The Interactive Fly
Zygotically transcribed genes
INDEX
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 sar1P1 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 sar1P1, and sar1EP3575Δ28 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 sar1T38N form in the trachea. In btl > sar1T38N-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 > sar1T38N-expressing embryos, zygotic sar1P1 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).
Brains are highly metabolically active organs, consuming 20% of a person's energy at resting state. A decline in glucose metabolism is a common feature across a number of neurodegenerative diseases. Another common feature is the progressive accumulation of insoluble protein deposits, it's unclear if the two are linked. Glucose metabolism in the brain is highly coupled between neurons and glia, with glucose taken up by glia and metabolised to lactate, which is then shuttled via transporters to neurons, where it is converted back to pyruvate and fed into the TCA cycle for ATP production. Monocarboxylates are also involved in signalling, and play broad ranging roles in brain homeostasis and metabolic reprogramming. However, the role of monocarboxylates in dementia has not been tested. This study found that increasing pyruvate import in Drosophila neurons by over-expression of the transporter bumpel, leads to a rescue of lifespan and behavioural phenotypes in fly models of both frontotemporal dementia and Alzheimer's disease. The rescue is linked to a clearance of late stage autolysosomes, leading to degradation of toxic peptides associated with disease. It is proposed that upregulation of pyruvate import into neurons as potentially a broad-scope therapeutic approach to increase neuronal autophagy, which could be beneficial for multiple dementias (Xu, 2023).
Autophagy is a lysosomal-dependent degradation process of eukaryotic cells responsible for breaking down unnecessary and damaged intracellular components. This study aimed to uncover new regulatory points where autophagy could be specifically activated and tested these potential drug targets in neurodegenerative disease models of Drosophila melanogaster. One possible way to activate autophagy is by enhancing autophagosome-lysosome fusion that creates the autolysosome in which the enzymatic degradation happens. The HOPS (homotypic fusion and protein sorting) and SNARE (Snap receptor) protein complexes regulate the fusion process. The HOPS complex forms a bridge between the lysosome and autophagosome with the assistance of small GTPase proteins. Thus, small GTPases are essential for autolysosome maturation, and among these proteins, Rab2 (Ras-associated binding 2), Rab7, and Arl8 (Arf-like 8) are required to degrade the autophagic cargo. For these experiments, Drosophila melanogaster was used as a model organism. Nerve-specific small GTPases were silenced and overexpressed. The effects were examined of these genetic interventions on lifespan, climbing ability, and autophagy. Finally, the activation of small GTPases was also studied in a Parkinson's disease model. The results revealed that GTP-locked, constitutively active Rab2 (Rab2-CA) and Arl8 (Arl8-CA) expression reduces the levels of the autophagic substrate p62/Ref(2)P in neurons, extends lifespan, and improves the climbing ability of animals during ageing. However, Rab7-CA expression dramatically shortens lifespan and inhibits autophagy. Rab2-CA expression also increases lifespan in a Parkinson's disease model fly strain overexpressing human mutant (A53T) α-synuclein protein. Data provided by this study suggests that Rab2 and Arl8 serve as potential targets for autophagy enhancement in the Drosophila nervous system (Szinyakovics, 2023).
Macroautophagy/autophagy is a major pathway for the clearance of protein aggregates and damaged organelles, and multiple intracellular organelles participate in the process of autophagy, from autophagosome formation to maturation and degradation. Dysregulation of the autophagy pathway has been implicated in the pathogenesis of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), however the mechanisms underlying autophagy impairment in these diseases are incompletely understood. Since the expansion of GGGGCC (G4C2) repeats in the first intron of the C9orf72 gene is the most common inherited cause of both ALS and FTD (C9-ALS-FTD), this study investigated autophagosome dynamics in Drosophila motor neurons expressing 30 G4C2 repeats (30 R). In vivo imaging demonstrates that expression of expanded G4C2 repeats markedly impairs biogenesis of autophagosomes at synaptic termini, whereas trafficking and maturation of axonal autophagosomes are unaffected. Motor neurons expressing 30 R display marked disruption in endoplasmic reticulum (ER) structure and dynamics in the soma, axons, and synapses. Disruption of ER morphology with mutations in Rtnl1 (Reticulon-like 1) or atl (atlastin) also impairs autophagosome formation in motor neurons, suggesting that ER integrity is critical for autophagosome formation. Furthermore, live imaging demonstrates that autophagosomes are generated from dynamic ER tubules at synaptic boutons, and this process fails to occur in a C9-ALS-FTD model. Together, these findings suggest that dynamic ER tubules are required for formation of autophagosomes at the neuromuscular junction, and that this process is disrupted by expanded G4C2 repeats that cause ALS-FTD (Sung, 2024).
Under normal physiological conditions, the mammalian brain contains very little glycogen, most of which is stored in astrocytes. However, the aging brain and the subareas of the brain in patients with neurodegenerative disorders tend to accumulate glycogen, the cause and significance of which remain largely unexplored. Using cellular models, a neuroprotective role for neuronal glycogen and glycogen synthase has been recently demonstrated in the context of Huntington's disease. To gain insight into the role of brain glycogen in regulating proteotoxicity, a Drosophila model of Huntington's disease was used, in which glycogen synthase is either knocked down or expressed ectopically. Enhancing glycogen synthesis in the brains of flies with Huntington's disease decreased mutant Huntingtin aggregation and reduced oxidative stress by activating auto-lysosomal functions. Further, overexpression of glycogen synthase in the brain rescues photoreceptor degeneration, improves locomotor deficits and increases fitness traits in this Huntington's disease model. This study, thus, provides in vivo evidence for the neuroprotective functions of glycogen synthase and glycogen in neurodegenerative conditions, and their role in the neuronal autophagy process (Onkar, 2023).
Inter-organelle contacts enable crosstalk among organelles, facilitating the exchange of materials and coordination of cellular events. This study demonstrated that, upon starvation, autolysosomes recruit Pi4KIIα (Phosphatidylinositol 4-kinase II α) to generate phosphatidylinositol-4-phosphate (PtdIns4P) on their surface and establish endoplasmic reticulum (ER)-autolysosome contacts through PtdIns4P binding proteins Osbp (Oxysterol binding protein) and cert (ceramide transfer protein). Sac1 (Sac1 phosphatase), Osbp, and cert proteins are required for the reduction of PtdIns4P on autolysosomes. Loss of any of these proteins leads to defective macroautophagy/autophagy and neurodegeneration. Osbp, cert, and Sac1 are required for ER-Golgi contacts in fed cells. These data establishes a new mode of organelle contact formation - the ER-Golgi contact machinery can be reused by ER-autolysosome contacts by re-locating PtdIns4P from the Golgi apparatus to autolysosomes when faced withstarvation (Liu, 2023).
Tauopathy, including Alzheimer Disease (AD), is characterized by Tau protein accumulation and autophagy dysregulation. Emerging evidence connects polyamine metabolism with the autophagy pathway, however the role of polyamines in Tauopathy remains unclear. The present study investigated the role of spermine synthase (SMS) in autophagy regulation and tau protein processing in Drosophila and human cellular models of Tauopathy. A previous study showed that Drosophila spermine synthase (dSms) deficiency impairs lysosomal function and blocks autophagy flux. Interestingly, partial loss-of-function of SMS in heterozygous dSms flies extends lifespan and improves the climbing performance of flies with human Tau (hTau) overexpression. Mechanistic analysis showed that heterozygous loss-of-function mutation of dSms reduces hTau protein accumulation through enhancing autophagic flux. Measurement of polyamine levels detected a mild elevation of spermidine in flies with heterozygous loss of dSms . SMS knock-down in human neuronal or glial cells also upregulates autophagic flux and reduces Tau protein accumulation. Proteomics analysis of postmortem brain tissue from AD patients showed a significant albeit modest elevation of SMS protein level in AD-relevant brain regions compared to that of control brains consistently across several datasets. Taken together, this study uncovers a correlation between SMS protein level and AD pathogenesis and reveals that SMS reduction upregulates autophagy, promotes Tau clearance, and reduces Tau protein accumulation. These findings provide a new potential therapeutic target of Tauopathy (Tao, 2023).
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 (Sac1ts), 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 (Sac1ts). Sac1ts 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 Sac1ts 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, Sac1ts 2°/3°pc contain an excess of intracellular Rst and Kirre due to impaired endo-lysosomal trafficking and degradation. Sac1ts 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 Sac1ts, loss of Mys at the basal grommets in Sac1ts 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 Sac1ts retinas at 24 h APF (Del Bel, 2018), which it is speculated could perturb basal trafficking. Alternatively, aberrant distribution of basal F-actin in Sac1ts 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 Sac1ts (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 Sac1ts retinas, which suggests a similar delay in autophagy. It is a compelling notion that increased PI4P levels in Sac1ts 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 Sac1ts, 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 Sac1ts 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 Sac1ts 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 Sac1ts 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 Sac1ts. 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 Sac1ts 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 Sac1ts 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).
Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. This study identified the HDS3 domain as essential for GBF1 membrane association in mammalian cells and documents the critical role of HDS3 during the development of Drosophila melanogaster. Upon binding to Golgi membranes, GBF1 undergoes conformational changes in regions bracketing the catalytic Sec7d. GBF1 interdomain arrangements were illuminated by negative staining electron microscopy of full-length human GBF1 to show that GBF1 forms an anti-parallel dimer held together by the paired central DCB-HUS core, with two sets of HDS1-3 arms extending outward in opposite directions. The catalytic Sec7d protrudes from the central core as a largely independent domain, but is closely opposed to a previously unassigned α-helix from the HDS1 domain. Based on tese data, models of GBF1 engagement on the membrane are proposed to provide a paradigm for understanding GBF1-mediated Arf activation required for cellular and organismal function (Meissner, 2023).
Ingression of the plasma membrane is an essential part of the cell topology-distorting repertoire and a key element in animal cell cytokinesis. Many embryos have rapid cleavage stages in which they are furrowing powerhouses, quickly forming and disassembling cleavage furrows on timescales of just minutes. Previous work has shown that cytoskeletal proteins and membrane trafficking coordinate to drive furrow Ingression, but where these membrane stores are derived from and how they are directed to furrowing processes has been less clear. This study identified an extensive Rab35/Rab4>Rab39/Klp98A>gt;trans-Golgi network (TGN) endocytic recycling pathway necessary for fast furrow ingression in the Drosophila embryo. Rab39 is present in vesiculotubular compartments at the TGN where it receives endocytically derived cargo through a Rab35/Rab4-dependent pathway. A Kinesin-3 family member, Klp98A, drives the movements and tubulation activities of Rab39, and disruption of this Rab39-Klp98A-Rab35 pathway causes deep furrow ingression defects and genomic instability. These data suggest that an endocytic recycling pathway rapidly remobilizes membrane cargo from the cell surface and directs it to the trans-Golgi network to permit the initiation of new cycles of cleavage furrow formation (Mio, 2023).
Compelling evidence indicates that defects in nucleocytoplasmic transport contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS). In particular, hexanucleotide (G4C2) repeat expansions in C9orf72, the most common cause of genetic ALS, have a widespread impact on the transport machinery that regulates the nucleocytoplasmic distribution of proteins and RNAs. It has been reported that the expression of G4C2 hexanucleotide repeats in cultured human and mouse cells caused a marked accumulation of poly(A) mRNAs in the cell nuclei. To further characterize the process, this study set out to systematically identify the specific mRNAs that are altered in their nucleocytoplasmic distribution in the presence of C9orf72-ALS RNA repeats. Interestingly, pathway analysis showed that the mRNAs involved in membrane trafficking are particularly enriched among the identified mRNAs. Most importantly, functional studies in cultured cells and Drosophila indicated that C9orf72 toxic species affect the membrane trafficking route regulated by ADP-Ribosylation Factor 1 GTPase Activating Protein (ArfGAP-1), which exerts its GTPase-activating function on the small GTPase ADP-ribosylation factor 1 to dissociate coat proteins from Golgi-derived vesicles. This study demonstrated that the function of ArfGAP-1 is specifically affected by expanded C9orf72 RNA repeats, as well as by C9orf72-related dipeptide repeat proteins (C9-DPRs), indicating the retrograde Golgi-to-ER vesicle-mediated transport as a target of C9orf72 toxicity (Rossi, 2023).
GM130 is a matrix protein that is conserved in metazoans and involved in the architecture of the Golgi apparatus. In neurons, Golgi apparatus and dendritic Golgi outposts (GOs) have different compartmental organizations, and GM130 localization is present in both, indicating that GM130 has a unique Golgi-targeting mechanism. This study investigated the Golgi-targeting mechanism of the GM130 homologue, dGM130, using in vivo imaging of Drosophila dendritic arborization (da) neurons. The results showed that two independent Golgi-targeting domains (GTDs) with different Golgi localization characteristics in dGM130, together determined the precise localization of dGM130 in both the soma and dendrites. GTD1, covering the first coiled-coil region, preferentially targeted to somal Golgi rather than GOs; whereas GTD2, containing the second coiled-coil region and C-terminus, dynamically targeted to Golgi in both soma and dendrites. These findings suggest that there are two distinct mechanisms by which dGM130 targets to the Golgi apparatus and GOs, underlying the structural differences between them, and further provides new insights into the formation of neuronal polarity (Cheng, 2023).
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 frcR29 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, frcRY34, the presence of which results in the death of most larvae during the second-instar stage, much earlier than the death induced by frcR29. Real-time PCR analysis revealed that the amount of frc transcripts in the second-instar larvae of frcRY34 or frcR29 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 frcRY34 allele. Together, these observations suggest that frcRY34 is essentially a null allele (Yano, 2005).
Clonal cells of the frcRY34 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 frcRY34 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 frcRY34 mutant. The frcRY34 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 frcRY34 mutant. The ectopic expression of Wg induced by the frcRY34 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 frcRY34 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 frcRY34 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 Cdc42T17N 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).
Sorting of integral membrane proteins plays crucial roles in establishing and maintaining the polarized structures of epithelial cells and neurons. However, little is known about the sorting mechanisms of newly synthesized membrane proteins at the trans-Golgi network (TGN). To identify which genes are essential for these sorting mechanisms, mutants were screened in which the transport of Rhodopsin 1 (Rh1), an apical integral membrane protein in Drosophila photoreceptors, was affected. Deficiencies in glycosylphosphatidylinositol (GPI) synthesis and attachment processes were found to cause loss of the apical transport of Rh1 from the TGN and mis-sorting to the endolysosomal system. Moreover, Na+K+-ATPase, a basolateral membrane protein, and Crumbs (Crb), a stalk membrane protein, were mistransported to the apical rhabdomeric microvilli in GPI-deficient photoreceptors. These results indicate that polarized sorting of integral membrane proteins at the TGN requires the synthesis and anchoring of GPI-anchored proteins. Little is known about the cellular biological consequences of GPI deficiency in animals in vivo. These results provide new insights into the importance of GPI synthesis and aid the understanding of pathologies involving GPI deficiency (Satoh, 2013).
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).
The golgi apparatus is optimized separately in different tissues for efficient protein
trafficking, little is known of how cell signaling shapes this organelle. This study finds
that the Abl tyrosine kinase signaling pathway controls the architecture of the golgi
complex in Drosophila photoreceptor (PR) neurons. The Abl effector, Enabled (Ena),
selectively labels the cis-golgi in developing PRs. Overexpression or loss-of-function of
Ena increases the number of cis and trans-golgi cisternae per cell, and Ena
overexpression also redistributes golgi to the most basal portion of the cell soma. Loss of Abl, or of its upstream regulator, the adaptor protein Disabled, lead to the same
alterations of golgi as does overexpression of Ena. The increase in golgi number in Abl
mutants arises in part from increased frequency of golgi fission events and a decrease in
fusions, as revealed by live imaging. Finally, it was demonstrated that the effects of Abl
signaling on golgi are mediated via regulation of the actin cytoskeleton. Together, these
data reveal a direct link between cell signaling and golgi architecture. Moreover, they
raise the possibility that some of the effects of Abl signaling may arise, in part, from
alterations of protein trafficking and secretion (Kannan, 2014).
The Abl tyrosine kinase signaling pathway controls golgi morphology and localization in Drosophila photoreceptors through its regulation of the actin cytoskeleton.
Ena, the main effector of Abl in morphogenesis, is associated with the cis-golgi
compartment, and it regulates golgi localization and dynamics under the control of Abl
and its interacting adaptor protein, Dab. Reducing the levels of Abl or Dab, or
overexpressing Ena, led to similar defects in golgi fragmentation state and subcellular
distribution. During golgi biogenesis, Abl increases the frequency of fusion of golgi
cisternae, and decreases fission events. Abl evidently controls golgi organization through
its regulation of actin structure, as the effect of Abl signaling on golgi could be blocked
by modulating actin structure genetically or pharmacologically. Collectively, these data
reveal an unexpected link between a fundamental tyrosine kinase signaling pathway in
neuronal cells and the structure of the golgi compartment (Kannan, 2014).
The data reported here suggest that the Abl signaling pathway controls golgi morphology and localization through its control of actin structure. This is consistent with previous reports that altering the levels of actin modulators perturbs the structure and function of the golgi apparatus. A variety of proteins that modulate actin dynamics have been localized to golgi. Ultra-structural studies established the association
of actin filaments with golgi membranes and the association of β and γ actin with the golgi. In cultured cell models, including neurons, actin
depolymerization leads to golgi compactness, fragmentation and altered subcellular
distribution. It is noted, moreover, that the
reported golgi-associated signaling proteins include several that have been linked to Abl
signaling, including the Abl target Abi, the Abi binding partner WAVE, and various
effectors of Rac GTPase including ADF/cofilin, WASH and Arp2/3. Thus, for example,
Abi and WAVE have been implicated in actin dependent golgi stack reorganization and
in scission of the golgi at cell division to allow faithful inheritance of golgi complex to
daughter cells in Drosophila S2 cell cycles (Kondylis, 2007). These data reinforce
the importance of actin-regulating signaling pathways for controlling golgi biogenesis (Kannan, 2014).
Two lines of evidence suggest that the increase observed in golgi number in Abl
pathway mutants is due primarily to net fragmentation of pre-existing golgi cisternae and
not to de novo synthesis of golgi. First, live imaging of golgi dynamics in neurons of
the Drosophila eye disc reveals that the steady-state number of golgi cisternae reflects an
ongoing balance of fusion and fission events, much as observed previously in yeast. Quantification of these events in
wildtype vs Abl mutant tissue demonstrated directly that loss of Abl significantly
increased the frequency of fission events, and reduced the frequency of fusions. Second,
the absolute volume of cis-golgi in Abl mutant photoreceptors was not substantially
greater than that in wildtype, as judged by direct measurement of the volume of GM130-
immunoreactive material in deconvoluted image stacks of photoreceptor clusters. While
a small apparent increase was observed in golgi volume in the mutants (~55%, based on
pixel counts), it is noted that golgi cisternae are small on the length scale of the point
spread function of visible light, such that the fluorescent signal from a single cisterna
extends into the surrounding cytoplasm. The increase in apparent golgi volume is
therefore within the range expected due simply to fluorescence 'spillover' from the
three-fold greater number of separate golgi cisternae in the mutants (Kannan, 2014).
It is striking that both increase and decrease of Ena led to net fragmentation of golgi.
Why might this be? It is known that both fission and fusion of membranes requires actin
dynamics: at scission, polymerization provides force for separating membranes, while in
fusion, actin polymerization is essential for bringing membranes together and for
supplying membrane vesicles, among other things. As a result, altering actin dynamics is apt to change the probabilities of multiple aspects of both fission and fusion events, making it impossible to
predict a priori how the balance will be altered by a given manipulation, just as either
increase or decrease of Ena can inhibit cell or axon motility, depending on the details of
the experiment, due to the non-linear nature of actin dynamics. Indeed, this study also observed net golgi fragmentation when actin was stabilized with jasplakinolide, just as was done from depolymerization with cytochalasin or latrunculin. More direct
experiments will be necessary to fully understand this dynamic, however.
deficits selectively disrupt dendritic morphogenesis but not axogenesis, and perhaps
consistent with this, Abl/Ena function is essential for dendrite arborization in these cells but has not been reported to affect their
axon patterning. Finally, in some contexts, neuronal development requires local
translation of guidance molecules in the growth cone rather than translation in the cell
soma. It is likely that the need for
actin dynamics to target different subcellular compartments in different cell types will be
reflected in different patterns of Abl/Ena protein localization (Kannan, 2014).
This study reports the role of Abl/Ena-dependent regulation of actin structure on overall golgi structure and localization but there may be more subtle effects on golgi function as
well. For example, recent evidence supports a role for actin-dependent regulation of the
specificity of protein sorting in the golgi complex. Preferential sorting of
cargos is achieved by nucleation of distinct actin filaments at the golgi complex. In Hela
cells, for example, Arp2/3 mediated nucleation of actin branches at cis-golgi regulates
retrograde trafficking of the acid hydroxylase receptor CI-MPR, while Formin family mediated nucleation of linear actin filaments at golgi regulates selective trafficking of the lysosomal enzyme cathepsin D. Similarly, the actin-severing protein ADF/cofilin, the mammalian ortholog of Drosophila twinstar, sculpts an actin-based sorting domain at the trans-golgi network for selective cargo sorting. It will be
important to investigate whether the effects of Abl/Ena on golgi morphology have
functional consequences on bulk secretion or protein sorting (Kannan, 2014).
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 tango12L3443 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 tango12L3443 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 tango12L3443 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 tango12L3443, 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 vps35MH20, TRPL is trapped in a Rab5-positive compartment. Evidenced by epistatic analysis in the double mutant PLD3.1 vps35MH20, 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, yataKE2.1, 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).
The mechanism surrounding chromosome inheritance during cell division has been well documented, however, organelle inheritance during mitosis is less understood. Recently, the Endoplasmic Reticulum (ER) has been shown to reorganize during mitosis, dividing asymmetrically in proneuronal cells prior to cell fate selection, indicating a programmed mechanism of inheritance. ER asymmetric partitioning in proneural cells relies on the highly conserved ER integral membrane protein, Jagunal (Jagn). Knockdown of Jagn in the compound Drosophila eye displays a pleotropic rough eye phenotype in 48% of the progeny. To identify genes involved in Jagn dependent ER partitioning pathway, a dominant modifier screen was performed of the 3rd chromosome for enhancers and suppressors of this Jagn RNAi-induced rough eye phenotype. this study screened through 181 deficiency lines covering the 3L and 3R chromosomes and identified 12 suppressors and 10 enhancers of the Jagn RNAi phenotype. Based on the functions of the genes covered by the deficiencies, genes were identified that displayed a suppression or enhancement of the Jagn RNAi phenotype. These include Division Abnormally Delayed (Dally), an heparan sulfate proteoglycan, the γ-secretase subunit Presenilin, and the ER resident protein Sec63. Based on current understanding of the function of these targets, there is a connection between Jagn and the Notch signaling pathway. Further studies will elucidate the role of Jagn and identified interactors within the mechanisms of ER partitioning during mitosis (Ascencio, 2023).
sease (Ascencio, 2023).
In Drosophila, the endoplasmic reticulum-associated protein degradation (ERAD) is engaged in regulating pleiotropic biological processes, with regard to retinal degeneration, intestinal homeostasis, and organismal development. The extent to which it functions in controlling the fly innate immune defense, however, remains largely unknown. This study shows that blockade of the ERAD in fat bodies antagonizes the Toll but not the IMD innate immune defense in Drosophila. Genetic approaches further suggest a functional role of Me31B in the ERAD-mediated fly innate immunity. Moreover, evidence is provided that silence of Xbp1 other than PERK or Atf6 partially rescues the immune defects by the dysregulated ERAD in fat bodies. Collectively, this study uncovers an essential function of the ERAD in mediating the Toll innate immune reaction in Drosophila (Zhu, 2022).
Translocon clogging at the endoplasmic reticulum (ER) as a result of translation stalling triggers ribosome UFMylation, activating translocation-associated quality control (TAQC) to degrade clogged substrates. How cells sense ribosome UFMylation to initiate TAQC is unclear. This study conducted a genome-wide CRISPR-Cas9 screen to identify an uncharacterized membrane protein named SAYSD1 that facilitates TAQC. SAYSD1 associates with the Sec61 translocon and also recognizes both ribosome and UFM1 directly, engaging a stalled nascent chain to ensure its transport via the TRAPP complex to lysosomes for degradation. Like UFM1 deficiency, SAYSD1 depletion causes the accumulation of translocation-stalled proteins at the ER and triggers ER stress. Importantly, disrupting UFM1- and SAYSD1-dependent TAQC in Drosophila leads to intracellular accumulation of translocation-stalled collagens, defective collagen deposition, abnormal basement membranes, and reduced stress tolerance. Thus, SAYSD1 acts as a UFM1 sensor that collaborates with ribosome UFMylation at the site of clogged translocon, safeguarding ER homeostasis during animal development (Wang, 2023).
Prolonged cellular activity may overload cell function, leading to high rates of protein synthesis and accumulation of misfolded or unassembled proteins, which cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) to re-establish normal protein homeostasis. Previous molecular work has demonstrated that sleep deprivation (SD) leads to ER stress in neurons, with a number of ER-specific proteins being upregulated to maintain optimal cellular proteostasis. This study investigated the transcriptional and ultrastructural ER and mitochondrial modifications induced by sleep loss. Gene expression analysis in mouse forebrains was used to show that SD was associated with significant transcriptional modifications of genes involved in ER stress but also in ER-mitochondria interaction, calcium homeostasis, and mitochondrial respiratory activity. Using electron microscopy, it was also shown that SD was associated with a general increase in the density of ER cisternae in pyramidal neurons of the motor cortex. Moreover, ER cisternae established new contact sites with mitochondria, the so-called mitochondria associated membranes (MAMs), important hubs for molecule shuttling, such as calcium and lipids, and for the modulation of ATP production and redox state. Finally, it was demonstrated that Drosophila male mutant flies (elav > linker), in which the number of MAMs had been genetically increased, showed a reduction in the amount and consolidation of sleep without alterations in the homeostatic sleep response to SD. This study has provided evidence that sleep loss induces ER stress characterized by increased crosstalk between ER and mitochondria. MAMs formation associated with SD could represent a key phenomenon for the modulation of multiple cellular processes that ensure appropriate responses to increased cell metabolism. In addition, MAMs establishment may play a role in the regulation of sleep under baseline conditions (Aboufares El Alaoui, 2023).
Aberrant immune responses and chronic inflammation can impose significant health risks and promote premature aging. Pro-inflammatory responses are largely mediated via reactive oxygen species (ROS) and reduction-oxidation reactions. A pivotal role in maintaining cellular redox homeostasis and the proper control of redox-sensitive signaling belongs to a family of antioxidant and redox-regulating thiol-related peroxidases designated as peroxiredoxins (Prx). Recent studies in Drosophila have shown that Prxs play a critical role in aging and immunity. This study identified two important 'hubs', the endoplasmic reticulum (ER) and mitochondria, where extracellular and intracellular stress signals are transformed into pro-inflammatory responses that are modulated by the activity of the Prxs residing in these cellular organelles. This study found that mitochondrial Prx activity in the intestinal epithelium is required to prevent the development of intestinal barrier dysfunction, which can drive systemic inflammation and premature aging. Using a redox-negative mutant, it ws demonstrated that Prx acts in a redox-dependent manner in regulating the age-related immune response. The hyperactive immune response observed in flies under-expressing mitochondrial Prxs is due to a response to abiotic signals but not to changes in the bacterial content. This hyperactive response, but not reduced lifespan phenotype, can be rescued by the ER-localized Prx (Odnokoz, 2023).
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).
Male reproductive glands secrete signals into seminal fluid to facilitate reproductive success. In Drosophila melanogaster, these signals are generated by a variety of seminal peptides, many produced by the accessory glands (AGs). One epithelial cell type in the adult male AGs, the secondary cell (SC), grows selectively in response to bone morphogenetic protein (BMP) signaling. This signaling is involved in blocking the rapid remating of mated females, which contributes to the reproductive advantage of the first male to mate. This paper shows that SCs secrete exosomes, membrane-bound vesicles generated inside late endosomal multivesicular bodies (MVBs). After mating, exosomes fuse with sperm (as also seen in vitro for human prostate-derived exosomes and sperm) and interact with female reproductive tract epithelia. Exosome release was required to inhibit female remating behavior, suggesting that exosomes are downstream effectors of BMP signaling. Indeed, when BMP signaling was reduced in SCs, vesicles were still formed in MVBs but not secreted as exosomes. These results demonstrate a new function for the MVB-exosome pathway in the reproductive tract that appears to be conserved across evolution (Corrigan, 2014).
Seminal fluid synthesized by male reproductive glands has a powerful influence on fertility, affecting multiple sperm activities and altering female behavior, in some cases directly conflicting with female reproductive interests. Several previous studies have revealed an important function for seminal peptides in Drosophila in these processes. However, this study presents the first in vivo evidence that exosomes also play a key role and identify a completely novel role for BMP signaling in regulating this process (Corrigan, 2014).
Exosome biogenesis, secretion, and uptake have been previously studied in Drosophila. However, the small size of exosomes, MVBs, and fly tissues makes these processes difficult to analyze in vivo. The AG contains only nanoliter volumes of secretions, making it impractical to use standard exosome analysis techniques, such as ultracentrifugation and Nanosight Tracking Analysis. Like other studies in flies, this study used genetic and imaging approaches to test the identity of SC-specific CD63-positive puncta. In addition, Western blot analysis of transferred seminal fluid and live imaging of giant MVBs in SCs were used to test the hypothesis that SCs produce exosomes (Corrigan, 2014).
The human CD63-GFP tetraspanin marker was used in this analysis. However, GFP-positive puncta were also observed in large secretory compartments of SCs expressing cytosolic GFP, and exosome-sized vesicles in MVBs and the AG lumen were observed in EM analysis of wild-type glands, confirming their presence in nontransgenic flies. Because exosomes can be loaded with many cellular components, the findings provide a potential explanation for the observation that AGs of several insects, including Drosophila, secrete intracellular proteins (Corrigan, 2014).
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).
Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in Drosophila were used to investigate the molecular pathway for biogenesis of the mucin-containing 'glue granules' that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1- and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules (Burgess, 2011).
Constitutive secretion of proteins and lipids from the trans-Golgi network (TGN) toward the cell surface is believed to operate in all cells. Constitutive secretion is characterized by the rapid deployment of newly synthesized cargo toward its final cellular destination. Specialized secretory cells such as endocrine, neuroendocrine, and exocrine cells contain an additional pathway termed the regulated secretory pathway. One hallmark of this pathway is the storage of regulated secretory proteins at high concentration in dense-core secretory granules that can be released in response to an external signal. How secreted proteins enter the regulated secretory pathway is a source of debate and may prove to be cargo and cell-type specific. In the case of endocrine and neuroendocrine cells, sorting of secreted cargo is believed to be content driven, with selective aggregation of regulated secretory proteins at the TGN playing a major role in secretory granule biogenesis (Burgess, 2011).
Little is known about the coat proteins that might be required on the cytoplasmic face to promote budding of lumenal regulated secretory cargo from the TGN. Initial studies in AtT20 pituitary cells noted that condensing secretory products accumulate in dilated regions of the TGN that are coated with clathrin. Similarly, in β-cells treated with monensin to perturb intracellular trafficking, proinsulin accumulates in a clathrin-coated compartment related to the TGN. These observations raise the possibility that the formation of regulated secretory granules might require clathrin at the TGN (Burgess, 2011).
Coat proteins selectively incorporate cargo into vesicles and provide a scaffold for vesicle formation. Clathrin and its associated heterotetrameric adaptor proteins (APs) make up a major class of vesicular coats. APs bind to sorting motifs found in the cytoplasmic tails of membrane cargo and function as links between vesicular cargo and the clathrin lattice, although some AP-3 and AP-4 coats lack clathrin. The four different AP complexes (AP-1-4) have distinct sites of action in the cell. Of these, the AP-1 complex has perhaps the most diverse roles, acting at the TGN to promote constitutive secretion (Chi, 2008), at the TGN and endosomes to sort mannose 6-phosphate receptors, and at immature secretory granules of specialized secretory cells to retrieve missorted proteins. Indeed, a coat composed of clathrin and AP-1 is required for maturation and condensation of regulated secretory granules. In contrast to granule maturation, the roles of AP-1 and clathrin in initial stages of secretory granule formation are less well established. AP-1 and clathrin were shown to be required for formation of Weibel-Palade bodies (Lui-Roberts, 2005), secretory organelles that store the hemostatic protein von Willebrand factor. However, a dominant-negative clathrin construct did not interfere with insulin granule production in neuroendocrine cells, suggesting these granules form through a clathrin-independent mechanism. Thus it is not clear how general a role AP-1 and clathrin play in granule biogenesis (Burgess, 2011).
The larval salivary gland in Drosophila provides an excellent system for molecular genetic analysis of factors required for formation of regulated secretory granules. During the last half of third-instar larval development, prior to pupariation, salivary gland cells initiate production of mucin-type secretory granules termed 'glue' granules. These granules contain highly glycosylated mucin-type glue proteins that are required to adhere the pupal case to a solid substrate during metamorphosis. Of the six known glue proteins (also called salivary gland secretion or Sgs proteins), Sgs1, Sgs3, and Sgs4 contain extended amino acid repeats that are likely sites of oligosaccharide linkage. These proteins, which are synthesized in response to a low-titer pulse of the steroid hormone ecdysone at the mid-third-instar larval stage, are stored until an additional high-titer pulse of ecdysone promotes their release at the onset of pupariation (Burgess, 2011).
Secreted mucin-type glycoproteins are ubiquitous in metazoans and serve important roles in animal physiology. This study analyzed the mechanism of mucin-type glue granule biogenesis in third-instar larval salivary gland cells. It was shown that AP-1 and clathrin localize to the TGN prior to glue production, colocalize with newly synthesized glue proteins during early stages of granule formation, and are found at later stages on maturing glue granules. Genetic disruption or knockdown of AP-1 subunits strongly reduces clathrin localization to the TGN. Moreover, AP-1 and clathrin are required for glue granule formation; loss of AP-1 causes glue cargo to accumulate at the TGN and in small, highly aberrant granules. These results reveal a requirement for AP-1 and clathrin in the formation of mucin-type secretory granules (Burgess, 2011).
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-47SHE-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-47SHE-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-47SHE-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-47SHE-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).
Steroid hormones are a large family of cholesterol derivatives regulating development and physiology in both the animal and plant kingdoms, but little is known concerning mechanisms of their secretion from steroidogenic tissues. This study presents evidence that in Drosophila, endocrine release of the steroid hormone ecdysone is mediated through a regulated vesicular trafficking mechanism. Inhibition of calcium signaling in the steroidogenic prothoracic gland (PG) results in the accumulation of unreleased ecdysone, and the knockdown of calcium-mediated vesicle exocytosis components in the gland caused developmental defects due to deficiency of ecdysone. Accumulation of synaptotagmin-labeled vesicles in the gland is observed when calcium signaling is disrupted, and these vesicles contain an ABC transporter that functions as an ecdysone pump to fill vesicles. It is proposed that trafficking of steroid hormones out of endocrine cells is not always through a simple diffusion mechanism as presently thought, but instead can involve a regulated vesicle-mediated release process (Yamanaka, 2015)
This study provides several lines of evidence demonstrating that the insect steroid hormone E is secreted from the PG not by simple diffusion, but rather through a calcium signaling-regulated vesicle fusion event. Three major points come from these findings: (1) Atet, an ABCG transporter, can facilitate E passage through membranes in an ATP-dependent manner, (2) GPCR-regulated calcium signaling in the PG promotes E release, and (3) the significance of steroid hormone release by vesicle exocytosis and its implication for other steroid hormone/cholesterol trafficking processes (Yamanaka, 2015)
Atet was originally cloned in Drosophila as an ABC transporter-encoding gene with unknown function. It was found to be highly expressed in embryonic trachea, leading to its name ABC transporter expressed in trachea or Atet. In an in situ hybridization experiment, however, this study found little expression of Atet in embryonic trachea, but instead saw specific high level expression in the PG, consistent with its expression pattern in the third instar larva. Since Atet has an atypical membrane topology and can transport E across membranes in vitro, renaming this gene Atypical topology ecdysone transporter is proposed, thereby retaining the Atet gene designation (Yamanaka, 2015)
Atet belongs to the ABCG subfamily of ABC transporters, members of which in mammals have been shown to transport cholesterol as well as other steroids, such as estrogens and their metabolites, in many biological systems. The atypical membrane topology, with the N-terminal ABC domain on the non-cytoplasmic side of the membrane, has not been reported for any ABC transporter to date. However, this topology may have a strong advantage in facilitating tight control on E release by preventing Atet from functioning on the plasma membrane, due to the lack of ATP in extracellular space. This configuration therefore prevents E transport directly through the plasma membrane and confines it to a vesicle-mediated fusion process, although it requires a separate molecular mechanism to transport ATP into the secretory vesicles. This mechanism remains unclear at this point, but it may involve a specific transporter like the recently described VNUT/SLC17A9. In this context, it is interesting to note that the human Atet orthologs ABCG1 and ABCG4 are also strongly predicted by membrane topology algorithms to position their N-terminal ABC domain on the non-cytoplasmic side. These transporters mediate cellular cholesterol efflux and have recently been shown to work not on the plasma membrane but in intracellular endosomes. Clearly, additional studies on the membrane topology of ABCG transporters are warranted (Yamanaka, 2015)
The results of the RNAi screening demonstrate that CG30054, a Gαq subunit, and Plc21C, a PLCβ class enzyme, are both required for proper PG function. These findings strongly implicate the existence of an unknown GPCR and cognate ligand as mediators of the calcium signaling event that is suggested to stimulates E release from the PG. On the other hand, it is known that the PTTH receptor is Torso, a receptor tyrosine kinase and its primary role is to promote E production by inducing E biosynthetic enzyme gene transcription. These observations suggest that, at least in Drosophila, E production and release are likely regulated separately. This machinery might help the GPCR ligand to generate large pulses of steroid in a timely fashion. The identification of the GPCR as well as its ligand is necessary to further pursue this possibility (Yamanaka, 2015)
The mechanism of steroid hormone transit through lipid membranes has not been well studied and in many physiology textbooks the issue is not even discussed. When this topic is mentioned, the explanation most often given is that they can freely diffuse through lipid membranes. Despite this prevailing assumption, there are only a few reports where such transbilayer transfer of steroids by free diffusion has been analyzed. In one theoretical study, it was shown in silico that a free energy of solvation-based mechanism can produce rapid flux of estradiol, testosterone, and progesterone through a simple membrane in concordance with measured rates. However, it is well known that steroid hormone transport across membranes can indeed be an active process in some situations: there are a number of reports on transporter involvement in either uptake or elimination of steroid hormones in eukaryotes ranging from yeast to human. These reports are suggestive enough to rationalize a potential mechanism that incorporates steroid hormones into secretory vesicles, which enables regulated secretion of steroid hormones from steroidogenic tissues (Yamanaka, 2015)
Historically, the possibility of vesicle-mediated steroid hormone release has been examined using ultrastructural and biochemical approaches in multiple biological systems, including the corpus luteum in sheep. The proposed vesicle-mediated progesterone release from the sheep corpus luteum, however, was later challenged, since the peptide oxytocin was shown to be present in dense granules by immuno-EM methods and release of oxytocin and progesterone responded differently to various secretagogues. Since that time, studies investigating the possibility of vesicle-mediated steroid release in any biological system have rarely been reported. One relevant and intriguing set of studies, however, involved ultrastructural localization of E in the PG of the waxworm Galleria mellonella using immuno-EM methods. These studies suggested that E in the PG is concentrated into what appear to be secretory granules that fuse with the plasma membrane, but once again no follow up studies have been reported in the literature (Yamanaka, 2015)
In considering the various models for steroid passage through membranes, it is important to note that steroids such as progesterone, testosterone, and estradiol are significantly more hydrophobic than E. Therefore, the free energy of solvation into a lipid bilayer of E is likely to be much more positive than for sex steroids; this may preclude the use of a simple diffusion mechanism for E. In this respect, E is more similar to bile acids, which are also highly hydrophilic and need active transporters to traverse lipid bilayers. Thus, depending on their specific physiochemical properties, different steroids might use either simple passive diffusion through the plasma membrane, active transporters or some combination of these mechanisms (Yamanaka, 2015)
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).
Many neurons influence their targets through co-release of neuropeptides and small molecule transmitters. Neuropeptides are packaged into dense-core vesicles (DCVs) in the soma and then transported to synapses, while small molecule transmitters such as monoamines are packaged by vesicular transporters that function at synapses. These separate packaging mechanisms point to activity, by inducing co-release, as the sole scaler of co-transmission. Based on screening in Drosophila for increased presynaptic neuropeptides, the receptor protein tyrosine phosphatase (Rptp) Ptp4E was found to post-transcriptionally regulate neuropeptide content in single DCVs at octopamine synapses. This occurs without changing neuropeptide release efficiency, transport and DCV size measured by both STED super-resolution and transmission electron microscopy. Ptp4E also controls presynaptic abundance and activity of the vesicular monoamine transporter (VMAT), which packages monoamine transmitters for synaptic release. Thus, rather than rely on altering electrical activity, the Rptp regulates packaging underlying monoamine-neuropeptide co-transmission by controlling vesicular membrane transporter and luminal neuropeptide content (Tao, 2019).
Synaptic complexity is enhanced by co-transmission with small molecules and bioactive peptides. The two transmitter classes differ in their postrelease distances traveled and durations of action, thus providing mechanisms for rapid point-to-point control and slow neuromodulation of circuits, development and behavior. Furthermore, from a cell biology perspective, transmission by small molecules and neuropeptides is distinguished by different vesicular loading mechanisms. The genetic results presented here are remarkable because (a) they reveal increased transmitter packaging, when past genetic screens have only yielded mutants that reduce vesicular packaging; (b) control of vesicular packaging varied between neuron subtypes based on differential Rptp expression, which represents a new mechanism for generating variation in co-transmission in the nervous system. Furthermore, this result is intriguing in the context of monoaminergic neurons because Ptp4E interacts genetically with α-synuclein toxicity in Drosophila. Given that synuclein is implicated in Parkinson's disease, DCV fusion pore dynamics and the early secretory pathway, the results here suggest that the mechanistic relationship between Rptps and synuclein may be broader than previously recognized; and (c) presynaptic abundance of a small-molecule vesicular membrane transporter and luminal neuropeptides are regulated in parallel. This shows that regulation of co-transmission is not limited to control of activity-induced vesicle exocytosis. Instead, an Rptp regulates vesicular packaging of both small-molecule and peptide neurotransmitters that underlies co-release (Tao, 2019).
How can a single Rptp simultaneously modify vesicular loading of both monoamines and neuropeptides? Peptidergic neurotransmission relies on packaging of neuropeptides in the soma, where they condense in the TGN and are sorted into DCVs. There is little DCV circulation in octopamine terminals because of their extensive axonal arbors and numerous boutons. Therefore, Rptp regulation of neuropeptide content of individual DCVs likely originates prior to axonal transport. VMAT is also processed in the TGN to be sorted into DCVs and small synaptic vesicles, rather than proceeding through the constitutive secretory pathway. A recent study found that knockdown of the TGN protein HID-1 reduces DCV luminal cargo and VMAT in DCVs by affecting sorting and DCV production. The coordinated effects on neuropeptides and VMAT are reminiscent of the results presented in this study, but the Ptp4E effect on packaging was not associated with a change in DCV number or transport. Therefore, the uncoupling of DCV number from packaging is indicative of a novel cell biological mechanism. With this in mind, a possible explanation for the effect of inhibiting Ptp4E is that the tyrosine phosphorylation stimulates TGN sorting of luminal and vesicle membrane content without changing DCV number or size. By this mechanism, Rptp regulation of vesicular packaging in the soma could scale co-release at the distal synaptic ending (Tao, 2019).
These results pose the question of the site of Ptp4E function. Rptps often mediate signaling triggered by cell-cell contacts. For example, the presynaptic Rptp Lar is activated by muscle Syndecan during development of the NMJ. By analogy, it is possible that presynaptic Ptp4E governs retrograde signaling (e.g., by interrupting tyrosine kinase-dependent mechanisms). In favor of this hypothesis, the closely related Rptp Ptp10D is found on axons in the embryo, where it is positioned to regulate axonal guidance during development. However, the potential involvement of vesicle biogenesis and the unknown localization of Ptp4E raise the possibility that somatic Ptp4E is responsible for synaptic effects. Novel tools to differentially control of Rptp activity by compartment (i.e. soma versus terminal) will be needed to distinguish between these possibilities. Regardless of the cellular location of Ptp4E signaling, the mechanism discovered here (i.e. coincident control of packaging of neuropeptides and small-molecule transmitters) represents a previously unknown cell biological strategy for regulating synaptic co-transmission (Tao, 2019).
Previous experiments have shown that increased VMAT leads to enlargement of vesicles and greater vesicular monoamine storage, but this effect was not seen in this study. Notably, the mechanistic basis of the VMAT expression effect on vesicle size is not understood because thermodynamics with a simple system suggests that maximal vesicular monoamine concentration should be reached even with one VMAT per vesicle. Therefore, to explain the previously observed effect on vesicle size, some other factor, such as monoamine leakage or membrane flexibility, must come into play. It is suggested that these parameters might differ in the synaptic terminals examined in this study. Alternatively, in contrast to the spherical vesicles studied previously, the DCVs in octopamine neurons are ovoid. Therefore, the current analysis of largest dimension cannot exclude that the narrow axis of these DCVs increased. According to the latter scenario, increases in vesicular volume and membrane surface area could have been undetected with the methodology used in the current study (Tao, 2019).
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).
Communication between distant cells can be mediated by extracellular vesicles (EVs) that deliver proteins and RNAs to recipient cells. Little is known about how EVs are targeted to specific cell types. This study identified the Drosophila cell-surface protein Stranded at second (Sas) as a targeting ligand for EVs. Full-length Sas is present in EV preparations from transfected Drosophila Schneider 2 (S2) cells. Sas is a binding partner for the Ptp10D receptor tyrosine phosphatase, and Sas-bearing EVs preferentially target to cells expressing Ptp10D. Co-immunoprecipitation and peptide binding to show that the cytoplasmic domain (ICD) of Sas binds to dArc1 and mammalian Arc. dArc1 and Arc are related to retrotransposon Gag proteins. They form virus-like capsids which encapsulate Arc and other mRNAs and are transported between cells via EVs. The Sas ICD contains a motif required for dArc1 binding that is shared by the mammalian and Drosophila amyloid precursor protein (APP) orthologs, and the APP ICD also binds to mammalian Arc. Sas facilitates delivery of dArc1 capsids bearing dArc1 mRNA into distant Ptp10D-expressing recipient cells in vivo (Lee, 2023).
Cell extrusion is a universal mode of cell removal from tissues, and it plays an important role in regulating cell numbers and eliminating unwanted cells. However, the underlying mechanisms of cell delamination from the cell layer are unclear. This study reports a conserved execution mechanism of apoptotic cell extrusion. Extracellular vesicle (EV) formation in extruding mammalian and Drosophila cells was found at a site opposite to the extrusion direction. Lipid-scramblase-mediated local exposure of phosphatidylserine is responsible for EV formation and is crucial for executing cell extrusion. Inhibition of this process disrupts prompt cell delamination and tissue homeostasis. Although the EV has hallmarks of an apoptotic body, its formation is governed by the mechanism of microvesicle formation. Experimental and mathematical modeling analysis illustrated that EV formation promotes neighboring cells' invasion. This study showed that membrane dynamics play a crucial role in cell exit by connecting the actions of the extruding cell and neighboring cells (Kira. 2023).
Neuronal dense-core vesicles (DCVs) contain neuropeptides and much larger proteins that affect synaptic growth and plasticity. Rather than using full collapse exocytosis that commonly mediates peptide hormone release by endocrine cells, DCVs at the Drosophila neuromuscular junction release their contents via fusion pores formed by kiss-and-run exocytosis. This study used fluorogen-activating protein (FAP) imaging to reveal the permeability range of synaptic DCV fusion pores and then shows that this constraint is circumvented by cAMP-induced extra fusions with dilating pores that result in DCV emptying. These Ca2+-independent full fusions require PKA-R2, a PKA phosphorylation site on Complexin and the acute presynaptic function of Rugose, the homolog of mammalian neurobeachin, a PKA-R2 anchor implicated in learning and autism. Therefore, localized Ca2+-independent cAMP signaling opens dilating fusion pores to release large cargoes that cannot pass through the narrower fusion pores that mediate spontaneous and activity-dependent neuropeptide release. These results imply that the fusion pore is a variable filter that differentially sets the composition of proteins released at the synapse by independent exocytosis triggers responsible for routine peptidergic transmission (Ca2+) and synaptic development (cAMP) (Bulgari, 2023).
At the trans-Golgi, complex traffic connections exist to the endolysosomal system additional to the main Golgi-to-plasma membrane secretory route. This study investigated three hits in a Drosophila screen displaying secretory cargo accumulation in autophagic vesicles: ESCRT-III component Vps20, SNARE-binding Rop, and lysosomal pump subunit VhaPPA1-1. Vps20, Rop, and lysosomal markers localize near the trans-Golgi. Furthermore, this study documents that the vicinity of the trans-Golgi is the main cellular location for lysosomes and that early, late, and recycling endosomes associate as well with a trans-Golgi-associated degradative compartment where basal microautophagy of secretory cargo and other materials occurs. Disruption of this compartment causes cargo accumulation in these hits, including Munc18 homolog Rop, required with Syx1 and Syx4 for Rab11-mediated endosomal recycling. Finally, besides basal microautophagy, this study shows that the trans-Golgi-associated degradative compartment contributes to the growth of autophagic vesicles in developmental and starvation-induced macroautophagy. These results argue that the fly trans-Golgi is the gravitational center of the whole endomembrane system (Zhou, 2023).
Exosomes are secreted nanovesicles with potent signalling activity that are initially formed as intraluminal vesicles (ILVs) in late Rab7-positive multivesicular endosomes, and also in recycling Rab11a-positive endosomes, particularly under some forms of nutrient stress. The core proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) participate in exosome biogenesis and ILV-mediated destruction of ubiquitinylated cargos. Accessory ESCRT-III components have reported roles in ESCRT-III-mediated vesicle scission, but their precise functions are poorly defined. They frequently only appear essential under stress. Comparative proteomics analysis of human small extracellular vesicles revealed that accessory ESCRT-III proteins, CHMP1A, CHMP1B, CHMP5 and IST1, are increased in Rab11a-enriched exosome preparations. These proteins are required to form ILVs in Drosophila secondary cell recycling endosomes, but unlike core ESCRTs, they are not involved in degradation of ubiquitinylated proteins in late endosomes. Furthermore, CHMP5 knockdown in human HCT116 colorectal cancer cells selectively inhibits Rab11a-exosome production. Accessory ESCRT-III knockdown suppresses seminal fluid-mediated reproductive signalling by secondary cells and the growth-promoting activity of Rab11a-exosome-containing EVs from HCT116 cells. It is concluded that accessory ESCRT-III components have a specific, ubiquitin-independent role in Rab11a-exosome generation, a mechanism that might be targeted to selectively block pro-tumorigenic activities of these vesicles in cancer (Marie, 2023).
Exocytosis is a fundamental cellular process by which cells secret cargos from their apical membrane into the extracellular lumen. Cargo release proceeds in sequential steps that depend on a coordinated assembly and organization of an actin cytoskeletal network. This study identified the conserved actin-crosslinking protein EFhD2/Swip-1 as a novel regulator controlling exocytosis of glue granules in the Drosophila salivary gland. Real-time imaging revealed that EFhD2/Swip-1 is simultaneously recruited with F-actin onto secreting granules in proximity to the apical membrane. EFhD2/Swip-1 is rapidly cleared at the point of secretory vesicle fusion and colocalizes with actomyosin network around the fused vesicles. Loss of EFhD2/Swip-1 function impairs secretory cargo expulsion resulting in strongly delayed secretion. Thus, these results uncover a novel role of EFhD2/Swip-1 in secretory vesicle compression and expulsion of cargo during regulated exocytosis. Remarkably, this function neither require calcium-binding nor dimerization of EFhD2/Swip-1. These data rather suggest that EFhD2/Swip-1 regulates actomyosin activity upstream of Rho-GTPase signaling to drive proper vesicle membrane crumpling and expulsion of cargo (Lehne, 2023).
Exocrine cells utilize large secretory vesicles (LSVs) up to 10 μm in diameter. LSVs fuse with the apical surface, often recruiting actomyosin to extrude their content through dynamic fusion pores. The molecular mechanism regulating pore dynamics remains largely uncharacterized. This study observed that the fusion pores of LSVs in the Drosophila larval salivary glands expand, stabilize, and constrict. Arp2/3 is essential for pore expansion and stabilization, while myosin II is essential for pore constriction. Several Bin-Amphiphysin-Rvs (BAR) homology domain proteins were identified that regulate fusion pore expansion and stabilization. The I-BAR protein Missing-in-Metastasis (MIM) localizes to the fusion site and is essential for pore expansion and stabilization. The MIM I-BAR domain is essential but not sufficient for localization and function. It is concluded that MIM acts in concert with actin, myosin II, and additional BAR-domain proteins to control fusion pore dynamics, mediating a distinct mode of exocytosis, which facilitates actomyosin-dependent content release that maintains apical membrane homeostasis during secretion (Biton, 2023).
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 myd3PM71 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).
The Drosophila melanogaster junctional neoplastic tumor suppressor, Lethal-2-giant larvae (Lgl), is a regulator of apicobasal cell polarity and tissue growth. Previous studies have shown in the developing Drosophila eye epithelium that, without affecting cell polarity, depletion of Lgl results in ectopic cell proliferation and blockage of developmental cell death due to deregulation of the Hippo signaling pathway. This study shows that Notch signaling is increased in lgl-depleted eye tissue, independently of Lgl's function in apicobasal cell polarity. The upregulation of Notch signaling is ligand dependent and correlates with accumulation of cleaved Notch. Concomitant with higher cleaved Notch levels in lgl- tissue, early endosomes (Avalanche [Avl+]), recycling endosomes (Rab11+), early multivesicular bodies (Hrs+), and acidified vesicles, but not late endosomal markers (Car+ and Rab7+), accumulate. Colocalization studies revealed that Lgl associates with early to late endosomes and lysosomes. Upregulation of Notch signaling in lgl- tissue requires dynamin- and Rab5-mediated endocytosis and vesicle acidification but is independent of Hrs/Stam or Rab11 activity. Furthermore, Lgl regulates Notch signaling independently of the aPKC-Par6-Baz apical polarity complex. Altogether, these data show that Lgl regulates endocytosis to restrict vesicle acidification and prevent ectopic ligand-dependent Notch signaling. This Lgl function is independent of the aPKC-Par6-Baz polarity complex and uncovers a novel attenuation mechanism of ligand-activated Notch signaling during Drosophila eye development (Parsons, 2014).
This study demonstrates a novel function for the cell polarity regulator lgl in regulation of endocytosis and Notch signaling. In the developing Drosophila eye epithelium that (1) Notch targets, E(spl)m8, CycA, and Rst, are upregulated in lgl- tissue; (2) Notch upregulation contributes to the lgl- mosaic adult eye phenotype; (3) Notch upregulation in lgl- clones is ligand dependent and requires endocytosis; (4) Lgl colocalizes with intracellular Notch and endocytic markers; (5) lgl- tissue accumulates Avl+ EEs, Hrs+MVBs, Rab11+ REs, endocytic compartments, and acidified vesicles, but not LE markers, Rab7, and Car; (6) Notch upregulation in lgl- clones is independent of the ESCRT-0 complex (Hrs/Stam) or Rab11, but it requires Rab5 function and acidification of endosomes; and (7) Notch upregulation in lgl- clones is independent of aPKC-Baz-Par6. Altogether, these data reveal a novel role for Lgl in attenuating ligand-activated Notch signaling via restricting the acidification of endocytic compartments, independently of its role in regulating the aPKC-Baz-Par6 cell polarity complex (Parsons, 2014).
The data have revealed that increased vesicle acidification in lgl- tissue is responsible for elevated Notch signaling. Because S3 cleavage (by γ-secretase) of Notch extracellular domain (Next) to form the intracellular domain (Nicd) depends on acidification of endosome, this suggests that increased vesicle acidification in lgl- tissue leads to aberrant γ-secretase activity and cleavage of Next to Nicd, resulting in upregulation of Notch signaling (Parsons, 2014).
The precise endocytic compartment in which the Notch receptor undergoes γ-secretase-mediated S3 proteolytic processing is controversial. In Drosophila epithelial tissues, V-ATPase function is implicated in Notch activation in the EEs or the MVBs; however, whether this is ligand dependent or independent is unclear. In contrast, ligand-independent generation of Nicd in the LE and/or lysosome compartment has been described. Because increased Notch signaling in lgl- tissue depends on Rab5 EE activity, but Rab7 and Car LE compartments were not perturbed in lgl- tissue, a model is favored in which Lgl restricts vesicle acidification and activation of Notch signaling in the EE and/or early MVB compartments (Parsons, 2014).
It is speculated that Lgl regulates Notch signaling by two possible mechanisms: (1) via direct regulation of vesicle acidification or (2) via regulation of endosomal maturation, which indirectly affects vesicle acidification. In the first model, Lgl might inhibit V-ATPase activity by regulating levels and/or subunit composition or the association and/or dissociation of the V-ATPase complex. In the second model, Lgl might regulate endosomal maturation, and alterations in this process subsequently lead to accumulation of acidic vesicles and ectopic Notch activation. The data showing that lgl- tissue accumulates EEs (Avl+), REs (Rab11+), and early MVBs (Hrs+), but not LEs (Rab7+ or Car+), suggest that Lgl regulates a specific step in endosome maturation after early MVB formation. Further studies are required to determine whether Lgl controls vesicle acidification by affecting the V-ATPase or endosome maturation to regulate Notch signaling (Parsons, 2014).
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 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).
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 iDelta20' 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 iDelta20' 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>NeurRNAi 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>NeurRNAi, Sara12/Df(2R)48 transheterozygote mutants, the number of supernumerary SOPs is increased by 35% with respect to the Pnr>NeurRNAi 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>NeurRNAi, 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>NeurRNAi 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>NeurRNAi need Sara function to perform asymmetric cell fate assignation: in Pnr>NeurRNAi, Sara12/Df(2R)48 and Pnr>NeurRNAi, 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).
Asymmetric cell division gives rise to two daughter cells that inherit different determinants, thereby acquiring different fates. Polarized trafficking of endosomes containing fate determinants recently emerged as an evolutionarily conserved feature of asymmetric cell division to enhance the robustness of asymmetric cell fate determination in flies, fish and mammals. In particular, polarized sorting of signalling endosomes by an asymmetric central spindle contributes to asymmetric cell division in Drosophila melanogaster. However, how central spindle asymmetry arises remains elusive. This study identified a moonlighting function of the Elongator complex-an established protein acetylase and tRNA methylase involved in the fidelity of protein translation-as a key factor for central spindle asymmetry. Elongator controls spindle asymmetry by stabilizing microtubules differentially on the anterior side of the central spindle. Accordingly, lowering the activity of Elongator on the anterior side using nanobodies mistargets endosomes to the wrong cell. Molecularly, Elongator regulates microtubule dynamics independently of its acetylation and methylation enzymatic activities. Instead, Elongator directly binds to microtubules and increases their polymerization speed while decreasing their catastrophe frequency. These data establish a non-canonical role of Elongator at the core of cytoskeleton polarity and asymmetric signalling (Planelles-Herrero, 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).