynaptic, endocytosis-independent function of Cip4 has been identified at the neuromuscular junction. This function also depends on an interaction with the Cdc42-WASP-Arp2/3 pathway but does not require a functional F-BAR domain (Zobel, 2015 and references therein).

The Drosophila genome contains an additional, not yet characterized gene encoding a Cip4-like F-BAR protein with highest similarities to human Nostrin (CG42388). Human Nostrin was originally identified as an interaction partner of the endothelial nitric oxide synthase (eNos). Cell culture studies further suggest that Nostrin regulates N-WASP and/or dynamin-dependent trafficking and the activity of endothelial nitric oxide synthase (eNos). However, an in vivo role of Nostrin in the regulation of eNos activity or endocytosis has not yet been found. A recent loss-of-function study in zebrafish and mice revealed a role of Nostrin in endothelial cell morphology during vascular development. Antisense morpholino oligonucleotide (MO)-mediated knockdown of Nostrin in developing zebrafish affects the migration of endothelial tip cells of intersegmental blood vessels. Remarkably, nostrin-knockout mice are viable and show only mild retinal angiogenesis defects. This suggests that other F-BAR proteins compensate for nostrin function in mutant mice (Zobel, 2015 and references therein).

As Drosophila contains only a single gene copy of each F-BAR subfamily, studies in flies are well-suited to address putative functional redundancies within and between F-BAR domain subfamilies. This study presents a functional analysis of CG42388, which encodes the Drosophila Nostrin protein, and its physiological relationship to Cip4. Flies that lack Nostrin are viable and fertile. However, loss of both nostrin and cip4 results in reduced viability and fertility. Double mutant flies show a strong multiple wing hair phenotype and females are semi-sterile. Egg chambers of double mutant flies show strong encapsulation defects that are likely to be caused by an impaired membrane turnover of E-cadherin. Cip4 and Nostrin, preferentially, bind similar membrane curvatures but localize at distinct subdomains of membrane structures in cells. These data suggest an important, non-redundant function of Cip4 and Nostrin in the regulation of membrane dynamics in epithelial morphogenesis (Zobel, 2015).

F-BAR proteins play an important role in the regulation of membrane curvatures in a sequential manner during endocytosis. Despite their pivotal functions in linking actin and membrane dynamics, recent single-knockout studies revealed that many F-BAR proteins are not essential for development and, thus, might have redundant or cooperative functions in vivo. In fission yeast, the Cip4-like F-BAR proteins Cdc15 and Imp2 are examples for such synergistic function of two F-BAR proteins. Cells deficient for either Cdc15 or Imp2 show mild defects in cytokinesis but are still able to divide. However, deletion of both C-terminal SH3 domains of the proteins completely restricts the division of the cells (Zobel, 2015 and references therein).

This study provides first evidence for cooperative function of the two F-BAR proteins Cip4 and Nostrin in the multicellular context of Drosophila development. Like Cip4, members of the Nostrin subfamily show a remarkably high evolutionary conservation and single orthologs can be found from porifera to humans. All the more surprising is the fact that nostrin loss-of-function mutant flies have no obvious phenotype. Only after removal of both cip4 and nostrin, was a strong enhancement found of the phenotypic traits already observed in cip4 single mutants. Double mutants show a substantial reduction in the number of offspring. Both mutant females and males display reduced fertility, indicating a common function of both F-BAR proteins in early morphogenesis. Female sterility of double mutant flies is caused by strong defects in egg chamber morphogenesis. The formation of compound egg chambers results from a defective encapsulation in the germarium, a phenotype that has not yet been described for many mutants. Most mutations that have been reported of so far, either affect gene functions directly through the regulation of cell division or control of follicle cell differentiation through Notch/Delta signaling. However, multicyst egg chambers in nost;;cip4 double mutants display neither defects in cell division nor in the differentiation of follicle cells (Zobel, 2015).

Interestingly, loss of Maelstrom (Mael), a high-mobility group box protein that regulates microtubule organization leads to egg chambers with cell division defects but also results in an encapsulation defect with misplaced oocytes that is similar to the one observed in nost;;cip4 double mutants. Mael forms a complex with the components of the microtubule-organizing center (MTOC) -- including centrosomin and γ-tubulin, which seems to be required not only for early oocyte determination but also egg chamber packing and oocyte positioning in the germarium. Interestingly, in mael mutant multicyst egg chambers E-cadherin is not enriched on the oocyte cortex and not apically concentrated in the follicle epithelium as in wild type. nost;;cip4 double mutant egg chambers show a similar E-cadherin mislocalization, suggesting that the microtuble cytoskeleton plays an important role in E-cadherin localization. Consistently, Nostrin mainly localizes to Rab11-positive vesicles that move along microtubules. Thus, Nostrin might act on Rab11-dependent E-cadherin trafficking along microtubules. A strong requirement for Rab11 in E-cadherin trafficking in germline stem cells (GSC) and in the maintenance of GSC identity has recently been identified. Mosaic egg chambers are severely disorganized, comprising mispositioned oocytes. Most importantly, compound egg chambers can be found that contain two or more germline cysts surrounded by a single continuous follicle epithelium, as was observed in this study for nost;;cip4 mutants. However, rab11-null follicle stem cells (FSC) give rise to the normal number of cells that enter polar, stalk and epithelial cell differentiation pathways. Like Rab11, Nostrin and Cip4 functions do not seem to be required in follicle cell differentiation (Zobel, 2015).

Given the dramatic switch in Nostrin expression in the germline cysts between region 2a and region 2b, when protein levels drop from highest to very low or no expression, an important function is proposed of Nostrin in germ cells rather than in somatic follicle cells. The germline cyst undergoes a dramatic change in shape within this region, as is reflected by a transformation from a spherical to a lens-shaped structure. This morphological transition might imply important changes in the adhesiveness mediated by homophilic E-cadherin cell-cell contacts between germ cells within the cyst. Thus, it is proposed that Nostrin and Cip4 are involved in the regulation of this transition by controlling E-cadherin endocytosis and vesicle recycling in germline cells. A failure of nost;;cip4 mutant cysts in adopting a lens-shaped morphology might interfere with their encapsulation by follicle cells, which results in the formation of compound egg chambers (Zobel, 2015).

Given the substantial mislocalization of E-cadherin that becomes obvious in double mutant egg chambers, an additional role of both F-BAR proteins in the maintenance of E-cadherin cell-cell contacts between germline and somatic follicle cells is suggested (Zobel, 2015).

Cip4 and Nostrin partially mark the same membrane structures but they localize to distinct subregions in vivo. Consistently, in vitro liposome studies showed that both proteins prefer defined membrane curvatures of similar diameter, i.e. both might associate with similarly shaped membrane compartments. However, the different appearance of Cip4- and Nostrin-decorated vesiculo-tubular structures might also reflect differences in lattice formation of these two F-BAR domain proteins. It is hypothesized that these regular arrays of electron-dense structures at liposomes represent regular Cip4 lattices formed upon self-association by head-to-tail and lateral interactions as previously supposed by real-space reconstruction. Because such patterns were not observed at tubular structures following incubation in the presence of Nostrin, it is suggested that Nostrin does not polymerize into rigid helical coats that are thought to be the structural basis for membrane invagination. Consistently, unlike Cip4, overexpression of Nostrin in S2R+ cells did not induce membrane tubulation. How do Cip4 and Nostrin cooperate in membrane remodeling and vesicle trafficking? In cells, Cip4 mainly localizes to Rab5-positive early endosomes, whereas Nostrin marks both Rab5- and Rab11-positive vesicles. Thus, a cooperative recruitment model is proposed, in which first Cip4 promotes membrane invagination, vesicle scission and motility of Rab5-positive membrane compartments by recruiting dynamin and the WASP-WAVE-Arp2/3 pathway. Nostrin will then be recruited to Cip4-positive membrane structures because Nostrin prefers the curvature induced by the highly organized Cip4 coats. Yet, at these membrane compartments, both proteins still occur in a spatially segregated manner, as visualized in EM analyses of Cip4- and Nostrin-coated membrane tubules. This segregation might reflect that Nostrin is not interacting with Cip4, and that Cip4 has the ability to bind PE - which Nostrin does not have. Furthermore Nostrin protein arrays seem less organized, as reflected by the broader range of curvatures induced in vitro and by the lack of regular structures of Nostrin-coated membranes. Strong formation of rigid lattices might explain why Cip4 tubulates membranes effectively in vitro and in vivo, and why anti-Cip4 labeling usually outlines tubular structures. In contrast, Nostrin is confined to Cip4-free segments of these structures and predominantly appears at the end of such Cip4-coated tubules because the end does not accommodate a Cip4 coat optimized for cylindrical surfaces (Zobel, 2015).

Interestingly, Kif13A (a kinesin motor that directly binds Rab11) is most enriched at the tips of membrane tubules. Moreover, Kif13A localizes to distinct Rab11-positive subdomains within sorting endosomes and is thought to initiate the formation of recycling endosomal tubules along microtubules through its motor activity. Interestingly, Nostrin directly interacts with the Drosophila Kif13A homologue Khc73 and colocalizes with Khc73-marked endosomes that move along microtubules. A close link between Nostrins and kinesin motors seem to be evolutionarily conserved and the interaction is likely mediated by a conserved bipartite tryptophan-based kinesin-1 binding motif. Thus, in the current model of cooperative recruitment, Cip4 stabilizes endosomal tubules, whereas Nostrin defines subdomains of recycling intermediates of endosomal tubules and makes contact with microtubules through the kinesin Khc-73 for long-range trafficking of recycling endosomes (Zobel, 2015).

A similar scenario might also take place during cell polarization of the wing epithelium. Here, Cip4 and Nostrin act together to control the polarized outgrowth of a single actin-rich protrusion called prehair, a process that also requires tight coupling of membrane trafficking and the cytoskeleton. The restriction of wing hair formation at the most distal apical vertex of each wing cell depends on the Frizzled-PCP signaling pathway. A key step in the cell polarization is the asymmetric localization of core PCP proteins at adjacent cell membranes within the plane of the epithelium. Thus, one of the central questions in understanding PCP signaling is how this asymmetric localization is achieved. Based on live-imaging studies, selective endocytosis and directional transport of Frizzled along polarized non-centrosomal microtubules have been proposed as possible mechanisms for asymmetric polarization. Previous studies that had used microtubule antagonists already revealed an important role of the microtubule cytoskeleton in order to localize prehair initiation to the cell. Disruption of the microtubule cytoskeleton resulted in the development of multiple prehairs along the apical cell periphery. Multiple pre-hair formation is also caused by overexpression of Frizzled, presumably through an ectopic activation of the pre-hair nucleation machinery. However, the multiple wing hair phenotype in nost;;cip4 double mutant wings does not seem to affect the asymmetric distribution of Frizzled. Interestingly, a similar Frizzled-independent multiple wing hair phenotype has recently been observed in mutants that affect casein kinase 1γ (CK1γ, also known as CSNK1G). Loss of CK1γ disrupts the apical localization of Rab11 at the base of prehairs, suggesting that Ck1γ regulates Rab11-mediated polarized vesicle trafficking that is required for prehair nucleation. Consistently, expression of either a dominant-negative or a dominant-active Rab11 variant strongly induces the formation of multiple wing hairs. Overexpression of Cip4 or Nostrin alone also phenocopies the multiple wing hair defect of nost;;cip4 double mutants. Like CK1γ and Rab11, Cip4 and Nostrin accumulate at the base of forming prehairs. Because Nostrin colocalizes with Rab11-positive vesicles that move along microtubules, it is proposed that Nostrin is involved in Rab11-mediated polarized vesicle trafficking in the developing wing (Zobel, 2015).

Polarized Rab11-dependent vesicle trafficking of E-cadherin is also needed for the hexagonal packing of wing cells. During this process, irregularly shaped cells adopt a hexagonal geometry by coordinated endocytosis and Rab11-dependent recycling of junctional E-cadherin. Hexagonal packing starts shortly after pupal molt and ends just before wing hair formation but, remarkably, also depends on components of the PCP pathway. The underlying molecular mechanism that links hexagonal packing and hair formation in the wing is unknown. However, both processes depend on vesicle trafficking because suppression of Rab11, Rab23 or the simultaneous knockdown of cip4 and nostrin results not only in multiple wing hairs but also affects the regular hexagonal array of wing epithelial cells. A similar E-cadherin-dependent process of cell packing and remodeling can also be observed in the dorsal thorax, an epithelium that originally derived from the fused proximal parts of two wing imaginal discs. A role in E-cadherin membrane turnover has already been reported for Cip4 in the developing thorax epithelium of Drosophila. In cells that express cip4 dsRNA, E-cadherin-GFP accumulates in apical punctate structures and elongated malformed tubules form at the cell cortex. These long and defective endocytic structures do not tolerate fixation and could only be observed in live-imaging experiments. This study observed an even stronger defect on E-cadherin membrane dynamics upon simultaneous downregulation of Cip4 and Nostrin. The number of elongated malformed tubules that form at the cell cortex is clearly increased. Moreover, knockdown of both cip4 and nostrin cause obvious defects in the formation of E-cadherin junctions, a phenotype that was never observed when suppressing either cip4 or nostrin. These strong junctional defects might be responsible for the lethality of late pupae following RNAi transgene expression by the aptereous-Gal4 driver. Thus, it is concluded that both F-BAR proteins play an important cooperative rather than a redundant function in E-cadherin trafficking and junction maintenance (Zobel, 2015).

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. This study shows that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. Vps8 forms a complex and acts together with Vps16A, Deep Orange/Vps18 and Carnation/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Light/Vps41. This study thus identifies a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila (Lorincz, 2016).

Cell death is a fundamental aspect of development, homeostasis, and disease; yet, understanding of non-apoptotic forms of cell death is limited. One such form is phagoptosis, in which one cell utilizes phagocytosis machinery to kill another cell that would otherwise continue living. A non-autonomous requirement of phagocytosis machinery has been identified for the developmental programmed cell death of germline nurse cells in the Drosophila ovary; however, the precise mechanism of death remained elusive. This study shows that lysosomal machinery acting in epithelial follicle cells is used to non-autonomously induce the death of nearby germline cells. Stretch follicle cells recruit V-ATPases and chloride channels to their plasma membrane to extracellularly acidify the germline and release cathepsins that destroy the nurse cells. These results reveal a role for lysosomal machinery acting at the plasma membrane to cause the death of neighboring cells, providing insight into mechanisms driving non-autonomous cell death (Mondragon, 2019).

Retromer, including Vps35, Vps26, and Vps29, is a protein complex responsible for recycling proteins within the endolysosomal pathway. Although implicated in both Parkinson's and Alzheimer's disease, understanding of retromer function in the adult brain remains limited, in part because Vps35 and Vps26 are essential for development. In Drosophila, this study finds that Vps29 is dispensable for embryogenesis but required for retromer function in aging adults, including for synaptic transmission, survival, and locomotion. Unexpectedly, in Vps29 mutants, Vps35 and Vps26 proteins are normally expressed and associated, but retromer is mislocalized from neuropil to soma with the Rab7 GTPase. Further, Vps29 phenotypes are suppressed by reducing Rab7 or overexpressing the GTPase activating protein, TBC1D5. With aging, retromer insufficiency triggers progressive endolysosomal dysfunction, with ultrastructural evidence of impaired substrate clearance and lysosomal stress. These results reveal the role of Vps29 in retromer localization and function, highlighting requirements for brain homeostasis in aging (Ye, 2020).

Active transport of organelles within axons is critical for neuronal health. Retrograde axonal transport, in particular, relays neurotrophic signals received by axon terminals to the nucleus and circulates new material among en passant synapses. A single motor protein complex, cytoplasmic dynein, is responsible for nearly all retrograde transport within axons: its linkage to and transport of diverse cargos is achieved by cargo-specific regulators. This study has identified Vezatin as a conserved regulator of retrograde axonal transport. Vertebrate Vezatin (Vezt) is required for the maturation and maintenance of cell-cell junctions and has not previously been implicated in axonal transport. However, a related fungal protein, VezA, has been shown to regulate retrograde transport of endosomes in hyphae. In a forward genetic screen,a loss-of-function mutation was identified in the Drosophila vezatin-like (vezl) gene. vezl loss prevents a subset of endosomes, including signaling endosomes containing activated BMP receptors, from initiating transport out of motor neuron terminal boutons. vezl loss also decreases the transport of endosomes and dense core vesicles (DCVs) but not mitochondria within axon shafts. vezl was disrupted in zebrafish; vezl loss specifically impairs the retrograde axonal movement of late endosomes, causing their accumulation in axon terminals. This work establishes a conserved, cargo-specific role for Vezatin proteins in retrograde axonal transport (Spinner, 2020).

Defective rhodopsin homeostasis is one of the major causes of retinal degeneration, including the disease Retinitis pigmentosa. To identify cellular factors required for the biosynthesis of rhodopsin, a genome-wide genetic screen was performed in Drosophila for mutants with reduced levels of rhodopsin. Loss-of-function alleles were investigated in endoplasmic reticulum membrane protein complex 3 (emc3), emc5, and emc6, each of which exhibited defective phototransduction and photoreceptor cell degeneration. EMC3, EMC5, and EMC6 were essential for rhodopsin synthesis independent of the ER associated degradation (ERAD) pathway, which eliminates misfolded proteins. Null mutations were generated for all EMC subunits; it was further demonstrated that different EMC subunits play roles in different cellular functions. Conditional knockout of the Emc3 gene in mice led to mislocalization of rhodopsin protein and death of cone and rod photoreceptor cells. These data indicate conserved roles for EMC subunits in maintaining rhodopsin homeostasis and photoreceptor function, and suggest that retinal degeneration may also be caused by defects in early biosynthesis of rhodopsin (Xiong, 2019).

Neuroendocrine cells communicate via neuropeptides to regulate behaviour and physiology. This study examines how STIM (Stromal Interacting Molecule), an ER-Ca2+ sensor required for Store-operated Ca2+ entry, regulates neuropeptides required for Drosophila development under nutrient restriction (NR). Two STIM-regulated peptides, Corazonin and short Neuropeptide F, were found to be required for NR larvae to complete development. Further, a set of secretory DLP (Dorso lateral peptidergic) neurons which co-express both peptides was identified. Partial loss of dSTIM caused peptide accumulation in the DLPs, and reduced systemic Corazonin signalling. Upon NR, larval development correlated with increased peptide levels in the DLPs, which failed to occur when dSTIM was reduced. Comparison of systemic and cellular phenotypes associated with reduced dSTIM, with other cellular perturbations, along with genetic rescue experiments, suggested that dSTIM primarily compromises neuroendocrine function by interfering with neuropeptide release. Under chronic stimulation, dSTIM also appears to regulate neuropeptide synthesis (Megha, 2019).

Metazoan cells commonly use ionic Ca2+ as a second messenger in signal transduction pathways. To do so, levels of cytosolic Ca2+ are dynamically managed. In the resting state, cytosolic Ca2+ concentration is kept low and maintained thus by the active sequestration of Ca2+ into various organelles, the largest of which is the ER. Upon activation, ligand-activated Ca2+ channels on the ER, such as the ryanodine receptor or inositol 1,4,5-trisphosphate receptor (IP3R), release ER-store Ca2+ into the cytosol. Loss of ER-Ca2+ causes STromal Interacting Molecule (STIM), an ER-resident transmembrane protein, to dimerize and undergo structural rearrangements. This facilitates the binding of STIM to Orai, a Ca2+ channel on the plasma membrane, whose pore then opens to allow Ca2+ from the extracellular milieu to flow into the cytosol. This type of capacitative Ca2+ entry is called Store-operated Ca2+ entry (SOCE). Of note, key components of SOCE include the IP3R, STIM and Orai, that are ubiquitously expressed in the animal kingdom, underscoring the importance of SOCE to cellular functioning. Depending on cell type and context, SOCE can regulate an array of cellular processes (Megha, 2019).

Neuronal function in particular is fundamentally reliant on the elevation of cytosolic Ca2+. By tuning the frequency and amplitude of cytosolic Ca2+ signals that are generated, distinct stimuli can make the same neuron produce outcomes of different strengths. The source of the Ca2+ influx itself contributes to such modulation as it can either be from internal ER-stores or from the external milieu, through various activity-dependent voltage gated Ca2+ channels (VGCCs) and receptor-activated Ca2+ channels or a combination of the two. Although the contributions of internal ER-Ca2+ stores to neuronal Ca2+ dynamics are well recognized, the study of how STIM and subsequently, SOCE-mediated by it, influences neuronal functioning, is as yet a nascent field (Megha, 2019).

Mammals have two isoforms of STIM, STIM1 and STIM2, both which are widely expressed in the brain. As mammalian neurons also express multiple isoforms of Orai and IP3R, it follows that STIM-mediated SOCE might occur in them. Support for this comes from studies in mice, where STIM1-mediated SOCE has been reported for cerebellar granule neurons and isolated Purkinje neurons, while STIM2-mediated SOCE has been shown in cortical and hippocampal neurons. STIM can also have SOCE-independent roles in excitable cells, that are in contrast to its role via SOCE. In rat cortical neurons and vascular smooth muscle cells, Ca2+ release from ER-stores prompts the translocation of STIM1 to ER-plasma membrane junctions, and binding to the L-type VGCC, CaV1.2. Here STIM1 inhibits CaV1.2 directly and causes it to be internalized, reducing the long-term excitability of these cells. In cardiomyocyte-derived HL1 cells, STIM1 binds to a T-type VGCC, CaV1.3, to manage Ca2+ oscillations during contractions. These studies indicate that STIM regulates cytosolic Ca2+ dynamics in excitable cells, including neurons and that an array of other proteins determines if STIM regulation results in activation or inhibition of neurons. Despite knowledge of the expression of STIM1 and STIM2 in the hypothalamus, the major neuroendocrine centre in vertebrates, studies on STIM in neuroendocrine cells are scarce. This study therefore used Drosophila melanogaster to address this gap (Megha, 2019).

Neuroendocrine cells possess elaborate machinery for the production, processing and secretion of neuropeptides (NPs), which perhaps form the largest group of evolutionarily conserved signalling agents. Inside the brain, NPs typically modulate neuronal activity and consequently, circuits; when released systemically, they act as hormones. Drosophila is typical in having a vast repertoire of NPs that together play a role in almost every aspect of its behaviour and physiology. Consequently, NP synthesis and release are highly regulated processes. As elevation in cytosolic Ca2+ is required for NP release, a contribution for STIM-mediated SOCE to NE function was hypothesized (Megha, 2019).

Drosophila possess a single gene for STIM, IP3R and Orai, and all three interact to regulate SOCE in Drosophila neurons. In dopaminergic neurons, dSTIM is important for flight circuit maturation, with dSTIM-mediated SOCE regulating expression of a number of genes, including Ral, which controls neuronal vesicle exocytosis. In glutamatergic neurons, dSTIM is required for development under nutritional stress and its' loss results in down-regulation of several ion channel genes which ultimately control neuronal excitability. Further, dSTIM over-expression in insulin-producing NE neurons could restore Ca2+ homeostasis in a non-autonomous manner in other neurons of an IP3R mutant, indicating an important role for dSTIM in NE cell output, as well as compensatory interplay between IP3R and dSTIM. At a cellular level, partial loss of dSTIM impairs SOCE in Drosophila neurons as well as mammalian neural precursor cells. Additionally, reducing dSTIM in Drosophila dopaminergic neurons attenuates KCl-evoked depolarisation and as well as vesicle release. Because loss of dSTIM specifically in dimm+ NE cells results in a pupariation defect on nutrient restricted (NR) media, this study used the NR paradigm as a physiologically relevant context in which to investigate STIM's role in NE cells from the cellular as well as systemic perspective (Megha, 2019).

This study employed an in vivo approach coupled to a functional outcome, in order to broaden understanding of how STIM regulates neuropeptides. A role for dSTIM-mediated SOCE in Drosophila neuroendocrine cells for survival on NR was previously established. The previous study offered the opportunity to identify SOCE-regulated peptides, produced in these neuroendocrine cells, that could be investigated in a physiologically relevant context (Megha, 2019).

In Drosophila, both Crz and sNPF have previously been attributed roles in many different behaviours. Crz has roles in adult metabolism and stress responses, sperm transfer and copulation, and regulation of ethanol sedation. While, sNPF has been implicated in various processes including insulin regulation circadian behaviour, sleeping and feeding. Thus, the identification of Crz and sNPF in coping with nutritional stress is perhaps not surprising, but a role for them in coordinating the larval to pupal transition under NR is novel (Megha, 2019).

A role for Crz in conveying nutritional status information is supported by this study. In larvae, Crz+ DLPs are known to play a role in sugar sensing and in adults, they express the fructose receptor Gr43a. Additionally, they express receptors for neuropeptides DH31, DH44 and AstA, which are made in the gut as well as larval CNS. Together, these observations and are strongly indicative of a role for Crz+ DLPs in directly or indirectly sensing nutrients, with a functional role in larval survival and development in nutrient restricted conditions (Megha, 2019).

Several neuropeptides and their associated signalling systems are evolutionarily conserved. The similarities between Crz and GnRH (gonadotrophin-releasing hormone), and sNPF and PrRP (Prolactin-releasing peptide), at the structural, developmental and receptor level therefore, is intriguing. Structural similarity of course does not imply functional conservation, but notably, like sNPF, PrRP has roles in stress response and appetite regulation. This leads to the conjecture that GnRH and PrRP might play a role in mammalian development during nutrient restriction (Megha, 2019).

dSTIM regulates Crz and sNPF at the levels of peptide release and likely, peptide synthesis upon NR. It is speculated that neuroendocrine cells can use these functions of STIM, to fine tune the amount and timing of peptide release, especially under chronic stimulation (such as 24hrs NR), which requires peptide release over a longer timeframe. Temporal regulation of peptide release by dSTIM may also be important in neuroendocrine cells that co-express peptides with multifunctional roles, as is the case for Crz and sNPF. It is conceivable that such different functional outcomes may require distinct bouts of NP release, varying from fast quantile release to slow secretion. As elevation in cytosolic Ca2+ drives NP vesicle release, neurons utilise various combinations of Ca2+ influx mechanisms to tune NP release. For example, in Drosophila neuromuscular junction, octopamine elicits NP release by a combination of cAMP signalling and ER-store Ca2+, and the release is independent of activity-dependent Ca2+ influx. In the mammalian dorsal root ganglion, VGCC activation causes a fast and complete release of NP vesicles, while activation of TRPV1 causes a pulsed and prolonged release. dSTIM-mediated SOCE adds to the repertoire of mechanisms that can regulate cytosolic Ca2+ levels and therefore, vesicle release. This has already been shown for Drosophila dopaminergic neurons and this study extends the scope of release to peptides. Notably, dSTIM regulates exocytosis via Ral in neuroendocrine cells, like in dopaminergic neurons (Megha, 2019).

In Drosophila larval Crz+ DLPs, dSTIM appears to have a role in both fed, as well as NR conditions. On normal food, not only do Crz+ DLPs exhibit small but significant levels of neuronal activity but also, loss of dSTIM in these neurons reduced Crz signalling. Thus, dSTIM regulates Ca2+ dynamics and therefore, neuroendocrine activity, under basal as well as stimulated conditions. This is consistent with observations that basal SOCE contributes to spinogenesis, ER-Ca2+ dynamics as well as transcription. This regulation appears to have functional significance only in NR conditions as pupariation of larvae, with reduced levels of dSTIM in Crz+ neurons, is not affected on normal food. In a broader context, STIM is a critical regulator of cellular Ca2+ homeostasis as well as SOCE, and a role for it in the hypothalamus has been poorly explored. Because STIM is highly conserved across the metazoan phyla, this study predicts a role for STIM and STIM-mediated SOCE in peptidergic neurons of the hypothalamus. There is growing evidence that SOCE is dysregulated in neurodegenerative diseases. In neurons derived from mouse models of familial Alzheimer's disease and early onset Parkinson's, reduced SOCE has been reported. How genetic mutations responsible for these diseases manifest in neuroendocrine cells is unclear. If they were to also reduce SOCE in peptidergic neurons, it's possible that physiological and behavioural symptoms associated with these diseases, may in part stem from compromised SOCE-mediated NP synthesis and release (Megha, 2019).

In dividing animal cells the endoplasmic reticulum (ER) concentrates around the poles of the spindle apparatus by associating with astral microtubules (MTs), and this association is essential for proper ER partitioning to progeny cells. The mechanisms that associate the ER with astral MTs are unknown. Because astral MT minus-ends are anchored by centrosomes at spindle poles, it is hypothesized that the MT minus-end motor dynein mediates ER concentration around spindle poles. Live in vivo imaging of Drosophila spermatocytes revealed that dynein is required for ER concentration around centrosomes during late interphase. In marked contrast, however, dynein suppression had no effect on ER association with astral MTs and concentration around spindle poles in early M-phase. In fact, there was a sudden onset of ER association with astral MTs in dynein RNAi cells, revealing activation of an M-phase specific mechanism of ER-MT association. ER redistribution to spindle poles also did not require non-claret disjunctional (ncd), the other known Drosophila MT minus-end motor, nor Klp61F, a MT plus-end motor that generates spindle poleward forces. Collectively, these results suggest that a novel, M-phase specific mechanism of ER-MT association that is independent of MT minus-end motors is required for proper ER partitioning in dividing cells (Karabasheva, 2019).

The endoplasmic reticulum (ER) cannot be formed by cells de novo and must be inherited during the process of cell division. While the essential roles of the ER in the biogenesis of proteins, lipids and steroid hormones, as well as calcium signaling, are well recognized, little is known about the molecular mechanisms that ensure proper partitioning of the ER to progeny cells. This knowledge is fundamental to understanding the role of the ER in cell division and stem cell biology, with important implications for proper development, tissue repair, and cancer. Specifically, recent findings indicate that asymmetric partitioning of the ER, and misfolded proteins that accumulate there, has a critical role in maintaining pluripotency in stem cells as they undergo rapid cycles of cell division. Related evidence that ER functions contribute to the proliferative capacity and drug resistance of cancer cells is under active investigation, with an objective of revealing novel therapeutic strategies for treating malignancies. The overall goal of the present study was to establish new understanding of the cellular mechanisms that guide the localization of the ER during cell division, bringing closer a full comprehension of the essential roles of the ER in normal physiology and disease (Karabasheva, 2019).

Recent data suggest that proper partitioning of the ER during cell division, or M-phase, depends on specific association of the organelle with astral microtubules (MTs) of the mitotic spindle in both symmetrically and asymmetrically dividing cells4. However, the specific factors that link the ER to astral MTs remain unknown. Identification of these factors is therefore an important next step in understanding mitotic ER partitioning. Most of the knowledge of ER-MT associations comes from non-dividing interphase cells, in which the ER is distributed throughout the cytoplasm with a distinct clustering or focus around centrosomes, the major MT organizing centers of cells. This distribution depends on MT motor-dependent movements of the ER toward both MT plus-ends and minus-ends, as well as stable attachments of the ER along MT filaments mediated by ER membrane proteins including REEPs, spastin, and CLIMP-6. Transport of the ER toward MT minus-ends is mediated by dynein motors, which are also responsible for focusing the ER around centrosomes where MT minus-ends are anchored. Conversely, MT plus-end transport is likely mediated by kinesins9, and also depends on association with growing MT tips mediated, at least in part, by ER membrane embedded STIM1 and STIM2 proteins10. Collectively these associations point to a carefully orchestrated interplay between MT plus-end and minus-end directed transport mechanisms that determine the cellular distribution of the ER (Karabasheva, 2019).

During the course of M-phase, there is a dramatic reorganization of the MT cytoskeleton, whereby the spindle apparatus forms with MT minus-ends anchored at the spindle poles by centrosomes. As this occurs, the majority of the ER becomes focused around the two centrosomes at the spindle poles and along astral MTs, and virtually none is found in the kinetochore region of the spindle where MT plus-ends reside. Thus, there appears to be a shift from the balanced MT plus- and minus-end directed ER distribution during interphase to predominantly minus-end directed localization around spindle poles during M-phase. Consistent with this conclusion, tracking of the ER with growing MT plus-ends is inhibited during cell division due to mitosis-specific phosphorylation of STIM1. This suppression of MT plus-end directed ER transport, and the totality of ER distribution toward MT minus-ends around spindle poles, suggests a predominant role for the MT-minus end motor dynein in M-phase specific ER distribution. Notably, dynein is highly localized to astral MTs and spindle poles in dividing cells and is required for the spindle pole localization of endosomes. However, despite this compelling case, a definitive role for dynein in M-phase ER distribution has never been directly established. Determining dynein's role in the dramatic redistribution of the ER to spindle poles in M-phase is therefore important to understanding the mechanisms that ensure proper ER partitioning to progeny cells (Karabasheva, 2019).

The primary goal of this study was to test the hypothesis that dynein is required for astral MT association and spindle pole focusing of the ER in dividing cells. This was accomplished by live timelapse imaging of Drosophila spermatocytes undergoing the first meiotic division of spermatogenesis in vivo. This experimental system is particularly well suited for this investigation, as Drosophila spermatocytes allow for the analysis of dividing cells in a physiologic environment and have contributed greatly to understanding of fundamental cell division mechanisms including spindle formation and cytokinesis. Moreover, Drosophila spermatocytes are large cells that exhibit clearly defined redistribution of the ER onto astral MTs early in meiosis. This study presents the surprising finding that although dynein is required for peri-centrosomal focusing of the ER during late interphase, it does not mediate the astral MT-dependent spindle pole focusing of the ER around centrosomes during M-phase. Surprisingly, the results further reveal that redistribution of the ER toward MT minus-ends in dividing cells is mediated by a mechanism of ER-MT association that is entirely specific to M-phase and does not operate during interphase. This report lays the groundwork for identification of this novel mechanism of ER-MT association that is essential for the process of ER inheritance (Karabasheva, 2019).

Proper distribution of the ER in dividing cells is critical, because it ensures that progeny cells receive necessary proportions of this essential organelle. Disruptions in this process may result in cells that are prone to accumulation of misfolded proteins and ER stress, as well as dysregulation of lipid homeostasis and calcium signaling, all of which are involved in a spectrum of diseases including cancer, neurodegeneration, and diabetes. However, the importance of mitotic ER distribution may extend beyond organelle inheritance. For example, it was recently demonstrated that the ER plays an essential role in restricting the distribution of damaged proteins in asymmetrically dividing stem cells. This mechanism, which depends on proper distribution of the ER around the mitotic spindle, may allow vital stem cells to protect themselves by asymmetrically shuttling damaged proteins to non-essential progeny cells. In addition, it has been suggested that the ER delivers highly localized calcium signals that are essential for proper function of the spindle apparatus. Thus, disruption of ER distribution around the mitotic spindle may impair the process of cell division itself with potentially devastating effects on development and tissue homeostasis. Conversely, therapeutic interventions that specifically target ER functions in cancer cells may prove to be effective treatments in neoplastic disease. Identification of the molecular mechanisms that regulate M-phase specific distribution of the ER is critical to understanding these multi-faceted roles for the organelle in dividing cells. The present investigation demonstrates the existence of a novel mechanism that mediates ER-MT association in a manner that is specific to M-phase and independent of dynein and other known MT motors (Karabasheva, 2019).

The first studies to examine ER distribution in dividing cells reported the organelle's distinct organization around the poles of the mitotic spindle, where MT minus-ends are clustered. Subsequent studies have confirmed this observation across multiple species, suggesting the existence of a unifying mechanism of ER partitioning that involves MT-dependent spindle pole association. Accordingly, involvement of a MT minus-end motor to move the ER toward spindle poles has been suggested in multiple studies, with dynein being the most likely candidate. Importantly, dynein associates with ER-rich microsomal fractions and is required for proper ER distribution in interphase cells and extracts, suggesting that the motor can associate with and transport ER membranes. Surprisingly however, the putative role for dynein in regulating ER distribution during cell division has not been directly tested. This question was addressed using meiotic Drosophila spermatocytes, which demonstrate a dramatic, near complete MT-dependent redistribution of the ER to spindle poles during cell division. The results clearly demonstrate a role for dynein in ER transport and localization in these cells, because ER distribution toward MT minus-ends and around centrosomes during late interphase was completely disrupted by dynein suppression. Surprisingly, however, dynein was dispensable for astral MT association and spindle pole focusing of the ER during meiosis. Importantly and consistent with previous reports, dynein suppression had dramatic effects on spindle formation and architecture in Drosophila spermatocytes including failure of centrosome migration to the nuclear envelope and complete dissociation of spindle poles from the focused kinetochore MT fibers. Nevertheless, despite these highly abnormal spindles, this study observed that early in meiosis the ER was still drawn onto the astral MTs and towards the centrosomes, despite the centrosomes' mislocalization at the cell cortex. Moreover, this redistribution of the ER occurred with the same kinetics and to the same extent as in control cells, suggesting that the mechanism of ER-astral MT association is completely independent of dynein. In this regard, data indicating that dynein association with membranous organelles is inhibited during M-phase of cell division suggests the existence of a dynamic mechanism that reciprocally regulates dynein association of the ER with MTs according to the stage of the cell cycle (Karabasheva, 2019).

An important outcome of dynein suppression in spermatocytes is that it allowed a clear separation of interphase versus M-phase mechanisms of ER association with centrosomal MTs, wherein the ER was completely excluded from centrosomal MTs during late interphase but suddenly moved along MTs toward the centrosomes early in M-phase. This reveals that an M-phase specific mechanism of ER-MT association that concentrates the ER around centrosomes is triggered at the onset of cell division, possibly due to specific cyclin/cyclin-dependent kinase activity. This conclusion is further supported by the observations that several of the mechanisms for ER-MT association that operate during interphase are in fact inhibited during M-phase, including those mediated by STIM1, CLIMP-63, and possibly dynein. Collectively, these findings suggest an enticing mechanism whereby most or all interphase mechanisms of ER-MT association are inhibited during M-phase, allowing an M-phase specific mechanism to predominate and ensure ER association with astral MTs and proper partitioning to daughter cells (Karabasheva, 2019).

The challenge moving forward is to identify the M-phase specific mechanism that associates the ER with astral MTs. Importantly, in dynein suppressed spermatocytes it was clear that the ER moved along astral MTs toward the minus-ends, suggesting that a MT minus-end motor distinct from dynein may be involved. However, the present findings indicate that neither ncd, the only other known bona fide MT minus-end motor in Drosophila, nor Klp61F, which generates pole-ward forces in the spindle, are involved in the unique M-phase distribution of ER with astral MTs. Thus, it is possible that an unidentified MT minus-end motor is required, or that a non-motor factor stably attaches ER membranes along MT fibers and tubulin flux then moves these membranes toward centrosomes. In this regard, it was recently demonstrated that human REEP3 and 4, members of the REEP1-4 family of ER membrane proteins, directly associate with MTs and play a role in spindle pole focusing of the ER37. However, whether REEP3/4 associate the ER specifically with astral MTs has yet to be determined. Drosophila have a single orthologue to human REEPs1-4, known as REEPA, and preliminary observations indicate that REEPA also is not required for astral MT association of the ER in meiotic Drosophila spermatocytes. It is also important to note that different mechanisms of ER-MT association may operate during cell division in different cell types. Thus, while the current results cannot rule out a role for dynein or other MT minus-end motors in ER partitioning in cells other than Drosophila spermatocytes, the findings do indicate that these factors are not universally required (Karabasheva, 2019).

In addition to identifying the molecular factors that link the ER to astral MTs during cell division, another important question is how does this association discriminate astral MTs from other MTs of the spindle apparatus? Certainly, suppression of interphase mechanisms plays a role, as expression of nonphosphorylatable STIM1, which remains associated with MTs during mitosis, results in mislocalization of the ER to kinetochore MTs. It is also possible that differences in tubulin post-translational modifications facilitate discrimination between different MT populations within the spindle, as demonstrated for kinesin-7 motors that carry chromosomes toward the spindle equator along detyrosinated MTs of the inner spindle (Karabasheva, 2019).

In conclusion, this study has demonstrated that an M-phase specific mechanism associates the ER with astral MTs and partitions the organelle to spindle poles in dividing cells. Surprisingly, and in contrast to many previous suggestions, this ER localization is not mediated by dynein or other known MT minus-end motors. Identification of the mechanisms involved is an important next step in better understanding the role of the ER in cell division, stem cell longevity and pluripotency, and tissue architecture. This knowledge will facilitate development of novel therapeutics for pathological processes underlying cancer and age-related tissue degeneration (Karabasheva, 2019).

The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. This study finds that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility (Espadas, 2019).

The endoplasmic reticulum (ER) comprises two uninterrupted domains, the nuclear envelope and the peripheral ER. The peripheral ER is composed of structural elements with different membrane curvature and topology, from flat sheets and reticulated tubules to complex fenestrated structures. These elements are distributed throughout the cytoplasm of the eukaryotic cell as a membrane network enclosing a single lumen. Network maintenance requires homotypic membrane fusion mediated by the atlastin family of dynamin-related GTPases. Suppression of atlastin fusogenic activity leads to ER fragmentation, thus revealing an endogenous mechanism aimed at the reduction of ER connectedness. The existence of this mechanism has been confirmed by several reports showing ER disassembly during mitosis, reversible fragmentation of the ER both in neurons and other cell types, and fragmentation of the ER prior to autophagic degradation. Furthermore, fission of individual ER branches was recently detected by using super-resolution live-cell imaging of the ER network. While no dedicated molecular machinery has been linked to ER fragmentation, few experimental observations suggest an involvement of reticulons, highly conserved integral ER membrane proteins implicated in shaping and stabilizing the tubular ER1. Notably, mutations in both Reticulon-2 and Atlastin-1 have been linked to the neurodegenerative disorder hereditary spastic paraplegia, corroborating their participation in coordinated functional and pathological pathways (Espadas, 2019).

Overexpression of members of the Yop1 and reticulon families of proteins has been reported to cause severe constriction of ER branches and ER fragmentation. Fragmentation could proceed via the breakage of ER tubules, implicating high local curvature stress and membrane fission. Fragmentation was also linked to the shedding of small vesicles, a process whose significance in ER fragmentation, however, is not understood. Tubule fission would naturally antagonize the fusogenic activity of atlastin in the ER, making fusion/fission balance a paradigm in intracellular organelle maintenance. Despite the reported association between reticulons and ER fragmentation, direct involvement of reticulons has not been shown and the mechanism(s) of fragmentation remains obscure. Furthermore, creation of membrane curvature by reticulons was mechanistically linked to construction, not fragmentation of the tubular ER network, both in vitro and in vivo. In agreement with involvement in formation rather than fragmentation of the tubular ER, purified reticulons reconstituted into lipid vesicles induced membrane curvatures insufficient to produce membrane fission (Espadas, 2019).

This study reveals the mechanism underlying reticulon membrane activity that unifies these seemingly contradictory observations. rosophila Reticulon (Rtnl1), while promoting ER tubulation and enhancing the total curvature of ER membranes, is also responsible for ER fragmentation via membrane fission. Fragmentation occurs both at endogenous levels of Rtnl1, when unchallenged due to the absence of atlastin, and upon Rtnl1 overexpression. Corroborating these in vivo results, purified Rtnl1 reconstituted into dynamic lipid nanotubes produces curvatures ranging from moderate, as reported earlier (Hu, 2008), to those causing spontaneous membrane fission. In vivo, this ability of Rtnl1 to induce membrane fission is counterbalanced by atlastin, with the interplay between these proteins exerting the core control on total curvature and connectedness of the ER network in a living organism (Espadas, 2019).

Ever since the discovery of homotypic fusion of ER membranes by atlastin there have been indications in the literature of the existence of an endogenous mechanism balancing unceasing fusion during ER network maintenance. Recent studies, both in vitro and in vivo, reiterated the physiological importance of ER fragmentation and linked it to the curvature-creating proteins operating in the ER15,49. Yet, the puzzle remained as to how proteins implicated in making the tubular ER network, such as reticulons, could also mediate fragmentation of the same network. The results demonstrate that these seemingly opposite functions can indeed exist in a single protein, Rtnl1, combining two different modes of curvature creation, static, and dynamic. The static mode, associated with local membrane bending by the membrane-inserting domains of reticulons, accounts for mechanical stabilization of membrane tubes. The dynamic mode, associated in this work with the increased viscosity of Rtnl1-containing membranes, accounts for friction-driven constriction of elongating membrane tubules, leading to their scission. Dynamic coupling between these two modes via curvature-driven sorting of Rtnl1 toward the nanotube is absolutely critical for fission to occur. Viscous drag alone would produce nanotube constriction only at elevated tensile stress and thus result in the mechanical rupture of the membrane. Dynamic accumulation of Rtnl1 in the curved nanotubes, however, critically amplifies constriction so that scission can happen at reduced tensile stress, via a hemi-fission mechanism. Thus, the hemi-fission curvature threshold can be reached at physiological elongation, speeds and forces, within a range of Rtnl1 concentration that creates only the moderate static curvatures required for ER tubule stabilization. Hence, in the dynamic ER network Rtnl1 readily combines its membrane curvature stabilization and fission activities without risking the leakage of the ER lumen contents into the cytoplasm (Espadas, 2019).

In ER maintenance, membrane fission by Rtnl1 must be balanced by atlastin-mediated membrane fusion. Fundamentally, this balance is described by a kinetic model which explicitly accounts for the two opposing functions of Rtnl1, static curvature stabilization and dynamic fission. The intrinsic sensitivity to membrane dynamics suggests a paradigm of dynamic regulation of ER topology linking membrane fusion and fission with membrane motility. This paradigm implies that ER fragmentation, a process crucial in physiological conditions, for example maintenance of ER morphology and ER-phagy, and likely involved in neuropathological processes can be implicitly controlled by multiple factors connected to ER motility and stresses, with Rtnl1 constituting the core element of the ER-specific membrane fission machinery (Espadas, 2019).

During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. This study examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, this study shows that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, it was shown that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle (Diaz, 2019)

Research over the last decade has highlighted the dramatic changes of the ER during cell division, however the factors that govern these mitotic changes are poorly understood. This study has focused on the dynamics of the highly conserved ER shaping protein, Rtnl1, during mitosis. Rtnl1 displays a steady enrichment at the nuclear envelope and spindle poles prior to bulk of the ER membrane at prometaphase. Using precise temporal inhibition, this study shows that microtubule dynamics are necessary for proper ER localization and partitioning during mitosis. Furthermore, the small molecule inhibitors, Binucleine 2 and BI 2536, which affect the formation of astral microtubules, leads to defects in ER localization at the spindle poles early in mitosis. Blocking cytoplasmic dynein both by small molecule injection and RNAi does not affect localization of Rtnl1 at the poles. This work highlights the mechanistic requirements of mitotic ER localization and provides a framework for ER partitioning during cell division (Diaz, 2019).

A general concern with the approach of using small molecule inhibitors to examine mitotic ER organization is that they can be broad acting and the defects observed can be attributed to indirect or downstream disruptions of the cell cycle. However, it is believed that the phenotypes observed for Rtnl1 are direct outcomes of disruption of the cytoskeletal networks. This is in large part to a prior study that showed when the cell cycle was arrested using small molecule inhibitors either in interphase or mitosis, ER structure initially was unaffected and only after 15-20 minutes of arrest, were any ER defects observed. The ER defects shown in this study were immediate, within 1-2 minutes after exposure to the small molecule indicating a more direct role (Diaz, 2019).

Research over the last decade has elucidated that the ER is a dynamic organelle drastically changing its shape and localization upon entry into mitosis. The mitotic factors responsible for these dramatic changes involving the ER remains an area of great interest. It has been well established that organization of the ER relies on the microtubule network during interphase allowing the ER to stretch from the nuclear envelope to the cell periphery, however it is unknown if the microtubule network performs a similar organizational role during mitosis. Studies involving ER movement during mitosis in S. cerevisiae and C. elegans have implicated the actin cytoskeleton network in mitotic ER dynamics. The ER shares a close localization with the mitotic spindle and poles and it has generally been thought that microtubules and associated motor proteins are responsible for mitotic ER organization and partitioning. To this end, an investigation into the ER transmembrane protein, STIM1 showed an interaction with the microtubule plus-end tracking protein (+TIP) EB1. Additionally, this interaction between the ER and microtubule network is regulated by phosphorylation of STIM1. However, sequence analysis between the mammalian STIM1 and the isoforms of the Drosophila homolog, dSTIM1, showed that dSTIM1 does not contain the identified EB1 binding site or the serine or threonine regulatory amino acids, indicating the existence of multiple mechanisms for ER / microtubule interactions. In support of the role of microtubules, there have been studies implicating the astral microtubule network in partitioning of the ER during asymmetric neuroblast divisions (Diaz, 2019).

The data display a strong localization of ER at the spindle poles during mitosis. It has been assumed that ER is transported towards the poles by the major cellular minus-end directed motor, dynein. In support of this, cytoplasmic dynein has been implicated in trafficking and transport through the secretory system, as well as in the structural support and localization of organelles during interphase. It is also well established that dynein is involved in several mitotic processes including nuclear envelope breakdown through pulling forces along the astral microtubule network, thereby leading to bipolar spindle formation at metaphase. In addition, dynein has also been implicated in the transport and localization of recycling endosomes at the spindle poles during early mitosis. This study shows that small molecule inhibition of dynein during mitosis, while disrupting proper spindle assembly, did not prevent ER localization at the spindle poles. This qualitative result of dynein independence is in line with a very recent study showing that dynein does not affect ER movement to the spindle poles in Drosophila spermatocytes. This result suggests that ER is not being transported or maintained at the minus-end of the microtubules by dynein. However, the quantitative approach carried out in this study suggests that a small amount of Rtnl1 movement is affected and largely lags along the nuclear envelope. While this lack of movement was shown to be significant, this can be explained by the known role of dynein involvement in the breakdown of the nuclear envelope thereby affecting timing of NEB and release of the mitotic kinase Cyclin A affecting ER reorganization rather than a direct connection of dynein with Rtnl1 (Diaz, 2019).

Gurel (2014), in a review focused on ER calcium sequestration suggested two models of microtubule-based ER transport, the sliding mechanism and / or the tip attachment complex (TAC). Sliding mechanism focuses on motor-based movement along an existing microtubule, while TAC model suggest that ER structural proteins would attach to a +TIP protein and movement would be connected to microtubule growth. While there is evidence in different systems for each, the current data suggest that there is a direct connection to microtubule dynamics of growth and stability of the ER. Furthermore, based on small molecule inhibition of the astral microtubule network, this suggests that astral microtubule dynamics also are key in proper mitotic localization of ER at the poles. Recently, a study also showed that the kinesin-14 family member of microtubule minus-end directed motor protein, non-claret disjunctional (ncd) or the kinesin 5 plus-end directed microtubule motor protein Klp61F did not affect ER movement to the spindle poles. Future studies should investigate other possible candidate proteins including the astral microtubule associated kinesin motor protein Khc-73 or the NUMA ortholog, Mushroom body defective (Mud) that associate with the spindle poles or along the astral microtubule network (Diaz, 2019).

Early investigations into the mechanism that regulates ER shape and structure identified a class of proteins, known as Reticulons. This protein family is not defined by sequence homology, but rather by the presence of two short hairpin transmembrane domains on the cytoplasmic leaflet of the ER. Recent studies have begun to elucidate the role of these Reticulon family members in both regulating the structural changes of the ER and connection to the cytoskeleton. Rtnl1 was the first reticulon family member identified in Drosophila, however several additional proteins with reticulon homology domains (RHD) have recently been identified in Drosophila and other systems including spastin, atlastin-1, DP1/YOP and REEPs. It has been shown that these proteins can oligomerize and form homomeric and heteromeric complexes in regards to ER tubule formation. Recently, the mammalian REEP3/4 proteins have been shown to contribute to membrane curvature changes in mitosis and interact with microtubules. However, it is unclear if the REEP proteins work in concert with the reticulons during mitosis. The current data demonstrate the importance of the microtubule network in organizing mitotic ER. Furthermore, it is believed that the disruption of the mitotic spindle and ER organization is a direct outcome and not a downstream consequence of affecting mitotic progression, as a previous study in the early embryo showed that ER dynamics are in frame with the cell cycle and were halted when cell cycle was blocked. However, even with these disruptions, the ER maintained its mitotic organization. Future directions regarding mitotic ER organization would be to identify additional targets that regulate ER organization and partitioning during mitosis. An interesting candidate is the microtubule severing enzyme, Spastin. Several studies have indicated an interaction between reticulons and spastin, however, spastin has recently been shown to mediate contacts between the ER and lysosomes. Future studies that investigate the biochemical interaction between the reticulon family members and the role that mitotic regulatory factors play in complex formation should provide insight into mitotic ER dynamics (Diaz, 2019).

Much of the research in cell biology focused on analysis of fixed or live images to explore and understand cellular function. However, this analysis has largely relied on the individual selecting the region of analysis, thereby giving a qualitative overview of any given phenotype. Furthermore, this type of qualitative analysis, while useful, is difficult to compare with predictions from in silico modeling. Moreover, high content screening efforts require numerical measures in order to allow statistical analysis of results and identification of hits. While automated high-content image analysis has been extensively employed in studies of mammalian cells in culture, quantitative and automated methods remain under-utilized in studies of the Drosophila embryo. In order to address a quantitative approach with respect to ER movement along the perispindle region and spindle poles during mitosis, a MATLAB code was developed that allowed for the unbiased selection of ROIs at spindle poles in the syncytial embryo, and this was applied to high-content data collected with Rtnl1-GFP and ReepB-GFP embryos injected with different small molecule inhibitors. This quantitative approach gave similar results that were in line with the qualitative injection analysis, with some minor exceptions. Seemingly contrary to the qualitative observation of embryos injected with the cytoplasmic dynein inhibitor, Ciliobrevin D, the quantitative analysis carried out in this study reveals less Rtnl1-GFP enrichment to spindles and more Rtnl1-GFP depletion from the cytoplasm. An observation for this discrepancy between observations and measurements is that Rtnl1-GFP is enriched around the nuclear envelope but fails to move toward spindles upon NEB and as mitosis progresses. Similarly, the quantitative approach showed a decrease in ReepB-GFP enrichment to spindles with an increase in cytoplasmic depletion in Ciliobrevin D treatment, . Furthermore, the use of a qualitative and quantitative analysis demonstrates the strength of using a mixed method approach which it is believed will greatly advance the field by providing key insights to the mechanistic underpinnings of complex cellular processes (Diaz, 2019).

Endoplasmic reticulum (ER) stress-induced apoptosis is a primary cause and modifier of degeneration in a number of genetic disorders. Understanding how genetic variation influences the ER stress response and subsequent activation of apoptosis could improve individualized therapies and predictions of outcomes for patients. This study finds that the uncharacterized, membrane-bound metallopeptidase CG14516 in Drosophila melanogaster, which was rename as SUPpressor of ER stress-induced DEATH (superdeath), plays a role in modifying ER stress-induced apoptosis. Loss of superdeath reduces apoptosis and degeneration in the Rh1(G69D) model of ER stress through the JNK signaling cascade. This effect on apoptosis occurs without altering the activation of the unfolded protein response (IRE1 and PERK), suggesting that the beneficial pro-survival effects of this response are intact. Furthermore, superdeath was was shown to function epistatically upstream of CDK5, a known JNK-activated pro-apoptotic factor in this model of ER stress. superdeath is not only a modifier of this particular model, but affects the general tolerance to ER stress, including ER stress-induced apoptosis. Finally, evidence is presented of Superdeath localization to the endoplasmic reticulum membrane. While similar in sequence to a number of human metallopeptidases found in the plasma membrane and ER membrane, its localization suggests that superdeath is orthologous to ERAP1/2 in humans. Together, this study provides evidence that superdeath is a link between stress in the ER and activation of cytosolic apoptotic pathways (Palu, 2020).

In eukaryotes, the double membranous nuclear envelope (NE) encloses the nucleoplasm and separates it from the cytoplasm. The inner nuclear membrane (INM) provides contact with chromatin and the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum (ER). The two bilayers are fused at nuclear pore complexes (NPCs) that form aqueous channels through which regulated transport of macromolecules occurs. NPCs consist of multiple copies of ~30 different nucleoporins (Nups) that are organized into biochemically distinct sub-complexes. Two such modules, the inner ring complex (also called Nup93 complex) and the Y-complex (also called Nup107 complex) constitute the NPC scaffold that is symmetric across the NE plane. FG-Nups (containing phenylalanine-glycine rich intrinsically disordered protein domains) dock onto the scaffold. They constitute the permeability barrier and interact with translocating cargo complexes. Some of them (e.g., Nup214/88, Nup358 [RanBP2], and Nup153) introduce asymmetry by specifically binding to the cytoplasmic or nuclear face of the NPC, respectively (Hampoelz, 2016).

Obviously, the sheer size and compositional complexity of NPCs renders its assembly and membrane insertion a very intricate task. Two distinct NPC assembly pathways that are temporally separated during the cell cycle have been described. First, during interphase, NPCs are assembled de novo onto an enclosed NE. Interphase assembly occurs ubiquitously throughout eukaryotes and strictly requires the fusion of the INM and ONM by a mechanism that is only partially understood. Second, no membrane fusion is required for NPC assembly at mitotic exit. This so-called postmitotic assembly mode is restricted to eukaryotes that disassemble their NPCs during mitosis into soluble sub-complexes after phosphorylation by mitotic kinases. In anaphase, de-phosphorylation of Nups is thought to trigger the ordered re-assembly onto the separated chromatids before or while membranes enclose daughter nuclei. Both insertion mechanisms rely on the stepwise recruitment of pre-assembled sub-complexes. An insertion of pre-assembled NPCs into the NE has not yet been described (Hampoelz, 2016).

NPCs not only reside within the NE but are also found in stacked cytoplasmic membranes termed annulate lamellae (AL) that are a subdomain of the ER. Based on two-dimensional (2D) transmission electron micrographs these membrane stacks have been perceived as parallel membrane sheets decorated with NPCs (hereafter called AL-NPCs) that morphologically appear similar to their counterparts on the nuclear envelope (NE-NPCs). AL appear in some but not all transformed cell lines and are highly abundant in germ cells and early embryos throughout animal phyla, including Xenopus, Caenorhabditis elegans, sea urchin, Drosophila, and also humans. A role of AL as a storage compartment for maternally deposited Nups that can be made available for meiosis and the rapid cell cycles during early embryogenesis has been suggested but not experimentally proven. Despite these fundamental and long-standing pretensions the function of AL remains elusive and controversial, primarily for two reasons: (1) it has been difficult to conceive how the insertion of parallel stacked membrane sheets containing pre-assembled and possibly pre-oriented NPCs is topologically possible; and (2) direct experimental evidence for a contribution of AL-NPCs to the pool of NE-NPCs has never been obtained. On the contrary a previous study in Drosophila embryos has detected large soluble pools of transport channel Nups and concluded that NPC insertion likely proceeds from soluble cytosolic Nups (Hampoelz, 2016).

This study addressed the function of AL in the physiological context of the Drosophila blastoderm embryo that is rich in AL, while it undergoes a series of 13 synchronized mitoses in a syncytium. Subsequently, the plasma membranes enclose the cortically aligned somatic nuclei in the extended 14th interphase, forming the first epithelial cell layer before the embryo initiates gastrulation. This occurs concomitantly with the broad onset of transcriptional activity on the zygotic genome, a major developmental transition present in all metazoan. In the syncytial blastoderm, cell-cycle progression is very rapid, with interphase durations of ~10 min during the early cell cycles. At least in mammalian cells, de novo NPC interphase assembly has been described to proceed with markedly slower kinetics. This led to a hypothesis that NPC assembly into a closed NE in Drosophila embryos might occur by a different, faster mechanism. By tracking NPCs in living embryos, this study demonstrates direct uptake of AL-NPCs into the NE, as the ER feeds nuclear expansion. A topological model was derived that explains how the INM becomes continuous with inserting membrane sheets from the ER. It is concluded that AL insertion to the NE is a previously unanticipated mode of NPC insertion that relies on pre-assembled, yet immature NPC scaffolds and operates prior to gastrulation (Hampoelz, 2016).

Collectively, the following scenario emerges from the data. AL are abundant in early Drosophila embryos and predominantly contribute to maintain the constant NE-NPC density in the expanding NE during interphase. The abundance of AL at the cortical nuclei layer thereby oscillates together with the progression of the consecutive interphases until the start of global transcription when AL disappear and the mode of NPC insertion changes. During each onset of early interphases, AL-NPCs are assembled similarly to NE-NPCs but since the combined nuclear surface of the two daughter nuclei is smaller as compared to the parental nucleus, they remain in the cytoplasm. As interphases progress, AL-NPCs feed into the pool of NE-NPCs alongside ER membranes that augment NE surface during rapid nuclear expansion. AL insertion is enabled by NE openings that might either persist from previous mitosis or form de novo by an unknown mechanism. Upon AL insertion, the NE permeability barrier remains unperturbed, likely because the NE openings are entirely surrounded by the ER network. The inserting NPCs comprise pre-assembled NPC scaffolds that recruit the full set of Nups only subsequent to insertion and only then establish transport competence (Hampoelz, 2016).

Why do the expanding nuclei of the syncytial blastoderm maintain a constant number of NPCs per surface area despite their transcriptional inactivity? One might surmise that this is due to mechanical properties but also temporal constraints. The insertion of NPCs might be crucial to enable the massive influx of material into the nucleoplasm during nuclear expansion (volume increase). Indeed, the strained configuration of nuclei is reflected by their strong mechanical response (NE tumbling) upon disruption of the NE and permeability barrier after laser puncture. Second, the batch transfer of entire NPC scaffolds as inherent parts of membrane sheets overcomes the described kinetic constrains of interphase assembly in mammalian cells, that are not compatible with the short interphases in the Drosophila syncytium. Given the abundance of AL-NPCs and the reported high insertion rate of NPCs into the NE of Xenopus leavis oocytes it appears likely that similar mechanisms operate in vertebrates. It remains unclear how sufficient amounts of AL are generated to globally feed nuclear surface expansion over multiple cell cycles until the start of transcription. However, Nups are maternally provided and AL are abundant not only at the cortical layer of nuclei but also within the interior of the embryo. Therefore, a possibility that needs to be considered is that a source of AL-NPCs already generated during oogenesis feeds nuclear growth throughout the syncytial blastoderm (Hampoelz, 2016).

In addition to their eminent role in transport, NE-NPCs organize the nuclear periphery by delineating zones of active euchromatin as compared to transcriptionally repressed heterochromatin in between pores. Crucial to this is that NPCs are laterally immobile within the NE, which was shown to depend on the nuclear lamina. Lamins are nuclear intermediate filament proteins and come in two major types: B-type Lamins are ubiquitous, while A-type Lamins are expressed exclusively when cells differentiate. Both proteins engage in distinct meshworks and also impact on NPC insertion rate. This work puts NPC organization and the mode of pore insertion into a developmental context. It is proposed that in Drosophila AL insertion is innate to earliest embryogenesis and diminishes when pores get laterally restricted and cluster at the NE. There are no A-type lamins expressed at that stage, and specifically expressed INM proteins could be crucial. Intriguingly, the formation of immobile pore clusters coincides with the transcriptional upregulation of hundreds of genes at zygotic induction, a developmental transition present in all metazoan that is accompanied by characteristic changes in chromatin signatures. This study revealed that the zygotically upregulated INM protein LBR, a developmentally controlled INM tether of peripheral heterochromatin, is sufficient to prematurely aggregate NPCs in blastoderm interphases, when artificially expressed earlier in embryogenesis. This also leads to larger AL likely because LBR counteracts AL insertion for which lateral NPC mobility is required. The data suggest a zygotically induced regulation that links pore insertion and organization, NE composition and ultimately also chromatin organization at the nuclear periphery. All of these events eventually contribute to the commitment of originally pluripotent somatic nuclei into distinct lineages (Hampoelz, 2016).

Developing cells and tissues in a growing animal need to sense food quality and integrate this information with on-going time-bound developmental programs. The integration of metabolism with development requires cellular and systemic coordination. This laboratory has focused on Ca(2+) signaling arising from the release of Ca(2+) stored in the endoplasmic reticulum (ER), which triggers store-operated Ca(2+) entry. A role is described for ER-store Ca(2+) that operates at the cellular level in various classes of neurons, and eventually drives the systemic coordination required to survive and complete development under conditions of nutritional deprivation. In the model system Drosophila melanogaster, a paradigm was developed to induce nutritional stress during the larval stage and used pupariation as a read-out for development. Applying the vast genetic tool kit available in Drosophila to this paradigm, this study has uncovered novel roles for intracellular Ca (2+) signaling in regulating neuronal activity, at the level of transcription in glutamatergic neurons, and translation in neuropeptidergic neurons. Such regulation of cellular processes is critical for integrating information across a neural circuit at multiple levels, starting from the point of sensing systemic and environmental levels of amino acids to finally connecting with neuropeptide secreting neurons, that communicate with the prothoracic gland, an organ that makes the key developmental hormone, ecdysone. This work underscores the importance of ER-store Ca(2+) for neuronal health, with consequences for animal development (Hasan, 2020).

Lipid droplet (LD) formation from the endoplasmic reticulum (ER) is accompanied by the targeting and accumulation of specific hydrophobic, membrane-embedded proteins on LDs. The determinants of this process are unknown. The hydrophobic membrane motifs of two Drosophila melanogaster proteins, GPAT4 and ALG14, that utilize this pathway were studied, and crucial sequence features were identified that mediate LD accumulation. Molecular dynamics simulations and studies in cells reveal that LD targeting of these motifs requires deeply inserted tryptophans that have lower free energy in the LD oil phase and positively charged residues near predicted hairpin hinges that become less constrained in the LD environment. Analyzing hydrophobic motifs from similar LD-targeting proteins, it appears that the distribution of tryptophan and positively charged residues distinguishes them from non-LD-targeting membrane motifs. These studies identify specific sequence features and principles of hydrophobic membrane motifs that mediate their accumulation on LDs (Olarte, 2020).

Genes for endoplasmic reticulum (ER)-shaping proteins are among the most commonly mutated in hereditary spastic paraplegia (HSP). Mutation of these genes in model organisms can lead to disruption of the ER network. To investigate how the physiological roles of the ER might be affected by such disruption, tools were developed to interrogate its Ca(2+) signaling function. GAL4-driven Ca(2+) sensors were developed targeted to the ER lumen to record ER Ca(2+) fluxes in identified Drosophila neurons. Using GAL4 lines specific for Type Ib or Type Is larval motor neurons, this study compared the responses of different lumenal indicators to electrical stimulation, in axons and presynaptic terminals. The most effective sensor, ER-GCaMP6-210, had a Ca(2+) affinity close to the expected ER lumenal concentration. Repetitive nerve stimulation generally showed a transient increase of lumenal Ca(2+) in both the axon and presynaptic terminals. Mutants lacking neuronal reticulon and REEP proteins, homologs of human HSP proteins, showed a larger ER lumenal evoked response compared to wild type; mechanisms are proposed by which this phenotype could lead to neuronal dysfunction or degeneration. These lines are useful additions to a Drosophila Ca(2+) imaging toolkit, to explore the physiological roles of ER, and its pathophysiological roles in HSP and in axon degeneration more broadly (Oliva, 2020).

The endoplasmic reticulum (ER) is a highly dynamic network whose shape is thought to be actively regulated by membrane resident proteins. Mutation of several such morphology regulators cause the neurological disorder Hereditary Spastic Paraplegia (HSP), suggesting a critical role of ER shape maintenance in neuronal activity and function. Human Atlastin-1 mutations are responsible for SPG3A, the earliest onset and one of the more severe forms of dominant HSP. Atlastin has been initially identified in Drosophila as the GTPase responsible for the homotypic fusion of ER membrane. The majority of SPG3A-linked Atlastin-1 mutations map to the GTPase domain, potentially interfering with atlastin GTPase activity, and to the three-helix-bundle (3HB) domain, a region critical for homo-oligomerization. This study examined the in vivo effects of four pathogenetic missense mutations (two mapping to the GTPase domain and two to the 3HB domain) using two complementary approaches: CRISPR/Cas9 editing to introduce such variants in the endogenous atlastin gene and transgenesis to generate lines overexpressing atlastin carrying the same pathogenic variants. Sll pathological mutations examined reduce atlastin activity in vivo although to different degrees of severity. Moreover, overexpression of the pathogenic variants in a wild type atlastin background does not give rise to the loss of function phenotypes expected for dominant negative mutations. These results indicate that the four pathological mutations investigated act through a loss of function mechanism (Montagna, 2020).

Friedreich ataxia (FRDA) is a neurodegenerative disorder characterized by neuromuscular and neurological manifestations. It is caused by mutations in the FXN gene, which results in loss of the mitochondrial protein frataxin. Endoplasmic Reticulum-mitochondria associated membranes (MAMs) are inter-organelle structures involved in the regulation of essential cellular processes, including lipid metabolism and calcium signaling. This study has analyzed in both, unicellular and multicellular models of FRDA, calcium management and integrity of MAMs. Function of MAMs was observed to be compromised in the cellular model of FRDA, which was improved upon treatment with antioxidants. In agreement, promoting mitochondrial calcium uptake was sufficient to restore several defects caused by frataxin deficiency in Drosophila melanogaster. Remarkably, the findings describe for the first time frataxin as a member of the protein network of MAMs, where interacts with two of the main proteins implicated in endoplasmic reticulum-mitochondria communication. These results suggest a new role of frataxin, indicate that FRDA goes beyond mitochondrial defects and highlight MAMs as novel therapeutic candidates to improve patient's conditions (Rodríguez, 2020).

Endoplasmic reticulum (ER) stress and its adaptive cellular response, the unfolded protein response (UPR), are involved in various diseases including neurodegenerative diseases, metabolic diseases, and even cancers. This study analyzed the novel function of ubiquitin-specific peptidase 14 (USP14) in ER stress. The overexpression of Drosophila USP14 protected the cells from ER stress without affecting the proteasomal activity. Null Hong Kong (NHK) and alpha-1-antitrypsin Z (ATZ) are ER-associated degradation substrates. The degradation of NHK, but not of ATZ, was delayed by USP14. USP14 restored the levels of rhodopsin-1 protein in a Drosophila model for autosomal dominant retinitis pigmentosa and suppressed the retinal degeneration in this model. In addition, it was observed that proteasome complex is dynamically reorganized in response to ER stress in human 293T cells. These findings suggest that USP14 may be a therapeutic strategy in diseases associated with ER stress (Park, 2020).

During phospholipase C-β (PLC-β) signalling in Drosophila photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER-PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER-PM MCS. This study reports that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function (Basak, 2021).

Missense mutations in Valosin-Containing Protein (VCP) are linked to diverse degenerative diseases including IBMPFD, amyotrophic lateral sclerosis (ALS), muscular dystrophy and Parkinson's disease. This study characterize a VCP-binding co-factor (SVIP) that specifically recruits VCP to lysosomes. SVIP is essential for lysosomal dynamic stability and autophagosomal-lysosomal fusion. SVIP mutations cause muscle wasting and neuromuscular degeneration while muscle-specific SVIP over-expression increases lysosomal abundance and is sufficient to extend lifespan in a context, stress-dependent manner. Multiple links between SVIP and VCP-dependent disease were established in a Drosophila model system. A biochemical screen identifies a disease-causing VCP mutation that prevents SVIP binding. Conversely, over-expression of an SVIP mutation that prevents VCP binding is deleterious. Finally, a human SVIP mutation was identified and the pathogenicity of this mutation was confirmed in the Drosophila model. A model is proposed for VCP disease based on the differential, co-factor-dependent recruitment of VCP to intracellular organelles (Johnson, 2021).

Mutations in the human ALS2 gene cause recessive juvenile-onset amyotrophic lateral sclerosis and related motor neuron diseases. Although the ALS2 protein has been identified as a guanine-nucleotide exchange factor for the small GTPase Rab5, its physiological roles remain largely unknown. This study demonstrates that the Drosophila homologue of ALS2 (dALS2) promotes postsynaptic development by activating the Frizzled nuclear import (FNI) pathway. dALS2 loss causes structural defects in the postsynaptic subsynaptic reticulum (SSR), recapitulating the phenotypes observed in FNI pathway mutants. Consistently, these developmental phenotypes are rescued by postsynaptic expression of the signaling-competent C-terminal fragment of Drosophila Frizzled-2 (dFz2). It was further demonstrated that dALS2 directs early to late endosome trafficking and that the dFz2 C terminus is cleaved in late endosomes. Finally, dALS2 loss causes age-dependent progressive defects resembling ALS, including locomotor impairment and brain neurodegeneration, independently of the FNI pathway. These findings establish novel regulatory roles for dALS2 in endosomal trafficking, synaptic development, and neuronal survival (Kim, 2021).

Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect from cancer treatment with no known method for prevention or cure in clinics. CIPN often affects unmyelinated nociceptive sensory terminals. Despite the high prevalence, molecular and cellular mechanisms that lead to CIPN are still poorly understood. In this study a genetically tractable Drosophila model and primary sensory neurons isolated from adult mouse to examine the mechanisms underlying CIPN and identify protective pathways. Chronic treatment of Drosophila larvae with paclitaxel caused degeneration and altered the branching pattern of nociceptive neurons, and reduced thermal nociceptive responses. It was further found that nociceptive neuron-specific overexpression of integrins (see Drosophila Myospheroid), which are known to support neuronal maintenance in several systems, conferred protection from paclitaxel-induced cellular and behavioral phenotypes. Live imaging and superresolution approaches provide evidence that paclitaxel treatment causes cellular changes that are consistent with alterations in endosome-mediated trafficking of integrins. Paclitaxel-induced changes in recycling endosomes precede morphological degeneration of nociceptive neuron arbors, which could be prevented by integrin overexpression. Primary dorsal root ganglia (DRG) neuron cultures to test conservation of integrin-mediated protection. Transduction of a human integrin β-subunit 1 also prevented degeneration following paclitaxel treatment. Furthermore, endogenous levels of surface integrins were decreased in paclitaxel-treated mouse DRG neurons, suggesting that paclitaxel disrupts recycling in vertebrate sensory neurons. Altogether, this study supports conserved mechanisms of paclitaxel-induced perturbation of integrin trafficking and a therapeutic potential of restoring neuronal interactions with the extracellular environment to antagonize paclitaxel-induced toxicity in sensory neurons (Shin, 2021).

While Delta non-autonomously activates Notch in neighboring cells, it autonomously inactivates Notch through cis-inhibition, the molecular mechanism and biological roles of which remain elusive. The wave of differentiation in the Drosophila brain, the 'proneural wave', is an excellent model for studying Notch signaling in vivo. This study shows that strong nonlinearity in cis-inhibition reproduces the second peak of Notch activity behind the proneural wave in silico. Based on this, Delta expression was demonstrated to induce a quick degradation of Notch in late endosomes and the formation of the twin peaks of Notch activity in vivo. Indeed, the amount of Notch is upregulated and the twin peaks are fused forming a single peak when the function of Delta or late endosomes is compromised. Additionally, this study showed that the second Notch peak behind the wavefront controls neurogenesis. Thus, intracellular trafficking of Notch orchestrates the temporal dynamics of Notch activity and the temporal patterning of neurogenesis (Wang, 2021).

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development (Shah, 2022).

The baculovirus envelope protein GP64 is an essential component of the budded virus and is necessary for efficient virion assembly. Little is known regarding intracellular trafficking of GP64 to the plasma membrane, where it is incorporated into budding virions during egress. To identify host proteins and potential cellular trafficking pathways that are involved in delivery of GP64 to the plasma membrane, this study developed and characterized a stable Drosophila cell line that inducibly expresses the AcMNPV GP64 protein and used that cell line in combination with a targeted RNA interference (RNAi) screen of vesicular protein trafficking pathway genes. Of the 37 initial hits from the screen, six host genes were validated and examined that were important for trafficking of GP64 to the cell surface. Validated hits included Rab GTPases Rab1 and Rab4, Clathrin heavy chain, clathrin adaptor protein genes AP-1-2β and AP-2&my;, and Snap29. Two gene knockdowns (Rab5 and Exo84) caused substantial increases (up to 2.5-fold) of GP64 on the plasma membrane. A small amount of GP64 is released from cells in exosomes, and tsome portion of cell surface GP64 is endocytosed, suggesting that recycling helps to maintain GP64 at the cell surface (Hodgson, 2022).

Pentameric ligand-gated ion channels (pLGICs) constitute a large protein superfamily in metazoa whose role as neurotransmitter receptors mediating rapid, ionotropic synaptic transmission has been extensively studied. Although the vast majority of pLGICs appear to be neurotransmitter receptors, the identification of pLGICs in non-neuronal tissues and homologous pLGIC-like proteins in prokaryotes points to biological functions, possibly ancestral, that are independent of neuronal signalling. This study reports the molecular and physiological characterization of a highly divergent, orphan pLGIC subunit encoded by the pHCl-2 (CG11340/Hodor) gene, in Drosophila melanogaster. pHCl-2 forms a channel that is insensitive to a wide array of neurotransmitters, but is instead gated by changes in extracellular pH. pHCl-2 is expressed in the Malpighian tubules, which are non-innervated renal-type secretory tissues. This study demonstrates that pHCl-2 is localized to the apical membrane of the epithelial principal cells of the tubules and that loss of pHCl-2 reduces urine production during diuresis. The data implicate pHCl-2 as an important source of chloride conductance required for proper urine production, highlighting a novel role for pLGICs in epithelial tissues regulating fluid secretion and osmotic homeostasis (Feingold, 2016).

Pentameric ligand-gated ion channels (pLGICs) constitute a superfamily of ionotropic neurotransmitter receptors that includes vertebrate Cys-loop nicotinic acetylcholine, GABA, glycine and 5HT3 receptors. pLGICs play a central role in mediating rapid ionotropic neurotransmission and are expressed in all characterized bilateria. These channels typically reside on postsynaptic membranes of excitable cells and open in response to the binding of neurotransmitter released from presynaptic axon terminals. Ligand binding induces allosteric changes to protein conformation that result in the opening of a transmembrane, ion-selective pore that initiates the flow of specific ions down their electrochemical gradients, altering the membrane potential of the postsynaptic cell. The subunits of pLGICs have a stereotypical tertiary structure that consists of three general domains: an amino-terminal extracellular ligand-binding domain, four transmembrane domains (M1-M4), which collectively form the ion-permeable channel pore, and an intracellular loop between M3 and M4. Functional channels can exist as homomers, or as heteromers, containing as many as five distinct channel subunits (Feingold, 2016).

Sequencing of invertebrate genomes has led to the recognition that the pLGIC subunit superfamily is much larger and more diverse than was previously realized based on work in vertebrate nervous systems. Vertebrate genomes encode five main classes of pLGICs that have been defined based on ligand specificity: the cation-selective nicotinic acetylcholine receptors, serotonin 5HT3 receptors and zinc-activated receptors and the anion-selective GABA and glycine receptors. Invertebrate genomes, in contrast, encode a greater assortment of channel types with a wider range of ligand specificities and ligand-ion combinations than those found in vertebrates (Dent, 2006). In addition to the nicotinic acetylcholine and GABA receptors found in vertebrates, invertebrate genomes encode anion-selective acetylcholine, glutamate, serotonin, dopamine, tyramine and pH channels, as well as cation-selective GABA and proton channels. Moreover, multiple putative invertebrate pLGICs have been identified that cannot be assigned to any neurotransmitter family based on sequence homology (Feingold, 2016).

The biological functions of pLGICs are also likely to be much more diverse than has generally been appreciated. For instance, the cation-selective, proton-activated PBO-5,-6 channel in Caenorhabditis elegans mediates an intercellular pH signal that stimulates muscle contraction. The proton signal is generated by a proton pump in the intestine rather than by synaptic release from neurons. The function of the Drosophila melanogaster pHCl channel, which is open under alkaline conditions, is not known but its expression in the nervous system and the hindgut suggests non-canonical roles in signalling and/or ion regulation. Finally, the discovery of the proton-gated channel from the cyanobacterium Gloeobacter violaceus suggests that pLGICs originally evolved to regulate ion homeostasis in response to environmental changes (Feingold, 2016).

This study shows that CG11340, a putative pLGIC subunit in D. melanogaster which this study has named pHCl-2, forms a pH-gated chloride channel that is expressed in the Malpighian tubules, which are non-innervated secretory tissues. pHCl-2 channels are localized to the apical (lumen-facing) membrane of Malpighian tubule principal cells, precluding a role in responding to humoral signals originating in the haemolymph. Evidence is presented that, instead, pHCl-2 regulates fluid secretion by the Malpighian tubules in response to the pH of urine by controlling chloride counter-ion availability. Based on these data, a new role is proposed for pLGICs in ion homeostasis and implicate pHCl-2 in a previously unrecognized mechanism regulating urine secretion, a mechanism that will enrich current models of insect secretion (Feingold, 2016).

Previous phylogenetic analysis identified pHCl-2 as a member of an arthropod-specific clade of divergent orphan Cys-loop pLGICs. In Drosophila, pHCl-2 groups with two other orphan pLGIC subunits, Secretory chloride channel and CG6927, which together most closely resemble the Drosophila pH-sensitive chloride channel (pHCl) and the pH-sensitive chloride channel in S. scabiei (SsCl). Clades of channel subunits orthologous to the clade of subunits defined by pHCl-2, CG7589 and CG6927 have been reported in other insects such as Apis mellifera, A. aegypti, Nasonia vitripennis and Tribolium castaneums and in non-insect arthropods such as the deer tick Ixodes scapularis, but not in nematodes, molluscs, annelids or chordates (Dent, 2006). The pH response of pHCl-2 closely resembles that of the two other characterized pH-sensitive arthropod pLGICs, Drosophila pHCl and Sarcoptes SsCl; both pHCl and SsCl are inhibited by protons and are increasingly activated by a rise in alkalinity, exhibiting half-maximal activity at pH 7.33±0.16 and 7.55±0.06, respectively. In contrast, the other well-characterized pH-gated pLGICs identified to date, the PBO-5,-6 heteromeric cation channel in C. elegans and GLIC from cyanobacterium G. violaceus, are inhibited by alkaline conditions, and instead, are increasingly activated by a rise in proton concentration, displaying half-maximal responses at pH 6.83±0.01 and 5.1±0.1, respectively. While this study has shown that standard neurotransmitters do not gate pHCl-2 channels expressed in oocytes, it cannot be ruled out that pHCl-2 channels are sensitive to other ligands that might be found in gastric fluid or urine (Feingold, 2016).

Fluid secretion in the Malpighian tubules is mediated by active transepithelial ion transport, which establishes the osmotic gradient necessary for the formation of the primary urine. This active transport is powered by an apically localized, electrogenic H+-ATPase that pumps protons into the tubule lumen and generates a net positive apical membrane potential. The proton gradient is used to drive alkali metal cation/H+ antiporters, which replace luminal protons with sodium and potassium. Chloride enters the lumen passively, following its electrochemical gradient, and is a critical regulator of secretion because, in the absence of this negative counter-ion, cation transport into the lumen would result in an increasingly positive apical membrane potential, which would oppose transport by the ATPase before a significant osmotic gradient has formed (Feingold, 2016).

A role for pHCl-2 as an important source of chloride conductance in the Malpighian tubules is supported by the observation that urine production is affected in pHCl-2 mutants. Loss of pHCl-2 did not obviously impair fluid secretion in unstimulated Malpighian tubules, consistent with alternative routes of chloride flow into the lumen, either via known channels in the stellate cells or through a putative paracellular route via septate junctions. However, upon stimulation of the Malpighian tubules with cAMP, a second messenger that enhances the output of the H+ ATPase, pHCl-2 mutant Malpighian tubules showed a significantly reduced diuretic response compared with wild-type. These data suggest that under conditions of enhanced cation transport into the lumen, pHCl-2 provides a necessary source of chloride conductance in the principal cells, without which maximal secretion rates are not achieved (Feingold, 2016).

The expression of pHCl-2 in the apical membrane of principal cells, together with electrophysiology data demonstrating that pHCl-2 forms a pH-sensitive channel, suggests that the pHCl-2-mediated chloride conductance may be regulated by the pH of the luminal environment. Luminal pH is strongly influenced by the relative activities of the proton ATPase and the cation/H+ antiporter, which transport protons into and out of the tubule lumen, respectively, and it is proposed that the gating of pHCl-2 by pH may reveal a homeostatic mechanism that maintains an appropriate balance between these two cation transporters. Under conditions where the activity of the ATPase is high relative to the antiporter, the pH of the lumen would drop because the rate of proton transport into the lumen would exceed proton removal by the antiporter. As secondary active transport of sodium and potassium into the tubule lumen by the antiporter is coupled to active proton transport, an accumulation of excess protons in the tubule lumen would reflect a decrease in the energy efficiency of sodium and potassium secretion. It is proposed that the presence of a pH-sensitive chloride channel like pHCl-2 would counteract such an imbalance. If the pH of the lumen were too acidic, pHCl-2 would be antagonized, chloride permeability would become rate limiting for secretion and the increasingly positive apical membrane potential generated by the electrogenic ATPase would oppose further transport of protons into the lumen. The proton gradient, however, would continue to drive the antiporter, increasing luminal pH, which would, in turn, increase pHCl-2 conductance, and decrease the electrical barrier opposing the ATPase, thus re-establishing homeostasis. pHCl-2 would therefore serve as a 'brake' on the H+-ATPase, providing an upper limit to which the ATPase can operate relative to the antiporter, thus minimizing the expenditure of ATP under conditions where protons are not being put to metabolically efficient use (Feingold, 2016).

If pHCl-2 is in fact inhibited by acidic luminal pH, then one might predict that the stimulatory effects of cAMP on the H+-ATPase would lead to inhibition of pHCl-2-mediated chloride conductance. cAMP signalling in the Malpighian tubules leads to the secretion of a more acidic urine, with pH decreasing from 7.8 to 7.4, consistent with an increase in proton transport by the ATPase. Based on its pH sensitivity in oocytes, the chloride conductance of pHCl-2 channels should decrease ∼80% in response to cAMP, and the normalized chloride conductance-limited secretion rate increase should be significantly smaller in the presence of a pH-sensitive channel (i.e. wild-type) than in its absence (i.e. pHCl-2 knockout). Yet, the opposite was observed: the normalized response of secretion to cAMP in the pHCl-2 knockout was smaller than in wild-type, indicating that a simple physiological model does not account for the pHCl-2 phenotype. Instead, the pHCl-2 knockout may have indirect feedback effects on lumen pH or membrane potential, for example by directly affecting the ability of the H+-ATPase to respond to cAMP (Feingold, 2016).

Another effect of down-regulating pHCl-2 may be to increase the relative chloride current through stellate cell chloride channels. Decreased luminal pH that inhibits pHCl-2, resulting in a rise in apical membrane potential, should increase the driving force for chloride through apically localized chloride channels in stellate cells, thus increasing the relative contribution of stellate cells to apical chloride current. The increased activity of stellate cell chloride channels could in turn drive anion exchange via the basal membrane-localized Cl-/HCO3- transporter in stellate cells, thereby alkalinizing the haemolymph. Interestingly, pharmacological block of the Cl-/HCO3- transporter in A. aegypti has little effect on resting secretion rate but inhibits stimulated secretion, similar to the pHCl-2 mutant phenotype (Piermarini, 2010). As bicarbonate (HCO3-) is thought to be produced by carbonic anhydrase in the principal cells and enter the stellate cells through intracellular junctions, the effect of the pHCl-2 mutant on stimulated transport may be an indirect effect on the Cl-/HCO3- balance in principal cells feeding back and affecting chloride transport through stellate cells (Feingold, 2016).

Recent work by Remnant (2016) demonstrated that pHCl-2 is expressed in the copper cells of the midgut and influences sensitivity to dietary copper: flies deficient for pHCl-2 display increased copper tolerance, whereas the opposite is observed when pHCl-2 is over-expressed. Copper cells are the principal site of acid secretion in the midgut, and, like the Malpighian tubules, are thought to transport protons via apically localized V-type H+-ATPases. While a clear relationship between H+-ATPase output and copper uptake has yet to be elucidated, it has been shown that copper uptake is impaired in flies whose copper cells are deficient in acid secretion, and that the copper cell midgut region is less acidic following copper feeding. Similar to the proposed model in the Malpighian tubules, pHCl-2 may influence ATPase output in copper cells by regulating chloride counter-ion availability, which, in turn, could affect the rate of copper uptake. Curiously, the role of pHCl-2 both in the Malpighian tubules and in the copper cells points to a somewhat counter-intuitive physiological model in which the activity of an alkaline-gated chloride channel provides the necessary counter-current for an acid-secreting transporter (Feingold, 2016).

Demonstration that pHCl-2 regulates secretion in the Malpighian tubules, a polarized epithelial tissue that is not directly associated with the nervous system, underscores the ability of pLGICs to function in a wide array of biological contexts beyond their canonical function in the nervous system. Previous work, for instance the discovery of pLGIC-like proteins in bacteria, has also hinted at possible roles for this ion channel superfamily that are entirely independent of neuronal signalling. There are also examples of pLGICs with well-characterized roles in the nervous system that appear to function in non-neuronal tissues. For example, the mammalian immune system is rich in pLGICs, including nicotinic acetylcholine (nACh), GABAA and glycine receptors. While such non-neuronal roles of the GABA and glycine receptors are poorly understood, the α7 nicotinic acetylcholine receptor (nAChR) is expressed in macrophages where it regulates tumour necrosis factor-α in response to acetylcholine released from spleen lymphocytes. nAChR function has also been reported in bronchial epithelia, where nAChRs expressed on the apical membrane respond to non-neuronal autocrine/paracrine ACh release to regulate chloride permeability through the cystic fibrosis transmembrane conductance regulator (CFTR) channel. Non-neural roles for nAChRs have also been identified in vascular endothelia and in keratinocytes (Feingold, 2016).

pHCl-2 represents an extreme in the evolution of pLGIC functions. Like epithelial nAChRs, it is expressed in non-innervated tissues, but pHCl-2 is unique in that it is not obviously responding to an autocrine/paracrine signal. Whether pHCl-2 has an additional function in the nervous system remains unclear; microarray data suggest that it is not expressed in the head, brain and eyes of adult flies, or in the larval central nervous system, but this broad survey would not necessarily detect expression in a small subset of neurons. Additionally, althoug pHCl-2 localizes to the apical membrane of principal cells, the possibility cannot be ruled out that it functions in apically enriched endosomal vesicles to regulate secretion, similar to CUP-4 in C. elegans, which is localized to endosomes and is necessary for endosomal trafficking, although its activating ligand, if any, is unknown. Nevertheless, whether it acts in endosomes or the apical membrane, characterization of pHCl-2 illustrates the remarkable ability of the pLGICs to evolve diverse physiological functions (Feingold, 2016).

Large-scale insecticide application is a primary weapon in the control of insect pests in agriculture. However, a growing body of evidence indicates that it is contributing to the global decline in population sizes of many beneficial insect species. Spinosad emerged as an organic alternative to synthetic insecticides and is considered less harmful to beneficial insects, yet its mode of action remains unclear. Using Drosophila, this study showed that low doses of spinosad antagonize its neuronal target, the nicotinic acetylcholine receptor subunit α 6 (nAChRα6), reducing the cholinergic response. The nAChRα6 receptors are transported to lysosomes that become enlarged and increase in number upon low doses of spinosad treatment. Lysosomal dysfunction is associated with mitochondrial stress and elevated levels of reactive oxygen species (ROS) in the central nervous system where nAChRα6 is broadly expressed. ROS disturb lipid storage in metabolic tissues in an nAChRα6-dependent manner. Spinosad toxicity is ameliorated with the antioxidant N-acetylcysteine amide. Chronic exposure of adult virgin females to low doses of spinosad leads to mitochondrial defects, severe neurodegeneration, and blindness. These deleterious effects of low-dose exposures warrant rigorous investigation of its impacts on beneficial insects (Martelli, 2022).

The endolysosomal system not only is an integral part of the cellular catabolic machinery that processes and recycles nutrients for synthesis of biomaterials, but also acts as signaling hub to sense and coordinate the energy state of cells with growth and differentiation. Lysosomal dysfunction adversely influences vesicular transport-dependent macromolecular degradation and thus causes serious problems for human health. In mammalian cells, loss of the lysosome associated membrane proteins LAMP1 and LAMP2 strongly affects autophagy and cholesterol trafficking. This study shows that the previously uncharacterized Drosophila Lamp1 is a bona fide ortholog of vertebrate LAMP1 and LAMP2. Surprisingly and in contrast to lamp1 lamp2 double-mutant mice, Drosophila Lamp1 is not required for viability or autophagy, suggesting that fly and vertebrate LAMP proteins acquired distinct functions, or that autophagy defects in lamp1 lamp2 mutants may have indirect causes. However, Lamp1 deficiency results in an increase in the number of acidic organelles in flies. Furthermore, Lamp1 mutant larvae were found to have defects in lipid metabolism as they show elevated levels of sterols and diacylglycerols (DAGs). Because DAGs are the main lipid species used for transport through the hemolymph (blood) in insects, these results indicate broader functions of Lamp1 in lipid transport. These findings make Drosophila an ideal model to study the role of LAMP proteins in lipid assimilation without the confounding effects of their storage and without interfering with autophagic processes (Chaudhry, 2022).

For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with a POI that harbors different tags are needed but generation of these strains is still labor-intensive work. To overcome this, a versatile Drosophila toolkit was developed with a genetically encoded single-chain variable fragment for the HA epitope tag: 'HA Frankenbody'. This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Mechanistically, lysosomal activity was downregulated at this stage, and endocytosis, but not autophagy, was indispensable for the swelling of lysosomes. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, it is proposed that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila (Murakawa, 2022).

Macroautophagy, the degradation and recycling of cytosolic components in the lysosome, is an important cellular mechanism. It is a membrane-mediated process that is linked to vesicular trafficking events. The sorting nexin (SNX) protein family controls the sorting of a large array of cargoes, and various SNXs impact autophagy. To improve understanding of their functions in vivo, all Drosophila SNXs were screened using inducible RNA interference in the fat body. Significantly, depletion of Snazarus (Snz) led to decreased autophagic flux. Interestingly, altered distribution of Vamp7-positive vesicles was observed with Snz depletion, and the roles of Snz were conserved in human cells. SNX25, the closest human ortholog to Snz, regulates both VAMP8 endocytosis and lipid metabolism. Through knockout-rescue experiments, it was demonstrated that these activities are dependent on specific SNX25 domains and that the autophagic defects seen upon SNX25 loss can be rescued by ethanolamine addition. The presence of differentially spliced forms of SNX14 and SNX25 was detected in cancer cells. This work identifies a conserved role for Snz/SNX25 as a regulator of autophagic flux and reveals differential isoform expression between paralogs (Lauzier, 2022).

Macroautophagy, hereafter termed autophagy, is a crucial homeostatic and stress-responsive catabolic mechanism. Autophagy is characterized by the formation of double-membrane structures, called phagophores, which expand and incorporate cytoplasmic proteins or organelles. These structures ultimately close to form autophagosomes. When mature, the autophagosomes fuse with lysosomes, and autophagosomal content is degraded by lysosomal enzymes and recycled. Hence, autophagy requires an intricate balance between various cellular processes to ensure appropriate cargo selection, and autophagosome formation, maturation and fusion (Lauzier, 2022).

Although the core signaling pathways controlling autophagy induction in response to stress were rapidly described and are now well understood, the molecular mechanisms controlling autophagosome sealing, maturation and fusion were only defined more recently. Findings in yeast and metazoans have shed light on the molecular machinery required for autophagosome-lysosome fusion and its regulation. Although different proteins are involved in autophagosome-vacuole fusion in yeast and autophagosome-lysosome fusion in metazoans, the overarching principle is conserved and requires the presence of specific soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs). In metazoans, syntaxin (STX) is recruited to mature autophagosomes by two hairpin regions, where it forms a Qabc complex with synaptosome associated protein 29 (SNAP29). The STX17-SNAP29 complex then forms a fusion-competent complex with lysosome-localized vesicle associated membrane protein (VAMP). More recently, the Qa SNARE YKT6 v-SNARE homolog (YKT6) was also found to mediate autophagosome-lysosome fusion. YKT6 is recruited to mature autophagosomes and associates with SNAP29. The YKT6-SNAP29 complex interacts with the lysosomal R-SNARE STX7 to mediate fusion . These fusion complexes are conserved, and flies also use these proteins for autophagosome-lysosome fusion. However, unlike in human cells, where STX17 and YKT6 act redundantly in parallel pathways, Ykt6 is epistatic to Syx17 and Vamp7 in flies. SNARE functions are supported by other intracellular factors, which ensure their specificity and rapid action. The small Rab GTPases Ras-related protein RAB7 and RAB2 are important determinants of fusion, as lysosome-localized RAB7 and autophagosome-localized RAB2 interact with the tethering homotypic fusion and vacuole protein sorting (HOPS) complex to bring autophagosomes and lysosomes in close proximity and enable SNARE-mediated fusion. Interestingly, a direct interaction has been observed between STX17 and the HOPS complex, favoring autophagosome-lysosome tethering. The lipid composition of autophagosomes and lysosomes is also an important determinant of fusion. Specific phosphoinositides [PtdIns(3)P, PtdIns(3,5)P2, PtdIns(4)P, and PtdIns(4,5)P2] impact fusion through different mechanisms. Low cholesterol levels affect autophagosome tethering to late endosomes/lysosomes, while increased saturated fatty acid levels or a high-fat diet in mice decrease fusion events. Recently, the phosphatidylserine:phosphatidylethanolamine ratio was also demonstrated to affect autophagosome-lysosome fusion (Lauzier, 2022).

It is clear that multiple inputs are integrated to regulate the final step of the autophagic process. Accordingly, trafficking events must properly regulate the trafficking of essential SNAREs involved in autophagosome-lysosome fusion, like VAMP8 and STX7, that also mediate various other membrane fusion events. This is also true for the dynamic regulation of the lipid composition of these organelles, given that inappropriate ratios of specific lipids affect autophagic flux. Hence, defining trafficking regulators coordinating the localization of SNAREs, as well as the lipid composition of autophagosomes and lysosomes, is of paramount importance for better understanding of the dynamic link between trafficking and autophagy (Lauzier, 2022).

One class of endosomal sorting regulators is the sorting nexin (SNX) family. These proteins have phox homology (PX) domains that interact with diverse phosphoinositide species. Many SNXs localize to early endosomes, where they are involved in sorting events. Importantly, a few SNXs play roles in autophagy. SNX18 and SNX4-SNX7 heterodimers control autophagy-related ATG9 trafficking to modulate autophagosome expansion, and SNX5 and SNX6 also indirectly regulate autophagy by modulating cation-independent mannose-6-phosphate receptor sorting, affecting lysosomal functions. In yeast, SNX4 regulates autophagosome-lysosome fusion by controlling endosomal phosphatidylserine levels. These reports highlight the multifaceted roles of SNXs in regulating autophagy. However, SNX involvement in SNARE protein trafficking has not been reported (Lauzier, 2022).

Using Drosophila as a simple system to screen genes involved in autophagy, this study has identified the sorting nexin Snazarus (Snz) and its human ortholog SNX25 as regulators of the localization and lipid metabolism of Vamp7 and VAMP8, respectively. Using RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-generated mutants, as well as ethanolamine supplementation, this study showed that loss of Snz decreases autophagic flux. Importantly, it was shown that this effect is independent of the endoplasmic reticulum (ER) localization of SNX25 and that it affects two independent processes - Vamp7/VAMP8 internalization and lipid homeostasis. Altogether, these findings identify Snz and SNX25 as regulators of autophagic flux (Lauzier, 2022).

This study has uncovered a conserved autophagic function for snz and its ortholog SNX25. Using both RNAi-mediated depletion and CRISPR/Cas9-generated KOs, it was shown that Snz and SNX25 are required for full autophagic flux. The impact on autophagy is unlikely to occur via lysosomal dysfunction, but potentially through a combination of inappropriate Vamp7 (in flies) and VAMP8 (in humans) internalization or trafficking and defective lipid metabolism. Interestingly, the SNX25 PX domain was necessary for VAMP8 uptake, while ER anchoring was dispensable. Furthermore, LC3 accumulation observed upon SNX25 loss could be rescued by SNX25 lacking either its PX/Nexin or ER anchoring domains, and by ETA supplementation. Altogether, the findings uncover the multifaceted effects of SNX25 loss on endocytosis and lipid metabolism, which ultimately affect autophagic flux (Lauzier, 2022).

To further refine the endosomal sorting regulators involved in autophagy, a targeted RNAi screen was performed of SNXs in the fly fat body and monitored autolysosome formation. Unexpectedly, most SNXs tested caused defects in autolysosome acidification. It is believed that this is a consequence of the wide range of cargos sorted or endocytosed by SNXs. The misrouting of specific cargos could directly or indirectly affect lysosomal function and therefore autolysosome acidification or formation. The results also reveal the potential for complementation between SNXs paralogs in mammalian cells, which may explain why autophagy defects were not observed for most SNXs in genome-wide screens (Lauzier, 2022).

SNX14 has three paralogs in mammals - SNX13, SNX19 and SNX25. In neural precursor cells derived from patients with SCAR20, SNX14 loss was associated with autophagosome clearance defects. Conversely, weak effects were observed in dermal fibroblasts from patients. As Drosophila have only a single ortholog of these proteins,it was possible to show through multiple approaches that loss of Snz affected autophagosome clearance and led to autophagosome and autophagic cargo [ref(2)P] accumulation. Data in HeLa cells also indicate defective autophagic flux in SNX14- and SNX25-KO cells. The differences between the current results and findings in patient fibroblasts might be due to differential regulation of either paralog expression or mRNA splicing between cell types. It is worth mentioning that, in HeLa cells, increased SNX14 expression was detected upon SNX25 KO (Fig. 2F). Furthermore, given the complementation of SNX25 KO by SNX14 expression, it is conceivable that SNX25 expression could be differentially modulated in various cell types and be able to rescue SNX14-linked autophagic defects (Lauzier, 2022).

The data indicate defects in the trafficking of Vamp7 and VAMP8 after depletion of Snz and SNX25, respectively. Since the YKT6-SNAP29-STX7 complex can also promote autophagosome-lysosome fusion, it is likely that this complex partially complements the loss of Snz and SNX25, which would explain why their loss did not completely abrogate autophagic flux. Along these lines, differential expression of SNARE complexes between cell types could also account for the variations in penetrance observed between SNX14 studies (Lauzier, 2022).

How exactly Snz/SNX25 regulates Vamp7/VAMP8 endocytosis or trafficking remains to be defined. It was not possible to directly test Vamp7 trafficking in flies; however, ectopic accumulation of GFP:Vamp7 puncta was observed near or at the PM, suggesting a potential uptake defect. To test this more directly, VAMP8 uptake was assessed in SNX25 KO cells. Interestingly, these cells showed decreased VAMP8 internalization that was dependent on the SNX25 PX domain, which interacts with diphosphorylated phosphoinositides like PtdIns(4,5)P2, which is highly abundant at the PM. Defects were not observed in clathrin-dependent or -independent endocytosis, nor were variations in clathrin recruitment at the PM. Hence, it is unlikely that SNX25 depletion results in VAMP8 trafficking defects by affecting PtdIns(4,5)P2 or PtdIns(3,4)P2 dynamics at the PM. Recently, Snz was demonstrated to bridge PM-ER contact sites to modulate LD formation. Therefore, SNX25 may fulfill a similar function in mammals, bridging PM-ER contact sites to favor VAMP8 internalization. A precedent for the involvement of ER-PM contact sites in endocytosis exists; however, it was possible to rescue VAMP8 internalization in SNX25 KO cells with a transgene lacking its ER-anchoring domains, implying that ER-PM proximity is not required for efficient VAMP8 uptake. This notion is consistent with the known requirement of PICALM for VAMP8 uptake. Surprisingly, no defects were detected in PICALM localization in SNX25 or SNX14/SNX25 KO cells, although close proximity between it and overexpressed SNX25 was observed. VAMP8 can also be internalized through a clathrin-independent pathway stimulated by Shiga toxin. This pathway is dependent on lipid organization and might be perturbed in SNX25 KO cells. An earlier study identified SNX25 as a regulator of transforming growth factor β receptor (TGFβR) endocytosis. However, this study erroneously characterized the ΔTM isoform of SNX25 and showed that overexpression of this short isoform increased TGFβR internalization, while SNX25 knockdown decreased uptake. Thus, Snz/SNX25 might affect the endocytosis of multiple cargos, in addition to Vamp7 and VAMP8 (Lauzier, 2022).

It is also worth mentioning that the yeast ortholog of snz and SNX25, MDM1, was originally identified as a regulator of endocytic trafficking, thus other aspects of trafficking could be impaired in Snz/SNX25 mutants and be sensitive to protein expression levels. Although the data illustrate decreased internalization of VAMP8 in SNX25 KO cells, the possibility remains that VAMP8, in addition to its uptake defect, could be misrouted on route to autolysosomes. Decreased colocalization was observed between VAMP8 and CD63 in SNX25 KO cells; therefore, defective trafficking cannot be ruled out. Moreover, co-expression of both SNX25 and VAMP8 led to the re-localization of both proteins to large internal vesicles. This effect required the TM region of SNX25, thus it is conceivable that although the short isoform is sufficient for VAMP8 internalization, the longer ER-associated isoform could regulate the endosomal sorting of VAMP8, through potential inter-organellar contact sites or by modulating lipid metabolism (Lauzier, 2022).

Recent studies have demonstrated important roles for SNX14 in lipid metabolism. SNX14 loss results in saturated fatty acid accumulation and increased sensitivity to lipotoxic stress. Moreover, SNX14, Snz and Mdm1, the yeast ortholog, all regulate LD formation. The functional domains required for SNX14 regulation of LD formation differ from the ones required in SNX25 for VAMP8 uptake; the TM and C-terminal nexin domains of SNX14 are essential for LD localization and regulation, while the PX domain of SNX25 is required for VAMP8 uptake, and its TM domains are dispensable. Interestingly, LD biogenesis, fatty acid trafficking and autophagy are known to intersect. In this context, it is tempting to speculate that Snz and its human orthologs SNX14 and SNX25 could bridge lipid stress and autophagy regulation. Further supporting this hypothesis is the finding that SNX25 loss can be rescued by SNX14 or by either SNX25ΔTM and SNX25ΔPX/Nexin. Moreover, ETA addition, which is predicted to result in higher intracellular phosphatidylethanolamine levels, rescued SNX25 deletion. These rescue experiments highlight that SNX25 loss causes independent phenotypes that culminate in decreased autophagic flux. The effects are likely more potent in flies, since they have a single ortholog and the data show that SNX14 can efficiently rescue SNX25 loss. Concerning the role of SNX25 in lipid metabolism, it is tempting to speculate that it is most probably linked to an effect on lipid saturation and LD biogenesis for four main reasons. First, the C-Nexin region of SNX14 was shown to mediate LD localization, and SNX25 loss could be rescued using a SNX25 mutant deleted of this region, arguing that LD recruitment of SNX25 is dispensable. Second, KO/rescue experiments in HeLa cells were performed in normal growth conditions, where LD biogenesis is minimal, and thus unlikely to affect autophagy. Third, recent findings in U2OS cells identified the PXA region of SNX14 as important in regulating lipid saturation and ER stress in response to saturated lipid accumulation. As the PXA was conserved in the two rescue constructs used for autophagy rescue, it is plausible that SNX25 somehow affects lipid homeostasis and thus autophagosome-lysosome fusion. Moreover, recent findings illustrated the importance of the PE ratio in membrane fusion, and SNX14 deletion leads to increased phosphatidylserine levels as in SNX4 yeast mutants (Ma et al., 2018). This intriguing possibility warrants further studies to identify the specific determinants that mediate the action of SNX25 in endocytosis versus lipid homeostasis (Lauzier, 2022).

Another possibility to consider is that SNX25 may encode ba lipid clustering or transport domain that could help concentrate lipids or move them between organelles in a manner that support functional autophagy. In support of this, recent work using Alphafold2 structural predictions suggest that the Nexin-C and PXA domains of the yeast SNX25 ortholog Mdm1 fold together to create a large spherical domain with a hydrophobic channel that could, in principle, ferry lipids between organelles at organelle contacts. Such a domain could enable SNX25 to localize to various intracellular sites, and cluster and/or transport lipids to support functional autophagy. SNX14 is predicted to contain this domain arrangement as well and this might explain why it can rescue SNX25 loss. In this model, loss of SNX25 would alter lipid homeostasis and subcellular distribution, leading to defects in Vamp7/VAMP8 trafficking and functional autophagy. The molecular details for this process, however, remain to be addressed (Lauzier, 2022).

The observation that various isoforms of SNX14 and SNX25 are expressed in cells is intriguing. This raises the possibility of functional pools of SNX14 and SNX25, with the longer ER-anchored isoform regulating LD biogenesis and the shorter isoforms regulating other processes, like trafficking and autophagy. It is worth noting, however, that although this study provides evidence from ddPCR experiments, it was not possible to demonstrate differential splicing at the protein level because of a lack of isoform-specific antibodies. Isoform expression may be controlled by modulating splicing in response to stress, as has been observed for multiple genes. Alternatively, different transcription factors may favor the expression of certain isoforms. RNA-sequencing datasets from Drosophila do not contain different Snz isoforms, suggesting that a single isoform regulates both LD biogenesis and autophagy (Lauzier, 2022).

In summary, this study has identified a new role for snz and its ortholog SNX25 in autophagy regulation through effects on Vamp7/VAMP8 internalization and lipid metabolism. Moreover, differentially expressed isoforms of SNX14 and SNX25 were described in cancer cells. Based on thesd results and those of previous studies,it is propose that Snz and SNX25 finetune the endocytosis/trafficking of Vamp7 and VAMP8 and potentially regulate the lipid composition of endolysosomes to coordinate the autophagy level with the demands of the cell. It will be interesting to define how these functions differ between various genes and isoforms, and how they are affected by different stressors (Lauzier, 2022).

HTT (huntingtin) is a 350-kDa protein of unknown function. While HTT moves bidirectionally within axons and HTT loss/reduction causes axonal transport defects, the identity of cargo-containing vesicles that HTT helps move remain elusive. Previous work found an axonal retrogradely moving HTT-Rab7 vesicle complex; however, its biological relevance is unclear. Using Drosophila genetics, in vivo microscopy, membrane isolation and pharmacological inhibition, this study identified that adaptors Hip1 and Rilpl aid the retrograde motility of LAMP1-containing HTT-Rab7 late endosomes, not autophagosomes. Reduction of Syx17 and chloroquine- or bafilomycin A1-mediated pharmacological inhibition, but not reduction of Atg5, disrupted the in vivo motility of these vesicles. Further, because HTT-Rab7 vesicles colocalized with long-distance signaling components (BMP signaling: tkv-wit, injury: wnd) and move in a retrograde direction after Drosophila nerve crush, it is proposed that these vesicles likely traffic damage signals following axonal injury. Together, these findings support a previously unknown role for HTT in the retrograde movement of a Rab7-LAMP1-containing signaling late endosome (Krzystek, 2022).

Gamete development ultimately influences animal fertility. Identifying mechanisms that direct gametogenesis, and how they deteriorate with age, may inform ways to combat infertility. Recentl work has shown that lysosomes acidify during oocyte maturation in Caenorhabditis elegans, suggesting that a meiotic switch in lysosome activity promotes female germ-cell health. Using Drosophila melanogaster, this study reports that lysosomes likewise acidify in male germ cells during meiosis. Inhibiting lysosomes in young-male testes causes E-cadherin accumulation and loss of germ-cell partitioning membranes. Notably, analogous changes occur naturally during aging; in older testes, a reduction in lysosome acidity precedes E-cadherin accumulation and membrane dissolution, suggesting one potential cause of age-related spermatocyte abnormalities. Consistent with lysosomes governing the production of mature sperm, germ cells with homozygous-null mutations in lysosome-acidifying machinery fail to survive through meiosis. Thus, lysosome activation is entrained to meiotic progression in developing sperm, as in oocytes, and lysosomal dysfunction may instigate male reproductive aging (Butsch, 2022).

Variants in TBC1D8B cause nephrotic syndrome. TBC1D8B is a GTPase-activating protein for Rab11 (RAB11-GAP) that interacts with nephrin, but how it controls nephrin trafficking or other podocyte functions remains unclear. A stable deletion was generated in TBC1D8B using microhomology-mediated end joining genome editing. Ex vivo functional assays utilized slit diaphragms in podocyte-like Drosophila nephrocytes. Manipulated endocytic regulators in transgenic mice provided a comprehensive functional analysis of TBC1D8B. A null allele of Drosophila TBC1D8B exhibited nephrocyte-restricted nephrin mislocalization, similar to patients with isolated nephrotic syndrome who have variants in the gene. The protein was required for rapid nephrin turnover in nephrocytes and for endocytosis of nephrin induced by excessive Rab5 activity. The protein expressed from TBC1D8B bearing the edited deletion predominantly localized to mature early endosomes and late endosomes and was required for endocytic cargo processing and degradation. Silencing Hrs, a regulator of endosomal maturation, phenocopied loss of TBC1D8B Low-level expression of murine TBC1D8B rescued loss of the Drosophila gene, indicating evolutionary conservation. Excessive murine TBC1D8B selectively disturbed nephrin dynamics. Finally, four novel TBC1D8B variants were discovered within a cohort of 363 FSGS patients, and functional impact was validated of two variants in Drosophila, suggesting a personalized platform for TBC1D8B-associated FSGS. It is concluded that variants in TBC1D8B are not infrequent among FSGS patients. TBC1D8B, functioning in endosomal maturation and degradation, is essential for nephrin trafficking (Milosavljevic, 2022).

Phagoptosis is a frequently occurring nonautonomous cell death pathway in which phagocytes eliminate viable cells. While it is thought that phosphatidylserine (PS) 'eat-me' signals on target cells initiate the process, the precise sequence of events is largely unknown. This study shows that in Drosophila testes, progenitor germ cells are spontaneously removed by neighboring cyst cells through phagoptosis. Using live imaging with multiple markers, it was demonstrated that cyst cell-derived early/late endosomes and lysosomes fused around live progenitors to acidify them, before DNA fragmentation and substantial PS exposure on the germ cell surface. Furthermore, the phagocytic receptor Draper is expressed on cyst cell membranes and is necessary for phagoptosis. Significantly, germ cell death is blocked by knockdown of either the endosomal component Rab5 or the lysosomal associated protein Lamp1, within the cyst cells. These data ascribe an active role for phagocytic cyst cells in removal of live germ cell progenitors (Zohar-Fux, 2022).

Mutations in DNAJC5/CSPα are associated with adult neuronal ceroid lipofuscinosis (ANCL), a dominant-inherited neurodegenerative disease featuring lysosome-derived autofluorescent storage materials (AFSMs) termed lipofuscin. Functionally, DNAJC5 has been implicated in chaperoning synaptic proteins and in misfolding-associated protein secretion (MAPS), but how DNAJC5 dysfunction causes lipofuscinosis and neurodegeneration is unclear. This study reports two functionally distinct but coupled chaperoning activities of DNAJC5, which jointly regulate lysosomal homeostasis: While endolysosome-associated DNAJC5 promotes ESCRT-dependent microautophagy, a fraction of perinuclear and non-lysosomal DNAJC5 mediates MAPS. Functional proteomics identifies a previously unknown DNAJC5 interactor SLC3A2/CD98hc that is essential for the perinuclear DNAJC5 localization and MAPS but dispensable for microautophagy. Importantly, uncoupling these two processes, as seen in cells lacking SLC3A2 or expressing ANCL-associated DNAJC5 mutants, generates DNAJC5-containing AFSMs resembling NCL patient-derived lipofuscin and induces neurodegeneration in a Drosophila ANCL model. These findings suggest that MAPS safeguards microautophagy to avoid DNAJC5-associated lipofuscinosis and neurodegeneration (Lee, 2022).

A total of 10-20% of plasma membrane proteins are anchored by glycosylphosphatidylinositol (GPI). GPI is attached to proteins by GPI transamidase (GPI-T), which contains five subunits named PIGK, PIGS, PIGT, PIGU, and GPAA1. It was previously reported that PIGT localizes near the nucleus in Drosophila. However, localizations of the other four subunits remain unknown. This study shows that a catalytic subunit of GPI-T, PIGK, mainly localizes to the endoplasmic reticulum (ER), while the other four subunits localize to the nuclear envelope (NE) and ER. The NE/ER localization ratio of PIGS differs between cell types and developmental stages. These results suggest that GPI-T catalyzes GPI attachment in the ER and the other four subunits may have other unknown functions in the NE (Kawaguchi, 2021).

VAMP-associated protein (VAP; see Drosophila Vap33) is an endoplasmic reticulum (ER) membrane protein that functions as a tethering protein at the membrane contact sites between the ER and various intracellular organelles. Mutations such as P56S in human VAPB cause neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, VAP functions in neurons are poorly understood. This study utilized Drosophila olfactory projection neurons with a mosaic analysis with a repressible cell marker (MARCM) to analyze the neuronal function of VAP33, a Drosophila ortholog of human VAPB. In vap33 null mutant clones, the dendrites of projection neurons exhibited defects in the maintenance of their morphology. The subcellular localization of the Golgi apparatus and mitochondria were also abnormal. These results indicate that Vap33 is required for neuronal morphology and organelle distribution. Additionally, to examine the impact of ALS-associated mutations in neurons, human VAPB-P56S was overexpressed in vap33 null mutant clones (mosaic rescue experiments) and found that, in aged flies, human VAPB-P56S expression caused mislocalization of Bruchpilot, a presynaptic protein. These results implied that synaptic protein localization and ER quality control may be affected by disease mutations. This study provides insights into the physiological and pathological functions of VAP in neurons (Kamemura, 2021).

The precise spatiotemporal characteristics of subcellular calcium (Ca(2+)) transients are critical for the physiological processes. This study reports a green Ca(2+) sensor called "G-CatchER(+)" using a protein design to report rapid local ER Ca(2+) dynamics with significantly improved folding properties. G-CatchER(+) exhibits a superior Ca(2+) on rate to G-CEPIA1er and has a Ca(2+)-induced fluorescence lifetimes increase. G-CatchER(+) also reports agonist/antagonist triggered Ca(2+) dynamics in several cell types including primary neurons that are orchestrated by IP(3)Rs, RyRs, and SERCAs with an ability to differentiate expression. Upon localization to the lumen of the RyR channel (G-CatchER(+)-JP45), a rapid local Ca(2+) release occurs that is likely due to calsequestrin. Transgenic expression of G-CatchER(+) in Drosophila muscle demonstrates its utility as an in vivo reporter of stimulus-evoked SR local Ca(2+) dynamics. G-CatchER(+) will be an invaluable tool to examine local ER/SR Ca(2+) dynamics and facilitate drug development associated with ER dysfunction (Reddish, 2021).

Fat stores are critical for reproductive success and may govern maturation initiation. This study reports signaling and sensing fat sufficiency for sexual maturation commitment requires the lipid carrier apolipophorin in fat cells and Sema1a in the neuroendocrine prothoracic gland (PG). Larvae lacking apolpp or Sema1a fail to initiate maturation despite accruing sufficient fat stores, and they continue gaining weight until death. Mechanistically, sensing peripheral body-fat levels via the apolipophorin/Sema1a axis regulates endocytosis, endoplasmic reticulum remodeling, and ribosomal maturation for the acquisition of the PG cells' high biosynthetic and secretory capacity. Downstream of apolipophorin/Sema1a, leptin-like upd2 triggers the cessation of feeding and initiates sexual maturation. Human Leptin in the insect PG substitutes for upd2, preventing obesity and triggering maturation downstream of Sema1a. Data shows how peripheral fat levels regulate the control of the maturation decision-making process via remodeling of endomembranes and ribosomal biogenesis in gland cells (Juarez, 2021).

Although nuclei are the defining features of eukaryotes, how the nuclear compartment is duplicated and partitioned during division is still do not fully understand. This is especially the case for organisms that do not completely disassemble their nuclear envelope upon entry into mitosis. In studying this process in Drosophila neural stem cells, which undergo asymmetric divisions, it was found that the nuclear compartment boundary persists during mitosis thanks to the maintenance of a supporting nuclear lamina. This mitotic nuclear envelope is then asymmetrically remodeled and partitioned to give rise to two daughter nuclei that differ in envelope composition and exhibit a >30-fold difference in volume. The striking difference in nuclear size was found to depend on two consecutive processes: asymmetric nuclear envelope resealing at mitotic exit at sites defined by the central spindle, and differential nuclear growth that appears to depend on the available local reservoir of ER/nuclear membranes, which is asymmetrically partitioned between the two daughter cells. Importantly, these asymmetries in size and composition of the daughter nuclei, and the associated asymmetries in chromatin organization, all become apparent long before the cortical release and the nuclear import of cell fate determinants. Thus, asymmetric nuclear remodeling during stem cell divisions may contribute to the generation of cellular diversity by initiating distinct transcriptional programs in sibling nuclei that contribute to later changes in daughter cell identity and fate (Roubinet, 2021).

Membrane contact sites are critical junctures for organelle signaling and communication. Endoplasmic reticulum-plasma membrane (ER-PM) contact sites were the first membrane contact sites to be described; however, the protein composition and molecular function of these sites is still emerging. This study leverage yeast and Drosophila model systems to uncover a novel role for the Hobbit (Hob) proteins at ER-PM contact sites. Hobbit was found to localize to ER-PM contact sites in both yeast cells and the Drosophila larval salivary glands, and this localization is mediated by an N-terminal ER membrane anchor and conserved C-terminal sequences. The C-terminus of Hobbit binds to plasma membrane phosphatidylinositols, and the distribution of these lipids is altered in hobbit mutant cells. Notably, the Hobbit protein is essential for viability in Drosophila, providing one of the first examples of a membrane contact site-localized lipid binding protein that is required for development (Neuman, 2021).

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the endoplasmic reticulum (ER) exit sites under the stress of amino acid starvation. This study addresses which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt-inducible kinases (SIKs; SIK2 and SIK3) by Na+ stress, which, when it is strong, is sufficient. The second is activation of IRE1 and PERK (also known as PEK in flies) downstream of ER stress induced by the absence of amino acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK, and IRE1 and PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work shows the role of SIKs in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino acids and salt (Zhang, 2021).

Cell compartmentalization is not only mediated by membrane-bound organelles. It also relies on non-membrane bound biomolecular condensates (so-called membraneless organelles) that populate the nucleus and the cytoplasm (Zhang, 2021).

The formation of membraneless organelles has been shown to occur through phase separation, which can be driven by stress (such as ER, oxidative, proteostatic or nutrient stress), resulting in the formation of stress assemblies. Those are mesoscale coalescence of specific and defined components that phase separate. For instance, nutrient stress leads to the formation of many biocondensates. Most of them are RNA based, such as stress granules and P-bodies, but some are not. This is the case for glucose-starved yeast where metabolic enzymes foci and proteasome storage granules form, as well as Drosophila S2 cells that form Sec bodies under conditions of amino acid starvation (Zhang, 2021).

Sec bodies are related to the inhibition of protein secretion in the early secretory pathway. The early secretory pathway comprises the endoplasmic reticulum (ER), where newly synthesized proteins destined to the plasma membrane and the extracellular medium are synthesized. Proteins exit the ER at the ER exit sites (ERES) to reach the Golgi. The ERES are characterized by the concentration of COPI-coated vesicles whose formation requires six proteins, including Sec12 and Sar1, the inner coat proteins Sec23 and Sec24, and the outer coat proteins Sec13 and Sec31 (Gomez-Navarro, 2016). In addition, a larger hydrophilic protein called Sec16, has been identified as a key regulator of the ERES organization and COPII vesicle budding. Many additional lines of evidence support the role of Sec16 in optimizing COPII-coated vesicle formation and export from the ER (Zhang, 2021).

Upon the stress of amino acid starvation in Krebs Ringer bicarbonate buffer (KRB), the ERES of Drosophila S2 cells are remodeled into large round non-membrane bound phase-separated Sec bodies. They are typically observed by immunofluorescence after staining of endogenous Sec16, Sec23 and expressed Sec24-GFP. Importantly, Sec bodies are very quickly resolved upon stress relief (addition of growth medium). Finally, they appear to protect the components of the ERES from degradation and they help cells to survive under conditions of amino acid shortage (Zhang, 2021).

Phase separation has been shown to be driven by specific components, the so-called drivers, either RNAs or proteins harboring structural features that become exposed or modified under certain conditions. In the case of Sec bodies, Sec24AB and Sec16 have been shown to drive Sec body coalescence in a manner that depends on a small stretch of 44 residues in Sec16 and on the mono-ADP-ribosylation enzyme by PARP16. This illustrates the critical role of post-translational modifications in phase separation (Zhang, 2021).

In parallel, changes in cytoplasmic biophysical properties have also been shown to be important in phase separation, such as a drop of cytoplasmic pH within minutes, without post-translational modifications (Zhang, 2021).

This study sought out to (1) identify the pathways elicited in S2 cells upon incubation in the starvation medium KRB that lead to Sec body formation, and (2) to assess whether changes in the cytoplasmic biophysical properties play a role in the phase transition leading to Sec body formation. Amino acid starvation in KRB is shown to stimulate ER stress and activation of two downstream kinases, IRE1 and PERK (also known as PEK in flies) leading to the stimulation of the unfolded protein response (UPR). However, the sole activation of the IRE1 and PERK does not lead to Sec body formation. To form Sec bodies in KRB, IRE1 and PERK activation needs to be combined with a moderate salt stress. Accordingly, KRB incubation is faithfully mimicked by cell incubation with dithiothreitol (DTT) and addition of 100 mM NaCl. Interestingly, a high-salt stress addition of 150 mM NaCl, which activates the salt-inducible kinases (SIKs; SIK2 and SIK3), is sufficient to efficiently drive Sec body formation. Importantly, it was found that a decrease in the cytoplasmic ATP concentration, a general RNA degradation and the stimulation of the UPR are factors strongly correlated to Sec body formation (Zhang, 2021).

This study shows that the Sec bodies that form in Drosophila S2 cells incubated in KRB are fully recapitulated by activation of SIKs, IRE1 and PERK (through SCH100 plus DTT), leading to the activation of a downstream UPR. Strikingly, the strong activation of SIKs in (SCH150) also induces the UPR and leads to Sec body formation. The resulting structures in each condition appear to be similar in size and number, and their formation is reversible. Whether their content is strictly similar has not been addressed in this study (Zhang, 2021).

Taken together, the results show that Sec body formation requires the stimulation of two main signaling pathways. The first is the salt stress pathway (addition of 150 mM NaCl), which activates the SIKs in a necessary and sufficient manner. It also does not lead to a change in the cytoplasmic ATP concentration. It does induce RNA degradation and it stimulates the UPR in an unexpected manner, given that PERK and IRE1 inhibitors do not alter SCH150 driven Sec body formation (Zhang, 2021).

The second pathway is the activation of IRE1 and PERK (but not ATF6), downstream of ER stress, which is partly induced by the absence of amino acids in KRB. Activation of either IRE1 or PERK is necessary but not sufficient. To form Sec bodies, this activation needs to be combined with a moderate salt stress. It is proposed that IRE1 and PERK activation combined with SIK activation occur in KRB, which is recapitulated by SCH100 plus DTT. This is associated with a decrease in the cytoplasmic concentration of ATP, with RNA degradation and with a stimulation of the UPR (Zhang, 2021).

Interestingly, both strong salt stress (SCH150) and KRB lead to the activation of the UPR (measured by the increase in Bip protein level), leading to the possibility that SIKs, IRE1 and PERK interact with and/or activate, each other. Either IRE1 and/or PERK activate the SIKs, or SIK activation activates IRE1 and/or PERK. This still needs to be refined. The prominent role of salt stress and SIKs in remodeling the cytoplasm Strong salt stress is induced by a 4-fold increase of Na+ in the medium combined with bicarbonate. This triggers an increase of Na+ in the cytoplasm that activates one or more SIK (as shown by the phosphorylation of the SIK target HDAC4). Accordingly, SIK inhibition decreases Sec body formation (Zhang, 2021).

Keeping intracellular Na+ as near as possible to physiological concentrations (5 mM) is critical for cellular life, and the cell spends 40% of its available ATP to extrude Na+ against K+ with the NaK ATPase. It is therefore not surprising that Na+ stress would elicit a cytoprotective response, such as prominent as Sec body formation (and stress granule formation in mammalian cells). This will need to be further elucidated, as many organisms and tissues are subjected to increased circulating Na+. This study shows, however, that it is not equivalent to an osmotic shock and that this addition of salt does not lead to a decrease in a cell volume. In contrast to P-bodies in yeast, osmotic stress does not induce Sec bodies. Interestingly, Na+/salt stress has recently been shown to induce the biogenesis of the lysosomal pathway (i.e. more endo/lysosomes as well as an increase in its activity) via TFEB and TOR (Zhang, 2021).

Increased Na+ activates the intracellular Na+-sensor network revolving around the SIKs. The SIKs belong to the family of AMPKs, and have been shown to be part of a nutrient-sensing mechanism so far revolving around glucose and unbalance of the ATP-to-ADP ratio. In mammals, there are three genes encoding SIK (SIK1-SIK3) but only 2 in Drosophila. Drosophila SIK2 is the ortholog of human SIK1 and SIK2, and has been shown to have a link to the fly Hippo pathway, possibly linking nutrient to growth. Drosophila SIK3 is required for glucose sensing in the fly. Which SIK is involved in Sec body formation has not been clarified, as overexpression of each SIK individually has not proven enough to trigger Sec body formation, even when combined to some excess salt (SCH84 or SCH100). However, at least two SIKs appear to change their intracellular localization in KRB, that is, SIK2 and the long SIK3 isoform, which appear to cluster near the plasma membrane and localize to the nuclear envelope. The role of SIKs in the formation of stress assemblies (here the Sec bodies) appears important and novel, and needs to be investigated further. Other members of the AMPK family do not appear to be involved and changing the ratio ADP-to-ATP did not alter Sec body formation in the SIC system (Zhang, 2021).

Although a high salt stress is sufficient to trigger Sec body formation, the Sec body formation observed during incubation in the amino acid starvation buffer KRB elicits another pathway, the ER stress pathway, leading to the activation of both IRE1 and PERK. Indeed, KRB-induced Sec body formation is entirely mimicked by a moderate salt stress (SCH100) combined with activation of IRE1 and PERK induced by DTT (SCH100 plus DTT) (Zhang, 2021).

Surprisingly, this study found that salt stress as well as KRB induces the UPR, which this study found is a common downstream event in all conditions inducing Sec body formation. How salt stress activates the UPR and the exact role of IRE1 and PERK is still not fully understood. It does not appear to be via SIKs, as HG does not modulate the UPR, yet strongly reduces Sec body formation. Conversely, IRE1 and PERK inhibitors do not influence SCH150-induced Sec body formation, so the exact link between IRE1, PERK, SIKs and the UPR remains to be further investigated (Zhang, 2021).

How UPR stimulation induces Sec body formation is not completely understood. It could occur through the clustering of IRE1 and PERK, two membrane kinases, and them forming a template in the plane of the ER membrane. In this regard, UPR stimulation has been linked to the MARylation enzyme Drosophila PARP16, which is also a transmembrane protein of the ER that undergoes a remodeling in KRB. What other roles are played by events downstream of the UPR, for example, Bip upregulation itself, and cytoplasmic changes, remains to be elucidated in detail. One interesting aspect is whether the activation of UPR might affect biophysical properties, membrane association dynamics, and conformation and post-translational modifications of Sec16 and COPII subunits, which would lead to their enhanced phase separation properties under stress (Zhang, 2021).

Lowering the cytoplasmic ATP concentration is one parameter that induces Sec body formation The lowering of the cytoplasmic ATP concentration occurs in KRB and SCH100 plus DTT. Forcing this lowering or preventing it has a strong incidence on Sec body formation. This finding is consistent with the hydrotropic property of ATP, that is, it being able to prevent phase separation in the cytoplasm. At least in vitro, addition of 8 mM ATP dissolves or prevents the phase separation of purified FUS and TAF15. This is a concentration matching physiological range of ATP level in mammalian cells (1-10 mM) and that is also compatible with the S2 cell ATP concentration (1.7 mM) (Zhang, 2021).

One possibility to explain the decrease of ATP concentration is the activation of kinases (among them, IRE1, PERK and SIK) that would consume the ATP pool. However, it is proposed that the decrease in the intracellular ATP concentration is upstream of the kinase activation. Indeed, incubating cells in KRB induces a ROS shock (as this study showed experimentally), which could damage mitochondrial respiration, resulting, in turn, in ER stress, and UPR activation. Taken together, although this study unraveled a strong causality between low ATP and Sec body formation, the noticeable exception of such formation in SCH150 suggests other possibilities (Zhang, 2021).

In conclusion, this work illustrates the complexity of amino acid starvation, the number of pathways it activates and how they interact with each other, as well as the different cellular cytoplasmic biophysical parameters it affects. It is proposed that the formation of Sec bodies depends on the activation of signaling pathways leading to activation of SIKs, IRE1 and PERK, altogether leading to the activation of the UPR, one of the common features of all pathways leading to Sec body formation (Zhang, 2021).

Cell competition is a context-dependent cell elimination via cell-cell interaction whereby unfit cells ('losers') are eliminated from the tissue when confronted with fitter cells ('winners'). Despite extensive studies, the mechanism that drives loser's death and its physiological triggers remained elusive. Through a genetic screen in Drosophila, this study found that endoplasmic reticulum (ER) stress causes cell competition. Mechanistically, ER stress upregulates the bZIP transcription factor Xrp1, which promotes phosphorylation of the eukaryotic translation initiation factor eIF2α via the kinase PERK, leading to cell elimination. Surprisingly, the genetic data show that different cell competition triggers such as ribosomal protein mutations or RNA helicase Hel25E mutations converge on upregulation of Xrp1, which leads to phosphorylation of eIF2α and thus causes reduction in global protein synthesis and apoptosis when confronted with wild-type cells. These findings not only uncover a core pathway of cell competition but also open the way to understanding the physiological triggers of cell competition (Ochi, 2021).

The endoplasmic reticulum (ER)-membrane protein complex (EMC) is a multi-protein transmembrane complex composed of 10 subunits that functions as a membrane-protein chaperone. Variants in EMC1 lead to neurodevelopmental delay and cerebellar degeneration. Multiple families with biallelic variants have been published, yet to date, only a single report of a monoallelic variant has been described, and functional evidence is sparse. Exome sequencing was used to investigate the genetic cause underlying severe developmental delay in three unrelated children. EMC1 variants were modeled in Drosophila, using loss-of-function (LoF) and overexpression studies. Glial-specific and neuronal-specific assays were used to determine whether the dysfunction was specific to one cell type. Exome sequencing identified de novo variants in EMC1 in three individuals affected by global developmental delay, hypotonia, seizures, visual impairment, and cerebellar atrophy. All variants were located at Pro582 or Pro584. Drosophila studies indicated that imbalance of EMC1-either overexpression or knockdown-results in pupal lethality and suggest that the tested homologous variants are LoF alleles. In addition, glia-specific gene dosage, overexpression or knockdown of EMC1 led to lethality, whereas neuron-specific alterations were tolerated. This study established de novo monoallelic EMC1 variants as causative of a neurological disease trait by providing functional evidence in a Drosophila model. The identified variants failed to rescue the lethality of a null allele. Variations in dosage of the wild-type EMC1, specifically in glia, lead to pupal lethality, which is hypothesized to result from the altered stoichiometry of the multi-subunit protein complex EMC (Marcogliese, 2022).

Membrane organelle function, localization, and proper partitioning upon cell division depend on interactions with the cytoskeleton. Whether membrane organelles also impact the function of cytoskeletal elements remains less clear. This study shows that acute disruption of the ER around spindle poles affects mitotic spindle size and function in Drosophila syncytial embryos. Acute ER disruption was achieved through the inhibition of ER membrane fusion by the dominant-negative cytoplasmic domain of atlastin. When centrosome-proximal ER membranes are disrupted, specifically at metaphase, mitotic spindles become smaller, despite no significant changes in microtubule dynamics. These smaller spindles are still able to mediate sister chromatid separation, yet with decreased velocity. Furthermore, by inducing mitotic exit, this study found that nuclear separation and distribution are affected by ER disruption. These results suggest that ER integrity around spindle poles is crucial for the maintenance of mitotic spindle shape and pulling forces. In addition, ER integrity also ensures nuclear spacing during syncytial divisions (Araujo, 2023).

Atlastin (ATL) GTPases undergo trans dimerization and a power strokelike crossover conformational rearrangement to drive endoplasmic reticulum membrane fusion. Fusion depends on GTP, but the role of nucleotide hydrolysis has remained controversial. For instance, nonhydrolyzable GTP analogs block fusion altogether, suggesting a requirement for GTP hydrolysis in ATL dimerization and crossover, but this leaves unanswered the question of how the ATL dimer is disassembled after fusion. The truncated cytoplasmic domain of wild-type Drosophila ATL (DATL) and a novel hydrolysis-deficient D127N variant were recently used in single turnover assays to reveal that dimerization and crossover consistently precede GTP hydrolysis, with hydrolysis coinciding more closely with dimer disassembly. Moreover, while nonhydrolyzable analogs can bind the DATL G domain, they fail to fully recapitulate the GTP-bound state. This predicted that nucleotide hydrolysis would be dispensable for fusion. This study reports that the D127N variant of full-length DATL drives both outer and inner leaflet membrane fusion with little to no detectable hydrolysis of GTP. However, the trans dimer fails to disassemble and subsequent rounds of fusion fail to occur. These findings confirm that ATL mediated fusion is driven in the GTP-bound state, with nucleotide hydrolysis serving to reset the fusion machinery for recycling (Crosby, 2022).

Mitochondria/a-ER contact sites (MERCs) orchestrate many important cellular functions including regulating mitochondrial quality control through mitophagy and mediating mitochondrial calcium uptake. This study identified and functionally characterized the Drosophila ortholog of the recently identified mammalian MERC protein, Pdzd8. Reducing pdzd8-mediated MERCs in neurons slows age-associated decline in locomotor activity and increases lifespan in Drosophila. The protective effects of pdzd8 knockdown in neurons correlate with an increase in mitophagy, suggesting that increased mitochondrial turnover may support healthy aging of neurons. In contrast, increasing MERCs by expressing a constitutive, synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation, accelerates age-related decline in locomotion, and reduces lifespan. Although depletion of pdzd8 prolongs the survival of flies fed with mitochondrial toxins, it is also sufficient to rescue locomotor defects of a fly model of Alzheimer's disease expressing Amyloid β(42) (Aβ(42)). Together, these results provide the first in vivo evidence that MERCs mediated by the tethering protein pdzd8 play a critical role in the regulation of mitochondrial quality control and neuronal homeostasis (Hewitt, 2022).

Pathways localizing proteins to their sites of action are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, how proteins, such as metabolic enzymes, target from the endoplasmic reticulum (ER) to cellular lipid droplets (LDs) is poorly understood. This study identified two distinct pathways for ER-to-LD protein targeting: early targeting at LD formation sites during formation, and late targeting to mature LDs after their formation. Using systematic, unbiased approaches in Drosophila cells, this study identified specific membrane-fusion machinery, including regulators, a tether and SNARE proteins, that are required for the late targeting pathway. Components of this fusion machinery localize to LD-ER interfaces and organize at ER exit sites. Multiple cargoes were identified for early and late ER-to-LD targeting pathways. These findings provide a model for how proteins target to LDs from the ER either during LD formation or by protein-catalysed formation of membrane bridges (Song, 2022).

Lipids are either taken up from food sources or produced internally in specialized tissues such as the liver. Among others, both routes of lipid metabolism involve cytochrome P450 monooxygenases (CYPs). This study sought to analyze the function of Cyp311a1 that has been shown to be expressed in the midgut of the fruit fly Drosophila melanogaster. Using a GFP-tagged version of CYP311A1 that is expressed under the control of its endogenous promoter, it was shown that Cyp311a1 localizes to the endoplasmic reticulum in epithelial cells of the anterior midgut. In larvae with reduced Cyp311a1 expression in the anterior midgut, compared to control larvae, the apical plasma membrane of the respective epithelial cells contains less and shorter microvilli. In addition, reduction of neutral lipids was observed in the fat body, the insect liver, and decreased phosphatidylethanolamine (PE) and triacylglycerols (TAG) amounts in the whole body of these larvae. Probably as a consequence, they cease to grow and eventually die. The microvillus defects in larvae with reduced Cyp311a1 expression are restored by supplying PE, a major phospholipid of plasma membranes, to the food. Moreover, the growth arrest phenotype of these larvae is partially rescued. Together, these results suggest that the anterior midgut is an import hub in lipid distribution and that the midgut-specific CYP311A1 contributes to this function by participating in shaping microvilli in a PE-dependent manner (Dong, 2022).

Miga-mediated endoplasmic reticulum-mitochondria contact sites regulate neuronal homeostasis

Endoplasmic reticulum (ER)-mitochondria contact sites (ERMCSs) are crucial for multiple cellular processes such as calcium signaling, lipid transport, and mitochondrial dynamics. However, the molecular organization, functions, regulation of ERMCS, and the physiological roles of altered ERMCSs are not fully understood in higher eukaryotes. This study found that Miga, a mitochondrion located protein, markedly increases ERMCSs and causes severe neurodegeneration upon overexpression in fly eyes. Miga interacts with an ER protein Vap33 through its FFAT-like motif and an amyotrophic lateral sclerosis (ALS) disease related Vap33 mutation considerably reduces its interaction with Miga. Multiple serine residues inside and near the Miga FFAT motif were phosphorylated, which is required for its interaction with Vap33 and Miga-mediated ERMCS formation. The interaction between Vap33 and Miga promoted further phosphorylation of upstream serine/threonine clusters in the Miga protein that fine-tuned Miga activity. Protein kinases CKI and CaMKII contribute to Miga hyperphosphorylation. MIGA2, encoded by the miga mammalian ortholog, has conserved functions in mammalian cells. A model is proposed that shows Miga interacts with Vap33 to mediate ERMCSs and excessive ERMCSs lead to neurodegeneration (Xu, 2020).

ERMCS mediates the crosstalk between ER and mitochondria. VAP proteins are ER membrane proteins that often serve as an 'entry point' to tether different organelles to ER and mediate the communications between ER and the tethered organelles. VAP proteins contains the MSP domain that binds to proteins with FFAT motifs. In ALS patients, a point mutation in the VAPB MSP domain was identified. The resultant mutant VAPB has reduced affinity to the FFAT motif containing proteins. Indeed, the disease mutation mimic Vap33 had less affinity to Miga than its wild type form. The reduction of organelle contacts including ERMCSs might contribute to the disease conditions in the ALS patients. This study found that increasing ERMCSs by enhancing the interaction between Miga and VAP proteins also led to neurodegeneration, suggesting that the proper amount of contacts and the right distance between ER and mitochondria are critical for neuronal homeostasis (Xu, 2020).

Although Miga overexpression greatly increased ERMCSs in both fly eyes and fat body tissues, the loss of Miga did not have obvious effects on the ERMCSs in fly fat body tissues. The ERMCSs (10-30 nm), especially the tight contacts (about 10 nm), are rare in the wild type fly fat body. It might be the reason for the difficulty to detect ERMCS reduction in this tissue. A Miga genomic rescue fragment with point mutations in the FFAT motif was introduced in Miga mutant animals in this study. Although it rescued the fatality of Miga mutant, the rescued animals were short lived and had eye degeneration. It suggested that the interaction between Miga and Vap33 had physiological importance. It is interesting that the rescued animals always losing R7/R8 upon aging but not other photoreceptor cells, while the loss of photoreceptor cells in Miga mutant animals during aging did not have preference to any particular photoreceptor cells. Drosophila has compound eyes composed with repeat units call ommatidia. Each ommatidia consists of eight photoreceptors: the outer photoreceptor cells R1-R6 and the inner photoreceptor cells R7 and R8. The R1-R6 cells express a single opsin Rh1 and are involved in image formation and motion detection. The R7 cells express one of two opsins Rh3 and Rh4. The R8 cells are localized beneath the R7 and express one of the three opsins: Rh3, Rh5, and Rh6. R7 and R8 cells are involved in color vision and polarized light detection. However, no obvious difference of ERMCSs between the outer and inner photoreceptor cells was observed. Further investigation is required to understand why the FFAT motif in Miga is particularly important for the inner photoreceptor cells. MigaFM rescued the lethality of Miga mutants, suggesting that at least some functions of Miga do not need its FFAT motif. The study in mammalian cells indicated that MIGA2 links mitochondria to lipid droplets (LD) through its C-terminal region and is required for adipocyte differentiation. The LD interaction region of MIGA2 is conserved in fly Miga protein. It needs further investigation whether Miga plays a role in lipid metabolism in Drosophila (Xu, 2020).

It has been reported that the ERMCSs marked the mitochondrial fission positions. However, presenting ERMCS is not sufficient to induce mitochondrial fission. It has been observed that both mitochondrial fission and fusion could happen at ERMCSs. When Miga is overexpressed, increased number was observed of mitochondria with ERMCSs, increased ERMCS length per mitochondrion, and decreased distance between ER and mitochondria at the ERMCSs. Instead of promoting mitochondrial fission, enhanced mitochondrial fusion and dramatically increased mitochondrial length were observed when Miga was overexpressed. Interestingly, when MigaFM was overexpressed, there is no increase of ERMCSs and the mitochondria length was also significantly shorter than that in the wildtype Miga overexpressed tissues. It has been suggested that Miga function in mitochondrial fusion might be coupled with its function in mediating ERMCSs (Xu, 2020).

This study found that Miga was hyperphosphorylated at multiple sites. The phosphorylation on the cluster V was essential for the interaction between Miga and VAP protein and the interaction mediated ERMCS establishment. Upon cell stress stimuli, such as starvation, the phosphorylation was increased and so was the ERMCSs. Therefore, the phosphorylation in the cluster V provides a switch to modulate ERMCSs and the subsequent communications between ER and mitochondria. In addition to the cluster V, Miga was also phosphorylated at multiple sites in the clusters I, II, and III (see Miga was phosphorylated at multiple clusters). The phosphorylation of these sites was enhanced by the interaction between Miga and Vap33. However, the phosphorylation of these sites did not affect Miga ability to form ERMCSs. The data indicated that the phosphorylation in the clusters I, II, and III could enhance Miga activity. It would be interesting to know how the phosphorylation affects Miga activity. Although phosphatase treatment abolished the higher molecular weight bands seen in western blot analysis, we cannot exclude that another unknown post-translation modification might also contribute to the molecular weight changes in Miga (Xu, 2020).

This study identified that CKI and CaMKII were required for Miga phosphorylation. ERMCSs play a key role in calcium flux between ER and mitochondria. The activity of CaMKII is regulated by Ca²+/calmodulin. Therefore, the local concentration of calcium might function as a trigger to modulate CaMKII activity and further modify the ERMCSs through the phosphorylation of Miga. We found that the reduction of mobility shift of Miga upon the RNAi of CaMKII was much smaller than that in the cluster V mutants, suggesting that other kinases are involved in the phosphorylation of the cluster V in Miga. Experiments were not performed to test whether CKI or CamKII could modify Miga overexpression phenotypes in fly eyes because of the shutdown of facilities during COVID-19 pandemic. It would be interesting to know whether these kinases are required for Miga overexpression mediated eye degeneration (Xu, 2020).

It has been reported that presenilins and γ-secretase activity are concentrated in the ERMCSs. Increased ERMCSs have also been observed in the fibroblasts from patients with sporadic Alzheimer's disease. However, whether ERMCS change is the cause or the consequence of the diseases was not known. These data provided evidence that enhanced ERMCSs by Miga overexpression or other means cause severe degeneration in neurons and muscles. It provided a direct evidence that enhanced ERMCSs is devastating to the cellular homeostasis and leads to neurodegeneration (Xu, 2020).

Mitochondrial malfunction and autophagy defects are often concurrent phenomena associated with neurodegeneration. This study shows that Miga, a mitochondrial outer-membrane protein that regulates endoplasmic reticulum-mitochondrial contact sites (ERMCSs), is required for autophagy. Loss of Miga results in an accumulation of autophagy markers and substrates, whereas PI3P and Syx17 levels are reduced. Further experiments indicated that the fusion between autophagosomes and lysosomes is defective in Miga mutants. Miga binds to Atg14 and Uvrag; concordantly, Miga overexpression results in Atg14 and Uvrag recruitment to mitochondria. The heightened PI3K activity induced by Miga requires Uvrag, whereas Miga-mediated stabilization of Syx17 is dependent on Atg14. Miga-regulated ERMCSs are critical for PI3P formation but are not essential for the stabilization of Syx17. In summary, this study identified a mitochondrial protein that regulates autophagy by recruiting two alternative components of the PI3K complex present at the ERMCSs (Xu, 2022).

Eukaryotic cells are compartmentalized into different organelles that execute distinct functions and communicate with each other through indirect signal transduction or direct organelle-organelle contacts. Mitochondria and the adjacent endoplasmic reticulum (ER) form contacts, which are characterized by a 10-30 nm distance between the two organelles. These contacts mediate lipid exchange and calcium flux between the ER and mitochondria. It has been reported that ER-mitochondrial contact sites (ERMCSs) are important platforms for regulating macroautophagy (hereafter referred to as autophagy) and mitophagy (Xu, 2022).

Autophagosome formation at the ERMCSs in mammalian cells has been reported. Upon starvation, the ER-resident SNARE protein syntaxin 17 (STX17 in mammals; Syx17 in flies) recruits the PI3K complex subunit Atg14 to the ERMCSs and triggers autophagosome formation. However, Syx17 was not required for autophagosome formation in flies , and the major role of Syx17 in both mammals and flies is to mediate the fusion between autophagosome and lysosome. In addition, VAPB and PTPIP51, a pair of ERMCS tethers, also regulate autophagy. Increased ERMCS formation facilitated by VAPB or PTPIP51 overexpression inhibits autophagy; conversely, the weakening of contact by knockdown of these tethers stimulates autophagosome formation. Recent studies have shown that autophagy occurs at ERMCSs to supply free fatty acids for mitochondrial energy metabolism, while mitochondrial respiratory chain activity supports autophagy through the regulation of ERMCS formation. In addition to regulating autophagy at the initiation stage, in a previous study, it was determined that mitochondria play a crucial role in the late stage of autophagy. The loss of Tom40, a key subunit of the mitochondrial protein import channel, results in blockage of autophagosome and lysosome fusion. It was also found that defects in several general mitochondrial metabolic processes, such as ATP production, mitochondrial protein synthesis, or the citrate cycle, do not cause the autophagy defects observed in Tom40-depleted tissues. This implied that the autophagy defects caused by blocking mitochondrial protein import are rather specific. It is therefore hypothesized that certain mitochondrial proteins regulate autophagy directly (Xu, 2022).

In the present study, it was demonstrated that Miga, a mitochondrial outer-membrane protein, is required for autophagy. Loss of Miga led to defects in autophagosome-lysosome fusion. Miga is an evolutionarily conserved protein, with orthologs from worms to humans. In a previous study, it was found to be localized on the mitochondrial outer membrane to regulate mitochondrial fusion by stabilizing MitoPLD. Miga interacts with the ER-localized VAP protein to establish ERMCSs. The interactions between Miga and VAP proteins are regulated by the phosphorylation of the FFAT motif in Miga. A recent study also reported that MIGA2 (the human ortholog of Miga) regulates ERMCSs and contacts between mitochondria and lipid droplets (LDs) . Loss of Miga led to the degeneration of photoreceptor cells in flies. Overexpression of Miga in fly eyes resulted in increased ERMCSs and severe eye degeneration. In mice, loss of MIGA2 led to anxiety-like behavior. This study found that Miga interacts with Atg14 and Uvrag to regulate PI3K activity and Syx17 stability, thereby modulating autophagy (Xu, 2022).

Defects in both mitochondria and autophagy are hallmarks of several types of neurodegenerative diseases. This study found that Miga establishes a direct link between mitochondria and autophagy to maintain cellular homeostasis (Xu, 2022).

It is striking that a mitochondrial protein directly regulates autophagy by interacting with the core components of the autophagy machinery. In the present study, it was found that the mitochondrial protein Miga forms complexes with Uvrag and Atg14 to regulate PI3P production and to stabilize Syx17 during autophagy (Xu, 2022).

Miga interacts with Vap33 to mediate formation of ERMCSs. Overexpression of wild-type Miga, but not MigaFM, led to increased PI3P levels, implying that Miga-induced ERMCSs are required for regulating PI3P formation. However, the ERMCS tether function of Miga is neither required for recruiting Uvrag nor for binding to Atg14 and Syx17 stabilization. It has been shown previously that Atg14 and other components of the PI3K complex, such as Atg16 and Vps34, are enriched in ERMCSs upon starvation. The question remains as to why the PI3K complex needs to be present. Phosphatidylinositol (PI) is a substrate required for the PI3K complex to produce PI3P. PI is synthesized on the ER, and ERMCSs are the sites for the transfer of PI between the ER and mitochondria. During autophagy, the PI3K complex promotes PI3P formation to facilitate autophagic processes, and ERMCSs represent platforms to access PI. It is believed that the enrichment of the PI3K complex at ERMCSs is needed to assess the supply of PI. The present study found that MigaFM failed to promote PI3P formation, although it was still able to recruit key PI3K components, such as Uvrag or Atg14. This implied that PI3P formation during autophagy not only requires the activity of the PI3K complex but also PI supplied from ERMCSs (Xu, 2022).

Previous studies reported that ERMCSs are required for the initiation of autophagy. In the current study, it was found that in Miga mutants, autophagic processes were blocked at the autophagosome-lysosome fusion stage, while autophagosome formation was largely unaffected. Lack of Miga led to a reduction in PI3P and Syx17 levels. Previous studies have demonstrated that PI3K is not only essential for autophagy initiation but is also recruited to the autophagosome together with the HOPS complex to facilitate autophagosome and lysosome fusion in mammalian cells. The remaining PI3P in Miga mutants is probably sufficient for autophagosome formation but not enough for the autophagosome-lysosome fusion process. This study found that the loss of Miga reduced co-localization of FYVE-GFP and Atg8a but not co-localization of FYVE-GFP and CathL. This suggests that the reduction of PI3P in autophagosomes, but not lysosomes, might contribute to fusion defects (Xu, 2022).

The fusion defects observed in Miga mutants were not identical to those found in mutants without Syx17 or HOPS components. The puncta of autophagosome markers are larger in Miga mutants than those in mutants without Syx17 or HOPS components, possibly due to the combined effects of reduction of PI3P and Syx17. In worms and mammalian cells, the lack of EPG5 prevents autophagosome maturation and induces the ectopic fusion of autophagosomes with various endocytic vesicles. The enlarged Atg8a-positive structures in Miga mutants might also be a result of the ectopic fusion of autophagosomes with other vesicles (Xu, 2022).

In mammals, both UVRAG and ATG14 are required for autophagy. In flies, Uvrag regulates PI3P formation under fed conditions, and Atg14 is required for PI3P-positive autophagosome formation. This study found that Miga overexpression induces PI3P formation; additionally, Uvrag, but not Atg14, is required during this process. It was also found that Miga overexpression leads to an upregulation of numerous autophagy markers, such as Atg9, Syx17, Atg18a, Rab7, and LAMP, among others. However, the expression levels or patterns of p62 and Atg8a did not change significantly upon Miga overexpression. This implied that Miga overexpression is not sufficient to fully activate autophagy (Xu, 2022).

STX17, the mammalian ortholog of Syx17, is an autophagosome-localized Q-SNARE that mediates autophagosome and lysosome fusion through interactions with SNAP29 and VAMP8/Vamp7. STX17 contains two tandem transmembrane domains that have low hydrophobicity but are required for autophagosome localization. In fed mammalian cells, STX17 reportedly localizes to the ER, mitochondria, and cytosol. STX17 was enriched in ERMCSs upon autophagy stimulation and was present on completely closed autophagosomes. The detailed translocation mechanism remains unclear. In flies, Syx17 shows diffusely dispersed patterns, and there is no mitochondria-specific localization under normal fed conditions. Syx17 forms puncta and co-localizes with Atg8-positive autophagosomes upon starvation. It was found that Miga is required for the stabilization of Syx17. Miga does not bind to Syx17 but stabilizes it through Atg14. It is puzzling why a mitochondrial protein would be required for the stabilization of a protein that functions in autophagosome maturation. It has been reported that there are three-way contacts among the ER, mitochondria, and late endosomes. It is possible that Miga, Vap33, and Atg14 mediate the contact between the ER, mitochondria, and autophagosomes. Autophagosome-associated Atg14 further stabilizes Syx17 to mediate the fusion between autophagosomes and lysosomes. GFP-Atg14 formed large puncta instead of decreasing in the Miga mutant clones. One possible explanation for this is that the overexpression of GFP-Atg14 overrides the requirement of Miga to stabilize it, but the overexpression of GFP-Atg14 per se is not sufficient to fully rescue the autophagy defects in the Miga mutant. Therefore, similar to other autophagy markers, GFP-Atg14 puncta accumulated in the Miga mutant clones (Xu, 2022).

In summary, this study identified a mitochondrial protein, Miga, that regulates autophagic processes by interacting with Atg14 and Uvrag. This delineates a link between mitochondria and macroautophagy. However, this study did not solve how Miga stabilizes Atg14 and Syx17. It is possible that Miga mediates the three-way contact between the ER, mitochondria, and autophagosomes. Miga interacts with Atg14 and stabilizes Atg14. Furthermore, Atg14 interacts with Syx17 to stabilize it. It is not clear how the relay is carried out during autophagy (Xu, 2022).

Both Atg14 and Uvrag interact with MigaN (1-252 aa), but there is no evident competition between Atg14 and Uvrag. The exact regions of Miga that bind to each protein were not identified in this study (Xu, 2022).

Sleep disruptions are quite common in psychological disorders, but the underlying mechanism remains obscure. Wolfram syndrome 1 (WS1) is an autosomal recessive disease mainly characterized by diabetes insipidus/mellitus, neurodegeneration and psychological disorders. It is caused by loss-of function mutations of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes an endoplasmic reticulum (ER)-resident transmembrane protein. Heterozygous mutation carriers do not develop WS1 but exhibit 26-fold higher risk of having psychological disorders. Since WS1 patients display sleep abnormalities, this study aimed to explore the role of WFS1 in sleep regulation so as to help elucidate the cause of sleep disruptions in psychological disorders. It was found in Drosophila that knocking down wfs1 in all neurons and wfs1 mutation lead to reduced sleep and dampened circadian rhythm. These phenotypes are mainly caused by lack of wfs1 in dopamine 2-like receptor (Dop2R) neurons which act to promote wake. Consistently, the influence of wfs1 on sleep is blocked or partially rescued by inhibiting or knocking down the rate-limiting enzyme of dopamine synthesis, suggesting that wfs1 modulates sleep via dopaminergic signaling. Knocking down wfs1 alters the excitability of Dop2R neurons, while genetic interactions reveal that lack of wfs1 reduces sleep via perturbation of ER-mediated calcium homeostasis. Taken together, a role is proposed for wfs1 in modulating the activities of Dop2R neurons by impinging on intracellular calcium homeostasis, and this in turn influences sleep. These findings provide a potential mechanistic insight for pathogenesis of diseases associated with WFS1 mutations (Hao, 2023).

Sleep disruptions are common in individuals with psychiatric disorders, and sleep disturbances are risk factors for future onset of depression. However, the mechanism underlying sleep disruptions in psychiatric disorders are largely unclear. Wolfram Syndrome 1 (WS1) is an autosomal recessive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, deafness and psychiatric abnormalities such as severe depression, psychosis and aggression. It is caused by homozygous (and compound heterozygous) mutation of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes wolframin, an endoplasmic reticulum (ER) resident protein highly expressed in the heart, brain, and pancreas. On the other hand, heterozygous mutation of WFS1 does not lead to WS1 but increase the risk of depression by 26 fold. A study in mice further confirmed that WFS1 mutation is causative for depression. Consistent with the comorbidity of psychiatric conditions and sleep abnormalities, WS1 patients also experience increased sleep problems compared to individuals with type I diabetes and healthy controls. It has been proposed that sleep symptoms can be used as a biomarker of the disease, especially during relatively early stages, but the mechanisms underlying these sleep disturbances are unclear. Considering that heterozygous WFS1 mutation is present in up to 1% of the population and may be a significant cause of psychiatric disorder in the general population, it was decided to investigate the role of wolframin in sleep regulation so as to probe the mechanism underlying sleep disruptions in psychiatric disorders (Hao, 2023).

Although the wolframin protein does not possess distinct functional domains, a number of ex vivo studies in cultured cells demonstrated a role for it in regulating cellular responses to ER stress and calcium homeostasis, as well as ER-mitochondria cross-talk. Mice that lack Wfs1 in pancreatic β cells develop glucose intolerance and insulin deficiency due to enhanced ER stress and apoptosis. Knocking out Wfs1 in layer 2/3 pyramidal neurons of the medial prefrontal cortex in mice results in increased depression-like behaviors in response to acute restraint stress. This is accompanied by hyperactivation of the hypothalamic-pituitary-adrenal axis and altered accumulation of growth and neurotrophic factors, possibly due to defective ER function. A more recent study in Drosophila found that knocking down wfs1 in the nervous system does not increase ER stress, but enhances the susceptibility to oxidative stress-, endotoxicity- and tauopathy-induced behavioral deficits and neurodegeneration (Sakakibara, 2018). Overall, the physiological function of wolframin in vivo, especially in the brain, remains elusive for the most part. This study identified a role for wolframin in regulating sleep and circadian rhythm in flies. Wfs1 deficiency in the dopamine 2-like receptor (Dop2R) neurons leads to reduced sleep, while inhibiting dopamine synthesis blocks the effect of wfs1 on sleep, implying that wfs1 influences sleep via dopaminergic signaling. It was further found that these Dop2R neurons function to promote wakefulness. Depletion of wfs1 alters neural activity, which leads to increased wakefulness and reduced sleep. Consistent with this, it was found that knocking down the ER calcium channel Ryanodine receptor (RyR) or 1,4,5-trisphosphate receptor (Itpr) rescues while knocking down the sarco(endoplasmic)reticulum ATPase SERCA synergistically enhances the short-sleep phenotype caused by wfs1 deficiency, indicating that wolframin regulates sleep by modulating calcium homeostasis. Taken together, these findings provide a potential mechanism for the sleep disruptions associated with WFS1 mutation, and deepen understanding of the functional role of wolframin in the brain (Hao, 2023).

Sleep problems have been reported in WS1 patients. Their scores on Pediatric Sleep Questionnaire are more than 3 times higher than healthy controls and doubled compared to individuals with type I diabetes, indicating that the sleep issues are not merely due to metabolic disruptions. Indeed, this study suggests that the sleep problems in human patients are of neural origin, specifically in the wake-promoting Dop2R neurons. Given that the rebound sleep is not significantly altered in wfs1 depleted flies, it is believed that lack of wfs1 does not shorten sleep duration by impairing the sleep homeostasis system. Instead, wfs1 deficiency leads to excessive wakefulness which in turn results in decreased sleep. Considering that heterozygous WFS1 mutation is present in up to 1% of the population, it would be interesting to examine whether these heterozygous mutations contribute to sleep disruptions in the general population (Hao, 2023).

In mouse, chick, quail and turtle, Wfs1 has been shown to be expressed in brain regions where dopamine receptor Drd1 is expressed. D1-like dopamine receptor binding is increased while striatal dopamine release is decreased in Wfs1-/- mice. The current results also implicate a role for wolframin in dopamine receptor neurons and that lack of wfs1 impacts dopaminergic signaling, as the effects of wfs1 deficiency on both sleep and mushroom body (MB) calcium concentration is blocked by the tyrosine hydroxylase inhibitor AMPT. Both Dop2RGAL4 and GoαGAL4 exhibit prominent expression in the MB, and to be more specific, in the α and β lobes of MB. Previous studies have shown that dopaminergic neurons innervate wake promoting MB neurons, and this study found Dop2R and Goα+ cells to be wake-promoting as well. Therefore, it is suspected that wolframin functions in MB Dop2R/Goα+ neurons to influence sleep. Taken together, these findings suggest an evolutionarily conserved role of wolframin within the dopaminergic system. As this system is also important for sleep/wake regulation in mammals, it is reasonable to suspect that wolframin functions in mammals to modulate sleep by influencing the dopaminergic tone as well (Hao, 2023).

MB neural activity appears to be enhanced in wfs1 deficient flies based on the results obtained using CaLexA and spH reporters. This elevated activity is consistent with behavioral data, as activation of Dop2R/Goα+ cells reduces sleep, similar to the effects of wfs1 deficiency. Moreover, silencing Dop2R neurons rescues the short-sleep phenotype of wfs1 mutants, while over-expressing wfs1 restores the decreased sleep induced by activation of Dop2R neurons. These findings suggest that wolframin functions to suppress the excitability of MB Dop2R neurons, which in turn reduces wakefulness and promotes sleep. Comparable cellular changes have been observed in SERCA mutant flies. Electric stimulation leads to an initial increase followed by prolonged decrease of calcium concentration in mutant motor nerve terminal compared to the control, while action potential firing is increased in the mutants. This series of results underpin the importance of ER calcium homeostasis in determining membrane excitability and thus neural function (Hao, 2023).

GCaMP6 monitoring reveals that wfs1 deficiency selectively reduces fluorescence signal in the MB both under baseline condition and after dopamine treatment, which should reflect a reduction of cytosolic calcium level that is usually associated with decreased excitability. Previous studies have shown that lack of wolframin leads to increased basal calcium level in neural progenitor cells derived from induced pluripotent stem (iPS) cells of WS1 patients and primary rat cortical neurons, but after stimulation the rise of calcium concentration is smaller in Wfs1 deficient neurons, resulting in reduced calcium level compared to controls. Similarly, evoked calcium increase is also diminished in fibroblasts of WS1 patients and MIN6 insulinoma cells with WFS1 knocked down. Notably, wolframin has been shown to bind to calmodulin (CaM) in rat brain, and is capable of binding with calcium/CaM complex in vitro and in transfected cells. This may undermine the validity of using GCaMP to monitor calcium level in wfs1 deficient animals and cells, and could potentially account for the contradictory data acquired using CaLexA vs GCaMP (Hao, 2023).

It is intriguing that in this study wfs1 deficiency appears to selectively impair the function of Dop2R/Goα+ neurons. It has been shown that in the rodent brain Wfs1 is enriched in layer II/III of the cerebral cortex, CA1 field of the hippocampus, central extended amygdala, striatum, and various sensory and motor nuclei in the brainstem. Wfs1 expression starts to appear during late embryonic development in dorsal striatum and amygdala, and the expression quickly expands to other regions of the brain at birth. It is suspect that in flies wfs1 may be enriched in Dop2R/Goα+ cells during a critical developmental period, and that sufficient level of wolframin is required for their maturation and normal functioning in adults. Another possibility is that these cells are particularly susceptible to calcium dyshomeostasis induced by loss of wfs1. Indeed, this is believed to be an important cause of selective dopaminergic neuron loss in Parkinson's Disease, as dopaminergic neurons are unique in their autonomic excitability and selective dependence on calcium channel rather than sodium channel for action potential generation. It is reasoned that Dop2R/Goα+ neurons may also be more sensitive to abnormal intracellular calcium concentration, making them particularly vulnerable to wfs1 deficiency. The pathogenic mechanism underlying the neurodegeneration of WS1 is quite complex, possibly involving brain-wide neurodegenerative processes and neurodevelopmental dis-regulations. The findings of this study provide some evidence supporting a role for altered dopaminergic system during development. Obviously, much more needs to be done to test these hypotheses (Hao, 2023).

The precise role of wolframin in ER calcium handling is not yet clear. It has been shown in human embryonic kidney (HEK) 293 cells that knocking down WFS1 reduces while over-expressing WFS1 increases ER calcium level. The authors concluded that wolframin upregulates ER calcium concentration by increasing the rate of calcium uptake. Consistently, this study found by genetic interaction that knocking down RyR or Itpr (which act to reduce ER calcium level and thus knocking down either one will increase ER calcium level) rescues the short-sleep phenotype caused by wfs1 mutation, while knocking down SERCA (which acts to increase ER calcium level and thus knocking down this gene will reduce ER calcium level) synergistically enhances the short-sleep phenotype. Based on the results of these genetic interactions, it is proposed that lack of wfs1 increases cytosolic calcium while decreasing ER calcium, leading to hyperexcitability of Dop2R neurons and thus reduced sleep. Knocking down RyR or Itpr decreases cytosolic calcium and increases ER calcium, counteracting the influences of wfs1 deficiency and thus rescuing the short-sleep phenotype. On the other hand, knocking down SERCA further increases cytosolic calcium and decreases ER calcium, rendering an enhancement of the short-sleep phenotype. In line with this, study conducted in neural progenitor cells derived from iPS cells of WS1 patients demonstrated that pharmacological inhibition of RyR can prevent cell death caused by WFS1 mutation. In addition, inhibiting the function of IP3R may mitigate ER stress in wolframin deficient cells. One caveat is that SERCA protein level is increased in primary islets isolated from Wfs1 conditional knock-out mice, as well as in MIN6 cells and neuroblastoma cell line with WFS1 knocked down. It is reasoned that this may be a compensatory increase to make up for the reduced ER calcium level due to wolframin deficiency. It is acknowledged that the hypothesis proposed in in the papert is not supported by GCaMP data, which indicates decreased cytosolic calcium level in Dop2R neurons of wfs1 deficient flies. It is suspected that since the sleep phenotype associated with lack of wfs1 is of developmental origin, it is possible there is an initial increase of cytosolic calcium during critical developmental period in wfs1 deficient flies and this influences the function of Dop2R neurons in adults. Clearly, further characterizations need to be done to fully elucidate this issue, and preferably another calcium indicator independent of the GCaMP system should be employed (Hao, 2023).

In conclusion, this study identified a role for wolframin in the wake-promoting Dop2R neurons. wfs1 depletion in these cells lead to impaired calcium homeostasis and altered neural activity, which in turn leads to enhanced wakefulness and reduced sleep. This study may provide some insights for the mechanisms underlying the sleep disruptions in individuals with WFS1 mutation, as well as for the pathogenesis of WS1 (Hao, 2023).

The major glycerophospholipid phosphatidylethanolamine (PE) in the nervous system is essential for neural development and function. There are two major PE synthesis pathways, the CDP-ethanolamine pathway in the endoplasmic reticulum (ER) and the phosphatidylserine decarboxylase (PSD) pathway in mitochondria. However, the role played by mitochondrial PE synthesis in maintaining cellular PE homeostasis is unknown. This study shows that Drosophila pect (phosphoethanolamine cytidylyltransferase) mutants lacking the CDP-ethanolamine pathway, exhibited alterations in phospholipid composition, defective phototransduction, and retinal degeneration. Induction of the PSD pathway fully restored levels and composition of cellular PE, thus rescued the retinal degeneration and defective visual responses in pect mutants. Disrupting lipid exchange between mitochondria and ER blocked the ability of PSD to rescue pect mutant phenotypes. These findings provide direct evidence that the synthesis of PE in mitochondria contributes to cellular PE homeostasis, and suggest the induction of mitochondrial PE synthesis as a promising therapeutic approach for disorders associated with PE deficiency (Zhou, 2020).

In a forward genetic screen to identify genes necessary for photoreceptor cell survival, this study isolated mutations in the gene pect, which encodes CTP:phosphoethanolamine cytidylyltransferase. In these mutants, light-evoked photoreceptor potentials persisted after 20-s light stimulation, indicating prolonged activation of the visual response. These mutants also exhibited light-independent degeneration of photoreceptor neurons, and lipidomic analysis revealed alterations in major phospholipid composition. Phospholipid composition was manipulated via comprehensive genetic interactions and it was concluded that pect mutant phenotypes resulted from PE deficiency. Strikingly, increasing PE synthesis through the PSD pathway effectively suppressed retinal degeneration in pect mutants. Finally, the Mitochondria Associated Membrane (MAM)-enriched proteins MFN and SERCA were required for PSD to rescue pect phenotypes. A model is proposed in which PE synthesized in the mitochondria through PSD is transported back to the ER through endoplasmic reticulum-mitochondria contact sites (ERMCS) when cellular PE is deficient (Zhou, 2020).

Membrane phospholipids, in particular PE, play key roles in regulating neuronal activity and integrity. Drosophila photoreceptor neurons utilize the fastest phospholipid signaling cascade and exhibit high rates of membrane trafficking. Therefore, it is particularly important for neurons to maintain phospholipid pools. As phototransduction is completely mediated by phospholipase C (PLC), maintaining levels of PIP2 and its product DAG are critical for visual responses. Mutations in the gene pect prevent the synthesis of PE from DAG, resulting in increased levels of PI and DAG, and prolonged visual responses. Reducing DAG levels by either introducing mutations in the Lazaro enzyme, which converts PA to DAG, or overexpressing the DAG kinase rdgA, did not suppress the prolonged afterpotentials in pect mutants. Moreover, inhibiting PI synthesis failed to suppress the retinal degeneration and defective ERG responses. In addition, TRP channels were constitutively active in rdgA mutant photoreceptors, and the over-activation and retinal degeneration of rdgA mutants were rescued in rdgA;trp double mutants. In the case of pect, loss of TRP channels did not affect the severity of neurodegeneration, indicating that RDGA and PECT function in different pathways. In conclusion, PECT regulates photoreceptor function and morphology independent of PI and DAG metabolism. Recent evidence suggested TRPs are mechanosensitive channels, and membrane physical properties are involved in channel activation. Indeed, the rhabdomere size was reduced in the 1-day-old pect mutants, indicating changes in the physical properties of the lipid bilayer. It is speculated that alterations in phospholipid composition in pect mutants may change the compact structure and membrane fluidity of rhabdomere, which leads to prolonged activation of TRP channels (Zhou, 2020).

Direct evidence is provided that maintaining cellular PE levels is critical for neuronal function and integrity. First, disruption of the PSD pathway by knocking down PSD enhanced retinal degeneration in pect mutants. More importantly, overexpression of PSD restored cellular PE levels, and thus greatly suppressed both the prolonged afterpotentials in response to light and retinal degeneration of pect mutants. Moreover, this suppression was fully reversed by down-regulating PSS. It has been reported that knockdown of Psd resulted in light-dependent retinal degeneration by preventing autophagy-dependent rhodopsin degradation since the abundance of PE could positively regulate autophagy. However, for pect mutants, rhodopsin turn-over is normal and degeneration is independent of light and TRP channel activity, suggesting that rhodopsin homeostasis does not cause pect-induced cell death. Furthermore, the light-independent retinal degeneration in pect mutants suggested that cellular PE homeostasis independently contributes to maintaining neuronal activity and integrity (Zhou, 2020).

The majority of mitochondrial PE is synthesized in situ in mitochondria via PSD. In contrast, only a small fraction of mitochondrial PE is made in the ER by the CDP-ethanolamine pathway. Maintaining mitochondrial PE levels is critical for mitochondrial respiratory capacity, morphology, and distribution, thus deletion of PSD impairs mitochondrial function, resulting in lethality. The pect mutant photoreceptor cells are dysfunctional, and both cellular and mitochondrial levels of PE are significantly reduced. Although it has been reported that < 30% depletion of mitochondrial PE by RNAi silencing of PSD alters mitochondrial morphology and function in mammalian cells, ~40% reduction in mitochondrial PE levels in pect²⁹ flies did not affect mitochondrial activity, morphology, or axonal localization. Overexpression of PSD, which completely rescued photoreceptor function and integrity in pect mutants, restored total PE levels but not levels of mitochondrial PE. Therefore, disruption of the CDP-ethanolamine PE synthesis pathway did not impair mitochondrial function. Disrupting PE synthesis through the CDP-ethanolamine pathway does not result in a mitochondrial phenotype because mitochondrial PE is synthesized locally through PSD, although the CDP-ethanolamine pathway does contribute to total mitochondrial PE levels (Zhou, 2020).

A recent study reported that yeast Psd1 localizes to both mitochondria and the ER through its transmembrane region (TMR). This forced the authors to consider that PSD in the ER may have restored cellular PE homeostasis in pect mutants. However, Drosophila PSD lacks the TMR sequence required for Psd1 to localize to the ER, and PSD remains in mitochondria when overexpression. ER-mitochondria connections are necessary for the efficient exchange of phospholipids between organelles. Several proteins, including the mitofusin MFN2 and the ER Ca2+ ATPase SERCA, have been implicated in maintaining ERMCS, thus facilitating phospholipid exchange. Consistent with previous reports, this study found that the fly proteins, MFN and SERCA, stabilize ER-mitochondrial contact sites. Importantly, disrupting ER-mitochondria contacts through mfnRNAi or sercaRNAi completely blocked the ability of PSD to rescue pect mutant phenotypes. This is also consistent with a recent study that described an unexpected role of MFN2 in PS transfer between the ER and mitochondria. However, as loss of SERCA induces UPR reaction, the possible role of inducing UPR in disruption of PE homeostasis cannot be ruled out. Taken together, these analyses prove that cellular PE homeostasis is maintained, in part, by synthesizing PE in mitochondria and then exporting this PE to cellular pools. Thus, mitochondria play a critical role in cellular phospholipid homeostasis (Zhou, 2020).

PE species generated from the CDP-ethanolamine and PSD pathways are different, especially fatty acids in the sn-2 position, where PE from the CDP-ethanolamine and PSD pathways prefer mono-/di-unsaturated and polyunsaturated fatty acids, respectively. It has been suggested that individual molecular species of PE may play specialized roles in cellular signaling, which explained why deletion of either Pisd or Pcyt2 causes embryonic lethality in mice. This study saw that as total PE levels decreased, the proportion of most PE species were unaffected. In contrast, the PE species PE38:1, PE36:4, and PE36:5 were down-regulated, and PSD overexpression fully restored levels of these three PE species, as well as total PE levels. This suggests that the same PE species are generated by the CDP-ethanolamine and PSD pathway in vivo, although they might prefer different substrates in vitro. These data further showed that PE generated in the mitochondria can compensate for cellular PE deficiency in the Drosophila visual system (Zhou, 2020).

A recent study also identified recessive lethal mutations in pect and demonstrated that the biosynthesis of specific phospholipids is linked to neurodegeneration and synaptic vesicle loss in adult Drosophila photoreceptors (Tsai, 2019). In pect^omb593 mutants levels of PE 34:1 and PE 36:2 were reduced, but the overall proportions of PE species were not significantly changed, whereas this study detected a significant reduction in total cellular PE levels. The different results of phospholipid composition in pect mutants may come from the different alleles analyzed. In contrast to the nonsense pect²⁹ mutation, the pect^omb593 contains a hypomorphic mutation with a single amino acid change (H55Y). Thus PECTH55Y might reduce PE levels to a much lesser extent than complete loss of pect, and this cellular PE reduction could be compensated by the alternative PSD pathway. Moreover, knocking down >srebp (regulatory element binding protein) reversed the loss of synaptic vesicles, but failed to suppress axonal or retinal degeneration in pect^omb593 mutants. Therefore, SREBP is a downstream factor of PE deficiency and specifically functions in maintaining the synaptic vesicle pools. This study demonstrated that increasing PE levels through induction of the PSD pathway restored the cellular PE levels and effectively suppressed retinal degeneration and defective phototransduction in pect mutants, supporting disruption of PE homeostasis is the major cause of photoreceptor cell degeneration and loss of synaptic transmission (Zhou, 2020).

Phospholipid composition is critical for cellular homeostasis, and alterations in the composition of major phospholipid PE are implicated in multiple diseases. Cellular PC/PE molar ratios can influence energy metabolism in numerous organelles and thus lead to disease conditions such as steatohepatitis, obesity, and muscular dystrophy. Cellular PC levels were elevated by 57% in pect²⁹ retinas compared with wild type, but inhibiting PC synthesis did not suppress the retinal degeneration, suggesting that PE levels, but not the PC/PE ratio, are crucial for neuronal function (Zhou, 2020).

Genetic mutations that affect PE synthesis have been identified in several human autosomal-recessive disorders. In particular, mutations in PISD, the human counterpart of fly PSD, cause Liberfarb syndrome, which is a multisystem disorder affecting the eyes, ears, bone, and brain. Studies in patient-derived fibroblasts revealed impaired phospholipid metabolism, altered mitochondrial respiration, and fragmentation of the mitochondrial network. Recently, mutations in PCYT2, the human counterpart of fly PECT, have been associated with hereditary spastic paraplegia. Lipidomic analysis of patient fibroblasts revealed profound lipid abnormalities impacting both neutral etherlipid and etherphospholipid metabolism. As mutations in fly pect affect phospholipid composition, resulting in defective photoresponse and severe retinal degeneration, this system represents a conserved model for studying diseases associated with PE deficiency. Moreover, these data provide direct genetic evidence that induction of mitochondrial PE synthesis can compensate for deficiencies in cellular PE, and suggest that mitochondrial phospholipid synthesis and trafficking represent a promising therapeutic target for treating disorders associated with defective phospholipid composition (Zhou, 2020).
Drosophila SPG12 ortholog, reticulon-like 1, governs presynaptic ER organization and Ca2+ dynamics

Neuronal endoplasmic reticulum (ER) appears continuous throughout the cell. Its shape and continuity are influenced by ER-shaping proteins, mutations in which can cause distal axon degeneration in Hereditary Spastic Paraplegia (HSP). It was therefore asked how loss of Rtnl1, a Drosophila ortholog of the human HSP gene RTN2 (SPG12), which encodes an ER-shaping protein, affects ER organization and the function of presynaptic terminals. Loss of Rtnl1 depleted ER membrane markers at Drosophila presynaptic motor terminals and appeared to deplete narrow tubular ER while leaving cisternae largely unaffected, thus suggesting little change in resting Ca2+ storage capacity. Nevertheless, these changes were accompanied by major reductions in activity-evoked Ca2+ fluxes in the cytosol, ER lumen, and mitochondria, as well as reduced evoked and spontaneous neurotransmission. Reduced STIM-mediated ER-plasma membrane was found to contact underlie presynaptic Ca2+ defects in Rtnl1 mutants. These results show the importance of ER architecture in presynaptic physiology and function, which are therefore potential factors in the pathology of HSP (Perez-Moreno, 2023).

Synapses, the computational units of nervous systems, are immensely adaptable. The ER is a site of some of the processes that tune synaptic strength. It is a major Ca2+ store of the cell, a site of lipid biosynthesis, and can regulate the physiology of other organelles through contact sites that support exchange of Ca2+ and lipids. Axonal ER comprises an extensive continuous network formed mainly of tubules that are shaped by proteins of the reticulon and REEP families, among others. These contain intramembrane hairpin domains that can curve ER membrane; loss of these proteins can reduce tubule number and curvature of axonal ER and disrupt network continuity (Perez-Moreno, 2023).

The ER network appears to influence axon survival and maintenance; mutations affecting ER-shaping proteins can lead to the axon degeneration disease, Hereditary Spastic Paraplegia (HSP). HSPs show degeneration of the distal regions of longer upper motor axons, consistent with 'dying back' degeneration from axon terminals, preferentially in the longest upper motor axons. The links between HSP and ER-shaping proteins suggest a critical relationship between ER architecture and axonal and presynaptic maintenance. However, a good model for how ER spatial organization affects axonal or presynaptic function and degeneration is lacking. One way in which ER might do this is as an intracellular Ca2+ store. ER Ca2+ stores can either contribute to presynaptic cytosolic Ca2+ responses or sequester excess Ca2+ from the cytosol in response to high frequency stimulation. ER might potentially also influence presynaptic physiology via its interactions with mitochondria. Like ER, mitochondria can also take up Ca2+ when cytosolic Ca2+ is elevated, although this process appears unable to buffer Ca2+ sufficiently to affect synaptic transmission; rather its role appears to be to stimulate ATP synthesis in response to the Ca2+ influx that occurs on synaptic activity. The cytosol, rather than ER, seems to be the main source of Ca2+ for mitochondria in presynaptic terminals (Perez-Moreno, 2023).

As well as being a bulk Ca2+ source or sink, the ER dynamically responds to lumenal Ca2+ levels. STIM (stromal interacting molecule) proteins in the ER membrane, when activated by depletion of ER Ca2+ stores, interact with effectors in the plasma membrane (PM), such as the Orai1 Ca2+ channel, leading to influx of extracellular Ca2+ into the cytosol (store-operated Ca2+ entry, SOCE) and refilling of ER stores. In primary hippocampal neurons, evoked neurotransmitter release correlates with ER Ca2+ content due to a feedback loop dependent on inhibition of voltage-gated Ca2+ channels in the plasma membrane by activated STIM1. Also in primary hippocampal neurons, SOCE, in a STIM2-dependent manner, can transiently increase presynaptic Ca2+ levels and robustly augment spontaneous glutamate vesicular release (Perez-Moreno, 2023).

Since mutations in several ER-shaping proteins can cause axon degeneration, altered ER architecture may elicit degenerative changes in axonal or presynaptic function. One mechanism could be local dysregulation of Ca2+—for example through reduced availability of Ca2+ for synaptic signaling or capacity to remove cytosolic Ca2+, or interference with regulatory effects of the ER STIM Ca2+ sensors. Presynaptic ER comprises a network of interconnected tubules with occasional cisternae While the presence of cisternae might indicate regions specialized in storing Ca2+, the predominance of narrow ER tubules suggests a local regulatory role for this organelle. To test how spatial organization of ER could impair synaptic function, Drosophila was used to study the effects of removing the tubular ER-shaping protein Rtnl1 on presynaptic ER organization, and on the Ca2+ fluxes between compartments during synaptic activity. Rtnl1 is one of two documented reticulons in Drosophila, orthologous to the four RTNs found in mammals (RTN1-4), including the HSP causative gene RTN2. It is distributed continuously in motor axons all the way to presynaptic termini, and its loss leads to partial depletion of axonal ER. Loss of Rtnl1 greatly decreases Excitatory Junction Potential (EJP) amplitude at the larval neuromuscular junction (NMJ), implying major effects on presynaptic physiology, although its effects on presynaptic ER and its role in presynaptic physiology has not been well defined (Perez-Moreno, 2023).

Loss of Rtnl1 depletes tubular ER but not cisternae or ER volume at NMJs, and how this loss significantly decreases ER, mitochondrial, and cytosolic evoked Ca2+ fluxes. This impact of Rtnl1 loss on presynaptic Ca2+ handling is caused by a decrease of STIM-mediated ER-PM contacts. These results suggest a feedback loop, whereby tubular ER and inactivated STIM control evoked Ca2+ entry into the presynaptic compartment and neurotransmitter release (Perez-Moreno, 2023).

While Rtnl1 loss reduces axonal ER levels mainly in longer motor axons (O'Sullivan, 2012; Yalcin., 2017), this study observed that it reduces presynaptic tubular ER independently of axonal length. This phenotype provides a unique model to study the contribution of the presynaptic tubular ER network and ER-shaping HSP proteins to presynaptic Ca2+ handling. These findings display a local role for presynaptic ER tubules, and how synaptic dysfunction might be a consequence of impairing functions of proteins that are mutated in HSP (Perez-Moreno, 2023).

From a body of work mostly in cultured non-neuronal cells, it is known that the ER controls Ca2+ handling. This role appears conserved in presynaptic ER, where both ER-PM contacts and the amount of ER are critical for neurotransmission. Given the relatively larger Ca2+ storage capacity of ER cisternae, are presynaptic ER tubules contributing to Ca2+ dynamics? In this work, the tubular ER-shaping protein Rtnl1 was disrupted and a decrease of presynaptic ER tubules was found. Rtnl1 loss results in a unique model to study the role of ER tubules at the presynaptic region. This model was used to analyze Ca2+ handling, which relates with neurotransmission and the biological basis of neurological disorders associated with tubular ER (Perez-Moreno, 2023).

Loss of either Rtnl1 or REEPs causes a loss of axonal ER preferentially in longer motor neurons of Drosophila larvae (Yalcin, 2017). In contrast, the decrease of presynaptic ER network in Rtnl1 mutants is independent of axon length. The tubular ER network in presynaptic terminals (mouse nucleus accumbens) is more extensive than in axons. It is therefore possible that presynaptic ER is more dependent on the function of tubular ER-shaping proteins than axonal ER. Regarding the continuity of the network, it is possible that neurons can better tolerate loss of ER tubules in presynaptic terminals than in axons, where there are relatively few tubules to begin with. This might explain why loss of Rtnl1 causes ER network discontinuity in axons (Yalcin, 2017), but not at the presynaptic region (Perez-Moreno, 2023).

Increasing the levels of axonal and presynaptic ER (by blocking neuronal autophagy) correlates with elevated Ca2+ release from ER, which in turn increases neurotransmission. In agreement with this, it was shown that lower ER levels correlate with decreased neurotransmission, indicating that the relationship works conversely. Interestingly, a severe decrease was found in the frequency of miniature neurotransmission in Rtnl1 mutants. Since presynaptic ER tubules, but not cisternae, appear to be reduced in Rtnl1 mutants, it is proposed that tubules help regulate Ca2+ handling across presynaptic compartments. Tubule loss abates Ca2+ handling in the ER, cytosol, and mitochondria, in both types of excitatory NMJs in Drosophila larvae. As neurotransmitter release is triggered by cytosolic Ca2+ influx through PM voltage-gated Ca2+ channels, the decrease of postsynaptic Ca2+ responses and presynaptic cytosolic influx corroborate with each other (Perez-Moreno, 2023).

ER-PM contacts have an inhibitory role in cytosolic Ca2+ influx in primary hippocampal neurons via activated STIM1 on emptying of ER stores; other studies in neurons have demonstrated that emptying of Ca2+ from ER stores (thereby activating STIM) promotes spontaneous (miniature), but not evoked vesicle release at excitatory terminals by the action of STIM2. At excitatory terminals it is not expected that STIM playis an inhibitory role to presynaptic Ca2+, as this study observed a decrease in STIM levels and foci in Rtnl1 mutants, as well as a decrease in evoked Ca2+ fluxes, which were rescued by overexpressing STIM. Work in non-neuronal cells indicate that STIM1/STIM2 promotes Ca2+ entry via coupling with Ca2+ channels on the PM, and in vivo data support a similar relationship in motor neurons for the Drosophila STIM1/STIM2 ortholog STIM. Accordingly, a model is proposed for presynaptic compartments, whereby narrow tubular ER modulates Ca2+ entry via STIM. Other components of the network with higher volume, such as cisternae, would work as a Ca2+ store, since both ER volume and resting ER Ca2+ levels are unaffected in Rtnl1 mutants. Evoked Ca2+ uptake by the ER was also decreased by loss of Rtnl1. While most lumenal GFP::HDEL or GCaMP, and hence Ca2+, appears to be in cisternae that would act as Ca2+ reservoirs, evoked ER Ca2+ uptake is reduced upon depletion of the tubular ER network in both phasic and tonic firing boutons. Moreover, this study saw the tubular network influencing the physiological profile of ER Ca2+, with ER Ca2+ release occurring on stimulation of WT Is boutons, and rarely on stimulation of WT Ib boutons. However, with loss of Rtnl1 and depletion of tubular ER, ER in Ib boutons released Ca2+ during neuronal activity in over half of NMJs tested, and thus caused it to act like smaller Is boutons. The results demonstrate a complex relationship between ER structure, and uptake and release of Ca2+ by the ER, suggesting that intact ER cisternae are not sufficient to maintain full levels of synaptic ER Ca2+ release and uptake, but that ER tubules are necessary as well (Perez-Moreno, 2023).

Rtnl1 loss also decreased evoked presynaptic Ca2+ uptake by mitochondria , similar to the ER and cytosolic compartments. As mitochondrial Ca2+ and ATP production are positively correlated, energy generation in both Type Is and Ib boutons might potentially be impaired on depletion of tubular ER. Mitochondria normally receive Ca2+ directly from ER, although synaptic mitochondria can take up Ca2+ from the cytosol. However, the comparative kinetics of the Ca2+ responses of WT cytosol, ER, and mitochondria do not allow distinguishing between mitochondrial Ca2+ uptake from the cytosol or from ER in this situation (Perez-Moreno, 2023).

Beyond the Ca2+ handling defects characterized in this work, it is proposed that Rtnl1 mutants are useful model to explore other biological roles of presynaptic ER, and to understand which presynaptic ER-related processes might be relevant to the mechanisms of HSP. In addition to Ca2+ handling, presynaptic ER depletion might affect other cellular processes such as autophagy, vesicle trafficking, glucose transport, or lipid metabolism. Although this study did not observe any PI(4,5)P2 alterations in Rtnl1 mutants, it is still possible that other ER-dependent lipids might be affected, since at least some ER-PM contacts seem to be reduced (according to the decreased levels observed for STIM1 foci). Failed lipid transfer at ER-PM contacts has been previously related with reduced expansion of the growth cone and with neuronal PM growth in general, but no any defect was observed in morphology of mature motor neurons in Rtnl1 mutants apart from a slight increase in the number of boutons (Perez-Moreno, 2023).

These results reveal at least three aspects of the Rtnl1 mutant phenotype that could be relevant to the disease mechanisms of HSPs caused by mutations in ER-shaping proteins. First, Rtnl1 mutants show a reduced frequency of miniature neurotransmission. This induces bouton fragmentation in young adult Drosophila so as to resemble that of aged Drosophila, whereas elevated minis can delay age-associated fragmentation and prolong motor ability in adult Drosophila]. Second, Rtnl1 mutants show decreased evoked cytosolic Ca2+ influx and neurotransmission at NMJs, which controls muscle contraction. Third, decreased evoked mitochondrial Ca2+ fluxes predict lower ATP generation capacity and potential energy deficits, although these may be mitigated by the reduced levels of synaptic transmission. These results suggest mechanisms whereby altered organization of ER tubules could be a potential mechanism for diseases including HSP by affecting synaptic Ca2+ handling, and thus synapse function (Perez-Moreno, 2023).

Toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs) sense microbial ligands and initiate signaling to induce inflammatory responses. Although the quality of inflammatory responses is influenced by internalization of TLRs, the role of endosomal maturation in clearing receptors and terminating inflammatory responses is not well understood. This study reports that Drosophila and mammalian Vps33B proteins play critical roles in the maturation of phagosomes and endosomes following microbial recognition. Vps33B was necessary for clearance of endosomes containing internalized PRRs, failure of which resulted in enhanced signaling and expression of inflammatory mediators. Lack of Vps33B had no effect on trafficking of endosomes containing non-microbial cargo. These findings indicate that Vps33B function is critical for determining the fate of signaling endosomes formed following PRR activation. Exaggerated inflammatory responses dictated by persistence of receptors in aberrant endosomal compartments could therefore contribute to symptoms of Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, a disease linked to loss of Vps33B (Akbar, 2016).

Synaptic vesicle (SV) exo- and endocytosis are tightly coupled to sustain neurotransmission in presynaptic terminals, and both are regulated by Ca(2+). Ca(2+) influx triggered by voltage-gated Ca(2+) channels is necessary for SV fusion. However, extracellular Ca(2+) has also been shown to be required for endocytosis. The intracellular Ca(2+) levels (<1 microM) that trigger endocytosis are typically much lower than those (>10 microM) needed to induce exocytosis, and endocytosis is inhibited when the Ca(2+) level exceeds 1 microM. This study identified and characterized a transmembrane protein associated with SVs that, upon SV fusion, localizes at periactive zones. Loss of Flower results in impaired intracellular resting Ca(2+) levels and impaired endocytosis. Flower multimerizes and is able to form a channel to control Ca(2+) influx. It is proposed that Flower functions as a Ca(2+) channel to regulate synaptic endocytosis and hence couples exo- with endocytosis (Yao, 2009).

Ca2+ influx triggers both SV exo- and endocytosis. Since SV retrieval requires much lower Ca2+ levels than those that elicit release, it was proposed that the Ca2+ levels needed to initiate endocytosis derived from diffusion of VGCC-dependent Ca2+ influxes from active zones. However, several reports have documented a requirement for extracellular Ca2+ during endocytosis. Furthermore, it has been proposed that a specific Ca2+ channel is required for SV endocytosis in Drosophila. The present study identified and characterized a SV- and presynaptic membrane-associated protein with three or four transmembrane domains that is evolutionarily conserved but has not been previously characterized in any species. Animals that lack Flower display the typical features of endocytic mutants. These include supernumerary boutons, a low number of SVs in boutons at rest, a severe depletion of SVs upon stimulation (except at active zones), enlarged SVs, a decrease in FM1-43 uptake, a rundown in neurotransmitter release upon repetitive stimulation, and an accumulation of endocytic intermediates (Yao, 2009).

flower mutants exhibit impaired Ca2+ handling, even when the boutons are at rest. This argues that Flower plays a role in Ca2+ homeostasis at rest as well as during endocytosis. Since Flower is associated with SVs and the presynaptic membrane, a reduction in Flower levels may cause lower resting Ca2+ levels because Ca2+ efflux from SVs or Ca2+ influx from the extracellular compartment is impaired. However, a role for Flower in SVs is unlikely. First, experimental evidence suggests that SVs have no or a very limited role in the sequestration of Ca2+ at NMJs in Drosophila. Second, although single SV fusions with the plasma membrane in flower mutants elicit larger amplitudes than in wild-type animals, these data suggest that this is due to the fact that the SVs in flower mutants are larger than wild-type SVs. Indeed, there is a near perfect correlation between the SV size and the size of the mEJPs, as observed in some other endocytic mutants. This suggests that the Flower protein present in the presynaptic membrane affects the resting Ca2+ levels, but does not exclude a role for the protein in SVs (Yao, 2009).

The second TM domain of Flower contains a 9 aa motif similar to the selectivity filters identified in Ca2+-gating TRPV channels. TRPV5 and 6 channels are homo- or multimeric channels that have been shown to form a pore lined by four negatively charged amino acids, similar to VGCCs. This study shows that Flower can form tetramers and higher order multimers and that its heterologous expression in salivary gland cells promotes Ca2+ influx. In addition, substitution of a conserved, negatively charged glutamate (E) residue in the second TM domain with a neutral amino acid (Q) abolishes this Ca2+ influx. Furthermore, the purified Flower protein in proteoliposomes can form a Ca2+-permeable cationic channel. A simple model is proposed to account for the data. SV exocytosis is triggered by VGCCs located at active zones. Subsequently, SVs, and hence Flower proteins are integrated in the presynaptic membrane, where they mostly localize to periactive zones, known sites for endocytosis). A homomultimeric Flower complex then promotes Ca2+ influx which triggers clathrin-mediated endocytosis. This is also supported by the observation that higher extracellular Ca2+ alleviates the endocytic defect. It is proposed that endocytosis removes most, but not all, of the Flower protein, thereby removing a key trigger for endocytosis. Thus, Flower may perform a simple autoregulatory role for itself during endocytosis. This model also addresses how exo-endocytosis coupling may be mediated at presynaptic terminals. The remainder of the Flower protein that is not endocytosed may help to regulate basal Ca2+ levels (Yao, 2009).

In principle, SPAIM could be applied to any fluorescent organelle. However, a dual scanner confocal microscope, which was used in this study, might not be ideal for detecting very dim signals. When optical sectioning is not required, this limitation could be overcome by using a single scanner system for initially photobleaching a region of interest and then for continually photobleaching newcomers while simultaneously imaging individual organelles with a sensitive camera. In fact, performing SPAIM experiments by coupling a camera with a common single scanner confocal microscope may be an attractive option to overcome both the insensitivity and the expense of dual scanner systems. With the appropriate setup, SPAIM could be used to study traffic in terminals, dendrites, cilia, and filopodia (Wong, 2012)

Distinct pools of the bone morphogenetic protein (BMP) Glass bottom boat (Gbb) control structure and function of the Drosophila neuromuscular junction. Specifically, motoneuron-derived Gbb regulates baseline neurotransmitter release, whereas muscle-derived Gbb regulates neuromuscular junction growth. Yet how cells differentiate between these ligand pools is not known. This study presents evidence that the neuronal Gbb-binding protein Crimpy (Cmpy) permits discrimination of pre- and postsynaptic ligand by serving sequential functions in Gbb signaling. Cmpy first delivers Gbb to dense core vesicles (DCVs) for activity-dependent release from presynaptic terminals. In the absence of Cmpy, Gbb is no longer associated with DCVs and is not released by activity. Electrophysiological analyses demonstrate that Cmpy promotes Gbb's proneurotransmission function. Surprisingly, the Cmpy ectodomain is itself released upon DCV exocytosis, arguing that Cmpy serves a second function in BMP signaling. In addition to trafficking Gbb to DCVs, it is proposed that Gbb/Cmpy corelease from presynaptic terminals defines a neuronal protransmission signal (James, 2014).

Local endosomal recycling at synapses is essential to maintain neurotransmission. Rab4 GTPase, found on sorting endosomes, is proposed to balance the flow of vesicles among endocytic, recycling, and degradative pathways in the presynaptic compartment. This study reports that Rab4-associated vesicles move bidirectionally in Drosophila axons but with an anterograde bias, resulting in their moderate enrichment at the synaptic region of the larval ventral ganglion. Results from FK506 binding protein (FKBP) and FKBP-Rapamycin binding domain (FRB) conjugation assays in rat embryonic fibroblasts together with genetic analyses in Drosophila indicate that an association with Kinesin-2 (mediated by the tail domain of Kinesin-2α/KIF3A/">KLP64D subunit) moves Rab4-associated vesicles toward the synapse. Reduction in the anterograde traffic of Rab4 causes an expansion of the volume of the synapse-bearing region in the ventral ganglion and increases the motility of Drosophila larvae. These results suggest that Rab4-dependent vesicular traffic toward the synapse plays a vital role in maintaining synaptic balance in this neuronal network (Dey, 2017).

The apical compartment of a neuronal cell extends into specific processes forming axons and synapses that are maintained by a distributed endosomal system. For example, vesicle recycling at the synapse, orchestrated by various RabGTPases and associated proteins, renews the readily releasable pool of synaptic vesicles sustaining the neurotransmission. Rab4 is thought to play an important role in balancing the vesicle traffic between the recycling and degradation pathways involved in various cellular processes such as metabolism, cell secretion, and antigen processing. Recruitment of Rab4 on sorting intermediate endosomes allows the transfer of vesicular cargoes from early endosomes to recycling endosomes in axons and at synapses. The activity of Rab4 has also been implicated in the progression of growth cone in Xenopus and the maintenance of dendritic spines in rat hippocampal neurons. Furthermore, endosomal abnormalities found in the cholinergic basal forebrain of patients with Alzheimer's disease correlate with elevated levels of Rab4. All these reports suggest that Rab4-dependent vesicle sorting is essential for the development and maintenance of the nervous system (Dey, 2017).

Vesicular cargoes, including the RabGTPases, originate at the trans-Golgi network, and microtubule-dependent motors transport them to different destinations within a cell. Specific association with motors ensures differential and dynamic subcellular localizations of various RabGTPases according to tissue-specific activities and metabolic demands. For instance, the anterograde trafficking of early, sorting, and late endosomes is dependent on the Kinesin-3 motors KIF13, KIF16B, and KIF1A/IBβ, respectively. Similarly, in Drosophila, Rab5-positive early endosomes associate with Khc-73, which is the ortholog of mammalian KIF13, showing a conserved machinery of Kinesin-Rab interaction. The same vesicles also associate with Dynein for their retrograde movement. It is further indicated that multiple RabGTPases can be grouped together and transported to a destination within a cell where they can engage in different functions. Although the long-range transport of certain presynaptic RabGTPases has been studied in cultured neurons, it is still unclear how the bidirectional movement of Rab-associated vesicles could localize them in distant compartments such as the synapse (Dey, 2017).

This study has shown that Rab4-associated vesicles are transported with an anterograde bias, induced by a specific interaction with the tail domain of Kinesin-2α subunit, which results in its moderate enrichment at the synapses. The rate of pre-synaptic inflow of Rab4 is positively correlated with its activation. Interestingly, this study found that a decreased flow of the Rab4-associated vesicles expanded the region occupied by synapses in the neuropil region of the larval ventral ganglion and enhanced larval motility. Altogether, these results indicate that the flow of Rab4-associated vesicles could maintain synaptic homeostasis in a neuronal network (Dey, 2017).

Heterotrimeric Kinesin-2, implicated in the trafficking of apical endosomes and late endosomal vesicles, was initially thought to bind to its cargoes through the accessory subunit KAP3. However, recent studies have reported that motor subunits could independently interact with soluble proteins through the tail domain. This study shows that the Kinesin-2α tail can also bind to a membrane-associated protein, Rab4. A preliminary investigation in the lab suggests that the interaction in not direct. The RabGTPases are known to recruit a variety of different motors to the membrane, directing intracellular trafficking. Although a previous report indicated that Rab4 could bind to Kinesin-2, this study found that such an interaction leads to the anterograde trafficking of Rab4-associated vesicles in the axon. Rab4 associates with early and recycling endosomes carrying a diverse range of proteins. The FRB-FKBP assay indicated that a particular subset of Rab4-only vesicles associates with Kinesin-2. These vesicles predominantly move toward the synapse in axons. Although Kinesin-2 is also found to bind and transport vesicles containing Acetylcholinesterase (AChE) in the same axon, it was observed that Rab4 does not mark them. Similarly, the post-Golgi vesicles carrying N-cadherin and β-catenin, which are known to associate with Kinesin-2, exclude Rab4. Therefore, the Rab4-associated vesicles transported in the axon are likely to contain a unique set of proteins (Dey, 2017).

An association between RabGTPases and putative transport proteins was identified using coimmunoprecipitation, pull-down, and yeast two-hybrid assays. While these assays provide information on the possible interactions, their nature in a cellular or physiological scenario is unknown. Long-range transport of the Rab27/CRMP2 complex by Kinesin-1 and the Rab3/DENN MADD complex by KIF1A/1Bβ are substantiated by genetic perturbations and imaging. The FKBP-FRB-inducible interaction system used in this study identified Kinesin-2α and KIF13A/B as the key motors for nascent Rab4-associated vesicles. The latter was also shown to associate with Rab5-marked early endosomes. The results excluded the possibility that the Rab4 effectors involved in this interaction could bind to Rab5 and Rab11. In vivo analysis of Rab4-associated vesicular traffic further confirmed that heterotrimeric Kinesin-2 plays a dominant role in the trafficking of nascent Rab4 vesicles toward the synapse (Dey, 2017).

Anterograde axonal movement of Rab4 vesicles is mostly mediated by Kinesin-2, which transports nascent Rab4 vesicles, whereas Kinesin-3 transports sorting endosomes. In a recent study, it was shown that combined activity of Kinesin-1 and Kinesin-2 steers AChE-containing vesicles in the axon (Kulkarni, 2016). A simultaneous association between these two motors was suggested to induce longer runs of AChE-containing vesicles. A similar collaboration between Khc-73 and Kinesin-2 could propel longer runs of Rab4 vesicles. Khc-73 has been implicated in neuronal functions. Khc-73 knockdown in lch5 neurons decreased the motility of Rab4-associated vesicles to a limited extent, which suggests that the motor is only engaged in transporting a relatively small fraction of Rab4-associated vesicles in axons. Khc-73 is a processive motor with an in vitro velocity of 1.54 ± 0.46 &mi;m/s, and this study found that Khc-73 RNAi particularly affected the longer runs. Thus, Khc-73 could support the relatively longer runs (3.05 &mi;m) of Rab4 vesicles in axons. It was previously shown that Rab5-associated vesicles bind to KIF13A/B/Khc-73. Hence, it is inferred that sorting endosomes, positive for both Rab4 and Rab5, can engage both Kinesin-2 and KIF13A/Khc-73 through the cognate adaptors of these two Rabs. However, one needs suitably tagged reagents to establish this conjecture through direct observation in vivo (Dey, 2017).

Local synaptic vesicle recycling mediated by RabGTPases is critical for the availability of fusion-ready synaptic vesicles to sustain neurotransmission. The machinery involved in the translocation of RabGTPases from cell bodies to the synapses is poorly understood. Certain RabGTPases such as Rab3 and Rab27 are known to associate with Kinesin-3 and Kinesin-1, respectively, for their anterograde axonal transport. Rab4 is a protein present on the endosomal sorting intermediates, and it allows for the transfer of cargoes from early endosomes to recycling endosomes. As Rab4 is present in both early and recycling endosomal populations, the logistics of Rab4 transport into the presynaptic terminal has been unclear. The current results show that Rab4-associated vesicles are present throughout the neuron and moderately enriched in the synapse, which is a consequence of a small anterograde bias in their transport (Dey, 2017).

The activity of Rab4 is critical for the physiology of the neuron-like extension of the growth cone. The overexpressed levels and activity of Rab4 found in conjunction with neuropathy, such as Alzheimer's disease, suggest that overactivation of the endosomal system could influence disease progression. In Drosophila, Rab4 is expressed in both neuronal and non-neuronal tissues throughout development with the exception of optic lobe neurons of the pupal brain. These studies project the idea that the expression and activity of Rab4 are crucial for the maintenance of neuronal physiology. This study has shown that the synaptic Rab4 is maintained through axonal transport by heterotrimeric Kinesin-2 and Kinesin-3. As the synaptic localization of Rab4 is dependent on its active form, other factors like PI3K are also implicated in this trafficking. The results further suggest that the function and localization of Rab4 at axon termini are critical for the maintenance of synaptic balance at ventral ganglion. Overactivation of Rab4 in cholinergic neurons reduced the synapse-bearing region of the ventral ganglion, although levels of the marker, Brp, remained unchanged. This observation suggests that local Rab4 activity at the axon termini suppresses synapse formation. Consistent with this conjecture, this study found that the synapse-bearing zone at the neuropil expands when Rab4DN is overexpressed. Although the current data are insufficient to explain the underlying mechanism, they indicate that alteration of the trafficking logistics driven by heterotrimeric Kinesin-2 and Kinesin-3 family motors might play an essential role in the development of the defect. Further analysis of larval behavior and neuronal stability in the ventral ganglion with aging would be useful in uncovering the mechanism (Dey, 2017).

Vesicular acetylcholine transporter (VAChT) function is essential for organismal survival, mediating the packaging of acetylcholine (ACh) for exocytotic release. However, its expression pattern in the Drosophila brain has not been fully elucidated. To investigate the localization of VAChT, an antibody against the C terminal region of the protein was developed; this antibody recognizes a 65KDa protein corresponding to VAChT on an immunoblot in both Drosophila head homogenates and in Schneider 2 cells. Further, the expression is reported of VAChT in the antennal lobe and ventral nerve cord of Drosophila larva, and the expression was confirmed of the protein in mushroom bodies and optic lobes of adult Drosophila. Importantly, it was shown that VAChT co-localizes with a synaptic vesicle marker in vivo, confirming previous reports of the localization of VAChT to synaptic terminals. Together, these findings help establish the vesicular localization of VAChT in cholinergic neurons in Drosophila and presents an important molecular tool with which to dissect the function of the transporter in vivo (Boppana, 2017).

Experimental evidence shows that neurotransmitter release, from presynaptic terminals, can be regulated by altering transmitter load per synaptic vesicle (SV) and/or through change in the probability of vesicle release. The vesicular acetylcholine transporter (VAChT) loads acetylcholine into SVs at cholinergic synapses. This study investigated how the VAChT affects SV content and release frequency at central synapses in Drosophila melanogaster by using an insecticidal compound, 5Cl-CASPP, to block VAChT and by transgenic overexpression of VAChT in cholinergic interneurons. Decreasing VAChT activity produces a decrease in spontaneous SV release with no change to quantal size and no decrease in the number of vesicles at the active zone. This suggests that many vesicles are lacking in neurotransmitter. Overexpression of VAChT leads to increased frequency of SV release, but again with no change in quantal size or vesicle number. This indicates that loading of central cholinergic SVs obeys the "set-point" model, rather than the "steady-state" model that better describes loading at the vertebrate neuromuscular junction. However, this study shows that expression of a VAChT polymorphism lacking one glutamine residue in a COOH-terminal polyQ domain leads to increased spontaneous SV release and increased quantal size. This effect spotlights the poly-glutamine domain as potentially being important for sensing the level of neurotransmitter in cholinergic SVs (Cash, 2016).

Using a Drosophila central synapse, this study has investigated in vivo how VAChT regulates cholinergic transmission. Decreased VAChT activity was shown to leads to decreased spontaneous quantal release frequency. Increased VAChT activity, by contrast, leads to increased frequency of spontaneous release with no change to amplitude or number of SVs at the active zone, suggestive of an increased probability of SV release (Cash, 2016).

Decreased functional VAChT causes a reduction in spontaneous quantal release frequency but not quantal size. This is in agreement with studies at Drosophila and snake NMJs, where decreased vesicular transporter results in decreased frequency but not amplitude of miniature excitatory junctional potentials. However, many studies in mice and rats also link decreased VAChT with decreased transmitter load. A possible explanation for this apparent difference is that Drosophila cholinergic SVs usually contain only one VAChT, and so each SV is either loaded with ACh or empty if the VAChT is blocked by 5Cl-CASPP. This stduy reports no change in vesicle number or size at the active zone, which suggests that there may be empty vesicles that undergo recycling, as has been previously reported. This is supported by a decrease in total quanta released after bafilomycin treatment (Cash, 2016).

When VAChT activity is increased, a clear increase is seen in frequency with no change in amplitude of spontaneous quantal release. An increase in mini frequency with increased VAChT is in good agreement with a study in Xenopus spinal neurons. Moreover, VGLUT overexpression at the Drosophila NMJ results in a modest increase in quantal release (Daniels, 2004). However, these studies, and another in rat, consistently report an increase in transmitter load of the SVs, which supports the steady-state model of SV filling. Based on the current observations, SV loading of ACh at central synapses in Drosophila is consistent with a set-point model, which caps the amount of uptake regardless of transporter activity (Williams, 1997). Williams suggests the set point model is achieved through a feedback mechanism. The observation that, following removal of a single glutamine residue from a 13-glutamine polyQ domain, mini amplitude is significantly increased indicates that this region may be involved in that feedback mechanism. An alternative is that VAChT-Q, which is a naturally occurring polymorphism, obeys the steady-state model. Either way, it would seem that the current data do not support one clear mechanism, raising the possibility that SV filling may differ between species and perhaps even within a species where such polymorphisms are expressed (Cash, 2016).

This study further showed that increased spontaneous release frequency is not likely caused by an increase in the number of vesicles at the active zone or active zone density. This corroborates previous work that shows VAChT plays a second role: facilitating SV mobilization or fusion. In Caenorhabditis elegans, an interaction between VAChT and SV release machinery has been reported (Sandoval, 2006). A glycine-to-arginine amino acid change at position 347 disrupts an interaction with synaptobrevin, a vesicle-associated membrane protein that is pivotal for exocytosis. The glycine at position 347 is well conserved and is present in Drosophila VAChT. However, a glycine-to-arginine substitution at this position in the Drosophila VAChT seemingly results in a nonfunctional protein that cannot rescue the VAChT null (Cash, 2016).

This study presents supporting data for a dual role for VAChT in SV filling and release. Moreover, it provides evidence to suggest that the polyQ domain of Drosophila VAChT has a role in determining SV transmitter load. Mammals, including humans, rats, and mice, have a di-leucine motif at residues 485-486 within the cytoplasmic COOH-terminal. This di-leucine motif has been reported to be important for localizing VAChT to the SV membrane and also to play a role in endocytosis after neurotransmitter release through an interaction with the AP-2 complex. Drosophila VAChT does not have a di-leucine motif, but, unlike the mammalian VAChT sequences mentioned, has a 13-residue polyQ domain at the COOH-terminal. Extended polyQ domains are associated with diseases such as Huntington's and spinocerebellar ataxia. Little is known about the normal function of polyQ domains, but functions may include protein-protein interactions, transcriptional regulation, and RNA binding and signaling. This poses the possibility that the polyQ domain may be responsible for VAChT localization and endocytosis in Drosophila. It has been suggested that the number of glutamines may be of importance. It is possible, therefore, that VAChT-Q may be transported more efficiently to the SV membrane. In D. melanogaster the VAChT-Q tested in this study is a naturally occurring polymorphism identified during cloning of the Drosophila VAChT and subsequently confirmed by sequencing PCR products from cDNA libraries (Cash, 2016 and references therein).

A BLAST search for similar amino acid sequences and predicted sequences of VAChT found that many Drosophila species contain polyQ domains in the same region as D. melanogaster, but that the length varied from 15 in D. pseudoobscura to 5 in D. grimshawi. Other insects that also have a polyQ domain in the same region include the housefly Musca domestica, which has a nine-residue polyQ domain, and three Anopheles mosquitoes (A. sinensis, A. gambiae, and A. darlingi with 7, 10, and 7 glutamines, respectively). Ant, moth, bee, and butterfly species found during the search did not contain a polyQ of more than two glutamines in the same region. The presence of the polyQ domain in three malaria-transmitting mosquito species but not other insects identifies this region as a possible target for insecticides to control these disease-carrying insects (Cash, 2016).

Throughout the animal kingdom, GABA is the principal inhibitory neurotransmitter of the nervous system. It is essential for maintaining the homeostatic balance between excitation and inhibition required for the brain to operate normally. Identification of GABAergic neurons and their GABA release sites are thus essential for understanding how the brain regulates the excitability of neurons and the activity of neural circuits responsible for numerous aspects of brain function including information processing, locomotion, learning, memory, and synaptic plasticity, among others. Since the structure and features of GABA synapses are critical to understanding their function within specific neural circuits of interest, this study developed and characterized a conditional marker of GABAergic synaptic vesicles for Drosophila, 9XV5-vGAT. 9XV5-vGAT is validated for conditionality of expression, specificity for localization to synaptic vesicles, specificity for expression in GABAergic neurons, and functionality. Its utility for GABAergic neurotransmitter phenotyping and identification of GABA release sites was verified for ellipsoid body neurons of the central complex. In combination with previously reported conditional SV markers for acetylcholine and glutamate, 9XV5-vGAT was used to demonstrate fast neurotransmitter phenotyping of subesophageal ganglion neurons. This method is an alternative to single cell transcriptomics for neurotransmitter phenotyping and can be applied to any neurons of interest represented by a binary transcription system driver. A conditional GABAergic synaptic vesicle marker has been developed and validated for GABA neurotransmitter phenotyping and subcellular localization of GABAergic synaptic vesicles (Certel, 2022).

Previous studies have demonstrated the striking mutational effects of the Drosophila planar cell polarity gene prickle (pk) on larval motor axon microtubule-mediated vesicular transport and on adult epileptic behavior associated with neuronal circuit hyperexcitability. Mutant alleles of the prickle-prickle (pk(pk)) and prickle-spiny-legs (pk(sple)) isoforms (hereafter referred to as pk and sple alleles, respectively) exhibit differential phenotypes. While both pk and sple affect larval motor axon transport, only sple confers motor circuit and behavior hyperexcitability. However, mutations in the two isoforms apparently counteract to ameliorate adult motor circuit and behavioral hyperexcitability in heteroallelic pk/pk/pk(sple) flies. The consequences of altered axonal transport was further investigated in the development and function of the larval neuromuscular junction (NMJ). Robust dominant phenotypes were uncovered in both pk and sple alleles, including synaptic terminal overgrowth (as revealed by anti-HRP and -Dlg immunostaining) and poor vesicle release synchronicity (as indicated by synaptic bouton focal recording). However, recessive alteration of synaptic transmission was observed only in pk/pk larvae, i.e. increased excitatory junctional potential (EJP) amplitude in pk/pk but not in pk/+ or sple/sple. Interestingly, for motor terminal excitability sustained by presynaptic Ca(2+) channels, both pk and sple exerted strong effects to produce prolonged depolarization. Notably, only sple acted dominantly whereas pk/+ appeared normal, but was able to suppress the sple phenotypes, i.e. pk/sple appeared normal. Thesd observations contrast the differential roles of the pk and sple isoforms and highlight their distinct, variable phenotypic expression in the various structural and functional aspects of the larval NMJ (Ueda, 2022).

Both peroxisomes and lipid droplets regulate cellular lipid homeostasis. Direct inter-organellar contacts as well as novel roles for proteins associated with peroxisome or lipid droplets occur when cells are induced to liberate fatty acids from lipid droplets. This study has shown a non-canonical role for a subset of peroxisome-assembly [Peroxin (Pex)] proteins in this process in Drosophila. Transmembrane proteins Pex3, Pex13 and Pex14 were observed to surround newly formed lipid droplets. Trafficking of Pex14 to lipid droplets was enhanced by loss of Pex19, which directs insertion of transmembrane proteins like Pex14 into the peroxisome bilayer membrane. Accumulation of Pex14 around lipid droplets did not induce changes to peroxisome size or number, and co-recruitment of the remaining Peroxins was not needed to assemble peroxisomes observed. Increasing the relative level of Pex14 surrounding lipid droplets affected the recruitment of Hsl lipase. Fat body-specific reduction of these lipid droplet-associated Peroxins caused a unique effect on larval fat body development and affected their survival on lipid-enriched or minimal diets. This revealed a heretofore unknown function for a subset of Pex proteins in regulating lipid storage (Ueda, 2022).

A number of studies have implicated genes that function in endocytosis and endosomal protein sorting as tumor suppressors in human cancers. Most well known is Tsg101, as early studies showed that downregulation of Tsg101 (see Drosophila TSG101) promotes the growth of mouse 3T3 fibroblasts in soft aga. When these cells were injected into nude mice, they formed metastatic tumors. However, later studies have shown conflicting results, and it is still unclear if Tsg101 functions as a tumor suppressor in metazoans. Importantly, a number of studies have shown changes in expression of ESCRT components in human cancer cells, including changes in expression of ESCRT-I components Tsg101 and Vps37A and ESCRT-III components Chmp1A and CHMP3. Since the primary proteins that function in endocytosis and endosomal trafficking are conserved from yeast to humans, it is likely that these findings in Drosophila may have important implications for human disease (Woodfield, 2013).

Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. This study shows that ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC) division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, this study shows that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. It is concluded that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo (Eikenes, 2015).

Cytokinesis is the final step of cell division that leads to the physical separation of the two daughter cells. It is tightly controlled in space and time and proceeds in multiple steps via sequential specification of the cleavage plane, assembly and constriction of the actomyosin-based contractile ring (CR), formation of a thin intercellular bridge and finally abscission that separates the two daughter cells. Studies in a variety of model organisms and systems have elucidated key machineries and signals governing early events of cytokinesis. However, the mechanisms of the final abscission step of cytokinesis are less understood, especially in vivo in the context of different cell types in a multi-cellular organism (Eikenes, 2015).

During the recent years key insights into the molecular mechanisms and spatiotemporal control of abscission have been gained using a combination of advanced molecular biological and imaging technologies. At late stages of cytokinesis the spindle midzone transforms to densely packed anti-parallel microtubules (MTs) that make up the midbody (MB) and the CR transforms into the midbody ring (MR, diameter of ~1-2 μm). The MR is located at the site of MT overlap and retains several CR components including Anillin, septins (Septins 1, 2 and Peanut in Drosophila melanogaster), myosin-II, Citron kinase (Sticky in Drosophila) and RhoA (Rho1 in Drosophila) and eventually also acquires the centralspindlin component MKLP1 (Pavarotti in Drosophila). In C. elegans embryos the MR plays an important role in scaffolding the abscission machinery even in the absence of MB MTs (Eikenes, 2015).

Studies in human cell lines, predominantly in HeLa and MDCK cells, have shown that components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins play important roles in mediating abscission. Abscission occurs at the thin membrane neck that forms at the constriction zone located adjacent to the MR. An important signal for initiation of abscission is the degradation of the mitotic kinase PLK1 (Polo-like kinase 1) that triggers the targeting of CEP55 (centrosomal protein of 55 kDa) to the MR. CEP55 interacts directly with GPP(3x)Y motifs in the ESCRT-associated protein ALIX (ALG-2-interacting protein X) and in the ESCRT-I component TSG101, thereby recruiting them to the MR. ALIX and TSG101 in turn recruit the ESCRT-III component CHMP4B, which is followed by ESCRT-III polymerization into helical filaments that spiral/slide to the site of abscission. The VPS4 ATPase is thought to promote ESCRT-III redistribution toward the abscission site. Prior to abscission ESCRT-III/CHMP1B recruits Spastin that mediates MT depolymerization at the abscission site. ESCRT-III then facilitates membrane scission of the thin membrane neck, thereby mediating abscission (Eikenes, 2015).

Cytokinesis is tightly controlled by the activation and inactivation of mitotic kinases at several steps to ensure its faithful spatiotemporal progression. Cytokinesis conventionally proceeds to completion via abscission, but is differentially controlled depending on the cell type during the development of metazoan tissues. For example, germ cells in species ranging from insects to humans undergo incomplete cytokinesis leading to the formation of germline cysts in which cells are interconnected via stable intercellular bridges. How cytokinesis is modified to achieve different abscission timing in different cell types is not well understood, but molecular understanding of the regulation of the abscission machinery has started giving some mechanistic insight (Eikenes, 2015).

The Drosophila female germline represents a powerful system to address mechanisms controlling cytokinesis and abscission in vivo. Each Drosophila female germline stem cell (fGSC) divides asymmetrically with complete cytokinesis to give rise to another fGSC and a daughter cell cystoblast (CB). Cytokinesis during fGSC division is delayed so that abscission takes place during the G2 phase of the following cell cycle (about 24 hours later). The CB in turn undergoes four mitotic divisions with incomplete cytokinesis giving rise to a 16-cell cyst in which the cells remain interconnected by stable intercellular bridges called ring canals (RCs). One of the 16 cells with four RCs will become specified as the oocyte and the cyst becomes encapsulated by a single layer follicle cell epithelium to form an egg chamber. Drosophila male GSCs (mGSCs) also divide asymmetrically with complete cytokinesis to give rise to another mGSC and a daughter cell gonialblast (GB). Anillin, Pavarotti, Cindr, Cyclin B and Orbit are known factors localizing at RCs/MRs and/or MBs during complete cytokinesis in fGSCs and/or mGSCs. It has been recently reported that Aurora B delays abscission and that Cyclin B promotes abscission in Drosophila germ cells and that mutual inhibitions between Aurora B and Cyclin B/Cdk-1 control the timing of abscission in Drosophila fGSCs and germline cysts. However, little is known about further molecular mechanisms controlling cytokinesis and abscission in Drosophila fGSCs (Eikenes, 2015).

This study has characterize the roles of ALIX and the ESCRT-III component Shrub during cytokinesis in Drosophila fGSCs. ALIX and Shrub are required for completion of abscission in fGSCs. They co-localize during this process, and their direct interaction is required for abscission with normal kinetics. This study thus shows that a complex between ALIX and Shrub is required for abscission in fGSCs and provide evidence of an evolutionarily conserved functional role of the ALIX/ESCRT-III pathway in mediating cytokinetic abscission in the context of a multi-cellular organism (Eikenes, 2015).

Loss of ALIX or/and Shrub function or inhibition of their interaction delays abscission in fGSCs leading to the formation of stem cysts in which the fGSC remains interconnected to chains of daughter cells via MRs. As abscission eventually takes place a cyst of e.g. 2 germ cells may pinch off and subsequently undergo four mitotic divisions to give rise to a germline cyst with 32 germ cells. Consistently, loss of ALIX or/and Shrub or interference with their interaction caused a high frequency of egg chambers with 32 germ cells during Drosophila oogenesis. It was also found that ALIX controls cytokinetic abscission in both fGSCs and mGSCs and thus that ALIX plays a universal role in cytokinesis during asymmetric GSC division in Drosophila. Taken together this study provides evidence that the ALIX/ESCRT-III pathway is required for normal abscission timing in a living metazoan tissue (Eikenes, 2015).

The results together with findings in other models underline the evolutionary conservation of the ESCRT system and associated proteins in cytokinetic abscission. Specifically, ESCRT-I or ESCRT-III have been implicated in abscission in a subset of Archaea (ESCRT-III), in A. thaliana (elch/tsg101/ESCRT-I) and in C. elegans (tsg101/ESCRT-I). In S. cerevisiae, Bro1 (ALIX) and Snf7 (CHMP4/ESCRT-III) have also been suggested to facilitate cytokinesis. In cultured Drosophila cells, Shrub/ESCRT-III mediates abscission and in human cells in culture ALIX, TSG101/ESCRT-I and CHMP4B/ESCRT-III promote abscission. ALIX and the ESCRT system thus act in an ancient pathway to mediate cytokinetic abscission (Eikenes, 2015).

Despite the fact that an essential role of ALIX in promoting cytokinetic abscission during asymmetric GSC division was found in the Drosophila female and male germlines, strong bi-nucleation directly attributed to cytokinesis failure was found in Drosophila alix mutants in the somatic cell types that were examined. This might have multiple explanations. One possibility is that maternally contributed alix mRNA may support normal cytokinesis and development. Whereas ALIX and CHMP4B depletion in cultured mammalian cells causes a high frequency of bi- and multi-nucleation it is also possible that cells do not readily become bi-nucleate upon failure of the final step of cytokinetic abscission in the context of a multi-cellular organism. Consistent with the observations of a high frequency of stem cysts upon loss of ALIX and Shrub in the germline, Shrub depletion in cultured Drosophila cells resulted in chains of cells interconnected via intercellular bridges/MRs due to multiple rounds of cell division with failed abscission. Moreover, loss of ESCRT-I/tsg101 function in the C. elegans embryo did not cause furrow regression. These and the current observations suggest that ALIX- and Shrub/ESCRT-depleted cells can halt and are stable at the MR stage for long periods of time and from which cleavage furrows may not easily regress, at least not in these cell types and in the context of a multi-cellular organism. It is also possible that redundant mechanisms contribute to abscission during symmetric cytokinesis in somatic Drosophila cells. Further studies should address the general involvement of ALIX and ESCRT-III in cytokinetic abscission in somatic cells in vivo (Eikenes, 2015).

Different cell types display different abscission timing, intercellular bridge morphologies and spatiotemporal control of cytokinesis. In fGSCs it was found that ALIX and Shrub co-localize throughout late stages of cytokinesis and abscission. In human cells ALIX localizes in the central region of the MB, whereas CHMP4B at first localizes at two cortical ring-like structures adjacent to the central MB region and then progressively distributes also at the constriction zone where it promotes abscission. ALIX and CHMP4B are thus found at discrete locations within the intercellular bridge as cells approach abscission in human cultured cells. In contrast, ESCRT-III localizes to a ring-like structure during cytokinesis in Archaea, resembling the Shrub localization at MRs was observed in Drosophila fGSCs. Moreover, ALIX and Shrub are present at MRs for a much longer time (from G1/S) prior to abscission (in G2) in fGSCs than in human cultured cells. Here, ALIX and CHMP4B are increasingly recruited about an hour before abscission and then CHMP4B acutely increases at the constriction zones shortly (~30 min) before the abscission event (Eikenes, 2015).

How may ALIX and Shrub be recruited to the MR/MB in Drosophila cells in the absence of CEP55 that is a major recruiter of ALIX and ultimately CHMP4/ESCRT-III in human cells? Curiously, a GPP(3x)Y consensus motif was detected within the Drosophila ALIX sequence (GPPPGHY, aa 808-814) resembling the CEP55-interacting motif in human ALIX (GPPYPTY, aa 800-806). Whether Drosophila ALIX is recruited to the MR/MB via a protein(s) interacting with this motif or other domains is presently uncharacterized. Accordingly, alternative pathways of ALIX and ESCRT recruitment have been reported, as well as suggested in C. elegans, where CEP55 is also missing. Further studies are needed to elucidate mechanisms of recruitment and spatiotemporal control of ALIX and ESCRT-III during cytokinesis in fGSCs and different cell types in vivo (Eikenes, 2015).

This study found that the direct interaction between ALIX and Shrub is required for completion of abscission with normal kinetics in fGSCs. This is consistent with findings in human cells in which loss of the interaction between ALIX and CHMP4B causes abnormal midbody morphology and multi-nucleation. Following ALIX-mediated recruitment of CHMP4B/ESCRT-III to cortical rings adjacent to the MR in human cells, ESCRT-III extends in spiral-like filaments to promote membrane scission. Due to the discrete localizations of ALIX and CHMP4B during abscission in human cells ALIX has been proposed to contribute to ESCRT-III filament nucleation. In vitro studies have shown that the interaction between ALIX and CHMP4B may release autoinhibitory intermolecular interactions within both proteins and promote CHMP4B polymerization. Specifically, ALIX dimers can bundle pairs of CHMP4B filaments in vitro [65]. Moreover, in yeast, the interaction of the ALIX homologue Bro1 with Snf7 (CHMP4 homologue) enhances the stability of ESCRT-III polymers. There is a high degree of evolutionary conservation of ALIX and ESCRT-III proteins and because ALIX and Shrub co-localize and interact to promote abscission in fGSCs it is possible that ALIX can facilitate Shrub filament nucleation and/or polymerization during this process (Eikenes, 2015).

The current findings indicate that accurate control of the levels and interaction of ALIX and Shrub ensure proper abscission timing in fGSCs. Their reduced levels or interfering with their complex formation caused delayed abscission kinetics. How cytokinesis is modified to achieve a delay in abscission in Drosophila fGSCs and incomplete cytokinesis in germline cysts is not well understood. Aurora B plays an important role in controlling abscission timing both in human cells and the Drosophila female germline. During Drosophila germ cell development Aurora B contributes to mediating a delay of abscission in fGSCs and a block in cytokinesis in germline cysts. Bam expression has also been proposed to block abscission in germline cysts. It will be interesting to investigate mechanisms regulating the levels, activity and complex assembly of ALIX and Shrub and other abscission regulators at MRs/MBs to gain insight into how the abscission machinery is modified to control abscission timing in fGSCs (Eikenes, 2015).

Intercellular bridge MTs in fGSC-CB pairs were degraded in G1/S when the fusome adopted bar morphology. Abscission in G2 thus appears to occur independently of intercellular bridge MTs in Drosophila fGSCs. This has also been described in C. elegans embryonic cells where the MR scaffolds the abscission machinery as well as in Archaea that lack the MT cytoskeleton]. In mammalian and Drosophila S2 cells in culture, on the other hand, intercellular bridge MTs are present until just prior to abscission (Eikenes, 2015).

It is interesting to note a resemblance of the stem cysts that appeared upon loss of ALIX and Shrub function to germline cysts in that the MRs remained open for long periods of time similar to RCs. Some modification of ALIX and Shrub levels/recruitment may thus contribute to incomplete cytokinesis in Drosophila germline cysts under normal conditions. Because stem cysts were detected in the case when ALIX weakly interacted with Shrub it is also possible that inhibition of their complex assembly/activity may contribute to incomplete cytokinesis in germline cysts. Abscission factors, such as ALIX and Shrub, may thus be modified and/or inhibited during incomplete cytokinesis in germline cysts. Such a scenario has been shown in the mouse male germline where abscission is blocked by inhibition of CEP55-mediated recruitment of the abscission machinery, including ALIX, to stable intercellular bridges. Altogether these data thus suggest that ALIX and Shrub are essential components of the abscission machinery in Drosophila GSCs, and it is speculated that their absence or inactivation may contribute to incomplete cytokinesis. More insight into molecular mechanisms controlling abscission timing and how the abscission machinery is modified in different cellular contexts will give valuable information about mechanisms controlling complete versus incomplete cytokinesis in vivo (Eikenes, 2015).

In summary, this study reports that a complex between ALIX and Shrub is required for completion of cytokinetic abscission with normal kinetics during asymmetric Drosophila GSC division, giving molecular insight into the mechanics of abscission in a developing tissue in vivo (Eikenes, 2015).

The conserved family of Hedgehog (Hh) proteins acts as short- and long-range secreted morphogens, controlling tissue patterning and differentiation during embryonic development. Mature Hh carries hydrophobic palmitic acid and cholesterol modifications essential for its extracellular spreading. Various extracellular transportation mechanisms for Hh have been suggested, but the pathways actually used for Hh secretion and transport in vivo remain unclear. This study shows that Hh secretion in Drosophila wing imaginal discs is dependent on the endosomal sorting complex required for transport (ESCRT). In vivo the reduction of ESCRT activity in cells producing Hh leads to a retention of Hh at the external cell surface. Furthermore, ESCRT activity in Hh-producing cells is required for long-range signalling. Evidence is provided that pools of Hh and ESCRT proteins are secreted together into the extracellular space in vivo and can subsequently be detected together at the surface of receiving cells. These findings uncover a new function for ESCRT proteins in controlling morphogen activity and reveal a new mechanism for the transport of secreted Hh across the tissue by extracellular vesicles, which is necessary for long-range target induction (Matusek, 2014).

The innate immune system needs to distinguish between harmful and innocuous stimuli to adapt its activation to the level of threat. How Drosophila mounts differential immune responses to dead and live Gram-negative bacteria using the single peptidoglycan receptor PGRP-LC is unknown. This study describes rPGRP-LC, an alternative splice variant of PGRP-LC that selectively dampens immune response activation in response to dead bacteria. rPGRP-LC-deficient flies cannot resolve immune activation after Gram-negative infection and die prematurely. The alternative exon in the encoding gene, here called rPGRP-LC, encodes an adaptor module that targets rPGRP-LC to membrane microdomains and interacts with the negative regulator Pirk and the ubiquitin ligase DIAP2. rPGRP-LC-mediated resolution of an efficient immune response requires degradation of activating and regulatory receptors via endosomal ESCRT sorting. It is proposed that rPGRP-LC selectively responds to peptidoglycans from dead bacteria to tailor the immune response to the level of threat (Neyen, 2016).

PGRP-LC has a clear role as the major signaling receptor sensing Gram-negative bacteria in flies, but its contribution to the resolution phase once bacteria are killed and release polymeric PGN has remained elusive. This study has uncovered a regulatory isoform of LC (rLC) that adjusts the immune response to the level of threat. rLC specifically downregulates IMD pathway activation in response to polymeric PGN, a hallmark of efficient bacterial killing. The data are consistent with a model whereby the presence of rLC leads to efficient endocytosis of LC and termination of signaling via the ESCRT pathway. Trafficking-mediated shutdown of LC-dependent signaling ensures that LC receptors are switched off once the balance is tipped in favor of ligands signifying dead bacteria, allowing Drosophila to terminate a successful immune response. Failure to do so results in over-signaling, leading to the death of the host despite bacterial clearance. Consistent with this model, defects were found in endosome maturation and in the formation of MVBs enhance immune activation and prevent immune resolution. In addition to regulating LC signaling via the ESCRT machinery, rLC can also inhibit LC signaling by forming signaling-incompetent rLC-LC heterodimers or rLC-rLC homodimers (Neyen, 2016).

Recent evidence from vertebrates also implicates the ESCRT machinery in suppressing spurious NF-κB activation: the TNFR superfamily member lymphotoxin-β receptor, which activates a signaling cascade that is functionally similar to IMD signaling, is degraded in an ESCRT-dependent manner in zebrafish and human cells. Thus ESCRT-mediated clearance of receptors upstream of NF-κB seems well conserved throughout evolution (Neyen, 2016).

Internalization of receptor-ligand complexes raises the question of whether peptidoglycan is fully degraded in the endolysosomal compartment or fragmented and released into the cytosol for sensing by PGRP-LE, as is the case for peptidoglycan sensing by cytoplasmic NOD2 receptors in mammalian cells. Mechanistic coupling of LC-dependent peptidoglycan endocytosis and PGRP-LE-dependent cytosolic sensing of exported peptidoglycan fragments would help explain the partial cooperation between the two receptors (Neyen, 2016).

Molecularly, rLC is characterized by a cytosolic PHD domain predicted to bind to phosphoinositides. The PHD domain targets rLC were found to be distinct membrane domains but it cannot be excluded that this localization relies on additional protein-protein interactions. Furthermore, the PHD domain also mediates binding of rLC to the cytosolic regulator Pirk and the ubiquitin ligase DIAP2. The combined capability to control membrane localization and to recruit downstream signaling modulators is reminiscent of the 'sorting-signaling adaptor paradigm' that is emerging for mammalian PRRs. Sorting adaptors are cytosolic signaling components with phosphoinositide-binding domains that are selectively recruited to defined subcellular locations and thereby shape the signaling output of the receptors they interact with. In vertebrate immune signaling, bacterial sensing modules (for example, TLRs), lipid-binding sorting modules (for example, TIRAP or TRIF) and signaling modules (for example, MyD88 and TRAM) are carried on separate molecules and assemble via transient interactions. Drosophila MyD88 combines sorting and signaling functions in a single molecule, bypassing the need for TIRAP. Notably, rLC merges features of sensing and signaling receptors and sorting adaptors into a single molecule. The fact that Drosophila rLC has no immediate homologs in vertebrates with PGN-sensing and PGN-signaling pathways suggests evolutionary uncoupling of sensing and sorting domains, possibly to increase the spectrum of signaling by combinatorial recruitment of adaptors to sensing receptors (Neyen, 2016).

Whether raising synaptic activity also promotes an increase in the flux of CTB retrograde transport was examined in vivo, using live Drosophila melanogaster larval preparations. In addition to allowing measurement. of the effect of activity on retrograde carrier trafficking in a complementary model system, this approach also facilitated tracing the effect of presynaptic activity on the complete transport of CTB from the neuromuscular junction to the soma located in the central nervous system of the larvae (see CTB axonal retrograde carrier flux is controlled by synaptic activity in D. melanogaster motor neurons. In the fibers of neurons from wild-type larvae a low, albeit consistent, rate of CTB-positive retrograde carriers was detected. To analyse the flux of retrograde carriers under conditions of high activity, transgenic larvae were employed expressing dominant-negative forms of Ether-è-go-go (Eag) and Shaker (Sh) voltage-gated potassium channels in motor neurons, upregulating synaptic transmission. Consistent with the data in primary hippocampal neurons, increased presynaptic activity at the neuromuscular junction of the eag, Sh mutant larvae also resulted in an increase in the frequency of retrograde CTB carriers. Furthermore, the net transport of CTB could be quantified by measuring the accumulation of fluorescent CTB in the somata located in the ventral nerve cord. These data confirmed that presynaptic activity controls the transport of CTB retrograde carriers as well as their accumulation in the cell bodies located in the central nervous system (Wang, 2016).

This study demonstrates that the level of activity at the presynapse in hippocampal neurons, whether it be low or high activity, can induce changes in the frequency of CTB-positive retrograde carriers undergoing transport from the nerve terminal to the soma. Greater than 50% of CTB carriers are TrkB-positive signalling endosomes, and there is coupling between the level of presynaptic activity and the flux of signalling endosomes undergoing retrograde axonal transport. Given that the amount of activated TrkB per signalling endosome has been shown to be quantal in nature, it is likely that it is the number of signalling endosomes reaching the cell body that is responsible for monitoring the trophic response of each neuron. Importantly, it was demonstrated that the transient TrkB activation that occurs when presynaptic activity is increased controls the activity-dependent increase in the flux of signalling endosomes in a BDNF-independent manner. This suggests that TrkB activation may play an active role in generating signalling endosomes with a retrograde fate independent of BDNF secretion. Finally, SIM and morphometric analyses reveal a high number of sub-diffraction-limited CTB- and TrkB-positive carriers that may be involved in delivering the survival message (Wang, 2016).

CTB is a widely used neuroanatomical tracer with the ability to undergo retrograde transport in neurons. CTB was found to enter nerve terminals in an activity-dependent manner, and following a 2-4 h chase, is found in retrograde axonal carriers that co-localize extensively with TrkB. Importantly, TrkB staining in resting conditions produced a low and surface-localized pattern, which is in sharp contrast with that observed in the CTB/TrkB vesicular carriers following stimulation. Considering that internalization of activated TrkB is elicited by neuronal activity in hippocampal neurons, this suggests that the retrograde carriers containing both TrkB and CTB are signalling endosomes, destined to deliver a survival signal from the terminal to the soma. The data are also consistent with the previous demonstration that CTB can be co-transported in the same retrograde carriers as the neurotrophin receptor p75NTR and TrkB receptors. It has been speculated that activated neurotrophin receptors internalized into terminals through different pathways may converge in the retrograde traffic and be packaged into 'common' compartments, presumably signalling endosomes, which then undergo microtubule-dependent retrograde trafficking to induce signalling cascades and gene expression in the soma (Wang, 2016).

The signalling endosome model has been proposed to describe the propagation of activated Trk signals from the axon terminal to the neuronal soma. Although the presence of signalling endosomes is widely accepted, the precise nature(s) and regulation of these organelles remain unknown. In particular, it is not known how presynaptic activity translates into an increase in signalling endosome-encoded cell survival. Recent studies using super-resolution microscopy have shown that individual signalling endosomes appear to contain a limited number of signalling receptors, suggesting that retrograde survival signalling could be quantal in nature. Consistent with this idea, recent analysis has demonstrated that signalling endosomes have a fixed number of phosphorylated tyrosine receptor kinases. The current findings suggest that it is the flux of signalling endosomes reaching the cell body that encodes the level of presynaptic activity, and translates this into a survival signal. The data reveal a clear increase in the frequency of retrograde carriers delivering both CTB and TrkB from the nerve terminal to the cell soma following a short, transient burst of activity, whereas BoNT/A pretreatment not only completely blocked this increased flux despite equivalent stimulation but also lowered the basal level of retrograde CTB flux in resting conditions. These results strongly support the hypothesis that the soma of a neuron with lower synaptic activity receives fewer signalling endosomes than that of a cell with high synaptic activity, with ramifications for survival (Wang, 2016).

Previous studies have clearly established that there is constitutive delivery of retrogradely transported neurotrophins and their receptors in different types of primary neurons. This study is the first to reveal a coupling between synaptic activity and the number of retrograde signalling endosomes in both hippocampal neurons and motor neurons from D. melanogaster larvae, suggesting that this coupling represents a conserved regulatory mechanism (Wang, 2016).

Genetic manipulations in Drosophila allowed motor neuron activity to be chronically increase, thereby demonstrating a correlation with increased retrograde signalling endosome transport. Retrograde axonal transport has previously been reported in Drosophila larval motor neurons. However, this process is not regulated by the activation of TrkB receptors in flies, but by neurotrophin-type factors such as BMP (Gbb) p150(Glued) and Toll. Investigating the respective contributions of each of these mechanisms will be required to unravel the mechanism(s) controlling the coupling between synaptic activity and the flux of retrogradely transported signalling endosomes (Wang, 2016).

Clathrin-mediated endocytosis and bulk endocytosis are the two main modes for synaptic vesicle retrieval. On the basis of the current data, internalized CTB is sorted into both synaptic vesicles and larger endosomes following high K+-induced synaptic activation. These are therefore likely to be the source of CTB-positive signalling endosomes. Indeed, it is noted that, following a 4 h chase, the number of CTB-labelled endosomes present in nerve terminals was clearly reduced, suggesting that they could contribute to the generation of retrograde carriers leaving the terminals. More work is needed to pinpoint the actual contribution of the different presynaptic endocytic pathways in the process of generating signalling endosomes and recycling synaptic vesicles. Using electron microscopy and super-resolution SIM on the unidirectional axon bundles, a high proportion was found of CTB-positive retrograde carriers with a diameter <150 nm, indicating that these may constitute a significant source of the signalling endosomes that reach the soma. Interestingly, it was also found that a number of these small CTB retrograde endosomes closely aligned with each other to form consecutive structures. These were reminiscent of single-molecule quantum dot BDNF-labelled compartments, previously described as elongated multiple vesicular bodies. Several different types of organelles contain endocytosed neurotrophin receptors, including electron-lucent endosomes ranging from 50 nm to >200 nm in diameter and multivesicular bodies. The results further confirm this morphological heterogeneity of signalling endosomes. Interestingly, they also show that the sub-diffraction-limited pool of retrograde carriers is increased by a pulse of stimulation. A future challenge will be to fully define these compartments, including their cargo, their regulation and their potential role in neuronal survival (Wang, 2016).

The activity-dependent increase in the flux of CTB retrograde carriers is similar to that observed with autophagosomes undergoing axonal retrograde transport. Genetic and pharmacological inhibition of TrkB prevented the activity-dependent increase in signalling endosomes but did not affect the number of autophagosomes undergoing retrograde transport. This result suggests a limited cross-link between these two pathways, and indicates the existence of a TrkB-dependent sorting mechanism that is selective for the generation of retrograde signalling endosomes and is upregulated by synaptic activity (Wang, 2016).

Although TrkB activation was required to couple synaptic activity with the flux of retrograde CTB carriers, this study found that BDNF collators (anti-BDNF blocking antibody and TrkB-Fc), as well as exogenously applied BDNF, failed to affect this coupling. These results suggest that BDNF may not be required to promote this pathway. First, it can be speculated that increased neuronal activity alone promotes the secretion of alternative TrkB activators, such as NT4. In addition, considering that two different BDNF receptors, p75NTR and TrkB, have been reported to have opposing actions in neurons, the observation that BDNF collators have no effect on retrograde transport or the phosphorylation of TrkB could be explained by the fact that these collators alleviate an inhibitory effect of p75NTR signalling on TrkB activation. Third, it is possible that TrkB receptors could be indirectly transactivated by adenosine or by the MAPK pathway, similar to other tyrosine kinase receptors. However, further experiments are required to pinpoint the precise cascade of molecular events leading to the encoding of the level of synaptic activity by the flux of retrograde signalling endosomes in a BDNF-independent and TrkB activation-dependent pathway (Wang, 2016).

The data show that internalized CTB is sorted into both synaptic vesicles and larger endosomal structures reminiscent of bulk endosomes. Bulk endosomes are therefore likely to contribute to the sorting events that lead to the generation of CTB-positive signalling endosomes. Indeed, the data demonstrate that, in response to stimulation, the number of nerve terminals containing bulk endosomes more than doubles, as does the number of endosomes per terminal. More strikingly, after a chase of 4 h, the number of bulk endosomes returns to control levels, suggesting that sorting has taken place and/or that these large endosomes have undergone retrograde trafficking. In support of this view larger CTB-positive structures inside the axon were observed by electron microscopy. Bulk endocytosis is therefore likely to be a key element of this pathway, not only by budding small synaptic-like vesicles in nerve terminals, but also by generating large endosomes that undergo retrograde trafficking. More work is needed to pinpoint the actual contribution of bulk endocytosis in the process of generating signalling endosomes and recycling synaptic vesicles. The fact that synaptic activity increases the flux of CTB retrograde carriers advocates for the presence of a number of regulatory mechanisms that effectively couple these two pathways. Whether both recycling synaptic vesicles and signalling endosomes bud from the same bulk endosomes will require further investigation (Wang, 2016).

In summary, this study has uncovered a coupling between synaptic activity, trophic signalling and the generation of axonal retrograde signalling endosomes, which contain TrkB receptors and nerve terminal-derived CTB as cargo, and have characterized their kinetic and morphological nature. The data reveal a novel role of presynaptic activity in controlling the flux of retrograde signalling endosomes that will eventually reach the neuronal cell body and deliver cell survival signals. These results suggest that the flux of signalling endosomes undergoing axonal transport constitutes a digital output of the level of synaptic activity experienced by nerve terminals. How the flux of signalling endosomes reaching the cell body is decoded to pursue or halt a survival signal warrants further investigation (Wang, 2016).

Proper function of cell signaling pathways is dependent upon regulated membrane trafficking events that lead to the endocytosis, recycling, and degradation of cell surface receptors. The endosomal complexes required for transport (ESCRT) genes play a critical role in the sorting of ubiquinated cell surface proteins. CHMP2B(Intron5) , a truncated form of a human ESCRT-III protein, was discovered in a Danish family afflicted by a hereditary form of frontotemporal dementia (FTD). Although the mechanism by which the CHMP2B mutation in this family causes FTD is unknown, the resulting protein has been shown to disrupt normal endosomal-lysosomal pathway function and leads to aberrant regulation of signaling pathways. This study has misexpressed CHMP2B(Intron5) in the developing Drosophila external sensory (ES) organ lineage; it was shown to be capable of altering cell fates. Each of the cell fate transformations seen is compatible with an increase in Notch signaling. Furthermore, this interpretation is supported by evidence that expression of CHMP2B(Intron5) in the notum environment is capable of raising the levels of Notch signaling. As such, these results add to a growing body of evidence that CHMP2B(Intron5) can act rapidly to disrupt normal cellular function via the misregulation of critical cell surface receptor function (Wilson, 2019).

Loss of ESCRT function in Drosophila imaginal discs is known to cause neoplastic overgrowth fuelled by mis-regulation of signalling pathways. Its impact on junctional integrity, however, remains obscure. To dissect the events leading to neoplasia, transmission electron microscopy (TEM)was used on wing imaginal discs temporally depleted of the ESCRT-III core component Shrub. A specific requirement for Shrub was found in maintaining Septate Junction (SJ) integrity by transporting the Claudin Megatrachea (Mega) to the SJ. In absence of Shrub function, Mega is lost from the SJ and becomes trapped on endosomes coated with the endosomal retrieval machinery Retromer. ESCRT function is required for apical localization and mobility of Retromer positive carrier vesicles, which mediate the biosynthetic delivery of Mega to the SJ. Accordingly, loss of Retromer function impairs the anterograde transport of several SJ core components, revealing a novel physiological role for this ancient endosomal agent (Pannen, 2020).

Developmental and physiological functions of epithelia rely on a set of cellular junctions, linking cells within the tissue to a functional unit. While E-cadherin-based adherens junctions (AJs) provide adhesion and mechanical properties, formation of the paracellular diffusion barrier depends on tight junctions (TJs). Proteins of the conserved claudin family play a key role in establishing and regulating TJ permeability in the intercellular space by homo- and heterophilic interactions with Claudins of neighboring cells. Arthropods, such as Drosophila, do not possess TJs but a functionally similar structure in ectoderm-derived epithelia termed pleated septate junction (pSJ, SJ hereafter), characterized by protein dense septa lining the intercellular space in electron micrographs. Structure and function of Drosophila SJs depend on a convoluted multiprotein complex containing at least a dozen components. Three claudins, among them Megatrachea (Mega), have been shown to be required for SJ formation and barrier function in flies. Besides claudins, several transmembrane proteins (TMPs) such as Neurexin-IV (NrxIV), Neuroglian (Nrg) or ATPα contribute to the formation of the stable SJ core complex, which is characterized by low mobility within the membrane. At the intracellular side of the junction, cytoplasmic proteins such as Coracle (Cora), Varicose (Vari), and Discs large (Dlg) associate with the transmembrane components, contributing to the formation of a stable fence-like scaffold . While junction formation during embryogenesis requires the SJ localized cytoplasmic protein Dlg, this basolateral cell polarity factor is not a structural part of the immobile junction core comple. This explains the functional separation of barrier formation and apicobasal polarity despite the close association of Dlg-complex components with the SJ. Albeit growing knowledge about the structural composition of SJs, the intracellular events required for assembly and maintenance of SJ complexes remain largely unknown. Specifically, how proliferative tissues, such as the imaginal disc epithelium, maintain SJ integrity is not well established (Pannen, 2020).

It was recently shown that newly synthesized SJ components integrate into the junction from the apical side (in between AJ and SJ) in a 'conveyor belt-like' fashion (Babatz, 2018; Daniel, 2018). In addition, SJ components are frequently associated with endosomal compartments, suggesting a role for the endosomal system in coordinating transport and turnover of SJ complexes. Consistently, endocytosis is required to concentrate SJ components at the junctional region during embryogenesis. This suggests that passage of SJ TMP components (or the whole SJ protein complex) through the endosomal system may be a requirement for SJ formation, with the underlying mechanisms remaining poorly characterized (Pannen, 2020).

The endosomal system fulfils a plethora of physiological functions by tightly regulating the intracellular transport of TMPs and membranes within the cell. Following endocytosis from the plasma membrane, TMPs enter the endosomal system where they undergo cargo specific sorting. This process provides separation of proteins destined for degradation from those that exit the endosomal system to be recycled. Two evolutionary conserved endosomal sorting machineries, the endosomal sorting complex required for transport (ESCRT) and the retromer complex, mediate cargo sorting into the degradative and recycling pathway, respectively. To coordinate these opposing transport activities, the endosomal system comprises a highly dynamic membrane network governing retromer-dependent tubulation for recycling and ESCRT-mediated generation of intraluminal vesicles (ILV) for degradation (Pannen, 2020).

Endocytosed proteins can evade ESCRT-dependent packaging into ILVs by exiting the maturing endosome (ME) through tubular retrieval domains induced by specialized recycling machineries such as retromer. Initially characterized as a regulator of endosome-to-Golgi cargo retrieval in yeast, this endosomal agent comprises two subcomplexes that cooperatively drive cargo sorting into tubular recycling carriers. Similar to the ESCRT machinery, cargo clustering and membrane deformation is performed by distinct functional units within the retromer pathway. Motif-based cargo recognition and aggregation is mediated by the endosomally localized Vps26:Vps29:Vps35 complex, which has been termed cargo-selective complex, considered to constitute the core functional component of retromer. Since the ancient CSC does not possess membrane bending activity, cooperation with tubulating factors such as proteins of the SNX-BAR (Sorting Nexin-Bin/Amphiphysin/Rvs) family is required for recycling carrier generation. Proteins containing the curved BAR-domain can assemble into regular helical coats on endosomes, thereby inducing cytoplasm faced tubulation. Concerted action of CSC stably complexed with SNX-BAR proteins to retrieve endosomal cargo was initially characterized as the classical retromer pathway in yeast. In metazoans however, retromer function is not restricted to SNX-BAR-dependent pathways. Specifically, cooperations of CSC with SNX3 or SNX27 (both lacking BAR-domains) emerged as alternative routes for endosomal retrieval. Proteomic data from mammalian cells suggest that surface levels of well over 100 TMPs depend on retromer and many of these proteins seemingly interact with CSC or SNX27. Recently, Drosophila has proven invaluable for assessing and confirming the physiological relevance of some of these putative retromer cargos in vivo (Pannen, 2020).

Cargo proteins within the endosomal system that do not undergo recycling can enter the degradative trafficking route starting with their sorting into ILVs. Generation of ILVs at the limiting membrane of MEs requires the canonical ESCRT function, which is performed by four in sequence acting complexes (ESCRT-0, -I, -II, III) and the ATPase Vps4. Ubiquitination of TMPs serves as the primary degradative sorting signal and sequestration of TMPs into ILVs is an essential prerequisite to complete lysosomal degradation. Several ESCRT components such as Vps27/Hrs (ESCRT-0) and Vps23/TSG101 (ESCRT-I) possess ubiquitin interacting motifs, which allow them to bind and cluster ubiquitinated TMPs. Consequently, local concentration of ubiquitinated cargo by ESCRT complexes establishes a degradative subdomain at the endosomal membrane that is spatially separated from the retrieval subdomain. While ESCRT-0-II complexes provide cargo recognition and clustering, the membrane-deforming activity required to bud and abscise ILVs into the endosomal lumen depends on ESCRT-III components, which polymerize into helical arrays at the endosomal membrane. The most abundant ESCRT-III component is the highly conserved yeast Snf7/Vps32, encoded by the gene shrub (shrb) in Drosophila. Unlike upstream ESCRT components, ESCRT-III proteins only transiently assemble into a heterooligomeric complex at the endosomal membrane. In consequence of ESCRT activity, the maturing endosome accumulates cargo-containing ILVs and is recognized in electron micrographs as a multi-vesicular body (MVB). The ESCRT/MVB pathway ends with Vps4-dependent dissociation of ESCRT-III components from the endosomal membrane. This step is required for the release of the nascent ILV and subsequent rounds of ILV formation. Loss of ESCRT function was initially studied in yeast cells in which it led to the emergence of an aberrant pre-vacuolar endosomal organelle, termed class E compartment. This defective endosomal structure is characterized by accumulation of degradative cargo and a failure to fuse with the vacuole/lysosome (Pannen, 2020).

The physiological relevance of ESCRT-mediated degradative TMP trafficking is particularly evident in Drosophila imaginal disc tissue. Here, loss of ESCRT function induces severe overgrowth, multilayering, apoptosis, and invasive behavior of the tissue; a phenotype attributed to mis-regulation of cellular signaling pathways, such as the Jak/Stat-, Jun-Kinase-, and Notch pathways. Consequently, ESCRT components were classified as endocytic neoplastic tumor suppressor genes (nTSG) in Drosophila. While induction of over-proliferation and apoptosis in nTSG mutants have been extensively characterized, the events leading to loss of cell polarity and ultimately neoplastic transformation of the tissue remain poorly understood (Pannen, 2020).

This study has analyzed the integrity of cellular junctions in an ESCRT-depleted wing imaginal disc epithelium to gain insight into the initial events leading to neoplastic transformation. Surprisingly, preceding neoplastic overgrowth, this study found a strong and specific reduction in the density of SJ. ESCRT and retromer functions are required for anterograde transport of SJ components. By dissecting the intracellular trafficking itinerary of the claudin Megatrachea, this study revealed that biosynthetic delivery of this core SJ component depends on a complex basal to apical transcytosis route relying on ESCRT and retromer functions (Pannen, 2020).

While transcytosis of SJ components has been shown to occur during the initial establishment of the SJ in the embryo, this study revealed that this mechanism is also continuously required during maintenance of the SJs in a rapidly proliferating epithelium. The data reveal that the retromer CSC functions downstream of ESCRT to export Mega from the endosome. A novel physiological role is proposed for the retromer CSC in regulating membrane levels of several SJ core components. While the data suggest that retromer fails to export Mega from aberrant endosomes induced by ESCRT depletion, the exact mechanism behind this remains to be determined (Pannen, 2020).

The data reveal a critical requirement for ESCRT in a transport pathway that depends on retromer-mediated transcytosis to deliver newly synthesized Mega to its apical destination. Defects in endosomal retrieval upon ESCRT inactivation have been previously described in other systems, such as yeast or mammalian cells and, thus, appear to represent a common feature of the pleiotropic ESCRT deficient phenotype. In yeast, the endosome-to-Golgi retrieval of the sorting receptor Vps10p and its cargo carboxypeptidase Y (CPY) depends on retromer function. ESCRT mutant strains accumulate CPY in class E compartments from which retrieval to the Golgi is blocked. Similarly, the mammalian retromer cargo mannose 6-phosphate receptor (M6PR) also failed to recycle from endosomes to the Golgi in HeLa cells depleted of TSG101/ESCRT-I function. In this study, it is suggested that generation of class E compartments occurs at the expense of endosomal tubules. Consistently, the retromer-associated tubulation factor SNX1 and its yeast homolog Vps5p were found on the rims of mammalian and yeast class E compartments, respectively. Together with the finding of CSC accumulation on Drosophila class E-like compartments, this suggests that ESCRT deficient endosomes remain coated with retromer components but fail to export specific cargo. (Pannen, 2020).

While the possibility cannot be ruled out of ESCRT components directly cooperating with retromer to form recycling tubules (note that SNX-BAR, Snx3, and Snx27 are not required for Mega transport), an indirect mechanism is favored linking ESCRT and retromer in this transport pathway. Analysis of the aberrant endosomal compartments induced upon Shrub depletion revealed that they are enriched in endosomal organizers such as Rab5 and Rab7, which could potentially interfere with retromer-dependent export when their activity at the limiting membrane is unrestrained. While the role of Rab7 in endosomal recruitment of the CSC is well established, the necessity for Rab7 GDP/GTP cycling during retromer-dependent carrier generation is still under debate. Rab7 and its GTPase-activating protein (GAP) Tbc1d5 are interaction partners of the CSC and can modulate its capability to retrieve endosomal cargo. For example, interfering with Rab7-GTP hydrolysis by Tbc1d5 depletion yielded defects in retromer-dependent transport in HeLa cells. Strikingly, under these conditions, retromer cargo was trapped in CSC-coated endosomes, paralleling the current observation of Mega subcellular localization upon ESCRT depletion. Similarly, by exposing the interplay between the CSC component Vps29, Tbc1d5, and Rab7 in adult Drosophila brains, the authors of a recent study reported the capability of endosomal Rab7 to interfere with retromer CSC function in vivo (Pannen, 2020).

While the exact mechanism rendering retromer dysfunctional at Drosophila class E compartments remains to be determined, the data support the mounting pool of evidence that ESCRT is required for multiple endosomal retrieval pathways. It is therefore likely that aspects of the pleiotropic ESCRT phenotype in metazoans stem from defective export of proteins from the endosomal system. For example, in Drosophila, leaky SJ could support ESCRT-mediated neoplastic transformation by permitting diffusion of signaling molecules within the imaginal disc tissue (Pannen, 2020).

This study found that biosynthetic delivery of Mega depends on a transcytosis-like mechanism from the basodistal to the apical plasma membrane. This long-distance transport required sequential action of endocytic (clathrin, dynamin, Rab5) and endosomal (ESCRT, retromer CSC) machineries. Importantly, the finding that overexpressed HA-Mega is unable to reach the SJ in absence of retromer and ESCRT function is in agreement with biosynthetic delivery of Mega relying on endosomal function. Therefore, although the possibility that Mega transiently passes the Golgi after endocytosis at the basodistal membrane cannot be excluded, the unconventional transcytosis model is favored. Strikingly, while retromer-dependent endosomal recycling has been extensively documented, only one mammalian cell culture study implicated retromer in transcytosis from one membrane domain to another. Thus, SJ delivery of Mega in imaginal discs represents a novel physiological role of retromer to study this process in vivo. The finding that CSC-mediated anterograde transport of Mega is independent of retromer-associated sorting nexins indicates that this transcytosis pathway is distinct from many established CSC-dependent routes and suggests that it may require unknown cofactors (or does not require endosomal tubulation) (Pannen, 2020).

Analysis of Vps35 clones in pupal wings or leg imaginal discs revealed that in these tissues, clones completely devoid of the SJ core component NrxIV occur frequently. Similarly, shrub mutant clones in the pupal notum were entirely lacking junctional ATPα (Roland Le Borgne, personal communication to Pannen, July 2020). This is in contrast to surface levels of SJ components in Vps35 mutant wing discs, which were consistently reduced by about 50%. This provokes the hypothesis that a parallel endosomal export pathway for SJ components may exist in wing discs that could partially compensate for loss of retromer. However, this is thought unlikely since overexpressed HA-Mega fails to reach the SJ not only upon Shrub but also upon Vps26 depletion. One has to keep in mind that this experiment specifically monitors delivery of newly synthesized HA-Mega while the Vps35 clonal analysis assesses the impact of retromer loss of function on pre-existing SJ. Thus, in a clonal situation, a 'thinning out' of junctions is expected with consecutive rounds of cell division, which could explain incomplete phenotypic expressivity in wing disc Vps35 clones. Nevertheless, it remains to be determined why in leg discs and pupal wings, SJ appear to be more sensitive toward CSC loss. During metamorphosis, wing imaginal disc cells undergo drastic morphogenetic changes to form the pupal wing epithelium, a process known to require AJ remodeling. It is therefore possible that analogously to AJ, SJ may also be actively remodeled during pupal wing formation. This could explain the strong requirement for retromer function in maintaining SJ integrity in a tissue undergoing morphogenetic changes (Pannen, 2020).

Paralleling the findings described in this paper, a previous study showed that embryonic SJ formation depends on endocytosis and subsequent redistribution of junction components from the lateral membrane to the SJ. Thus, transcytosis of SJ components is a mechanism likely required for both initial SJ formation as well as maintenance of SJ integrity in non-embryonic tissues in Drosophila. While the roles of ESCRT and retromer CSC in initial SJ formation were not assessed, it is conceivable that they are already required for transporting SJ components during embryogenesis. Consistently, shrub mutant embryos display a defective epithelial barrier function, suggesting they fail to form functional SJx (Pannen, 2020).

The reason for SJ maintenance to rely on such an elaborate trafficking of its components remains to be determined. It has been suggested that SJ components form stable complexes prior to integration into the junction. Potentially, essential post-translational modifications of certain SJ components required for complex formation may occur exclusively at the basodistal membrane or during the passage through the endosomal system. If transient localization of SJ components at the basodistal membrane is a prerequisite for efficient SJ core complex formation, depositing transcripts for structural components such as Mega in the basal cytoplasm would shorten the route individual SJ components need to pass prior to SJ complex formation at the basodistal membrane domain. Alternatively, the finding that Mega mRNA predominantly localizes in the basal cytoplasm provides the foundation for another hypothesis: It is widely accepted that apical and basolateral cargos undergo motif-based sorting leading to secretion toward the respective membrane domains. Basal subcellular localization of Mega transcripts could potentially reflect an apical/basolateral sorting divergence at the mRNA level. Accordingly, basal translation and exocytosis of Mega (induced by a putative basal/basolateral sorting signal) may lead to its targeting toward the basodistal membrane, despite the fact that the SJ resides apically in wing disc cells. Thus, transcytosis may serve as an adaptation for redistribution of cargos to their destined membrane domain when they are initially secreted to a different one due to early sorting signals. Since the columnar wing imaginal disc cells have a very elongated shape and possess Golgi stacks all along the apicobasal axis, it is conceivable that certain Golgi stacks residing at the apical and basal poles are specialized in secreting apical and basolateral cargos, respectively. Although this is highly hypothetical, systematic analysis of transcript localization for apical (e.g., E-cad) and basolateral (e.g., SJ components) cargos could reveal a potential spatial separation of distinct secretory routes already at the mRNA level (Pannen, 2020).

In wing imaginal discs, a similarly complex transcytosis route (albeit with an apical to basal direction) has been described for the signaling molecule Wingless, which is translated apically, transiently presented at the apical membrane and finally transcytosed toward the basal membrane where it is secreted (Yamazaki, 2016). Thus, distinct transcytosis pathways in the wing disc epithelium provide a mechanism for targeting certain proteins to their site of action, specifically when the protein is translated far away from its terminal destination (Pannen, 2020).

This study has unravel a novel physiological retromer function in regulating surface levels of a claudin and other structural SJ components (e.g., Nrg, ATPα, Lac, NrxIV, Cont) in several Drosophila tissues. Currently, it is not known how the SJ components are selected for this retromer-dependent pathway, and whether it requires physical interaction with CSC components. Since SJ proteins may traverse the endosomal system in complex, the vast number of different components brings about a plethora of possible interaction sites. Importantly, a mass-spectrometry-based study of the Mega interactome did not detect any retromer CSC components or associated factors but confidently found SJ core components as well as clathrin. While the interaction mode of CSC and SJ proteins remains to be determined, the data reflect the assumption that several SJ core components represent novel putative retromer cargos in Drosophila (Pannen, 2020).

Strikingly, among the proteins affected by retromer loss of function, many possess mammalian homologs (e.g., NrxIV/CNTNAP2, ATPα/ATP1A1, Nrg/NRCAM). This suggests they could represent a novel set of conserved retromer cargos. Indeed, several lines of evidence suggest that ESCRT/retromer-mediated transport of SJ components may be evolutionary conserved from Drosophila to mammals. Depletion of ESCRT-I component TSG101 in mammalian epithelial cells led to a reduction of trans-epithelial resistance (TER), indicating defects in TJ-mediated barrier function. Additionally, Claudin-1, an essential TJ component, continuously underwent endocytosis and recycling back to the plasma membrane in several mammalian cell lines in a process requiring ESCRT function (Pannen, 2020).

Importantly, the mechanism behind reduced recycling and entrapment of Claudin-1 in ubiquitin-positive aberrant endosomes upon interference with ESCRT function remained elusive. Thus, it is unknown how the export of Claudin-1 from the endosomal system in mammalian cells is achieved. By revealing an ESCRT-dependent function of the retromer CSC in claudin endosomal export in Drosophila, the data may provide an explanation for a possibly conserved trafficking pathway of claudins. In support of this, Claudin-1 and Claudin-4 membrane levels were significantly reduced in a mass spectrometry-based surface proteome study of Vps35-depleted human cells. Furthermore, the TJ protein Zonula occludens-2 (ZO-2) was strongly enriched in a Vps26 interactome, suggesting that a presumptive retromer function in TJ maintenance in mammalian cells may not be limited to claudins, similar to the findings in Drosophila presented in this study. It remains to be determined whether a physiological role of retromer in mammalian TJ maintenance occurs also in vivo. An increasing amount of tools, such as conditional Vps35 knockout mice, will enable analysis of this putatively conserved retromer function in mammalian systems and reveal any possible implications in development and/or disease (Pannen, 2020).

A major task of the endosomal sorting complex required for transport (ESCRT) machinery is the pinching off of cargo-loaded intraluminal vesicles (ILVs) into the lumen of maturing endosomes (MEs), which is essential for the complete degradation of transmembrane proteins in the lysosome. The ESCRT machinery is also required for the termination of signalling through activated signalling receptors, as it separates their intracellular domains from the cytosol. The ESCRT-III complex is required for an increasing number of processes where membrane regions are abscised away from the cytosol. The core of ESCRT-III, comprising four members of the CHMP protein family, organises the assembly of a homopolymer of CHMP4, Shrub in Drosophila, that is essential for abscission. The tumour-suppressor lethal (2) giant discs (Lgd)/CC2D1 is a physical interactor of Shrub/CHMP4 in Drosophila and mammals, respectively. This study shows that the loss of function of lgd constitutes a state of reduced activity of Shrub/CHMP4/ESCRT-III. The forced incorporation in ILVs of lgd mutant MEs suppresses the uncontrolled and ligand-independent activation of Notch. Moreover, the analysis of Su(dx) lgd double mutants clarifies their relationship and suggests that they are not operating in a linear pathway. Since lgd mutants can be rescued to normal adult flies if extra copies of shrub (or its mammalian ortholog CHMP4B) are added into the genome, it is concluded that the net activity of Shrub is reduced upon loss of lgd function. In solution, CHMP4B/Shrub exists in two conformations. LGD1/Lgd binding does not affect the conformational state of Shrub, suggesting that Lgd is not a chaperone for Shrub/CHMP4B. These results suggest that Lgd is required for the full activity of Shrub/ESCRT-III. In its absence, the activity of the ESCRT machinery is reduced. This reduction causes the escape of a fraction of cargo, among it Notch, from incorporation into ILVs, which in turn leads to an activation of this fraction of Notch after fusion of the ME with the lysosome. These results highlight the importance of the incorporation of Notch into ILV not only to assure complete degradation, but also to avoid uncontrolled activation of the pathway (Baeumers, 2020).

The ESCRT pathway is evolutionarily conserved across eukaryotes and plays key roles in a variety of membrane remodeling processes. A new Drosophila mutant recovered in a forward genetic screens for synaptic transmission mutants mapped to the vps24 gene encoding a subunit of the ESCRT-III complex. Molecular characterization indicated a loss of VPS24 function, however the mutant is viable and thus loss of VPS24 may be studied in a developed multicellular organism. The mutant exhibits deficits in locomotion and lifespan and, notably, these phenotypes are rescued by neuronal expression of wild-type VPS24. At the cellular level, neuronal and muscle cells exhibit marked expansion of a ubiquitin-positive lysosomal compartment, as well as accumulation of autophagic intermediates, and these phenotypes are rescued cell-autonomously. Moreover, VPS24 expression in glia suppressed the mutant phenotype in muscle, indicating a cell-nonautonomous function for VPS24 in protective intercellular signaling. Ultrastructural analysis of neurons and muscle indicated marked accumulation of the lysosomal compartment in the vps24 mutant. In the neuronal cell body, this included characteristic lysosomal structures associated with an expansive membrane compartment with a striking tubular network morphology. These findings further define the in vivo roles of VPS24 and the ESCRT pathway in lysosome homeostasis and their potential contributions to neurodegenerative diseases characterized by defective ESCRT or lysosome function (Florian, 2021).

In many vertebrate and invertebrate organisms, gametes develop within groups of interconnected cells called germline cysts formed by several rounds of incomplete divisions. This study found that loss of the deubiquitinase USP8 gene in Drosophila can transform incomplete divisions of germline cells into complete divisions. Conversely, overexpression of USP8 in germline stem cells is sufficient for the reverse transformation from complete to incomplete cytokinesis. The ESCRT-III proteins CHMP2B and Shrub/CHMP4 are targets of USP8 deubiquitinating activity. In Usp8 mutant sister cells, ectopic recruitment of ESCRT proteins at intercellular bridges causes cysts to break apart. A Shrub/CHMP4 variant that cannot be ubiquitinated does not localize at abscission bridges and cannot complete abscission. These results uncover ubiquitination of ESCRT-III as a major switch between two types of cell division (Mathieu, 2022).

Autophagy targets cytoplasmic materials for degradation and influences cell health. Organelle contact and trafficking systems provide membranes for autophagosome formation, but how different membrane systems are selected for use during autophagy remains unclear. This study reports a novel function of the endosomal sorting complex required for transport (ESCRT) in the regulation of endoplasmic reticulum (ER) coat protein complex II (COPII) vesicle formation that influences autophagy. The ESCRT functions in a pathway upstream of Vps13D to influence COPII vesicle transport, ER-Golgi intermediate compartment (ERGIC) assembly, and autophagosome formation. Atg9 functions downstream of the ESCRT to facilitate ERGIC and autophagosome formation. Interestingly, cells lacking either ESCRT or Vps13D functions exhibit dilated ER structures that are similar to cranio-lenticulo-sutural dysplasia patient cells with SEC23A mutations, which encodes a component of COPII vesicles. These data reveal a novel ESCRT-dependent pathway that influences the ERGIC and autophagosome formation (Wang, 2022).

The ESCRT regulates autophagy in multiple ways, including AP membrane sealing and autolysosome (ALY) formation. This study reveals a previously undescribed function of the ESCRT in ER trafficking and autophagy. The findings indicate that the ESCRT is required for COPII vesicle-derived compartments and AP formation. Loss of ESCRT components influence COPII vesicle marker formation and ER morphology. This study also shows that Vps13D co-localizes with markers of different stages of COPII vesicle maturation between the ER and Golgi apparatus, including markers of ERGIC formation. In the absence of ESCRT and Vps13D function, ER structure is altered in a manner that is similar to CLSD patient cells with SEC23A mutations. In addition, the ESCRT impacts Atg9 distribution. Vps13D and Atg9 are required for autophagy and appear to influence the ERGIC for the induction of autophagy. Therefore, this study identifies previously unrecognized steps in a regulatory pathway for autophagy, including novel roles for the ESCRT and Vps13D in COPII-derived and ERGIC-associated AP formation (Wang, 2022).

Autophagy involves a core mechanism, but some autophagy regulatory mechanisms vary in different cell contexts in animals. During Drosophila midgut development, most of the core autophagy genes are required, but Atg7 and Atg3 are not required for cell size reduction, protein, and organelle clearance. Instead, the ubiquitin activating enzyme Uba1 and ubiquitin function in autophagy. Vps13D contains a conserved ubiquitin-binding UBA domain and is required for developmental autophagy in the fly intestine. Thus, Vps13D has properties of an autophagy receptor and therefore may link ubiquitin, autophagy proteins, and their autophagic cargo substrates. Alternatively, ubiquitin may have cargo-receptor-independent functions in autophagy during intestine development (Wang, 2022).

Tsg101 and Vps36 both possess ubiquitin-binding domains and are required for autophagy in the fly intestine. While previous studies have shown that the ESCRT functions in AP membrane sealing and ALY formation, studies of Tsg101, Vps36, and all other ESCRT components tested indicate a novel role in ER trafficking for AP formation in the intestine. This study systematically compared ESCRT function in different cell contexts in Drosophila. Consistent with previous studies, ESCRT mutant fat body cells accumulate Atg8a puncta indicating that autophagy is blocked following ALY formation. By contrast, the ESCRT is required for Atg8a puncta formation in intestine cells. Therefore, the ESCRT machinery functions in a cell context-specific manner to regulate autophagy (Wang, 2022).

ESCRT machinery regulates multiple dynamic membrane changes, including vesicle abscission, membrane sealing, and repair. The function of the ESCRT in the COPII pathway is novel but also has similarities to the essential function of the ESCRT in membrane remodeling. During Drosophila development, Hrs is distributed near ER and co-localized with Ergic53 when autophagy is induced. In ESCRT mutant cells, both COPII and Ergic53 puncta were decreased and dissociated, indicating that COPII vesicles derived from ER are inhibited without the ESCRT. Multiple membrane remodeling processes require the ESCRT, including vesicle budding and internalization. Thus, it appears that the ESCRT affects COPII-coated ER exit sites to shed vesicles and influences ERGIC and AP assembly in a cell-specific manner. Decreased function of ESCRT regulatory genes influences Vps13D levels. By contrast, Vps13D regulates COPII regulatory protein localization, suggesting that Vps13D functions downstream of the ESCRT to influence COPII. It should be noted, however, that it is difficult to exclude the possibility that the ESCRT may also participate in either AP membrane sealing or fusion between AP and lysosome at a later stage in the autophagy process. In addition, we cannot exclude a possible indirect influence of the ESCRT on the ER and COPII maturation that is independent of Vps34 (Wang, 2022).

The roles of the regulators of COPII vesicle and ERGIC formation in autophagy may be Drosophila intestine cell context specific. However, COPII and the ERGIC have been implicated in the regulation of autophagy in both yeast and mammalian cells. Atg9 interacts with membrane compartments from the secretory pathway, endocytic vesicles, and Golgi apparatus in both yeast and mammals. Atg9 interacts with the COPII component Sec24 in yeast, and co-fractionates with ERGIC components in mammalian cells. Thus, it is likely that membranes from COPII compartments are utilized for autophagy induction. Atg9 is conserved in Drosophila, and the current analyses indicate that Atg9 is downstream of the ESCRT, Vps13D, and the ERGIC. Therefore, the ERGIC is also a potential membrane source for Atg9 to regulate the induction of autophagy in Drosophila, consistent with ERGIC function in mammalian cells. Thus, it is possible that the roles of COPII, Vps13D, and the ERGIC are more prevalent in the regulation of autophagy than previously recognized. Future studies should reveal how broadly these factors function in autophagy, as well as the role of the ESCRT in COPII vesicle maturation in other cell types (Wang, 2022).

Homeostatic signaling systems stabilize neural function through the modulation of neurotransmitter receptor abundance, ion channel density, and presynaptic neurotransmitter release. Molecular mechanisms that drive these changes are being unveiled. In theory, molecular mechanisms may also exist to oppose the induction or expression of homeostatic plasticity, but these mechanisms have yet to be explored. In an ongoing electrophysiology-based genetic screen, 162 new mutations were tested for genes involved in homeostatic signaling at the Drosophila NMJ. This screen identified a mutation in the rab3-GAP gene. This study shows that Rab3-GAP is necessary for the induction and expression of synaptic homeostasis. Evidence is provided that Rab3-GAP relieves an opposing influence on homeostasis that is catalyzed by Rab3 and which is independent of any change in NMJ anatomy. These data define roles for Rab3-GAP and Rab3 in synaptic homeostasis and uncover a mechanism, acting at a late stage of vesicle release, that opposes the progression of homeostatic plasticity (Müller, 2011).

The function of Rab3-GAP and Rab3 have been analyzed extensively, both biochemically and genetically, in systems ranging from yeast to the mammalian central nervous system, and there are several (~10 on average) copies of Rab3-GTP on an individual synaptic vesicle. It has also been shown that Rab3 binds to several presynaptic proteins, most often in its GTP-bound form. Rab3-GAP is required to promote hydrolysis of Rab3-GTP to Rab3-GDP. It is unknown precisely when and where Rab3-GAP acts upon Rab3-GTP, but evidence suggests that this interaction may occur at the synapse. For example, Rab3-GTP is on the vesicle and delivered to the synapse, where it is found bound to the active zone associated protein RIM. Biochemical data indicate that clathrin-coated vesicles lack Rab3-GTP. In combination, these data place Rab3-GAP activity at or near the release site (Müller, 2011).

The data presented in this study are consistent with a model in which Rab3-GTP acts, directly or indirectly, to inhibit the progression of synaptic homeostasis at a late stage of vesicle release, and that Rab3-GAP functions to inactivate this action of Rab3-GTP. Loss of Rab3-GAP was shown to block both the rapid induction and sustained expression of synaptic homeostasis. Rab3 mutations alone do not block synaptic homeostasis and synaptic homeostasis proceeds normally in the rab3–rab3-GAP double mutant. Genetically, these data indicate that the presence of Rab3 is required for the block of synaptic homeostasis observed in the rab3-GAP mutant. Thus, Rab3 is likely to be the cognate GTPase for Rab3-GAP. Furthermore, in genetic terms, Rab3 functions to oppose the progression of synaptic homeostasis and, when it is removed, homeostasis proceeds (Müller, 2011).

The possibility is considered that homeostasis proceeds in the absence of Rab3 because another, redundant Rab takes the place of Rab3. In yeast membrane trafficking, there is evidence for semiredundant Rab function. However, this seems to be an exception because Rabs are hypothesized to have unique binding affinities for downstream effector proteins that are essential for their ability to define discrete membrane domains within membrane trafficking and secretory systems. In C. elegans, Rab3 and Rab27 are both involved in synaptic vesicle release and they can be activated by a common exchange factor. However, based upon available genetic data, these Rabs do not appear to function redundantly during release. In Drosophila, loss of Rab3 causes a dramatic change in active zone size and organization (Graf, 2009). Thus, a redundant Rab would have to selectively and completely replace Rab3 function during synaptic homeostasis without rescuing synapse development, and this seems unlikely. Finally, since Rab3 is required for the complete block of synaptic homeostasis observed in the rab3-GAP mutant, it is concluded that Rab3 itself participates in mechanisms that determine whether or not synaptic homeostasis will proceed (Müller, 2011).

Next, the possibility is considered that Rab3 accumulates in a GTP-bound form at the synapse in the rab3-GAP mutant background, and this accumulation could block homeostatic plasticity. This model is attractive because it could explain why loss of Rab3 does not block homeostatic plasticity. However, a direct test of this model failed to provide supporting evidence. A constitutively active rab3 transgene was expressed in the rab3 mutant background. This experiment should mimic the accumulation of Rab3-GTP in a rab3-GAP mutant background. It was found that the constitutively active rab3 transgene (rab3^CA) is trafficked to the NMJ and localizes in a manner that is indistinguishable from overexpressed wild-type Rab3. Furthermore, rab3^CA has activity at the synapse because it rescues the defects in active zone organization that are caused by loss of rab3. In addition, constitutively active Rab3A was shown to biochemically interact with Rab3-GAP. However, the expression of rab3^CA did not disrupt synaptic homeostasis. Therefore, aberrant accumulation of Rab3-GTP is not the cause of impaired synaptic homeostasis in the rab3-GAP mutant and another model should therefore be considered (Müller, 2011).

Another activity of Rab3-GAP that could be relevant to synaptic homeostasis is its ability to physically bind Rab3-GTP. It is known that Rab3-GTP can bind several synaptic proteins including RIM and Rabphillin. Moreover, this study provides evidence that Rab3's GTPase activity is not limiting during synaptic homeostasis. Therefore, it is proposed that Rab3-GAP competes for Rab3-GTP binding with another protein. If this other protein inhibits synaptic homeostasis when bound to the synaptic vesicle, then displacement by Rab3-GAP binding would be a required step for synaptic homeostasis to proceed. This model can explain all of the experimental data. First, Rab3-GAP would be necessary for synaptic homeostasis. Second, synaptic homeostasis would proceed normally in the rab3 mutant because the homeostatic inhibitor would no longer localize to the synaptic vesicle. Third, overexpression of rab3^CA in the rab3 mutant background would not block synaptic homeostasis because Rab3-GAP would still be able to compete for Rab3-GTP binding, displace the homeostatic inhibitor, and allow homeostatic plasticity to proceed (Müller, 2011).

This model is consistent with a conserved function of Rab proteins throughout the membrane trafficking and secretory pathways of organisms ranging from yeast to mammals. Rab proteins, in their GTP-bound state, function to nucleate the assembly of 'effector' protein complexes that define membrane microdomains. However, throughout the literature, more is known about the assembly of these Rab-dependent complexes than is known about their disassembly. The model assumes that the binding of Rab3-GAP to Rab3 and the Rab3^CA mutant protein is sufficient to disrupt effector binding (including the proposed homeostatic inhibitor). It is generally believed that Rab-GAPs interact with their cognate GTPases with lower affinity than the effector proteins. However, although Rab3-GAP has a relatively low affinity for Rab3A, Rab3-GAP effectively competes with an effector (Rabphillin) for binding to Rab3A and this is true even when Rab3A harbors the Q81L mutation. These data support the possibility that Rab3-GAP could compete for effector binding and disrupt a Rab3-GTP dependent scaffold. This is not the only manner in which Rab3-GAP differs from other Rab-GAPs. Rab3-GAP is somewhat unique in that it does not contain additional protein-protein interaction motifs. Thus, unlike IQ-GAP proteins for instance, it seems unlikely that Rab3-GAP has unique functions that are independent of Rab3, consistent with the double-mutant analysis of rab3 and rab3-GAP (Müller, 2011).

Finally, the possibility is considered that the rab3-GAP mutation could create a ceiling effect where baseline transmission is normal but release cannot be potentiated under any condition. Several pieces of data argue against this possibility. First, by elevating extracellular calcium, quantal content can be increased in the rab3-GAP mutant, indicating that there is no restriction on the absolute number of quanta that can be released. Second, during a stimulus train (20 Hz, 0.4 mM extracellular calcium), the rab3-GAP mutant plateaus at a higher EPSP amplitude compared to wild-type. Again, there is no evidence for a ceiling effect in rab3-GAP. Finally, it has been demonstrated that mutations that cause a severe defect in baseline transmission can still undergo homeostatic compensation. Taken together, these data argue against a simple ceiling effect and support the conclusion that Rab3-GAP is directly involved in the mechanisms of synaptic homeostasis (Müller, 2011).

A question that cannot be addressed is which step in the model is modified during the induction of synaptic homeostasis. One interesting possibility is that the interaction of Rab3-GTP with the homeostatic repressor is regulated. For example, if this interaction is stabilized, then homeostatic plasticity would be opposed and, conversely, if the interaction is weakened, then homeostasis would be allowed to proceed. A recent study in C. elegans provided evidence that an unknown retrograde signal, from muscle to motoneuron, causes increased expression of YFP-Rab3 at the presynaptic terminal. Although no evidence was found for a similar phenomenon at the Drosophila NMJ during synaptic homeostasis, these data support the possibility that the Rab3/Rab3-GAP signaling complex could be a downstream, regulated target of a homeostatic, retrograde signal at the NMJ (Müller, 2011).

Interpreting the current data requires consideration of a recent study examining the effects of a rab3 mutation on synapse organization at the Drosophila NMJ (Graf, 2009). In the Graf study, it was discovered that a rab3 mutation causes a dramatic accumulation of both the active zone-associated protein Bruchpilot (Brp, T-bars, the Drosophila homolog of CAST/ELKS) and presynaptic calcium channels at a subset of active zones (Graf, 2009). Based on these and other data it was suggested that Rab3 promotes the nucleation of new active zones, and without this activity, active zones coalesce (Graf, 2009). It was possible to clearly dissociate any morphological reorganization of the NMJ from a blockade of synaptic homeostasis. The rab3 mutants have altered NMJ morphology, but normal synaptic homeostasis, whereas rab3-GAP mutants have normal NMJ morphology and a defect in synaptic homeostasis) (Müller, 2011).

It is also important to consider why rab3 mutants do not show excessive homeostatic compensation. It is predicted that Rab3 and Rab3-GAP will not control the magnitude of the homeostatic response, just whether or not it is allowed to proceed. Additional negative feedback signaling mechanisms would be responsible for determining the magnitude of the homeostatic response. This would explain why no excessive homeostatic compensation was observed in the absence of Rab3. Thus, Rab3 and Rab3-GAP provide an additional layer of control on synaptic homeostasis, ensuring that modulation of release probability only occurs when, and perhaps where, appropriate (Müller, 2011).

Ultimately, it would be important to understand how presynaptic vesicle release is modulated during homeostatic plasticity. It is known from previously published data that a homeostatic increase in vesicle release is due to a change in presynaptic release probability without a change in active zone number. Mechanistically, the full functionality of presynaptic calcium channels is necessary for synaptic homeostasis. However, it remains unknown whether synaptic homeostasis involves a change in calcium channel number versus calcium channel function. Given these prior data, one possibility is that the homeostatic signaling system, identified in this study, acts upon presynaptic calcium channels to prevent a change in calcium influx. In this respect, the involvement of RIM and RIM binding protein are intriguing since RIM binds to Rab3-GTP and has been proposed to influence calcium-channel function (Müller, 2011 and references therein).

Rab3-GAP is the first protein to be implicated in the homeostatic modulation of presynaptic release that directly interacts with a resident synaptic vesicle protein. This fact, and analysis of baseline synaptic transmission in the rab3-GAP mutant raise the possibility that the homeostatic modulation of presynaptic release also includes mechanisms that are independent of increased calcium influx. For example, a defect was observed in presynaptic release probability in the rab3-GAP mutant that occurs only when recording is performed in low extracellular calcium. This defect could reveal a function of Rab3-GAP during vesicle release, or it could reflect the activity of the proposed homeostatic repressor on baseline synaptic transmission. One possibility that could explain the decrease in release probability is that synaptic vesicles reside at a greater physical distance from the calcium channel in the rab3-GAP mutant. When recording in low extracellular calcium, the calcium microdomains at the active zone would not effectively trigger the release of these more distant vesicles. This model would suggest that enhanced coupling of the synaptic vesicle and the calcium channel is part of the homeostatic modulation of presynaptic release. By extension, the action of the homeostatic repressor would be to prevent a tight association of the synaptic vesicle with the calcium channel. It is interesting to speculate that the homeostatic repressor could be Rabphillin. It has been shown that Rabphillin can compete with Rab3-GAP for binding to Rab3-GTP. Rabphillin has two C2 domains that could confer calcium-dependence to this protein-protein interaction and, by extension, homeostatic plasticity. Ultimately, a molecular change that influences the functionality of the calcium sensor for vesicle fusion cannot be ruled out. Regardless, the data identify a homeostatic mechanism that functions at a late stage of vesicle release to modulate presynaptic release probability (Müller, 2011).

In combination with a previously published genetic screen, thirteen mutations have now been identified that disrupt the expression of synaptic homeostasis without severely altering baseline synaptic transmission. Among these genes are rab3-GAP and dysbindin. The mutant phenotypes forrab3-GAP and dysbindin are remarkably similar. In both cases, loss of function mutations have little effect on baseline transmission under standard recording conditions (0.4 mM extracellular calcium). However, decreasing extracellular calcium reveals a significant decrease in release probability. In agreement, an increase was also observed in short-term synaptic facilitation in both mutations. Furthermore, neither mutation has an effect on synapse morphology or active zone number. In dysbindin mutants, these effects were shown to be downstream or independent of presynaptic calcium influx. It is tempting to place Dysbindin into the proposed model for homeostatic plasticity. One possibility is that Dysbindin functions to stabilize the close association of synaptic vesicles with the presynaptic calcium channel. The absence of Dysbindin would therefore phenocopy the rab3-GAP mutant but function through a different set of molecular interactions on the synaptic vesicle. The similarity between the phenotypes of dysbindin and rab3-GAP are also interesting because dysbindin has been linked to schizophrenia in human. The intriguing possibility that Dysbindin interacts with Rab3-Rab3-GAP signaling will be the subject of future studies (Müller, 2011).

The signaling activity of cell surface localized membrane proteins occurs primarily while these proteins are located on the plasma membrane but is, in some cases, not terminated until the proteins are degraded. Following internalization and movement through the endocytic pathway en route to lysosomes, membrane proteins transit a late endosomal organelle called the multivesicular body (MVB). MVBs are formed by invagination of the limiting membrane of endosomes, resulting in an organelle possessing a limiting membrane and containing internal vesicles. The molecular machinery that regulates the separation of membrane proteins destined for degradation from those resulting in surface expression is not well understood. This study reconstituted an endosomal sorting event under cell-free conditions. Advantage was taken of the itinerary of a prototypical membrane protein, the Epidermal growth factor receptor (EGFR) and a biochemical monitor was designed for cargo movement into internal MVB vesicles that is generally modifiable for other membrane proteins. Since is it not known how internal vesicle formation is related to cargo sorting, morphological examination using transmission electron microscopy (TEM) allows separate monitoring of vesicle formation. This study determined that MVB sorting is dependent on cytosolic components, adenosine triphosphate (ATP), time, temperature, and an intact proton gradient. This assay reconstitutes the maturation of late endosomes and allows the morphological and biochemical examination of vesicle formation and membrane protein sorting (Giraud, 2015).

Developmental axon pruning is essential for wiring the mature nervous system, but its regulation remains poorly understood. This study shows that the endosomal-lysosomal pathway regulates developmental pruning of Drosophila mushroom body γ neurons. The UV radiation resistance-associated gene (Uvrag) functions together with all core components of the phosphatidylinositol 3-kinase class III (PI3K-cIII; see Phosphotidylinositol 3 kinase 59F) complex to promote pruning via the endocytic pathway. By studying several PI₃P binding proteins, this study found that Hrs, a subunit of the ESCRT-0 complex, required for multivesicular body (MVB) maturation, is essential for normal pruning progression. Thus, the existence of an inhibitory signal that needs to be downregulated is hypothesized. Finally, the data suggest that the Hedgehog receptor, Patched, is the source of this inhibitory signal likely functioning in a Smo-independent manner. Taken together, this in vivo study demonstrates that the PI3K-cIII complex is essential for downregulating Patched via the endosomal-lysosomal pathway to execute axon pruning (Issman-Zecharya, 2014).

Neuronal remodeling is an essential step of nervous system development in both vertebrates and invertebrates. One mechanism used to remodel neuronal circuits is by the elimination of long stretches of axons in a process known as axon pruning. With a few exceptions, the current dogma is that axon pruning of long stretches of axons occurs via local axon degeneration while axon pruning of short stretches occurs via retraction. While in some cases remodeling is directly affected by experience or neural activity, in cases of stereotypical pruning the identity of the axon that is destined to be pruned does not depend on experience or neural activity. Because of mechanistic similarities to Wallerian degeneration and dying back neurodegenerative diseases, understanding the molecular mechanisms of axon pruning should result in a broader insight into axon fragmentation and elimination during development and in disease (Issman-Zecharya, 2014).

The neuronal remodeling of the Drosophila mushroom body (MB) during development is a unique model system to study the molecular aspects of axon pruning. The stereotypic temporal and spatial occurrence of MB axon pruning combined with mosaic analyses provide a platform to perform genetic screens and molecular dissections of these processes in unprecedented resolution. The MB is comprised of three types of neurons that are sequentially born from four identical neuroblasts per hemisphere. Out of the three MB neuronal types, only the γ neurons undergo axon pruning, indicating that the process is cell-type specific. During the larval stage, γ neurons project a bifurcated axon to the dorsal and medial lobes. At the onset of metamorphosis, the dendrites of the γ neurons as well as specific parts of the axons are eliminated by localized fragmentation in a process that peaks at about 18 hr after puparium formation. Subsequently, γ neurons undergo developmental axon regrowth, which is distinct from initial axon outgrowth, to occupy the adult specific lobe (Issman-Zecharya, 2014).

Axon pruning of MB γ neurons depends on the cell-autonomous expression of the nuclear steroid hormone receptor, ecdysone receptor B1 (EcR-B1). The expression of EcR-B1 is regulated by at least three distinct pathways: the cohesin complex, the TGF-β pathway, and a network of nuclear receptors comprised of ftz-f1 and Hr39. While expression of EcR-B1 is required for pruning, it is not sufficient to drive ectopic pruning either in γ neurons or in other MB neurons that do not undergo remodeling. This raises two possible nonmutually exclusive scenarios: (1) additional molecules are required to initiate pruning and (2) an inhibitory signal needs to be attenuated in the MB for pruning to occur. Additionally, the ubiquitin pathway is also cell-autonomously required in γ neurons for pruning, but the target that must be ubiquitinated remains unknown. Thus, while understanding of the cellular sequence of events culminating in the elimination of specific axonal branches is quite detailed, understanding of the molecular mechanisms remains incomplete (Issman-Zecharya, 2014).

In a forward genetic screen, this study identified a cell-autonomous role for the UV radiation resistance-associated gene (UVRAG) in MB γ neuron pruning. UVRAG was originally identified based on its ability to confer UV resistance to nucleotide excision repair deficient cells. It was later shown to function as a tumor suppressor gene deleted in various types of cancers including colon and gastric carcinomas. UVRAG interacts with Atg6 (also known as Beclin1), another tumor suppressor gene, and together they promote autophagy in vitro. Their tumor suppression capabilities were first attributed to their autophagy-promoting function. However, a mutant form of UVRAG isolated from colon carcinomas promoted autophagy normally in cell culture. Both UVRAG and Atg6 are subunits in the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex, involved in autophagy and endocytosis. Recent studies have found that UVRAG mediates endocytosis in an Atg6-dependent manner suggesting that as part of the PI3K-cIII complex, both proteins regulate various aspects of vesicle trafficking. Two studies have recently identified new and seemingly unrelated functions for UVRAG in regulating DNA repair in response to UV-induced damage and ER to Golgi trafficking. Finally, an in vivo study has shown that UVRAG affects organ rotation in Drosophila by regulating Notch endocytosis in what seemed to be an Atg6-independent manner. A unifying understanding of the various aspects of UVRAG physiological function in vivo is still lacking. Likewise, although the PI3K-cIII complex has been extensively studied and implicated in autophagy, cytokinesis and endocytosis, its physiological roles during the normal course of development are not known (Issman-Zecharya, 2014).

This study reports that UVRAG and the PI3K-cIII complex mediate the endosome-lysosome degradation of Ptc to promote axon pruning. Furthermore, the results suggest that Ptc represses pruning via a Smo- and Hh-independent manner. This study provides evidence for the existence of a pruning inhibitory pathway originating at the membrane of MB neurons (Issman-Zecharya, 2014).

This study shows that the endosomal-lysosomal pathway is cell-autonomously required for developmental axon pruning of mushroom body (MB) γ neurons. Genetic loss-of-function experiments indicate that UVRAG, a tumor suppressor gene previously linked to both endocytosis and autophagy, promotes pruning as part of the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex and that UVRAG is required in MB neurons for the formation of phosphatidylinositol ₃-phosphate (PI₃P). The ESCRT-0 complex, which is recruited to the PI₃ moiety on endosomal membranes, is required for pruning, indicating that endosome to multivesicular body maturation is critical for the normal progression of axon pruning and suggesting that it involves receptor downregulation. Genetic loss-of-function and gain-of-function experiments suggest that downregulation of the Hedgehog receptor Patched (Ptc) by the endocytic machinery is instrumental in promoting pruning. Finally, the results suggest that Ptc inhibits pruning in a smo-independent and likely also hh-independent manner (Issman-Zecharya, 2014).

A recent study suggested that UVRAG is required for Notch endocytosis during organ rotation in Drosophila in an Atg6-independent manner. While the current study shows that Atg6 is required for pruning, these seemingly contradicting results can be easily explained by specific allele differences. The Atg6⁰⁰⁰⁹⁶ allele, used in the previous study, is a P element insertion about 100 bp upstream of the Atg6 gene that does not necessarily create a null allele. Indeed, this study could also not see any effect of this allele on axon pruning. This study used an Atg61 null allele created by homologous recombination resulting in a strong effect on pruning. Furthermore, the data clearly show that the entire PI3K-cIII complex is required for axon pruning (Issman-Zecharya, 2014).

The PI3K-cIII complex has been implicated in a wide variety of membrane trafficking processes ranging from autophagy to endocytosis to cytokinesis. How the PI3K-cIII is regulated to participate in these different processes and its physiological roles in vivo are not well understood. While its role in promoting autophagy is supported by several studies, deleting the catalytic unit, Vps34, in sensory neurons does not affect autophagy, but rather endocytosis. Whether this is a common feature of PI3K-cIII function in neurons remains to be further elucidated. One attractive hypothesis is that the PI3K-cIII function is determined by its complex composition. Indeed, it appears that in vitro, UVRAG and Atg14 are mutually exclusive subunits defining two distinct populations of the PI3K-cIII complex (Funderburk, 2010; Itakura, 2009). The current study is consistent with these findings, suggesting that UVRAG may define an endocytosis-specific PI3K-cIII complex at least in neurons. The full spectrum of the various PI3K-cIII complexes physiological roles in vivo remains to be further studied (Issman-Zecharya, 2014).

The PI3K-cIII complex phosphorylates PI to form PI₃P on endosomal membranes. Indeed, this study found that UVRAG is essential for efficient PI₃P formation and that PI₃P is abundant throughout development. It is thus hypothesized that a PI₃P binding protein mediates the effect of UVRAG and the PI3K-cIII complex on axon pruning. This study has identified Hrs, a subunit of the ESCRT-0 complex and a PI₃P binding protein, as required for axon pruning. The role of ESCRT-0 in MVB maturation led to a hypothesis that the endolysosomal pathway is required to downregulate a signal that originates at the plasma membrane. While signaling can still occur in the early endosome, it is terminated at the MVB (Issman-Zecharya, 2014).

What is the identity of this transmembrane protein? Using genetic loss-of-function and gain-of-function experiments, it is suggested that Patched (Ptc) is at least one of the transmembrane proteins that is responsible for mediating the PI3K-cIII pruning defect. Strikingly, mutating ptc on the background of a Atg6 mutant significantly suppressed its pruning defect. Furthermore, overexpression of Ptc in WT brains resulted in a weak to mild pruning defect, depending on the Gal4 driver. Finally, overexpressing Ptc on the background of an endosomal defect significantly exacerbated the pruning defect. Together, these data suggest that Ptc mediates an inhibitory signal that needs to be attenuated for the normal progression of pruning. Interestingly, Ptc inactivation by endocytosis followed by lysosomal degradation was proposed before as a mechanism to activate the Hh pathway. What is the nature of this signal? Ptc is known to be the Hedgehog (Hh) receptor. Binding of Hh to Ptc relieves the Ptc-induced suppression of another transmembrane protein, Smoothened (Smo). Once derepressed, Smo initiates the intracellular Hh signal that culminates in the expression of specific nuclear transcription factors. Therefore this study tested the role of Smo and Hh in developmental axon pruning and, to surprisingly, demonstrated that both molecules seem to be irrelevant for pruning. Overexpressing Ptc mutant transgenes within MB neurons to identify the domains that are important for pruning inhibitions confirmed that Smo inhibition was not required to inhibit pruning. In contrast, the results suggest that the ligand binding domain is important. Because the results suggest that Hh is not required for pruning inhibition, it will be interesting to investigate in the future what other ligands might bind to Ptc. In this regard it is interesting to mention that a recent study has shown that Ptc is a lipoprotein receptor. The precise mechanism of Ptc action in MB neurons remains to be further elucidated in future studies (Issman-Zecharya, 2014).

This study has uncovered a role for the endocytic machinery in downregulating an inhibitory signal that is dependent on Ptc during MB axon pruning. A recently published study has shown that the Rab5/ESCRT endocytic pathways are required to downregulate neuroglian (Nrg) to promote dendrite pruning of sensory neurons in Drosophila. Both studies highlight that a combination of both promoting and inhibitory signals during developmental pruning is likely important to provide fail-safe mechanisms to regulate the process in a temporal, spatial, and cell-type specific resolution (Issman-Zecharya, 2014).

The lysosomal enzyme receptor protein (LERP) of Drosophila is the ortholog of the mammalian cation-independent mannose 6-phosphate (Man 6-P) receptor, which mediates trafficking of newly synthesized lysosomal acid hydrolases to lysosomes. However, flies lack the enzymes necessary to make the Man 6-P mark, and the amino acids implicated in Man 6-P binding by the mammalian receptor are not conserved in LERP. Thus, the function of LERP in sorting of lysosomal enzymes to lysosomes in Drosophila is unclear. This study analyzed the consequence of LERP depletion in S2 cells and intact flies. RNAi-mediated knockdown of LERP in S2 cells had little or no effect on the cellular content or secretion of several lysosomal hydrolases. A novel Lerp null mutation, LerpF6, was generated that abolishes LERP protein expression. Lerp mutants have normal viability and fertility and display no overt phenotypes other than reduced body weight. Lerp mutant flies exhibit a 30-40% decrease in the level of several lysosomal hydrolases, and are hypersensitive to dietary chloroquine and starvation, consistent with impaired lysosome function. Loss of LERP also enhances an eye phenotype associated with defective autophagy. These findings implicate Lerp in lysosome function and autophagy (Hasanagic, 2015).

FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, Drosophila models were generated of FIG4-related diseases. Fig4 null mutant flies are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS (Bharadwaj, 2015).

GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are known to cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GlcNAc-1-phosphotransferase activity remains unclear. Mammalian GlcNAc-1-phosphotransferases are α2β2γ2 hexamers in which the core catalytic α- and β-subunits are derived from the GNPTAB (N-acetylglucosamine-1-phosphate transferase subunits alpha and beta) gene. This study presents the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB. Four conserved regions were found located far apart in the sequence that fold into the catalytic domain, which exhibits structural similarity to that of the UDP-glucose glycoprotein glucosyltransferase. Comparison with UDP-glucose glycoprotein glucosyltransferase also revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Finally, this study showed that DmGNPTAB forms a homodimer that is evolutionarily conserved and that perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GlcNAc-1-phosphotransferase function and related diseases (Duk 2022, 202).

TORC1 (see Drosophila Tor) is a master regulator of metabolism in eukaryotes that responds to multiple upstream signaling pathways. The GATOR complex is a newly defined upstream regulator of TORC1 that contains two sub-complexes, GATOR1, which inhibits TORC1 activity in response to amino acid starvation and GATOR2, which opposes the activity of GATOR1. The genome of Drosophila contains a single Sea2/Wdr24 homolog encoded by the gene CG7609 that shares 25% identity and 44% similarity to yeast Sea2 and 37% identity and 54% similarity to the human homolog WDR24. This study defines the in vivo role of the GATOR2 component Wdr24 in Drosophila. Wdr24 was shown to have both TORC1 dependent and independent functions in the regulation of cellular metabolism. Through the characterization of a null allele, it was shown that Wdr24 is a critical effector of the GATOR2 complex that promotes the robust activation of TORC1 and cellular growth in a broad array of Drosophila tissues. Additionally, epistasis analysis between wdr24 and genes that encode components of the GATOR1 complex revealed that Wdr24 has a second critical function, the TORC1 independent regulation of lysosome dynamics and autophagic flux. Notably, it was found that two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. Wdr24 was also shown to promotes lysosome acidification and autophagic flux in mammalian cells. Taken together these data support the model that Wdr24 is a key effector of the GATOR2 complex, required for both TORC1 activation and the TORC1 independent regulation of lysosomes (Cai, 2016).

In metazoans multiple conserved signaling pathways control the integration of metabolic and developmental processes. TORC1 is an evolutionarily conserved multi-protein complex that regulates metabolism and cell growth in response to an array of upstream inputs including nutrient availability, growth factors and intracellular energy levels. The catalytic component of TORC1 is the serine/threonine kinase Target of Rapamycin (TOR). When nutrients are abundant, TORC1 activity promotes translation, ribosome biogenesis as well as other pathways associated with anabolic metabolism and cell growth. However, when nutrients or other upstream activators are limiting, TORC1 activity is inhibited triggering catabolic metabolism and autophagy (Cai, 2016).

The Seh1 associated/GTPase-activating protein toward Rags (SEA/GATOR) complex is a newly identified upstream regulator of TORC1 that can be divided into two putative sub-complexes GATOR1 and GATOR2 (Bar-Peled, 2013; Dokudovskaya, 2011; Panchaud, 2013). The GATOR1 complex, known as the Iml1 complex or the Seh1 Associated Complex Inhibits TORC1 (SEACIT) in yeast, inhibits TORC1 activity in response to amino acid limitation. SEACIT/GATOR1 contains three proteins Npr2/Nprl2, Npr3/Nprl3 and Iml1/DEPDC5. Recent evidence, from yeast and mammals, indicates that the components of the SEACIT/GATOR1 complex function through the Rag GTPases to inhibit TORC1 activity. Notably, Nprl2 and DEPDC5 are tumor suppressor genes while mutations in DEPDC5 are a leading cause of hereditary focal epilepsies (Cai, 2016).

The GATOR2 complex, which is referred to as Sevh1 Associated Complex Activates TORC1 (SEACAT) in yeast, activates TORC1 by opposing the activity of GATOR1. The SEACAT/GATOR2 complex is comprised of five proteins, Seh1, Sec13, Sea4/Mio, Sea2/WDR24, and Sea3/WDR59. Computational analysis indicates that multiple components of the GATOR2 complex have structural features characteristic of coatomer proteins and membrane tethering complexes. In line with the structural similarity to proteins that influence membrane dynamics, in Drosophila the GATOR2 subunits Mio and Seh1 localize to multiple endomembrane compartments including lysosomes, the site of TORC1 regulation, and autolysosomes. In metazoans, members of the Sestrin and Castor family of proteins bind to and inhibit the GATOR2 complex in response to leucine and arginine starvation respectively. This interaction is proposed to inhibit TORC1 activity through the derepression of the GATOR1 complex. However, how GATOR2 opposes GATOR1 activity, thus allowing for the robust activation of TORC1, remains unknown. Additionally, the role of the GATOR2 complex in the regulation of both the development and physiology of multicellular animals remains poorly defined (Cai, 2016).

Recent evidence from Drosophila indicates that the requirement for the GATOR2 complex may be context specific in multicellular animals. In Drosophila, null alleles of the GATOR2 components mio and seh1 are viable but female sterile. Surprisingly, somatic tissues from mio and seh1 mutants exhibit little if any reductions in cell size and have nearly normal levels of TORC1 activity. In contrast, TORC1 activity is dramatically decreased in ovaries from mio and seh1 mutant females. This decrease in TORC1 activity is accompanied by the activation of catabolic metabolism in the female germ line, a dramatic reduction in egg chamber growth and difficulties maintaining the meiotic cycle. Thus, there is a surprising tissue specific requirement for the GATOR2 components Mio and Seh1 during oogenesis. However, the in vivo role of the other members of the GATOR2 complex in the regulation of cellular metabolism remains undefined (Cai, 2016).

This study defines the in vivo requirement for the GATOR2 component Wdr24 in Drosophila. Wdr24 was found to have two distinct functions. First, Wdr24 is a critical effector of the GATOR2 complex that promotes TORC1 activity and cellular growth in a broad array of tissues. Second, Wdr24 is required for the TORC1 independent regulation of lysosome function and autophagic flux. Notably, two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. Taken together these data support the model that multiple components of the GATOR2 complex have both TORC1 dependent and independent roles in the regulation of cellular metabolism (Cai, 2016).

This study describes a dual role for the GATOR2 component Wdr24 in the regulation of TORC1 activity and lysosome dynamics. Wdr24 is a critical effector of the GATOR2 complex that promotes TORC1 activity in both germline and somatic tissues. This lies in contrast to the GATOR2 components Mio and Seh1, which have a limited role in the regulation of TORC1 activity in many cell types. Surprisingly, a second function of Wdr24 was identified that is independent of TORC1 status, the regulation of lysosome acidification and autophagic flux. Taken together these data support the model that the GATOR2 complex regulates both the response to amino acid starvation and lysosome function (Cai, 2016).

Whole animal studies often reveal tissue-specific and/or metabolic requirements for genes that are not readily observed in cell culture. In mammalian and Drosophila tissue culture cells, RNAi based depletions of the GATOR2 components Mio, Seh1, Wdr59, and Wdr24 result in decreased TORC1 activity in return to growth assays (Bar-Peled, 2013, Wei, 2014). These data have resulted in the model that all components of the GATOR2 complex are generally required for TORC1 activation (Wei, 2014). However, the characterization of mio and seh1 null mutants in Drosophila, demonstrated that Mio and Seh1 are critical for the activation of TORC1 and inhibition of autophagy in the female germ line, but play a relatively small role in the regulation of TORC1 activity and autophagy in somatic tissues under standard culture conditions. Thus, the requirement for at least a subset of GATOR2 complex components is tissue and/or context specific (Cai, 2016).

This study reports that the GATOR2 component Wdr24 is required for the full activation of TORC1 in both germline and somatic cells of Drosophila. Consistent with the global down-regulation of TORC1 activity in the absence of Wdr24, wdr24 mutant adults are notably smaller than controls and are female sterile. Depleting the GATOR1 components nprl2 and nprl3 in the wdr24 mutant background rescued the low TORC1 activity, growth defects, and female sterility of wdr24 mutants. Thus, the GATOR2 component Wdr24 is required to oppose GATOR1 activity in both germline and somatic cells of Drosophila. From these results it is proposed that Wdr24 is a key effector of the GATOR2 complex required for the full activation of TORC1 in most cell types (Cai, 2016).

There are several potential models to explain the differential requirement for individual GATOR2 proteins in Drosophila. First, there may be tissue specific requirements for individual GATOR2 subunits. In this model the different phenotypes observed in the seh1 and mio versus wdr24mutants reflects a qualitative difference in the requirement for these proteins in different tissues. However, an alternative model is favored in which Wdr24 is the core effector of GATOR2 activity, with Mio and Seh1 functioning primarily as positive regulators of GATOR2 activity. In this second model, the differential phenotypes observed in the seh1 and mio versus wdr24 mutants reflects a quantitative difference in the requirement for GATOR2 activity in different tissues. The distinction between these two models awaits the identification of the molecular mechanism of Wdr24 and GATOR2 action (Cai, 2016).

A novel TORC1 independent role has been identified for Wdr24 in the regulation of lysosome dynamics and function. In wdr24 mutants, the down-regulation of TORC1 activity and the accumulation of autolysosomes occur independent of nutrient status. It was initially hypothesized that in the absence of the GATOR2 component Wdr24, the deregulation of the GATOR1 complex results in low TORC1 activity, triggering the constitutive activation of autophagy and the accumulation of autolysosomes. Surprisingly, however, epistasis analysis determined that the accumulation of lysosomes could be decoupled from both the chronic inhibition of TORC1 activity and the activation of autophagy. Raising TORC1 activity in the wrd24 mutant background, by depleting either components of the GATOR1 or TSC complex, failed to rescue the accumulation of abnormal lysosomal structures. Notably, it was determined that two additional members of the GATOR2 complex, Mio and Seh1, also regulate lysosomal behavior independent of both GATOR1 and the down-regulation of TORC1 activity. From these data it is inferred that multiple components of the GATOR2 complex have a TORC1 independent role in the regulation of lysosomes (Cai, 2016).

An increased number of autolysosomes is often associated with reduced autophagic flux due to diminished lysosomal degradation. Consistent with reduced autophagic flux, in Drosophila wrd24-/- mutants accumulated enlarged autolysosomes filled with undegraded material. Moreover, lysosomes in the wrd24-/- mutants failed to quench the GFP fluorescence of a GFP-mCherry-Atg8a protein. These phenotypes are consistent with decreased lysosomal pH and degradative capacity. In order to examine in detail the role of Wdr24 in the regulation of lysosome function a wrd24-/- knockout HeLa cell line was generated that recapitulated the phenotypes observed in Drosophila wrd24-/- mutants. Specifically, wrd24-/- HeLa cells had have decreased TORC1 activity and accumulate a large number of autolysosomes. Using multiple assays it was determined that wrd24-/- lysosomes had reduced degradative capacity and autophagic flux and thus accumulate proteins that are normally degraded by lysosomal enzymes such as p62, LC3II and Cathepsin D. Additionally, it was determined that wrd24-/-lysosomes have increased pH relative to wild-type cells, again consistent with reduced lysosomal function. Taken together these data confirm that Wdr24 plays a key role in the regulation of lysosomal activity (Cai, 2016).

This study shows that components of the GATOR2 complex function in the regulation of TORC1 activity and in the TORC1 independent regulation of lysosomal dynamics and autophagic flux. These two functions suggest that the GATOR2 complex may regulate cellular homeostasis by coordinating TORC1 activity with the dynamic regulation of lysosomes during periods of nutrient stress. Intriguingly, several recent reports describe a very similar dual function for the RagA/B GTPases in both mice and zebrafish (Kim, 2014; Shen,2016). RagA/B play a critical role in the activation of TORC1 in the presence of amino acids (Kim, 2008; Sancak, 2008). Surprisingly, however, TORC1 activity was not found to be significantly decreased in cardiomyocytes of RagA/B knockout mice (Cai, 2016).

Nevertheless, the RagA/B mutant cardiomyocytes have decreased autophagic flux and reduced lysosome acidification. From published data, it was concluded that the RagA/B GTPases regulate lysosomal function independent of their role in the regulation of TORC1 activation in some cell types. Similarly, RagA is required for proper lysosome function and phagocytic flux in microglia. Notably, Mio, a component of the GATOR2 complex is found associated with RagA (Bar-Peled, 2013). Thus, in the future it will be important to determine if components of the GATOR2 complex function in a common pathway with the Rag GTPases to regulate lysosomal function (Cai, 2016).

In Saccharomyces cerevisiae single mutants of wrd24/sea2/ and wdr59/sea3 do not exhibit defects in TORC1 regulation but do have defects in vacuolar structure. Moreover, several recently identified genes that regulate the GATOR2-GATOR1-TORC1 pathway in response to amino acid limitation are restricted to metazoans. These data make it tempting to speculate that the ancestral function of the GATOR2 complex maybe the regulation of lysosome/vacuole function and autophagic flux. Indeed, the finding that GATOR2 components regulate lysosome dynamics is particularly intriguing in light of the observation that GATOR2 complex is comprised of proteins with characteristics of coatomer proteins and membrane tethering complexes. Notably, the GATOR2 complex components Mio, Seh1 and Wdr24 localize to lysosomes and autolysosomes. Similarly, these proteins associate with the vacuolar membrane in budding yeast. Thus, going forward it will be important to examine if the GATOR2 complex acts directly on lysosomal membranes to regulate their structure and/or function. More broadly, future studies on the diverse roles of the SEACAT/GATOR2 complex will further understanding of the complex relationship between cellular metabolism and the regulation of endomembrane dynamics in both development and disease (Cai, 2016).

This study evaluated the mechanisms underlying the neurodevelopmental deficits in Drosophila and mouse models of lysosomal storage diseases (LSDs). Lysosomes promote the growth of neuromuscular junctions (NMJs) via Rag GTPases and mechanistic target of rapamycin complex 1 (MTORC1). However, rather than employing S6K/4E-BP1, MTORC1 stimulates NMJ growth via JNK, a determinant of axonal growth in Drosophila and mammals. This role of lysosomal function in regulating JNK phosphorylation is conserved in mammals. Despite requiring the amino-acid-responsive kinase MTORC1, NMJ development is insensitive to dietary protein. This paradox is attributed to anaplastic lymphoma kinase (ALK), which restricts neuronal amino acid uptake, and the administration of an ALK inhibitor couples NMJ development to dietary protein. These findings provide an explanation for the neurodevelopmental deficits in LSDs and suggest an actionable target for treatment (Wong, 2015).

Mucolipidosis type IV (MLIV) and Batten disease are untreatable lysosomal storage diseases (LSDs) that cause childhood neurodegeneration. MLIV arises from loss-of-function mutations in the gene encoding TRPML1, an endolysosomal cation channel belonging to the TRP superfamily. The absence of TRPML1 leads to defective lysosomal storage and autophagy, mitochondrial damage, and macromolecular aggregation, which together initiate the protracted neurodegeneration observed in MLIV). Batten disease arises from the absence of a lysosomal protein, CLN3), and results in psychomotor retardation. Both diseases cause early alterations in neuronal function. For instance, brain imaging studies revealed that MLIV and Batten patients display diminished axonal development in the cortex and corpus callosum, the causes of which remain unknown (Wong, 2015).

To better understand the etiology of MLIV in a genetically tractable model, flies were generated lacking the TRPML1 ortholog. The trpml-deficient (trpml¹) flies have led to insight into the mechanisms of neurodegeneration and lysosomal storage (Wong, 2015).

This study reports that trpml¹ larvae exhibit diminished synaptic growth at the NMJ, a well-studied model synapse. Lysosomal function supports Rag GTPases and MTORC1 activation, and this is essential for JNK phosphorylation and synapse development (Wong, 2015).

Drosophila larvae and mice lacking CLN3 also exhibit diminished Rag/ MTORC1 and JNK activation, suggesting that alterations in neuronal signaling are similar in different LSDs and are evolution- arily conserved. Interestingly, the NMJ defects in the two fly LSD models were suppressed by the administration of a high-protein diet and a drug that is currently in clinical trials to treat certain forms of cancer. These findings inform a pharmacotherapeutic strategy that may suppress the neurodevelopmental defects observed in LSD patients (Wong, 2015).

This study shows that lysosomal dysfunction in Drosophila MNs results in diminished bouton numbers at the larval NMJ. Evidence is presented that lysosomal dysfunction results in decreased activation of the amino-acid-responsive cascade involving Rag/MTORC1, which are critical for normal NMJ development (Wong, 2015).

Despite the requirement for MTORC1 in NMJ synapse development, previous studies and the current findings show that bouton numbers are independent of S6K and 4E-BP1. Rather, MTORC1 promotes NMJ growth via a MAP kinase cascade culminating in JNK activation. Therefore, decreasing lysosomal function or Rag/MTORC1 activation in hiw^ND8 suppressed the associated synaptic overgrowth. However, the 'small-bouton' phenotype of hiw^ND8 was independent of MTORC1. Thus, MTORC1 is required for JNK-dependent regulation of bouton numbers, whereas bouton morphology is independent of MTORC1. Furthermore, although both rheb expression and hiw loss result in Wnd-dependent elevation in bouton numbers, the supernumerary boutons in each case show distinct morphological features. Additional studies are needed for deciphering the complex interplay between MTORC1-JNK in regulating the NMJ morphology (Wong, 2015).

Biochemical analyses revealed that both JNK phosphorylation and its transcriptional output correlated with the activity of MTORC1, which are consistent with prior observations that cln³ overexpression promotes JNK activation and that tsc1/tsc2 deletion in flies result in increased JNK-dependent transcription. These findings point to the remarkable versatility of MTORC1 in controlling both protein trans lation and gene transcription (Wong, 2015).

Using an in vitro kinase assay, this study demonstrates that Wnd is a target of MTORC1. Because axonal injury activates both MTORC1 and DLK/JNK, these findings imply a functional connection between these two pathways. Interestingly, the data also suggest that MTORC1 contains additional kinases besides MTOR that can phosphorylate Wnd. One possibility is that ULK1/Atg1, which associates with MTORC1, could be the kinase that phosphorylates Wnd. Consistent with this notion, overexpression of Atg1 in the Drosophila neurons has been shown to promote JNK signaling and NMJ synapse overgrowth via Wnd) (Wong, 2015).

This study also found that developmental JNK activation in axonal tracts of the CC and pJNK levels in cortical neurons were compromised in a mouse model of Batten disease. Thus, the signaling deficits identified in Drosophila are also conserved in mammals. The activity of DLK (the mouse homolog of Wnd) and JNK signaling are critical for axonal development in the mouse CNS. Therefore, decreased neuronal JNK activation during development might underlie the thinning of the axonal tracts observed in many LSDs (Wong, 2015).

Although the findings of this study demonstrate a role for an amino-acid- responsive cascade in the synaptic defects associated with lysosomal dysfunction, simply elevating the dietary protein content was not sufficient to rescue these defects. These findings were reminiscent of an elegant study that showed that the growth of Drosophila neuroblasts is uncoupled from dietary amino acids owing to the function of ALK, which suppresses the uptake of amino acids into the neuroblasts (Cheng, 2011). Indeed, simultaneous administration of an ALK inhibitor and a high-protein diet partially rescued the synaptic growth defects associated with the lysosomal dysfunction, and improved the rescue of pupal lethality associated with trpml¹. Although these studies do not causally link the defects in synapse development with pupal lethality, they do raise the intriguing possibility that multiple phenotypes associated with LSDs could be targeted using ALK inhibitors along with a protein-rich diet (Wong, 2015).

Although LSDs result in lysosomal dysfunction throughout the body, neurons are exceptionally sensitive to these alterations. The cause for this sensitivity remains incompletely understood. Given the findings of this study that mature neurons do not efficiently take up amino acids from the extracellular medium, lysosomal degradation of proteins serves as a major source of free amino acids in these cells. Therefore, disruption of lysosomal degradation leads to severe shortage of free amino acids in neurons, regardless of the quantity of dietary proteins, thus explaining the exquisite sensitivity of neurons to lysosomal dysfunction (Wong, 2015).

Degradation of cellular material by autophagy is essential for cell survival and homeostasis, and requires intracellular transport of autophagosomes to encounter acidic lysosomes through unknown mechanisms. This study identified the PX domain-containing kinesin Klp98A as a novel regulator of autophagosome formation, transport and maturation in Drosophila. Depletion of Klp98A caused abnormal clustering of autophagosomes and lysosomes at the cell center and reduced the formation of starvation-induced autophagic vesicles. Reciprocally, overexpression of Klp98A redistributed autophagic vesicles toward the cell periphery. These effects were accompanied by reduced autophagosome-lysosome fusion and autophagic degradation. In contrast, depletion of the conventional kinesin heavy chain caused a similar mislocalization of autophagosomes without perturbing their fusion with lysosomes, indicating that vesicle fusion and localization are separable, independent events. Klp98A-mediated fusion required the endolysosomal GTPase Rab14, which interacted and colocalized with Klp98A and required Klp98A for normal localization. Thus, Klp98A coordinates the movement and fusion of autophagic vesicles by regulating their positioning and interaction with the endolysosomal compartment (Mauvezin, 2016).

The role of Rab2 in the fusion of lysosomes with other vesicles is also supported by the autolysosomal localization of its active form and by its binding to the Vps39-containing end of HOPS, the tethering complex required for autophagosomal, endosomal, and biosynthetic transport to lysosomes. Consistently, Rab2 recruits HOPS to Rab7-positive vesicles in cultured Drosophila cells. Expression of Rab2^GTP increases degradative autolysosome and pigment granule size, suggesting that it is rate limiting during these fusion reactions, unlike Rab7. This is supported by low levels of wild-type Rab2 on these organelles, unlike wild-type Rab7 that is abundant on autophagosomes, late endosomes, and lysosomes. Consistent with this, it has been recently shown that expression of RAB2A^GTP also increases Rab7 vesicle size in human cells. Based on binding of Rab2 to one end of HOPS, an updated model is proposed of lysosomal fusions in animal cells. It is hypothesized that GTP-loaded Rab2 is transported on Golgi-derived carrier vesicles toward Rab7 positive vesicles, and its interaction with Vps39 promotes fusions. Vps41 located on the other end of HOPS may bind Rab7 vesicles via adaptors such as PLEKHM1. These interactions help the tethering and fusion of autophagic, endocytic, and lysosomal vesicles to generate degrading compartments. Lysosomal membranes may contain active Rab2 for only a short period of time, and it likely dissociates upon GTP hydrolysis to limit organelle size. Rab asymmetry is also observed during homotypic vacuole fusion in yeast: GTP-bound Ypt7/Rab7 is necessary on only one of the vesicles, and its nucleotide status is irrelevant on the opposing membrane. Importantly, Rab7 directly interacts with both ends of HOPS in the absence of a Rab2 homolog in yeast. This difference may explain why yeast cells contain one large vacuole instead of the many smaller lysosomes seen in animal cells. Collectively, these data indicate that Rab2 and Rab7 coordinately promote autophagic and endosomal degradation and lysosome function (Lorincz, 2017).

Lysosomes are the major catabolic compartment within eukaryotic cells, and their biogenesis requires the integration of the biosynthetic and endosomal pathways. Endocytosis and autophagy are the primary inputs of the lysosomal degradation pathway. Endocytosis is specifically needed for the degradation of membrane proteins whereas autophagy is responsible for the degradation of cytoplasmic components. The deubiquitinating enzyme UBPY/USP8 has been identified as being necessary for lysosomal biogenesis and productive autophagy in Drosophila. Because UBPY/USP8 has been widely described for its function in the endosomal system, it was hypothesized that disrupting the endosomal pathway itself may affect the biogenesis of the lysosomes. This study blocked the progression of the endosomal pathway at different levels of maturation of the endosomes by expressing in fat body cells either dsRNAs or dominant negative mutants targeting components of the endosomal machinery: Shibire, Rab4, Rab5, Chmp1 and Rab7. Inhibition of endosomal trafficking at different steps in vivo was observed to be systematically associated with defects in lysosome biogenesis, resulting in autophagy flux blockade. These results show that the integrity of the endosomal system is required for lysosome biogenesis and productive autophagy in vivo (Jacomin, 2016).

Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Lipocalin Apolipoprotein D (ApoD) is an extracellular lipid binding protein endowed with antioxidant capacity. This study performed a comprehensive analysis of ApoD intracellular traffic and demonstrates its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. ApoD was shown to be endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins (see Glial Lazarillo). These results set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular functions, critical for the outcome of a wide variety of neurodegenerative diseases (Pascua-Maestro, 2017).

How multicellular organisms maintain immune homeostasis across various organs and cell types is an outstanding question in immune biology and cell signaling. In Drosophila, blood cells (hemocytes) respond to local and systemic cues to mount an immune response. While endosomal regulation of Drosophila hematopoiesis is reported, the role of endosomal proteins in cellular and humoral immunity is not well-studied. This study demonstrated a functional role for endosomal proteins in immune homeostasis. The ubiquitous trafficking protein ADP Ribosylation Factor 1 (ARF1) and the hemocyte-specific endosomal regulator Asrij differentially regulate humoral immunity. Asrij and ARF1 play an important role in regulating the cellular immune response by controlling the crystal cell melanization and phenoloxidase activity. ARF1 and Asrij mutants show reduced survival and lifespan upon infection, indicating perturbed immune homeostasis. The ARF1-Asrij axis suppresses the Toll pathway anti-microbial peptides (AMPs) by regulating ubiquitination of the inhibitor Cactus. The Imd pathway is inversely regulated- while ARF1 suppresses AMPs, Asrij is essential for AMP production. Several immune mutants have reduced Asrij expression, suggesting that Asrij co-ordinates with these pathways to regulate the immune response. This study highlights the role of endosomal proteins in modulating the immune response by maintaining the balance of AMP production. Similar mechanisms can now be tested in mammalian hematopoiesis and immunity (Khadilkar, 2017).

A balanced cellular and humoral immune response is essential to achieve and maintain immune homeostasis. In Drosophila, aberrant hematopoiesis and impaired hemocyte function can both affect the ability to fight infection and maintain immune homeostasis. Endosomal proteins are known to regulate Drosophila hematopoiesis. This study shows an essential function for endosomal proteins in regulating immunity (Khadilkar, 2017).

Altered hemocyte number and distribution as a result of defective hematopoiesis, can also lead to immune phenotypes like increased melanization or phagocytosis. This study shows that perturbation of normal levels of endocytic molecules ARF1 or Asrij leads to aberrant hematopoiesis, affecting the circulating hemocyte number. This in turn leads to an impaired cellular immune response. The aberrant hematopoietic phenotypes with pan-hemocyte tissue-specific depletion of ARF1 using e33cGal4 or HmlGal4 are comparable to the phenotypes observed in the case of asrij null mutant. Hence this study has compared Gal4-mediated ARF1 knockdown to asrij null mutant (Khadilkar, 2017).

In addition, it was also shown that ARF1 and Asrij have a direct role in humoral immunity by regulating AMP gene expression. This is likely to be a contribution from the hemocyte compartment which is primarily affected upon perturbation of Asrij or ARF1. It is well established that hemocytes, apart from acting as the cellular arm of the immune response, also act as sentinels and relay signals to the immune organs that mount the humoral immune response. Hemocytes have been shown to produce ligands like Spaetzle and upd3 that activate immune pathways and induce anti-microbial peptide secretion from the fat body or gut. Asrij or ARF1 could also be affecting the production of such ligand molecules thereby affecting the target immune-activation pathways (Khadilkar, 2017).

Considering the involvement of Asrij and ARF1 in both the arms of immune response, a model is proposed for the role of the ARF1-Asrij axis in maintaining immune homeostasis that can be used for testing additional players in the process (Khadilkar, 2017).

It is known that ARF1 is involved in clathrin coat assembly and endocytosis and has a critical role in membrane bending and scission. In this context it is also intriguing to note that ARF1, like Asrij, does not seem to have an essential role in phagocytosis. This suggests that hemocytes could be involved in additional mechanisms beyond phagocytosis in order to combat an infection (Khadilkar, 2017).

Both ARF1 and Asrij control hemocyte proliferation as their individual depletion leads to an increase in the total and differential hemocyte counts. Also, both mutants have higher crystal cell numbers due to over-activation of Notch as a result of endocytic entrapment. This suggests that increased melanization accompanied by increase in phenoloxidase activity upon ARF1 or Asrij depletion is a consequence of aberrant hematopoiesis and not likely due to a cellular requirement in regulating the melanization response. Constitutive activation of the Toll pathway or impaired Jak/Stat or Imd pathway signaling in various mutants also leads to the formation of melanotic masses. Thus the phenotypes seen on Asrij or ARF1 depletion could either be due to the defective hematopoiesis which directly affects the cellular immune response or leads to a mis-regulation of the immune regulatory pathways (Khadilkar, 2017).

Regulation of many signaling pathways, including the immune regulatory pathways takes place at the endosomes. For example, endocytic proteins Mop and Hrs co-localize with the Toll receptor at endosomes and function upstream of MyD88 and Pelle, thus indicating that Toll signalling is regulated by endocytosis. This study shows that loss of function of the ARF1-Asrij axis leads to an upregulation of some AMP targets of the Toll pathway. Upon depletion of ARF1-Asrij endosomal axis, increased ubiquitination of Cactus, a negative regulator of the Toll pathway, was found in both hemocytes and fat bodies. This suggests non-autonomous regulation of signals by the ARF1-Asrij axis, which is in agreement with an earlier model of signalling through this route. Thus the endosomal axis may systemically control the sorting and thereby degradation of Cactus, which in turn promotes the nuclear translocation of Toll effector, Dorsal. This could explain the significant increase in Toll pathway reporter expression such as Drosomycin-GFP. Interestingly the effect of ARF1 depletion on the Toll pathway is more pronounced than that of Asrij depletion. This is not surprising as ARF1 is a ubiquitous and essential trafficking molecule that regulates a variety of signals. This suggests that ARF1 is likely to be involved with additional steps of the Toll pathway and may also interact with multiple regulators of AMP expression (Khadilkar, 2017).

ARF1 and Asrij show complementary effects on IMD pathway target AMPs. While ARF1 suppresses the production of IMD pathway AMPs, Asrij has a discriminatory role. Asrij seems to promote transcription of AttacinA and Drosocin, whereas it represses Cecropin. However in terms of AMP production only Drosocin and Diptericin are affected, but not to the extent of ARF1. In addition, Relish shows marked nuclear localization in fat body cells of hemocyte-specific arf1 knockdown larvae whereas there is no significant difference in the localization in Asrij depleted larval fat bodies. This indicates that ARF1-Asrij axis exerts differential control over the Imd pathway. Thus ARF1 causes strong generic suppression of the Imd pathway while the role of Asrij could be to fine tune this effect. Mass spectrometric analysis of purified protein complexes indicates that ARF1 and Imd interact. Hence it is very likely that ARF1 regulates Imd pathway activation at the endosomes. Whether this interaction involves Asrij or not remains to be tested and will give insight into modes of differential activation of immune pathways (Khadilkar, 2017).

This analysis shows that Asrij is the tuner for endosomal regulation of the humoral immune response by ARF1 and provides specialized tissue- specific and finer control over AMP regulation. This is in agreement with earlier data showing that Asrij acts downstream of ARF127. Since ARF1 is expressed in the fat body it could communicate with the hemocyte- specific molecule, Asrij, to mediate immune cross talk (Khadilkar, 2017).

As reduced Asrij expression is seen in Toll and Jak/Stat pathway mutants such as Rel E20 and Hop Tum1, it is likely that these effectors also regulate Asrij, setting up a feedback mechanism to modulate the immune response. Earlier work has shown that ARF1-Asrij axis modulates different signalling outputs like Notch by endosomal regulation of NICD (Notch Intracellular Domain) transport and activity and JAK/STAT by endosomal activation of Stat92e. Further, ARF1 along with Asrij regulates Pvr signaling in order to maintain HSC's. ARF1 acts downstream of Pvr. Surprisingly, Asrij levels are downregulated in the Pvr mutant. Hence it is likely that the ARF1-Asrij axis regulates trafficking of the Pvr receptor, which then also regulates Asrij levels thus providing feedback regulation. While active modulation of signal activity and outcome at endosomes could be orchestrated by ARF1 and Asrij, their activities in turn need to be modulated. The data suggest that targets of Asrij endosomal regulation may in turn regulate Asrij expression at the transcript level. Further, upon Gram positive infection in wild type flies, asrij transcript levels decrease with a concomitant increase in suppressed AMPs such as Cecropin. This indicates additional regulatory loops such as that mediated by the IMD pathway effector NFκB may regulate asrij transcription. Using bioinformatics tools, presence of binding sites for NFκβ and Rel family of transcription factors are seen in the upstream regulatory sequence (1kb upstream) of asrij and arf1. Hence, feedback regulation is proposed of Asrij and ARF1 by the effectors of the Toll and Imd pathway respectively. This is reflected in the regulation of Asrij expression by these pathways. This also implies multiple modes of regulation of asrij and arf1, which are likely important in its role as a tuner of the generic immune response, thereby allowing it to discriminate between AMPs that were thought to be uniformly regulated, such as those downstream of IMD. Thus this analysis gives insight into additional complex regulation of the Drosophila immune response that can now be investigated further (Khadilkar, 2017).

Asrij and ARF1 being endocytic proteins are likely to interact with a number of molecules that regulate different cell signalling cascades. Due to endosomal localization, molecular interactions may be favored that further translate into signalling output. Hence, it is not surprising that Asrij and ARF1 genetically interact with multiple signalling pathways and can aid crosstalk to regulate important developmental and physiological processes like hematopoiesis or immune response. It is quite likely that Asrij and ARF1 are themselves also part of different feedback loops or feed-forward mechanisms as their levels need to be tightly regulated. Evidence for this is found with respect to the Toll, JAK/STAT and Pvr pathway as described earlier. Hence it is proposed that the Asrij-ARF1 endosomal signalling axis genetically interacts with various signalling components thereby regulating blood cell and immune homeostasis (Khadilkar, 2017).

AMP transcript level changes upon ARF1 or Asrij depletion also correspond to reporter-AMP levels seen after infection. This suggests that although ARF1 is known to have a role in secretion, mutants do not have an AMP secretion defect. Hence aberrant regulation of immune pathways on perturbation of the ARF1-Asrij axis is most likely due to perturbed endosomal regulation (Khadilkar, 2017).

ARF1 has a ubiquitous function in the endosomal machinery and is well-positioned to regulate the interface between metabolism, hematopoiesis and immunity in order to achieve homeostasis. Along with Asrij and other tissue-specific modulators, it can actively modulate the metabolic and immune status in Drosophila. In this context, it is interesting to note that Asrij is a target of MEF253, which is required for the immune-metabolic switch in vivo. Thus Asrij could bring tissue specificity to ARF1 action, for example, by modulating insulin signalling in the hematopoietic system (Khadilkar, 2017).

It is likely that in Asrij or ARF1 mutants, the differentiated hemocytes mount a cellular immune response and perish as in the case of wild type flies where immunosenescence sets in with age and the ability of hemocytes to combat infection declines. Since their hematopoietic stem cell pool is exhausted, they may fail to replenish the blood cell population, thus compromising the ability to combat infections. Alternatively, mechanisms that downregulate the inflammatory responses and prevent sustained activation may be inefficient when the trafficking machinery is perturbed. This could result in constitutive upregulation thus compromising immune homeostasis (Khadilkar, 2017).

In summary, this study shows that in addition to its requirement in hematopoiesis, the ARF1-Asrij axis can differentially regulate humoral immunity in Drosophila, most likely by virtue of its endosomal function. ARF1 and Asrij bring about differential endocytic modulation of immune pathways and their depletion leads to aberrant pathway activity and an immune imbalance. In humans, loss of function mutations in molecules involved in vesicular machinery like Amphyphysin I in which clathrin coated vesicle formation is affected leads to autoimmune disorders like Paraneoplastic stiff-person syndrome. Synaptotagmin, involved in vesicle docking and fusion to the plasma membrane acts as an antigenic protein and its mutation leads to an autoimmune disorder called Lambert-Eaton myasthenic syndrome. Mutations in endosomal molecules like Rab27A, β subunit of AP3, SNARE also lead to immune diseases like Griscelli and Hermansky-Pudlak syndrome. Mutants of both ARF1 and Asrij are likely to have drastic effects on the immune system. Asrij has been associated with inflammatory conditions such as arthritis, thyroiditis, endothelitis and tonsillitis, whereas the ARF family is associated with a wide variety of diseases. ARF1 has been shown to be involved in mast cell degranulation and IgE mediated anaphylaxis response. Generation and analysis of vertebrate models for these genes such as knockout and transgenic mice will provide tools to understand their function in human immunity (Khadilkar, 2017).

Clearance of bacteria by macrophages involves internalization of the microorganisms into phagosomes, which are then delivered to endolysosomes for enzymatic degradation. These spatiotemporally segregated processes are not known to be functionally coupled. This study shows that lysosomal degradation of bacteria sustains phagocytic uptake. In Drosophila and mammalian macrophages, lysosomal dysfunction due to loss of the endolysosomal Cl- transporter ClC-b/CLCN7 delayed degradation of internalized bacteria. Unexpectedly, defective lysosomal degradation of bacteria also attenuated further phagocytosis, resulting in elevated bacterial load. Exogenous application of bacterial peptidoglycans restored phagocytic uptake in the lysosomal degradation-defective mutants via a pathway requiring cytosolic pattern recognition receptors and NF-κB. Mammalian macrophages that are unable to degrade internalized bacteria also exhibit compromised NF-κB activation. These findings reveal a role for phagolysosomal degradation in activating an evolutionarily conserved signaling cascade, which ensures that continuous uptake of bacteria is preceded by lysosomal degradation of microbes (Wong, 2017).

In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulate both lysosomal function and organ development. However, it is not clear whether these 2 processes are interconnected. This study shows that Mitf, the single TFEB and MITF ortholog in Drosophila, controls expression of vacuolar-type H+-ATPase pump (V-ATPase) subunits. Remarkably, it was also found that expression of Vha16-1 and Vha13, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, Vha16-1 expression follows differentiation of proneural regions of the disc. These regions, that will form sensory organs in the adult, appear to possess a distinctive endo-lysosomal compartment and Notch (N) localization. Modulation of Mitf activity in the disc in vivo alters endo-lysosomal function and disrupts proneural patterning. Similar to these findings in Drosophila, in human breast epithelial cells, it was observed that the impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endo-lysosomal compartment and depletion of TFEB reduces ligand-independent N signaling activity. These data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate (Tognon, 2016).

The molecular mechanisms underlying the interdependence between intracellular trafficking and epithelial cell polarity are poorly understood. This study shows that inactivation of class III phosphatidylinositol-3-OH kinase (CIII-PI3K), which produces phosphatidylinositol-3-phosphate (PtdIns3P) on endosomes, disrupts epithelial organization. This is caused by dysregulation of endosomally localized LKB1, also known as STK11, which shows delocalized and increased activity accompanied by dysplasia-like growth and invasive behaviour of cells provoked by JNK pathway activation. CIII-PI3K inactivation cooperates with Ras(V12) to promote tumour growth in vivo in an LKB1-dependent manner. Strikingly, co-depletion of LKB1 reverts these phenotypes and restores epithelial integrity. The endosomal, but not autophagic, function of CIII-PI3K controls polarity. The CIII-PI3K effector, WD repeat and FYVE domain-containing 2 (WDFY2), was identified an LKB1 regulator in Drosophila tissues and human organoids. Thus, this study defines a CIII-PI3K-regulated endosomal signalling platform from which LKB1 directs epithelial polarity, the dysregulation of which endows LKB1 with tumour-promoting properties (O'Farrell, 2017).

This study shows that Mask, an Ankyrin-repeat and KH-domain containing protein, plays a key role in promoting autophagy flux and mitigating degeneration caused by protein aggregation or impaired ubiquitin-proteasome system (UPS) function. In Drosophila eye models of human tauopathy or amyotrophic lateral sclerosis diseases, loss of Mask function enhanced, while gain of Mask function mitigated, eye degenerations induced by eye-specific expression of human pathogenic MAPT/TAU or FUS proteins. The fly larval muscle, a more accessible tissue, was then used to study the underlying molecular mechanisms in vivo. Mask was found to modulate the global abundance of K48- and K63-ubiquitinated proteins by regulating macroautophagy/autophagy-lysosomal-mediated degradation, but not UPS function. Indeed, upregulation of Mask compensated the partial loss of UPS function. It was further demonstrated that Mask promotes autophagic flux by enhancing lysosomal function, and that Mask is necessary and sufficient for promoting the expression levels of the proton-pumping vacuolar (V)-type ATPases in a TFEB-independent manner. Moreover, the beneficial effects conferred by Mask expression on the UPS dysfunction and neurodegenerative models depend on intact autophagy-lysosomal pathway. These findings highlight the importance of lysosome acidification in cellular surveillance mechanisms and establish a model for exploring strategies to mitigate neurodegeneration by boosting lysosomal function (Zhu, 2017).

Misfolded protein aggregates in and outside of cells in the central nervous system are pathological hallmarks of many neurodegenerative disorders including Alzheimer (AD), Parkinson (PD), Huntington (HD) diseases and amyotrophic lateral sclerosis (ALS). Interestingly, many of the aggregated proteins (such as MAPT (TAU) and APP for Alzheimer disease, SNCA/α-synuclein for Parkinson disease, HTT (Huntingtin) for Huntington disease, FUS, SOD1 and TARDBP/TDP-43 for ALS) can serve as seeds for 'prion-like' spreading of the aggregation within and among cells. It is not entirely clear whether these aggregates are the causes or the results of progressive and cell-type-specific neurodegeneration. However, mounting evidence suggests that clearance and prevention of these toxic protein aggregates are beneficial for meliorating degeneration (Zhu, 2017).

Two major pathways collaborate in regulating intracellular protein degradation: the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system. Under the normal conditions, UPS serves as the primary route for rapid protein turnover while autophagy mainly degrades long-lived proteins and large cellular organelles under basal conditions and can be robustly induced in face of stresses such as starvation, organelle damage or accumulation of misfolded proteins. However when it comes to degradation of damaged proteins in diseased states, autophagy has been shown to play at least an equally important role as UPS.5. Many of the neurodegenerative disease-related proteins are delivered to autophagic vacuoles and degraded by the autophagy pathway. Meanwhile, impairment of autophagy in the mouse brain causes neurodegeneration associated with ubiquitin-positive protein aggregation. These data suggest that UPS and autophagy are both indispensable in maintaining cellular protein homeostasis. Furthermore, recent studies indicate that UPS and autophagy pathways coordinate with each other to prevent accumulation of toxic protein aggregates, so that enhanced activity of one pathway can compensate if the other is compromised (Zhu, 2017).

Both UPS and autophagy degradation systems are complex processes consisting of chains of sequential events orchestrated by a large group of proteins. To understand their coordinated action, it is necessary to identify novel players that are necessary and sufficient to mediate the compensatory function between the two systems. This study shows Mask, a conserved protein with Ankyrin repeats and a KH domain, as a novel and critical player in such a context. Initially identified as a modulator of receptor tyrosine signaling during Drosophila development (Smith, 2002), Mask has recently been shown to function as a cofactor of the Hippo pathway effector Yorkie and together they regulate target gene transcription with another transcription cofactor (Scalloped) during cell proliferation (Sansores-Garcia, 2013; Sidor, 2013). The human ortholog of Mask, ANKHD1, is highly expressed in several cancer cell lines. Loss of mask function rescues the mitochondrial defects and muscle degeneration observed with pink1 and park mutants (Zhu, 2015). This study shows that in MAPT- and FUS-induced eye degeneration fly models, loss of Mask function enhances degeneration, while gain of Mask function suppresses degeneration. By enhancing V-type ATPase expression, Mask promotes lysosome acidification and autophagic flux; Mask is necessary and sufficient to mediate a compensatory effect for partial loss of UPS function, to increase clearance of ubiquitinated proteins, and to protect against degeneration induced by aggregation-prone mutations (Zhu, 2017).

Autophagy, an evolutionarily conserved cellular mechanism that preserves metabolic homeostasis during nutrient unavailability, is traditionally regarded as a self-eating degradative process with limited selectivity. However, mounting evidence suggests that both micro- and macro-autophagy can play cytoprotective roles to specifically target damaged and toxic organelles and proteins for clearance under pathological conditions. The mechanism of selective autophagy is unclear. There is some evidence that autophagy receptors can recognize ubiquitin-dependent and ubiquitin-independent signals for selective degradation. Autophagy is a multistep process including nucleation, autophagosome formation and fusion with lysosomes and each step can be regulated to enhance degradation of damaged cellular components. Research has emerged showing TFEB is a potent regulator of the autophagy-lysosomal pathway whose activation can promote lysosomal function and mitigate disease in a range of neurodegenerative disorders. This study shows that Mask acts in a TFEB-independent manner to boost the expression of V-ATPase subunits. This study provides novel evidence that lysosome function is not only required for the normal clearance of ubiquitinated and misfolded proteins, but its activity can also be boosted potential through enhanced lysosomal acidification, to mitigate cellular degeneration caused by toxic protein aggregation (Zhu, 2017).

Mask is well positioned to regulate lysosome-mediated clearance of ubiquitinated and misfolded proteins. As a positive regulator of several V-type ATPase V1 subunits expression, Mask function is necessary and sufficient to promote lysosomal acidification and autophagosome degradation in a cell-autonomous manner. When the UPS function is impaired, increased Mask expression is sufficient to increase autophagic flux, which in turn compensates the partial loss of the proteasome-mediated degradation. Interestingly, even when UPS function is intact, levels of Mask activity impact the abundance of UPS-dependent (K48) and -independent (such as K63) ubiquitin-conjugated proteins, suggesting that autophagy and lysosome-mediated degradation plays an important role for basal protein homeostasis. Under pathological conditions such as UPS inactivation or excessive accumulation of disease proteins, upregulation of Mask activity substantially suppressed the cellular degeneration phenotypes in both muscles and photoreceptors, potentially through Mask-mediated increase of autophagy and lysosome activities and subsequent degradation of harmful protein aggregates, as suggested by the current biochemical and genetic analyses. In support of this notion, upregulation of Mask promotes autophagic flux in larval muscles, adult eyes and adult brains (Zhu, 2017).

This work in the Drosophila model organism yielded new insight into Mask-mediated cellular protective mechanisms that regulate lysosomal function in normal and stressed conditions caused by misfolding-prone disease proteins or impaired UPS. Such mechanisms may provide a therapeutic approach for the treatment of a group of neurodegenerative disorders caused by intracellular inclusions (Zhu, 2017).

The autophagy-lysosome pathway plays a fundamental role in the clearance of aggregated proteins and protection against cellular stress and neurodegenerative conditions. Alterations in autophagy processes, including macroautophagy and chaperone-mediated autophagy (CMA), have been described in Parkinson disease (PD). CMA is a selective autophagic process that depends on LAMP2A (Lysosomal associated membrane protein 2A), a mammal and bird-specific membrane glycoprotein that translocates cytosolic proteins containing a KFERQ-like peptide motif across the lysosomal membrane. Drosophila reportedly lack CMA and use endosomal microautophagy (eMI) as an alternative selective autophagic process. This study reports that neuronal expression of human LAMP2A protected Drosophila against starvation and oxidative stress, and delayed locomotor decline in aging flies without extending their lifespan. LAMP2A also prevented the progressive locomotor and oxidative defects induced by neuronal expression of PD-associated human SNCA (synuclein alpha) with alanine-to-proline mutation at position 30 (SNCA(A30P)). LAMP2A expression stimulated selective autophagy in the adult brain and not in the larval fat body. Noteworthy, neurally expressed LAMP2A markedly upregulated levels of Drosophila Atg5, a key macroautophagy initiation protein, and of the Atg5-containing complex, and that it increased the density of Atg8a/LC3-positive puncta, which reflects the formation of autophagosomes. Furthermore, LAMP2A efficiently prevented accumulation of the autophagy defect marker Ref(2)P/p62 in the adult brain under acute oxidative stress. These results indicate that LAMP2A can promote autophagosome formation and potentiate autophagic flux in the Drosophila brain, leading to enhanced stress resistance and neuroprotection (Issa, 2018).

Almost all animals contain mitochondria of maternal origin only, but the exact mechanisms underlying this phenomenon are still vague. This stuy investigated the fate of Drosophila paternal mitochondria after fertilization. The sperm mitochondrial derivative (MD) is rapidly eliminated in a stereotypical process dubbed paternal mitochondrial destruction (PMD). PMD is initiated by a network of vesicles resembling multivesicular bodies and displaying common features of the endocytic and autophagic pathways. These vesicles associate with the sperm tail and mediate the disintegration of its plasma membrane. Subsequently, the MD separates from the axoneme and breaks into smaller fragments, which are then sequestered by autophagosomes for degradation in lysosomes. Evidence is provided for the involvement of the ubiquitin pathway and the autophagy receptor p62 in this process. Finally, it was shown that the ubiquitin ligase Parkin is not involved in PMD, implying a divergence from the autophagic pathway of damaged mitochondria (Politi, 2014).

The small GTPase Arl8 has emerged as a major regulatory GTPase on lysosomes. Studies in mammalian cells have shown that it regulates both fusion with late endosomes and also lysosomal motility. In its active GTP-bound state, it recruits to lysosomes the HOPS (homotypic fusion and protein sorting) endosomal tethering complex and also proteins that link lysosomes to microtubule motors such as the kinesin adaptor PLEKHM2. To gain further insights into Arl8 biology, this study examined the single Drosophila ortholog. Drosophila Arl8 is essential for viability, and mitotic clones of mutant cells are able to continue to divide but show perturbation of the late endocytic pathway. Progeny-lacking Arl8 die as late larvae with movement-paralysis characteristic of defects in neuronal function. This phenotype was rescued by expression of Arl8 in motor neurons. Examination of these neurons in the mutant larvae revealed smaller synapses and axons with elevated levels of carriers containing synaptic components. Affinity chromatography revealed binding of Drosophila Arl8 to the HOPS complex, and to the Drosophila ortholog of RILP, a protein that, in mammals, recruits dynein to late endosomes, with dynein being known to be required for neuronal transport. Thus Drosophila Arl8 controls late endocytic function and transport via at least two distinct effectors (Rosa-Ferreira, 2018).

The small GTPase Arl8 is known to be involved in the periphery-directed motility of lysosomes. However, the overall importance of moving these vesicles is still poorly understood. This study shows that Drosophila Arl8 is required not only for the proper distribution of lysosomes, but also for autophagosome-lysosome fusion in starved fat cells, endosome-lysosome fusion in garland nephrocytes, and developmentally programmed secretory granule degradation (crinophagy) in salivary gland cells. Moreover, proper Arl8 localization to lysosomes depends on the shared subunits of the BLOC-1 and BORC complexes, which also promote autophagy and crinophagy. In conclusion, this study demonstrates that Arl8 is responsible not only for positioning lysosomes but also acts as a general lysosomal fusion factor (Boda, 2019).

Two related multisubunit tethering complexes promote endolysosomal trafficking in all eukaryotes: Rab5-binding class C core vacuole/endosome tethering (CORVET) that was suggested to transform into Rab7-binding homotypic fusion and vacuolar protein sorting (HOPS). Previous work has identified miniCORVET, containing Drosophila Vps8 and three shared core proteins that are required for endosome maturation upstream of HOPS in highly endocytic cells. This study shows that Vps8 overexpression inhibits HOPS-dependent trafficking routes including late endosome maturation, autophagosome-lysosome fusion, crinophagy and lysosome-related organelle formation. Mechanistically, Vps8 overexpression abolishes the late endosomal localization of HOPS-specific Vps41/Light and prevents HOPS assembly. Proper ratio of Vps8 to Vps41 is thus critical because Vps8 negatively regulates HOPS by outcompeting Vps41. Endosomal recruitment of miniCORVET- or HOPS-specific subunits requires proper complex assembly, and Vps8/miniCORVET is dispensable for autophagy, crinophagy and lysosomal biogenesis. These data together indicate the recruitment of these complexes to target membranes independent of each other in Drosophila, rather than their transformation during vesicle maturation (Lorincz, 2019).

Lipids in extracellular matrices (ECM) contribute to barrier function and stability of epithelial tissues such as the pulmonary alveoli and the skin. In insects, skin waterproofness depends on the outermost layer of the extracellular cuticle termed envelope that contains cuticulin, an unidentified water-repellent complex molecule composed of proteins, lipids and catecholamines. Based on live-imaging analyses of fruit fly larvae, this study found that initially envelope units are assembled within putative vesicles harbouring the ABC transporter Snu (CG9990) and the extracellular protein Snsl (CG2837). In a second step, the content of these vesicles is distributed to cuticular lipid-transporting nanotubes named pore canals and to the cuticle surface in dependence of Snu function. Consistently, the surface of snu and snsl mutant larvae is depleted from lipids and cuticulin. By consequence, these animals suffer uncontrolled water loss and penetration of xenobiotics. These data allude to a two-step model of envelope i.e. barrier formation. The proposed mechanism in principle parallels the events occurring during differentiation of the lipid-based ECM by keratinocytes in the vertebrate skin suggesting establishment of analogous mechanisms of skin barrier formation in vertebrates and invertebrates (Zuber, 2017).

Peroxisomes are ubiquitous membrane-enclosed organelles involved in lipid processing and reactive oxygen detoxification. Mutations in human peroxisome biogenesis genes (Peroxin, PEX or Pex) cause developmental disabilities and often early death. Pex5 and Pex7 are receptors that recognize different peroxisomal targeting signals called PTS1 and PTS2, respectively, and traffic proteins to the peroxisomal matrix. This study characterized mutants of Drosophila melanogaster Pex5 and Pex7 and found that adult animals are affected in lipid processing. Pex5 mutants exhibited severe developmental defects in the embryonic nervous system and muscle similar to what is observed in humans with PEX5 mutations, while Pex7 fly mutants were weakly affected in brain development, suggesting different roles for fly Pex7 and human PEX7. Of note, although no PTS2-containing protein has been identified in Drosophila, Pex7 from Drosophila can function as a bona fide PTS2 receptor because it can rescue targeting of the PTS2-containing protein thiolase to peroxisomes in PEX7 mutant human fibroblasts (Di Cara, 2018).

The fruit fly Drosophila melanogaster is a valuable model organism for the discovery and characterization of innate immune pathways, but host responses to virus infection remain incompletely understood. This study describes a novel player in host defense, Sgroppino (Sgp; CG13091). Genetic depletion of Sgroppino causes hypersensitivity of adult flies to infections with the RNA viruses Drosophila C virus, cricket paralysis virus, and Flock House virus. Canonical antiviral immune pathways are functional in Sgroppino mutants, suggesting that Sgroppino exerts its activity via an as yet uncharacterized process. Sgroppino localizes to peroxisomes, organelles involved in lipid metabolism. In accordance, Sgroppino-deficient flies show a defect in lipid metabolism, reflected by higher triglyceride levels, higher body mass, and thicker abdominal fat tissue. In addition, knock-down of Pex3, an essential peroxisome biogenesis factor, increases sensitivity to virus infection. Together, these results establish a genetic link between the peroxisomal protein Sgroppino, fat metabolism, and resistance to virus infection (Merkling, 2019).

Peroxisomes are subcellular organelles that are essential for proper function of eukaryotic cells. In addition to being the sites of a variety of oxidative reactions, they are crucial regulators of lipid metabolism. Peroxisome loss or dysfunction leads to multi-system diseases in humans that strongly affect the nervous system. In order to identify previously unidentified genes and mechanisms that impact peroxisomes, a genetic screen was conducted on a collection of lethal mutations on the X chromosome in Drosophila. Using the number, size and morphology of GFP tagged peroxisomes as a readout, a screen was carried out for mutations that altered peroxisomes based on clonal analysis and confocal microscopy. From this screen, eighteen genes were identified that cause increases in peroxisome number or altered morphology when mutated. The human homologs were examined of these genes, and they were found to be involved in a diverse array of cellular processes. Interestingly, the human homologs from the X-chromosome collection are under selective constraint in human populations and are good candidate genes particularly for dominant genetic disease. This in vivo screening approach for peroxisome defects allows identification of novel genes that impact peroxisomes in vivo in a multicellular organism and is a valuable platform to discover genes potentially involved in dominant disease that could affect peroxisomes (Graves, 2019).

Peroxisomes are organelles in eukaryotic cells responsible for processing several types of lipids and managing reactive oxygen species. A conserved family of peroxisome biogenesis (Peroxin, Pex) genes encode proteins essential to peroxisome biogenesis and function. In yeast and mammals, PEROXIN7 (PEX7) acts as a cytosolic receptor protein that targets enzymes containing a peroxisome targeting sequence 2 (PTS2) motif for peroxisome matrix import. The PTS2 motif is not present in the Drosophila melanogaster homologs of these enzymes. However, the fly genome contains a Pex7 gene (CG6486) that is very similar to yeast and human PEX7. Pex7 is expressed in tissue-specific patterns analogous to differentiating neuroblasts in D. melanogaster embryos. This is correlated with a requirement for Pex7 in this cell lineage as targeted somatic Pex7 knockout in embryonic neuroblasts reduced survival. Pex7 over-expression in the same cell lineages caused lethality during the larval stage. Targeted somatic over-expression of a Pex7 transgene in neuroblasts of Pex7 homozygous null mutants resulted in a semi-lethal phenotype similar to targeted Pex7 knockout. These findings suggest that D. melanogaster has tissue-specific requirements for Pex7 during embryo development (Pridie, 2020).

Epithelial-repair-dependent mucosal healing (MH) is associated with a more favorable prognosis for patients with inflammatory bowel disease (IBD). MH is accomplished via repair and regeneration of the intestinal epithelium. However, the mechanism underlying MH is ill defined. This study found a striking upregulation of peroxisomes in the injured crypts of IBD patients. By increasing peroxisome levels in Drosophila midguts, it was found that peroxisome elevation enhanced RAB7-dependent late endosome maturation, which then promoted stem and/or progenitor-cell differentiation via modulation of Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT)-SOX21A signaling. This in turn enhanced ISC-mediated regeneration. Importantly, RAB7 and SOX21 were upregulated in the crypts of IBD patients. Moreover, administration of drugs that increased peroxisome levels reversed the symptoms of dextran sulfate sodium (DSS)-induced colitis in mice. This study demonstrates a peroxisome-mediated epithelial repair mechanism, which opens a therapeutic avenue for the enhancement of MH in IBD patients (Du, 2020).

Inflammatory bowel disease (IBD) encompasses a range of complex, long-lasting, and relapsing-remitting disorders, of which ulcerative colitis (UC) and Crohn's disease (CD) are the two most prevalent manifestations. Currently, the incidence of IBD is increasing globally, and the prevalence has increased to just below 0.5% in the Western population. Although genetic predisposition, inappropriate immune responses, and environmental factors have been reported to be closely associated with IBD, its exact etiology and pathogenesis remain poorly understood (Du, 2020).

A series of recent clinical studies indicated that complete regeneration of the intestinal mucosa (also called 'mucosal healing') is associated with a more favorable prognosis for patients with IBD. In the mammal intestine, pluripotent intestinal stem cells (ISCs), located at the base of the epithelial crypts, are responsible for the repair of the damaged epithelium by differentiating into multiple epithelial progenies. Thus, a better understanding of cellular and molecular mechanisms that underlie ISC-mediated epithelial repair could not only provide insight into the etiology but also the therapeutic targets of IBD (Du, 2020).

To identify the mechanism with which peroxisomes regulate the differentiation of ISC progenies during midgut regeneration, RNA sequencing (RNA-seq) was performed on dissected midguts from the Pex10-null and WT flies. This study showed that Sox21a, a transcription factor essential for ISC-to-EC differentiation, had a significantly lower level in mutant midguts compared with that in WT midguts after injury. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed genes, which only appeared in WT midguts but not in Pex10-null midguts during regeneration, showed significant enrichment of genes involved in the endocytosis process. A number of these endocytosis genes have been reported to regulate stem- and/or progenitor-cell differentiation in diverse animal models, such as polo, vap, Pcyt1, and Ubi-p63E. To confirm the results of the RNA-seq analyses, real-time quantitative PCR (qPCR) analyses were performed using sorted ISCs and EBs (esg-GFP+ cells). The results of real-time qPCR analyses of the selected genes, including Sox21a, vap, and rl/ERK showed similar expression patterns to those of RNA-seq analysis during regeneration. Thus, peroxisomes may regulate the ISC-to-EC differentiation by promoting the expression of Sox21a and/or that of specific endocytosis-related genes during regeneration (Du, 2020).

Since the Sox21a mutant has a similar differentiation defect (Zhai, 2015, Zhai, 2017, Chen, 2016) to Pex10 and Pex2 mutants, first, it was verified whether peroxisomes regulate the differentiation of ISCs and EBs through Sox21a-mediated signaling. The endogenous SOX21A reporter line Sox21a-HA was generated using a CRISPR-Cas9 knockin system. As expected, prior to injury, the expression of the SOX21A-HA protein in Pex10-null ISCs and EBs was similarly weak compared with its expression in control ISCs and EBs. However, after injury, while SOX21A expression was dramatically upregulated in control ISCs and EBs, it still retained a relatively low level in Pex10-null ISCs and EBs. Flip-out RNAi clone analysis consistently showed that peroxisomes promoted SOX21A expression after injury (Du, 2020).

More importantly, forced expression of either Sox21a or GATAe (one of the downstreams of SOX21A, which also showed a similar expression change pattern with Sox21a in Pex10-null mutant clone cells partially rescued the differentiation defect of ISC progenies in Pex10-null clones as evidenced by the generation of Pdm1+ cells. Consistently, forced expression of either Sox21a or GATAe in ISCs and EBs also partially restored the defects of esg-GFP+ cell and pH3+ cell accumulation in Pex10-null midguts (Du, 2020).

Since previous studies have shown that the JAK and STAT signaling functions upstream of Sox21a in ISCs and EBs to promote EC formation, this study tested the genetic interaction between peroxisomes and the JAK and STAT signaling. After injury, while the activity of the JAK and STAT signaling was significantly upregulated in control midguts as indicated by the mRNA expression of the STAT target Socs36E and the 10XSTAT-GFP transcriptional reporter, it still retained a relatively low level in Pex10-null midguts. More importantly, forced expression of a constitutively active version of JAK (hopscotch [hop]) (UAS-hopTUM) significantly restored the expression defect of SOX21A in Pex10-depleted cells and the differentiation defect of ISCs and EBs in Pex10 RNAi clones as evidenced by the generation of Pdm1+ cells. In addition, depletion of PEX10 did not affect the activity of Notch signaling, and overexpression of constitutively active Notch (Notchintra) did not show obvious rescue of the ISC-to-EC differentiation defect of Pex10 mutant flies (Du, 2020).

Similar to the human intestine, the adult midgut in Drosophila uses resident ISCs to replenish damaged and lost epithelial cells after injury. The Drosophila ISCs divide to self-renew and produce non-dividing enteroblasts (EBs) or enteroendocrine mother cells (EMCs) depending on Notch activity. Post-mitotic EBs with high levels of Notch signaling further differentiate into absorptive enterocytes (ECs). The EMCs divide once to produce a pair of secretory enteroendocrine cells (EEs). In response to Drosophila midgut injury, ISCs transiently increase their proliferation rates and initiate the production of differentiated ECs to compensate for the damaged and lost cells. This mechanism lasts for a few days until the damaged midgut recovers its normal morphology. In addition to the activation of numerous signals, intestinal damage results in clear changes of cellular structures including the organelles of stem and/or progenitor cells. Although the signaling requirements for the stem cell function in intestinal regeneration have been reported in detail, the contribution of organelle dynamics in stem cells to intestinal repair remains poorly understood (Du, 2020).

Among different organelles, peroxisomes are remarkably plastic with the capability to change their composition, abundance, and morphology in response to environmental stimuli, which in turn may play a role in intestinal repair. By analyzing samples from human patients, utilizing the Drosophila midgut model, and leveraging the mouse IBD model, this study elucidates a conserved molecular pathway with which peroxisome proliferation induces stem-cell-mediated intestinal repair. Furthermore, a new therapeutic approach for the treatment of IBD has been suggested (Du, 2020).

To meet various cellular requirements, organelles in one cell do not function as isolated or static units but rather form dynamic contacts between each other. The findings of this study show that peroxisomes likely modulate the maturation of late endosomes via modulation of vascular transportation, which had been reported to regulate several types of signaling, such as Notch and JAK and STAT signaling. Further studies should aim to understand the detailed mechanism of how peroxisomes modulate the maturation of late endosomes during tissue regeneration (Du, 2020).

This study showed that administration of peroxisome-proliferating agents, NaPB and fenofibrate, which are two drugs approved by the food and drug administration (FDA), effectively reversed the symptoms of DSS-induced colitis in mice. Since NaPB and fenofibrate have a long history of use for disease treatment in human patients, they can be applied to determine their therapeutic effect of enhancing mucosal healing in patients with IBD. Since the peroxisome enhances intestinal repair by promoting stem cell differentiation, it will likely not cause stem cell over-proliferation and tumor formation. These findings not only have important implications for deciphering the functions of the peroxisome in stem cells, tissue regeneration, and injury-induced human diseases but also suggest the peroxisome as a promising therapeutic target of IBD (Du, 2020).

Phagocytosis, signal transduction, and inflammatory responses require changes in lipid metabolism. Peroxisome have key roles in fatty acid homeostasis and in regulating immune function. Drosophila macrophages lacking peroxisomes have perturbed lipid profiles, which reduce host survival after infection. Using lipidomic, transcriptomic, and genetic screens, we determine that peroxisomes contribute to the cell membrane glycerophospholipid composition necessary to induce Rho1-dependent signals, which drive cytoskeletal remodeling during macrophage activation. Loss of peroxisome function increases membrane phosphatidic acid (PA) and recruits RhoGAPp190 during infection, inhibiting Rho1-mediated responses. Peroxisome-glycerophospholipid-Rho1 signaling also controls cytoskeleton remodeling in mouse immune cells. While high levels of PA in cells without peroxisomes inhibit inflammatory phenotypes, large numbers of peroxisomes and low amounts of cell membrane PA are features of immune cells from patients with inflammatory Kawasaki disease and juvenile idiopathic arthritis. These findings reveal potential metabolic markers and therapeutic targets for immune diseases and metabolic disorders (Nath, 2022).

Both peroxisomes and lipid droplets regulate cellular lipid homeostasis. Direct inter-organellar contacts as well as novel roles for proteins associated with peroxisome or lipid droplets occur when cells are induced to liberate fatty acids from lipid droplets. This study has shown a non-canonical role for a subset of peroxisome-assembly [Peroxin (Pex)] proteins in this process in Drosophila. Transmembrane proteins Pex3, Pex13 and Pex14 were observed to surround newly formed lipid droplets. Trafficking of Pex14 to lipid droplets was enhanced by loss of Pex19, which directs insertion of transmembrane proteins like Pex14 into the peroxisome bilayer membrane. Accumulation of Pex14 around lipid droplets did not induce changes to peroxisome size or number, and co-recruitment of the remaining Peroxins was not needed to assemble peroxisomes observed. Increasing the relative level of Pex14 surrounding lipid droplets affected the recruitment of Hsl lipase. Fat body-specific reduction of these lipid droplet-associated Peroxins caused a unique effect on larval fat body development and affected their survival on lipid-enriched or minimal diets. This revealed a heretofore unknown function for a subset of Pex proteins in regulating lipid storage (Ueda, 2022).

Mutations in the glucosylceramidase beta (GBA) gene are strongly associated with neurodegenerative diseases marked by protein aggregation. GBA encodes the lysosomal enzyme glucocerebrosidase, which breaks down glucosylceramide. A common explanation for the link between GBA mutations and protein aggregation is that lysosomal accumulation of glucosylceramide causes impaired autophagy. This study tested this hypothesis directly by measuring protein turnover and abundance in Drosophila mutants with deletions in the GBA ortholog Gba1b. Proteomic analyses revealed that known autophagy substrates, which had severely impaired turnover in autophagy-deficient Atg7 mutants, showed little to no overall slowing of turnover or increase in abundance in Gba1b mutants. Striking changes were found in the turnover and abundance of proteins associated with extracellular vesicles (EVs), which have been proposed as vehicles for the spread of protein aggregates in neurodegenerative disease. Western blotting of isolated EVs confirmed the increased abundance of EV proteins in Gba1b mutants, and nanoparticle tracking analysis revealed that Gba1b mutants had six times as many EVs as controls. Genetic perturbations of EV production in Gba1b mutants suppressed protein aggregation, demonstrating that the increase in EV abundance contributed to the accumulation of protein aggregates. Together, these findings indicate that glucocerebrosidase deficiency causes pathogenic changes in EV metabolism and may promote the spread of protein aggregates through extracellular vesicles (Thomas, 2018).

Mutations in the gene encoding the lysosomal enzyme glucocerebrosidase, glucosylceramidase beta (GBA), are associated with neurodegeneration and brain protein aggregation (Gegg, 2018; Nalls, 2013). Homozygous mutations in GBA cause the lysosomal storage disorder Gaucher disease, which in some cases includes devastating neurological symptoms, while heterozygous GBA mutations are the strongest risk factor for both Parkinson disease (PD) and the related disorder dementia with Lewy bodies. Up to 10% of individuals with nonfamilial PD carry a GBA mutation. In addition, PD patients with a GBA mutation have faster progression of both motor and cognitive symptoms. To study the mechanisms underlying the association between GBA mutations and neurodegeneration, this study created a Drosophila model of glucocerebrosidase (GCase) deficiency. Drosophila has two GBA homologs, designated Gba1a and Gba1b. The Gba1a gene is expressed exclusively in the midgut, and deletion of this gene does not appear to confer deleterious phenotypes. By contrast, the Gba1b gene is ubiquitously expressed, and Gba1b deletion causes marked abnormalities. Previously work has shown that Gba1b null mutants exhibit phenotypes including shortened lifespan, locomotor and memory deficits, neurodegeneration, accumulation of the autophagy adaptor Ref(2)P (p62/SQSTM1), and accumulation of ubiquitinated protein aggregates (Davis, 2016). Similar phenotypes were subsequently seen in an independently generated Gba1b null mutant (Kinghorn, 2016; Thomas, 2018 and references therein).

The protein aggregation and elevated Ref(2)P levels in Gba1b mutants suggested that they had impaired autophagy, as did morphological changes in the autolysosomal system noted by Kinghorn (2016). These findings are consistent with previous reports of autolysosomal impairment upon loss of GCase activity. Based on such findings, it has been hypothesized that lysosomal accumulation of glucosylceramide, the normal substrate of GCase, leads to impairment of autophagy. However, none of the work implicating autophagy in the pathogenic effects of GCase deficiency has yet established that GCase loss of function causes global impairment of autophagic degradation (Thomas, 2018).

To investigate the autophagy failure model of GBA pathogenesis, this study used proteomics-based techniques to measure protein turnover and abundance in Gba1b mutants and controls, as well as in flies with mutations in key autophagy (Atg7) or mitophagy (parkin) genes. While Atg7 mutants showed marked and widespread slowing of autophagy substrate turnover, Gba1b mutants did not. The effects of Gba1b mutation on the turnover and abundance of autophagy substrates also failed to correlate with those of Atg7 or parkin mutations. Moreover, no deficits were detected in turnover mediated by the proteasome, microautophagy, or endocytosis. However, high incidences were found of faster turnover and increased abundance among proteins associated with extracellular vesicles (EVs), which have been previously suggested as a mechanism for the spread of protein aggregates in neurodegenerative disease. Biochemical studies confirmed increased abundance of EV marker proteins in isolated EVs from Gba1b mutants, and nanoparticle tracking analysis showed that the mutants had markedly increased numbers of EVs. Genetic manipulations to reduce EV production decreased the accumulation of ubiquitinated protein aggregates and Ref(2)P in Gba1b mutants, supporting the model that excessive EV abundance promotes the accumulation of protein aggregates. Together, these findings suggest that the most important pathological consequence of Gba1b loss of function is not failure of autophagic protein degradation but excessive production of extracellular vesicles (Thomas, 2018).

Impairment of autolysosomal degradation is widely thought to explain the increased risk of neurodegeneration associated with mutations in GBA, which encodes the lysosomal enzyme glucocerebrosidase (GCase), and multiple studies have found hallmarks of impaired autophagy associated with GCase loss of function. These hallmarks have included accumulation of ubiquitinated protein aggregates, increased abundance of autophagic flux markers such as p62/SQSTM1 and LC3-II, impairment of autophagosome-lysosome fusion, and changes in the size and number of autophagosomes and lysosomes. These indications that GCase deficiency leads to autophagy impairment have been found in diverse experimental systems, including multiple animal models, cultured cells, iPSC-derived human neuronal models, and postmortem patient samples. Initial characterization of Drosophila Gba1b mutants, which revealed extensive ubiquitinated protein aggregates and markedly elevated levels of the p62 ortholog Ref(2)P, also appeared to support the model that GCase deficiency impairs autophagic degradation. In the current work, however, proteomic measurement of protein turnover and abundance showed no evidence that degradation of autophagy substrates was globally impaired in Gba1b mutants. The mutants also showed no evidence of failure in other protein degradation pathways. Instead, faster turnover and increased abundance of proteins associated with extracellular vesicles (EVs) was found. Followup experiments on isolated EVs confirmed increased abundance of EV marker proteins and revealed a strikingly increased number of EVs. Furthermore, genetic manipulations that reduced EV formation suppressed both the increased protein aggregation and the increased Ref(2)P abundance observed in Gba1b mutants. These findings suggest that dysregulation of extracellular vesicles, rather than failure of autophagic degradation, may be the primary mechanism by which GCase deficiency leads to protein aggregation and neurodegeneration (Thomas, 2018).

Although the many previous reports of autophagy impairment in GCase-deficient organisms appear incompatible with the current protein turnover findings, it is not believed that the current findings contradict previous work. When common markers of autolysosomal function such as Ref(2)P/p62 and insoluble ubiquitinated protein were measured, Drosophila Gba1b mutants show results comparable to those seen in vertebrate models of GCase deficiency. Proteomic measurements of protein abundance in the current study are also consistent with previous reports of increased lysosomal mass in GCase deficiency. The abundance of the lysosomal marker Lamp1 was nearly tripled in Gba1b mutants, and 41% of lysosomal proteins were significantly increased in abundance. Nevertheless, protein turnover measurements reveal that the overall rates of degradation through lysosomal processes are not grossly altered. Thus, one possible explanation of the current findings is that the efficiency of autolysosomal degradation is decreased, with lower throughput per unit of autolysosomal mass, but that the organism has compensated by increasing the amount of autolysosomal machinery available. Because this compensation is sufficient to maintain degradation rates, Gba1b mutants could be described as being under autolysosomal stress rather than in autolysosomal failure. Over time, the degree of stress may exceed the capacity to compensate, and aged Gba1b mutants may show overt failure of lysosomal degradation. Even if this is the case, late failure of autolysosomal degradation cannot explain the behavioral and biochemical abnormalities that begin in early adulthood (Thomas, 2018).

Another explanation for the apparent discrepancy between the current findings of normal autophagic substrate turnover and previous reports of impaired autophagy is that commonly used autophagy markers are not solely representative of autophagic flux. This is especially true of Ref(2)P, or p62, which has multiple nonautophagic functions and is transcriptionally upregulated by stress. In addition, p62 and LC3 have recently been detected in mammalian EVs, and this study found increased levels of oligomeric Ref(2)P in EVs from Gba1b mutants. It is therefore possible that the increased Ref(2)P levels detected in Gba1b mutants result from a combination of stress response and EV dysregulation (Thomas, 2018).

This work leaves unanswered the question of how GCase deficiency results in increased EV abundance, but does suggest two possible explanations. Increased production of EVs could be caused either by lysosomal stress or by changes in membrane lipid composition. Lysosomal stress has been shown in cultured cells to promote the release of exosomes, a major type of EV. Exosomes are generated when a multivesicular endosome (MVE) fuses with the plasma membrane rather than the lysosome, releasing its intraluminal vesicles into extracellular space. Lysosomal blockade increases the probability that an MVE will fuse with the plasma membrane. If lysosomal stress rather than outright failure is sufficient to trigger increased exosome release, it could account for the overabundance of EVs in Gba1b mutants (Thomas, 2018).

A second explanation for increased EVs in GCase-deficient animals is that abnormal membrane lipid composition may directly alter EV biogenesis. Lipid composition determines membrane fluidity and curvature, and thus controls the size, shape, and fusion kinetics of EVs. In fact, lipid rafts, particularly those enriched in ceramide, are required for formation of at least one type of EV. Membrane changes such as those caused by GCase deficiency, including accumulation of glucosylceramide and altered ceramide levels could alter EV functioning at any stage from formation to internalization by a recipient cell. Either increased or decreased probability of ceramide-dependent EV formation could lead to increased overall EV production, as suppression of one type of EV has been shown to cause overproduction of another type (Thomas, 2018).

While understanding the mechanism by which GCase deficiency causes increased EV release is an important goal of future work, an equally important question is how increased EV abundance in Gba1b mutants promotes the accumulation of protein aggregates. EVs have been increasingly implicated in the pathogenesis of neurodegenerative disease. Many disease-associated proteins, including prion protein, α-synuclein, β-amyloid, and tau, are detected in EVs, which have been proposed as vehicles for the well-documented progressive spread of protein aggregates from one brain region to another. In support of this model, toxic forms of these disease-associated proteins are more abundant in EVs from humans with neurodegenerative diseases such as Alzheimer disease, dementia with Lewy bodies, and Parkinson disease (PD), and EVs from these patients can induce protein aggregation in recipient cells under experimental conditions. However, progression of these diseases has not yet been conclusively demonstrated to be mediated by EVs. Perhaps the strongest evidence that EVs promote the spread of protein aggregates has been found for prion protein. Stimulating the release of EVs increased the cell-to-cell spread of misfolded prion protein, and decreasing EV release reduced the spread. The current findings appear to follow the same pattern: genetic interference with EV production suppressed protein aggregation in Gba1b mutants. If the same holds true for other aggregation-prone proteins, conditions that increase EV release could promote the spread of protein aggregates and thus be risk factors for neurodegenerative disease (Thomas, 2018).

This model is illustrated in the paper (see Increased EV production promotes the spread of protein aggregates in glucocerebrosidase-deficient organisms). When GCase activity is normal, EVs travel between cells, carrying both factors that promote protein aggregation (e.g., disease-associated proteins such as α-synuclein) and factors that oppose it (e.g., chaperones). Some cells likely generate more aggregates than others, and may therefore release more aggregate-promoting factors, including small aggregate 'seeds.' Quality control mechanisms in recipient cells successfully combat protein aggregation, and aggregates accumulate only slowly with age. If GCase activity is absent or reduced, however, more EVs are generated: this results in greater cell-to-cell transfer of aggregate-prone proteins, perhaps simply because these proteins are normally part of EV cargo. In particular, they may be normal cargo of ESCRT-dependent EVs, given the finding that knockdown of ESCRTs in Gba1b mutants ameliorated the mutants' protein aggregation phenotype. Alternatively, GCase deficiency may alter cargo selection so that more aggregation-prone proteins are loaded into EVs. The net effect of the EV changes is transfer of aggregation-producing factors in quantities that overwhelm quality control mechanisms, leading to excessive accumulation of ubiquitin-protein aggregates in recipient cells (Thomas, 2018).

Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. This study demonstrates a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. This study observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface. Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington's disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain (Dar, 2021).

Extracellular vesicles (EVs) comprise diverse types of cell-released membranous structures that are thought to play important roles in intercellular communication. While the formation and functions of EVs have been investigated extensively in cultured cells, studies of EVs in vivo have remained scarce. This study reports that EVs are present in the developing lumen of tracheal tubes in Drosophila embryos. Two distinct EV subpopulations are defined, one of which contains the Munc13-4 homologue Staccato (Stac) and is spatially and temporally associated tracheal tube fusion (anastomosis) events. The formation of Stac-positive luminal EVs depends on the tracheal tip-cell-specific GTPase Arl3, which is also required for the formation of Stac-positive multivesicular bodies, suggesting that Stac-EVs derive from fusion of Stac-MVBs with the luminal membrane in tip cells during anastomosis formation. The GTPases Rab27 and Rab35 cooperate downstream of Arl3 to promote Stac-MVB formation and tube fusion. It is proposed that Stac-MVBs act as membrane reservoirs that facilitate tracheal lumen fusion in a process regulated by Arl3, Rab27, Rab35, and Stac/Munc13-4 (Camelo, 2022).

This paper reports the presence of membranous EVs in the lumen of developing tracheal tubes in Drosophila embryos. A subset of these EVs is associated with tracheal tube fusion sites and carries the Munc13-4 ortholog Stac. Stac associates with lysosome-related organelles (LROs) that display features of MVBs, the formation of which, as well as of Stac-EVs, depends on the fusion-cell-specific GTPase Arl3. Moreover, we identified Rab27 and Rab35 as partially redundant regulators of Arl3-dependent Stac-MVB formation and tracheal tube fusion. It is proposed that tracheal anastomosis formation involves fusion of Stac-MVBs with the luminal membranes of FCs, resulting in release of Stac-EVs. Attempts to visualize tracheal EV exocytosis using the pH-sensitive fluorescent proteins pHluorin fused to the lysosomal transmembrane protein Lamp1 were unsuccessful. However, it was possible to detect EVs that emerged at and remained associated with anastomosis sites upon completion of dorsal trunk (DT) fusion. Moreover, the absence of Stac-EVs from fusion-defective tracheal metameres in shot mutant embryos supports the idea that Stac-EVs are released during tube fusion (Camelo, 2022).

How is MVB formation regulated in FCs? Consistent with the finding that Rab27 and Rab35 act downstream of Arl3 to promote Stac-MVB formation, Rab27a, Rab27b and Rab35 regulate MVB-dependent exosome secretion in mammalian cells. Moreover, the Stac homolog Munc13-4 is a key effector of Rab27a in LRO docking in hematopoietic cells and melanocytes, and regulates MVB maturation and exosome release in cancer cells. Whereas Rab27 and Rab35 are required for Stac-MVB formation or for anchoring Stac at MVBs, Rab39 is dispensable for FC-specific Stac-LRO formation, suggesting that it acts in a successive step, such as transport or plasma membrane docking of LROs. While the current findings define a core pathway of MVB maturation and EV release in tracheal cells, additional players, such as ESCRT complex components, are likely to be involved. MVBs also act as intermediates in intracellular lumen formation in tracheal terminal cells, which resemble FCs in forming seamless tubes. Interestingly, Rab35 directs transport of apical components to promote terminal cell lumen growth. Whether Rab35 also participates in MVB formation in tracheal terminal cells remains to be tested (Camelo, 2022).

Despite substantial progress in characterizing EVs and their mode of action in cultured cells, studies of physiological functions of EVs in vivo have remained scarce. Exosomes are released into the seminal fluid in Drosophila testis accessory glands and modulate female reproductive behavior. Glia-derived exosomes stimulate growth of motor neurons and tracheae in Drosophila larvae through exosomal microRNA-dependent gene regulation in target cells. Moreover, cardiomyocyte-derived EVs are taken up by macrophages and endothelial cells in zebrafish embryos, and may play a role in trophic support of endothelial cells during vasculogenesis. It will be exciting to explore possible functions of tracheal EVs, including potential roles in long-range intercellular communication across the tracheal lumen. However, the luminal EVs that this study discovered in late-stage embryos are unlikely to participate in such intercellular communication, as the presence at this stage of a luminal matrix appears to constrain EV mobility, and the cuticle covering the apical tracheal cell surface presumably prevents fusion of luminal EVs with the plasma membrane. Rather, Stac-EVs are likely to constitute remnants of MVB-plasma membrane fusion events during anastomosis formation. Stac-MVBs could serve as membrane reservoirs that facilitate lumen fusion. ILVs might back-fuse with the MVB limiting membrane, resulting in a rapid increase of membrane material available at the luminal membrane interfaces in FCs. Such a mechanism has been described in dendritic cells, where MVBs carrying major histocompatibility complex class II (MHC II) in ILVs are reorganized after stimulation, resulting in tubular membrane extensions and transport of MHCII to the plasma membrane. Recruitment of ILVs to the MVB limiting membrane will increase the surface area and may lead to surface exposure of ILV membrane proteins that could promote membrane fusion. Exploring how MVB exocytosis is regulated in tracheal cells, and whether similar mechanisms contribute to vascular anastomosis formation in vertebrates, will be exciting subjects for future studies (Camelo, 2022).

Morphogens are secreted molecules that regulate and coordinate major developmental processes, such as cell differentiation and tissue morphogenesis. Depending on the mechanisms of secretion and the nature of their carriers, morphogens act at short and long range. This study investigated the paradigmatic long-range activity of Hedgehog (Hh), a well-known morphogen, and its contribution to the growth and patterning of the Drosophila wing imaginal disc. Extracellular vesicles (EVs) contribute to Hh long-range activity; however, the nature, the site, and the mechanisms underlying the biogenesis of these vesicular carriers remain unknown. Through the analysis of mutants and a series of Drosophila RNAi-depleted wing imaginal discs using fluorescence and live-imaging electron microscopy, including tomography and 3D reconstruction, this study demonstrated that microvilli of the wing imaginal disc epithelium are the site of generation of small EVs that transport Hh across the tissue. Further, it was shown that the Prominin-like (PromL) protein is critical for microvilli integrity. Together with actin cytoskeleton and membrane phospholipids, PromL maintains microvilli architecture that is essential to promote its secretory function. Importantly, the distribution of Hh to microvilli and its release via these EVs contribute to the proper morphogenesis of the wing imaginal disc. These results demonstrate that microvilli-derived EVs are carriers for Hh long-range signaling in vivo. By establishing that members of the Prominin protein family are key determinants of microvilli formation and integrity, these findings support the view that microvilli-derived EVs conveying Hh may provide a means for exchanging signaling cues of high significance in tissue development and cancer (Hurbain, 2022).

While the primary role of vesicular transporters is to load neurotransmitters into synaptic vesicles (SVs), accumulating evidence suggests that these proteins also contribute to additional aspects of synaptic function, including vesicle release. This study extend the role of the VAChT, which is nested within the first intron of the choline acetyltransferase gene (ChaT), to include regulating the transmitter content of SVs. Manipulation of a C-terminal poly-glutamine (polyQ) region in the Drosophila VAChT is sufficient to influence transmitter content, and release frequency, of cholinergic vesicles from the terminals of premotor interneurons. Specifically, it was found that reduction of the polyQ region, by one glutamine residue (13Q to 12Q), results in a significant increase in both amplitude and frequency of spontaneous cholinergic miniature EPSCs (mEPSCs) recorded in the aCC and RP2 motoneurons. Moreover, this truncation also results in evoked synaptic currents that show increased duration: consistent with increased ACh release. By contrast, extension of the polyQ region by one glutamine (13Q to 14Q) is sufficient to reduce mEPSC amplitude and frequency and, moreover, prevents evoked SV release. Finally, a complete deletion of the polyQ region (13Q to 0Q) has no obvious effects to mEPSCs, but again evoked synaptic currents show increased duration. The mechanisms that ensure SVs are filled to physiologically-appropriate levels remain unknown. This study identifies the polyQ region of the insect VAChT to be required for correct vesicle transmitter loading and, thus, provides opportunity to increase understanding of this critical aspect of neurotransmission (Vernon, 2019).

Vesicular loading of synaptic vesicles (SVs) is dependent on initial acidification mediated by the vATPase pump. This pump generates both a pH gradient (ΔpH) and a voltage gradient (ΔΨ) across the SV membrane. The relative requirement for these two components for loading is dependent on neurotransmitter: anionic transmitters such as glutamate rely more heavily on ΔΨ. Zwitterionic transmitters require both gradients, whereas, cationic transmitters (e.g., ACh) rely predominantly on ΔpH. Transport of ACh into a SV involves the exchange of two protons in an antiporter system using the proton-electrochemical gradient. The current model suggests that one proton is used to transport ACh into the SV lumen while the second proton is needed to re-orientate the VAChT substrate binding site back toward the cytoplasm (2H+ for 1ACh+). In situ, acidified SVs exhibit a pH ~1.4 units less than un-acidified SVs. Theoretically, the cholinergic SV lumen has the capacity to concentrate ACh by 100-fold relative to cytoplasmic levels (which range between 1 and 4 mM). However, the maximal reported accumulation of ACh in SVs has been found to saturate at ~4 mM, suggesting a rather dramatic (and unknown) limiting factor impedes loading (Vernon, 2019).

A key limiting factor may be copy number of functional transporter per SV. Murine and Drosophila NMJ and mammalian cell culture models suggests vesicular loading is altered following either genetic and/or pharmacological manipulation of transporter activity. However, it is notable that upregulation of VAChT expression fails to show effects to quantal size at either snake NMJ or Drosophila motoneurons that receive cholinergic excitation (Cash, 2016). An inability of increased transporter to affect SV loading is consistent with a set-point model of filling. This model posits that SVs fill to a predetermined level, independent of filling rate, which changes following manipulation of transporter expression level. It has been previously reported that transgenic expression of VAChT, which carries a single glutamine truncation in a C-terminal poly-glutamine (polyQ) region (13Q to 12Q), results in increased quanta of spontaneously released SVs at identified interneuron to motoneuron synapses (Cash, 2016). This region, therefore, may contribute to the mechanism that regulates SV loading (Vernon, 2019).

This study use electrophysiological characterization of cholinergic release at Drosophila larval and embryonic interneuron->motoneuron synapses to investigate the physiologic implications to SV loading when the VAChT C-terminal polyQ region is manipulated. In agreement with previously published literature, this study found that expression of a single glutamine truncation VAChT^12Q increases both amplitude and frequency of spontaneously released cholinergic miniature EPSCs (mEPSCs; i.e., individual SV release) recorded from aCC and RP2 motoneurons. Evoked synaptic currents also show an increased duration consistent with an increased ACh load. Conversely, this study further shows that CRISPR induced single amino acid extension of the polyQ region (VAChT^14Q) results in the opposite effect: reduced mEPSC amplitude and frequency and, moreover, an inability to support evoked release. CRISPR mediated deletion of the polyQ region (VAChT^ΔQ) has no effect on mEPSC kinetics suggesting that elongation or truncation of the VAChT polyQ region is more detrimental to cholinergic functioning than its removal (Vernon, 2019).

It is notable that although mEPSC amplitude is increased following expression of VAChT^12Q the effect to evoked spontaneous rhythmic current (SRCs) is limited to increased duration. It is speculated that this may be indicative that the postsynaptic nAChR receptor field is already fully saturated under endogenous conditions and heightened cholinergic tone, through VAChT^12Q upregulation, is thus restricted to increasing SRC duration. Similarly, it is speculated on why increased SRC duration is accompanied by a decrease in SRC frequency. A possible explanation is a homeostatic-type negative feedback mechanism which acts to dampen the activity of presynaptic interneurons that form the central pattern generator controlling locomotor output. Future experiments will be required to clarify these issues (Vernon, 2019).

The results suggest that the length of the polyQ domain is both deterministic for SV filling and for the probability of SV release. Reducing glutamines by one residue is sufficient to increase SV load and release probability and vice versa. Moreover, addition of a glutamine (14Q) is sufficient to remove the ability of the CNS to generate a rhythmic fictive locomotor pattern, which is reliant on evoked release. It is rationalized that VAChT^14Q disrupts cholinergic loading, generating partially-filled SVs that, in turn, prevent evoked synaptic release. By contrast, increasing SV loading (12Q) results in evoked release events of longer duration. These observations are in agreement with recent work using a light activated vATPase pump (pHoenix) localized to SVs (Rost, 2015). Rost used this tool to show that glutamatergic vesicles are only 'nearly full' under normal conditions (i.e., can be further filled) and, moreover, show thatvesicle load is proportional to release probability (Rost, 2015). The current data are supportive of this observation: only increased SV loading supports evoked release. Moreover, the results are also indicative of a set point model, in which vesicles can only release once they surpass a threshold load. This hypothesis proposes two distinct models of SV loading. The set-point model proposes a mechanism restricting the amount of neurotransmitter per vesicle to a fixed maximum, whereas, the steady state model suggests the amount of neurotransmitter that enters a SV is offset by leakage, but that both are independent variables that can autonomously change to produce SVs with variable levels of filling. The set point model is consistent with observations at the snake NMJ and Drosophila central neurons. Whereas, the steady state model better describes loading at murine and Drosophila NMJ and in mammalian cell culture models (Vernon, 2019).

Analysis of related Drosophila species reveal polyQ regions of differing lengths (e.g., nine in D. willistoni, 11 in D. simulans, and 15 in D. pseudoobscura). It is tempting to speculate that evolution may have manipulated the length of the polyQ region to alter SV content in these related species. However, recordings from aCC/RP2 in these related species show mEPSC amplitude is remarkably conserved. Thus, the predicted effect of SV loading due to change in polyQ length, across these related species, may have been abrogated by compensatory mutations in other regions of the VAChT. A comparative analysis may thus be useful to identify such regions for future study (Vernon, 2019).

The VAChT polyQ region is specific to insects. A BLAST search comparison shows no other insect neuronal vesicular transporter possesses a C-terminal polyQ domain. Mammalian VAChT possesses a di-leucine motif in the same approximate location to the insect polyQ domain. The di-leucine motif is well established as a trafficking region. Removal of the mammalian VAChT C-terminal tail, or specific mutation of the di-leucine motif, results in mislocalization of the transporter to the neuronal membrane. Mutant Htt protein containing a polyQ expansion from 20Q to 120Q was found to preferentially bind to SVs in murine axon terminals and, further, to displace the binding of Huntington-associated protein (HAP1) usually co-localized to SVs. 120Q mutants were also shown to reduce glutamate release suggesting a direct interaction between extended polyQ domains and synaptic release. HAP1 has also been shown to bind synapsin 1 which is critical for SV pool mobilization and formation. It is therefore theorized that the polyQ region in VAChT may play a similar role in trafficking the transporter to the SV, plasma membrane and/or SV pool formation (Vernon, 2019).

It is notable that complete removal of the VAChT polyQ region does not influence mEPSCs, although it does alter SRC kinetics (increasing their duration). This dichotomy may mirror an increasingly accepted molecular distinction between spontaneous (mEPSCs) and synchronous (SRC/EPSC) release modalities. Other work has shown, for example, that mEPSC release is maintained in the absence of the vesicle associated SNARE protein synaptobrevin, while evoked release is halted. Munc-13 has also be shown to influence the spatial localization of evoked release while having no effect on mEPSCs at C. elegans NMJ. These observations are predictive of a model in which multiple fusion complexes are physiologically separate and dependent on the modality of release. Moreover, a role for VAChT in SV release is indicated by a reported interaction between synaptobrevin and VAChT. A glycine to arginine substitution (G342R) in VAChT is sufficient to reduce cholinergic mediated larval motility in C. elegans, an effect that is rescued by a complimentary substitution of an isoleucine to an aspartate in synaptobrevin (Vernon, 2019).

VAChT^ΔQ mutants show early larval mortality (L1) despite being able to produce SRCs. This is further confused by the similarity in SRC kinetics with chaB19>VAChT^12Q which produce viable L3 larvae and adults. Early VAChT^ΔQ mortality is attributed to the lack of wild-type transporter present in the VAChT^ΔQ genetic background and may be consistent with cholinergic deficiencies presented in wider physiologic function. In humans, ChAT immunoreactivity and nAChR/mAChR expression is observed in non-neuronal epithelial, endothelial, mesothelial and immune cells and are shown to modulate multiple cellular processes including but not exclusive to, cellular migration and apoptosis, proliferation, anti/proinflammatory responses and histamine release. In insects, non-neuronal ACh has been shown to be heavily influential in reproduction and larval development and so it remains possible that VAChT modulation may alter wider, and currently unknown, physiologic aspects of larval development (Vernon, 2019).

The effects reported in this study relating to expression of VAChT^12Q (truncation) versus VAChT^14Q (expansion) were achieved using different experimental conditions. VAChT^12Q was tested using Gal4-based overexpression in an otherwise wild-type VAChT background, while VAChT^14Q was tested using a CRISPR mutant. This was because an attempt to make VAChT^12Q via CRISPR was unsuccessful. Thus, the results reported in this study must be tempered. Indeed, the co-presence of wild-type VAChT in VAChT^12Q upregulation may, to some extent, reduce the observed phenotype. Moreover, protein level, nor protein localization, was measured and thus the possibility remains that the VAChT^14Q mutation may affect expression levels and/or vesicular localization, which makes it difficult to reach firm conclusions about results obtained. However, it is not believed this detracts from the interpretation of the data presented within this study (Vernon, 2019).

Since the first demonstration of fixed quanta that describes spontaneous release of SVs, a key question of 'how does a SV know when it is full' remains to be answered. The polyQ region of the Drosophila VAChT, that is reported in this study, seemingly orchestrates the filling of cholinergic SVs at central synapses. Future studies to identify the function of this region, including identification of binding partners, provide optimism for understanding how SVs monitor their fill state (Vernon, 2019).

Pseudomonas aeruginosa can cause nosocomial infections, especially in ventilated or cystic fibrosis patients. Highly pathogenic isolates express the phospholipase ExoU, an effector of the type III secretion system that acts on plasma membrane lipids, causing membrane rupture and host cell necrosis. This study used a genome-wide screen to discover that ExoU requires DNAJC5, a host chaperone, for its necrotic activity. DNAJC5 is known to participate in an unconventional secretory pathway for misfolded proteins involving anterograde vesicular trafficking. This study shows that DNAJC5-deficient human cells, or Drosophila flies knocked-down for the DNAJC5 orthologue (Cysteine string protein), are largely resistant to ExoU-dependent virulence. ExoU colocalizes with DNAJC5-positive vesicles in the host cytoplasm. DNAJC5 mutations preventing vesicle trafficking (previously identified in adult neuronal ceroid lipofuscinosis, a human congenital disease) inhibit ExoU-dependent cell lysis. These results suggest that, once injected into the host cytoplasm, ExoU docks to DNAJC5-positive secretory vesicles to reach the plasma membrane, where it can exert its phospholipase activity (Deruelle, 2021).

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