InteractiveFly: GeneBrief

Phosphotidylinositol 3 kinase 59F: Biological Overview | References

BIOLOGICAL OVERVIEW

The class III phosphatidylinositol-3 kinase [PI3K (III)] regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate [PI(3)P]. While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. This study, in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) were examined in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15, a protein serine/threonine kinase, resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein beta-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane-localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of beta-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes (Abe, 2009).

Cell polarity along the apical-basal axis is essential for the function of epithelial cells. This polarity is formed and maintained by distinct localization of membrane spanning and associated proteins, to apical, lateral or basal membrane domains. Membrane proteins localized to the apical or basolateral plasma membrane are endocytosed into early and apical or basolateral endosomes. For example, horseradish peroxidase (HRP) administered to the apical cell surface is incorporated into the apical early endosome. By contrast, HRP or dimeric IgA administered to the basolateral cell surface or transferring receptor (TfR) in the basolateral domain are internalized into the basolateral early endosome, which remain distinct. Sorting of proteins for transcytosis, recycling and degradation takes place in these early endosomes. The proteins, incorporated into apical and basolateral early endosomes, meet in common endosomes, a process that can be observed within 15 min after the onset of internalization in MDCK cells. The significance of keeping the apical and basolateral early endosomes distinct is thought to ensure that proteins from the apical and basolateral plasma membrane remain apart before the sorting processes proceeds. Although it is plausible that the trafficking of proteins in distinct membrane domains is regulated differently, the factors involved in such a differential regulation remain elusive (Abe, 2009).

One of the key molecules regulating membrane trafficking is PI3K (III), a heterodimer of Vps34p and Vps15p/p150, which produces phosphatidylinositol-3-phosphate (PI(3)P) (Herman, 1990; Hiles, 1992; Schu, 1993; Stack, 1993). PI(3)P is found to localize with early endosome and internal vesicles of multivesicular bodies (MVBs) in mammalian cells in culture (Gillooly, 2000). Genetic and pharmacological analysis, using yeast and mammalian cells in culture, suggests that PI3K (III) is required for five distinct processes. These are: (1) the fusion of clathrin-coated vesicles and early endosomes as well as the fusion between early endosomes (Christoforidis, 1999; Jones, 1995; Li, 1995; Spiro, 1996); (2) the recycling from early endosomes back to the Golgi complex or other destinations (Burda, 2002; Tuma, 2001); (3) the entry of proteins into the lysosomal degradation pathway; (4) the formation of internal vesicles of MVBs and (5) autophagy (Kihara, 2001; Petiot, 2000). Moreover, inactivation of PI3K (III) by Vps34 mutation leads to an expansion of the outer nuclear membrane and an abnormal reduction of the LDL receptor at the apical membrane in C. elegans (Roggo, 2002). In Drosophila, dVps34 mutation results in defective endocytosis of the apical membrane protein Notch and a defective onset of autophagy (Juhasz, 2008). It has been suggested that PI3K (III) utilizes different effectors at apical and basolateral endosomes (Tuma, 2001). However, the role of PI3K (III) in the regulation of protein localization at different membrane domains has remained unclear (Abe, 2009 and references therein).

To understand the various functions of PI3K (III), it is crucial to clarify which downstream effectors are involved in each of the processes it regulates. PI3K (III) is thought to exert its function through the recruitment of proteins that contain PI(3)P-binding motifs such as FYVE or PX domains. Among such proteins, Rabenosyn-5 (Rbsn-5) has been shown to contribute to endosome fusion and recycling processes in mammalian cells. Genetic studies on C. elegans and Drosophila also show that Rbsn-5 is essential for receptor-mediated endocytosis and endosome fusion (Gengyo-Ando, 2007; Morrison, 2008), although it is not clear whether or not Rbsn-5 is involved in other PI3K (III)-related phenomena (Abe, 2009).

To determine how the proteins in distinct membrane domains are regulated by PI3K (III) and its effector Rbsn-5 this study analyzed Drosophila wing development. This provides a good model since wing primordial cells have a clear polarity along the apical-basal axis. In addition a number of membrane proteins are known to be transported in an organized manner along the apical-basal axis. For example DE-cadherin, a cell adhesion protein and Fmi, a planar cell polarity (PCP) core protein, are localized in the apical junctions or zonula adherens (ZA), whereas the cell adhesion molecules FasIII and β-integrin are localized in lateral and basal membranes, respectively. This study found that inactivation of PI3K (III) in the wing primordial cells by knockdown of dVps15 affects the localization of these membrane proteins differently. In particular, it was found that dVps15 knockdown results in the accumulation of FasIII at the lateral membrane, whereas it results in intracellular accumulation of DE-cadherin, Fmi and β-integrin. Importantly, inactivation of Rbsn-5 shows accumulation of FasIII and β-integrin at the lateral membrane and intracellular vesicles, respectively, but no effects of DE-cadherin and Fmi localization (see in contrast Mottola, 2010). These results provide evidence for a differential regulation of protein localization by PI3K (III) and Rbsn-5 at distinct membrane domains (Abe, 2009).

This study demonstrated that PI3K (III) differentially regulates the localization of proteins at distinct membrane domains. The intracellular accumulation of Fmi, DE-cadherin and β-integrin induced by the dVps15 knockdown might be due to defects in the degradation pathway, since the maturation of MVBs and the lysosomal trafficking were defective in these cells. However, unlike these proteins, Fas III did not accumulate in the intracellular compartments, but rather accumulated at the surface of the lateral plasma membrane. It is possible that PI3K (III) regulates proteins at the lateral membrane differently from those localized at other membrane domains. It is also possible that PI3K (III) regulates Fas III in a different way, irrespective of the membrane domain to which it is localized. Whichever is the case it will be important to elucidate the mechanism underlying this difference in a future study (Abe, 2009).

Rbsn-5, a FYVE domain-containing protein, shares a part of the functions of PI3K (III), in that it is necessary for the regulation of Fas III and β-integrin localization, but not that of DE-cadherin and Fmi localization. Although the Rbsn-5^C241 null mutant clones may not completely lack Rbsn-5 activity, the requirement of Rbsn-5, or at least the requirement of an appropriate amount, differs between these proteins with respect to normal trafficking. It appears that Rbsn-5 preferentially controls the events at the basolateral regions, given that Rbsn-5 is necessary for the formation of large endosomes at the basal region, whereas it is indispensable for the formation of actin bundles at the apical surface (Abe, 2009).

PI3K (III) has been implicated in the differential regulation of vesicle trafficking at apical and basolateral regions. For instance, a reduction of PI(3)P dissociates EEA1, a FYVE-domain containing protein essential for early endosome fusion, selectively from basolateral endosomes (Tuma, 2001). However, which proteins, including EEA1, regulate the different trafficking pathways downstream of PI3K (III) has remained unknown. Rbsn-5 has been proposed to be a PI3K (III) effector, since Rbsn-5 harbors a FYVE domain. The current results provide further evidence supporting a possible functional interaction between these two molecules, based on their genetic interaction on the wing morphogenesis and the PI3K (III)-dependent Rbsn-5 immunostaining. Importantly, the different requirement of Rbsn-5 for trafficking at apical junction and basolateral membrane domains suggests that Rbsn-5 may a selective regulator under the control of PI3K (III) (Abe, 2009).

The PI3K class III complex promotes axon pruning by downregulating a Ptc-derived signal via endosome-lysosomal degradation

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

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

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

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

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

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

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

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

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

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

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

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

The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila

Using the Drosophila larval neuromuscular junction, this study shows a PI3K-dependent pathway for synaptogenesis (a pro-syaptogenesis pathway) which is functionally connected with other previously known elements including the Wit receptor, its ligand Gbb, and the MAPkinases cascade. Based on epistasis assays, the functional hierarchy within the pathway was determined. Wit seems to trigger signaling through PI3K, and Ras85D also contributes to the initiation of synaptogenesis. However, contrary to other signaling pathways, PI3K does not require Ras85D binding in the context of synaptogenesis. In addition to the MAPK cascade, Bsk/JNK undergoes regulation by Puc and Ras85D which results in a narrow range of activity of this kinase to determine normalcy of synapse number. The transcriptional readout of the synaptogenesis pathway involves the Fos/Jun complex and the repressor Cic. In addition, an antagonistic pathway (an anti-synaptogenesis pathway) was identified that uses the transcription factors Mad and Medea and the microRNA bantam to down-regulate key elements of the pro-synaptogenesis pathway. Like its counterpart, the anti-synaptogenesis signaling uses small GTPases and MAPKs including Ras64B, Ras-like-a, p38a and Licorne. Bantam downregulates the pro-synaptogenesis factors PI3K, Hiw, Ras85D and Bsk, but not AKT. AKT, however, can suppress Mad which, in conjunction with the reported suppression of Mad by Hiw, closes the mutual regulation between both pathways. Thus, the number of synapses seems to result from the balanced output from these two pathways (Jordan-Alvarez, 2017).

The epistasis assays have determined the in vivo functional links between PI3K and other previously known pro-synaptogenesis factors. Epistasis assays are based on the combined expression of two or more UAS constructs. Several double combinations in this study have produced a phenotype in spite of the apparent ineffectiveness of the single constructs. This type of results underscores the necessity to use epistasis assays in order to reveal functional interactions in vivo, hence, biologically relevant. In addition to the pro-synaptogenesis signaling, the study has revealed an anti-synaptogenesis pathway that composes a signaling equilibrium to determine the actual number of synapses. The magnitude of the synapse number changes elicited by the factors tested here are mostly within the range of 20%-50%. Are these values significant to cause behavioral changes? Reductions in the order of 30% of excitatory or inhibitory synapses in adult Drosophila local olfactory interneurons transform perception of certain odorants from attraction to repulsion and vice versa. In schizophrenia patients, a 16% loss of inhibitory synapses in the brain cortex has been reported. In Rhesus monkeys, the pyramidal neurons in layer III of area 46 in dorsolateral prefrontal cortex show a 33% spine loss, and a significant reduction in learning task performance during normal aging. Thus, it seems that behavior is rather sensitive to small changes in synapse number irrespective of the total brain mass (Jordan-Alvarez, 2017).

The signaling interactions analyzed here were chosen because they were reported in other cellular systems and species previously. Some of these interactions have been confirmed (e.g., Gbb/Wit), while others have proven ineffective in the context of synaptogenesis (e.g., Ras85D/PI3K binding). Likely, the two signaling pathways, pro- and anti-synaptogenesis, are not the only ones relevant for synapse formation. For example, in spite of the null condition of the gbb and wit mutant alleles used here, the resulting synaptic phenotypes are far less extreme than expected if these two factors would be the only source of signaling for synaptogenesis. Although it could be argued that the incomplete absence of synapses in the mutant phenotypes could result from maternal perdurance, Wit is not part of the oocyte endowment while Gbb is. Three alternative possibilities may be considered, additional ligands for Wit, additional receptors for Gbb, and a combination of the previous two. Beyond the identity of these putative additional ligands and receptors, the stoichiometry between ligands and receptors may certainly be relevant. Actually, Gbb levels are titrated by Crimpy. An equivalent quantitative regulation could operate on Wit. The reported data on Wit illustrate already the diversity of the functional repertoire of this receptor. Wit can form heteromeric complexes with Thick veins (Tkv) or Saxophone (Sax) receptors to receive Dpp/BMP4 or Gbb/BMP7 as ligands. However, the same study also showed that Wit could dimerize with another receptor, Baboon, upon binding of Myoglianin to activate a different and antagonistic signaling pathway, TGFβ/activin-like (Jordan-Alvarez, 2017).

The Gbb/Wit/PI3K signaling analyzed in this study is likely not the only pro-synaptogenesis pathway in flies and vertebrates. The ligand Wingless (Wg), member of the Wnt family, and the receptors Frizzled have been widely documented as relevant in neuromuscular junction development, albeit data on synapse number are scant. Interestingly, however, the downstream intermediaries can be as diverse as those mentioned above for Wit. Although generally depicted as linear pathways, a more realistic image would be a network of cross-interacting signaling events whose in situ regulation and cellular compartmentalization remains fully unexplored (Jordan-Alvarez, 2017).

The quantitative regulation of receptors is most relevant to understand their biological effects. In that context, is worth noting that Tkv levels are distinctly regulated from those of Wit and Sax through ubiquitination in the context of neurite growth. On the other hand, although the receptor Wit is considered a RSTK type, the functional link with PI3K is a feature usually associated to the RTK type instead. The link of Wit with a kinase has a precedent with LIMK1 that binds to, and is functionally downstream from, Wit in the context of synapse stabilization. Thus, Wit should be considered a wide spectrum receptor in terms of its ligands, co-receptor partners and, consequently, signaling pathways elicited. Actually, the Wit amino acid sequence shows both, Tyr and Ser/Thr motifs justifying its initial classification as a 'dual' type of receptor. In this report this study did not determine if Wit heterodimerizes with other receptors, as canonical RSTKs do, or if it forms homodimers, as canonical RTKs do. However, the lack of synaptogenesis effects by the putative co-receptors, Tkv and Sax, and the phenotypic similarity with the manipulation of the standard RTK signaling effector Cic, leaves open the possibility that Wit could play RTK-like functions, at least in the context of synaptogenesis (Jordan-Alvarez, 2017).

Consistent with the proposal of a dual mechanism for Wit, its activation seems to be a requirement to elicit two independent signaling steps, PI3K and Ras85D, that could reflect RTK and RSTK mechanisms, respectively. Both steps are independent because the mutated form of PI3K unable to bind Ras85D, PI3KΔRBD, is as effective as the normal PI3K to elicit synaptogenesis. PI3K and Ras85D signaling, however, seem to converge on Bsk revealing a novel feature of this crossroad point. The activity level of Bsk is known to be critical in many signaling processes. The peculiarities of Bsk/JNK activity include its coordinated regulation by p38a and Slpr in the context of stress heat response without interference on the developmental context. Another modulator, Puc, was described as a negative feed-back loop in the context of oxidative stress. The Puc mediated loop is operative also for synaptogenesis, while that of p38a/Slpr is relevant for p38a only, as shown here. Further, Ras85D represents an additional regulator in the neural scenario. The triple regulation of Bsk/JNK by Ras85D, Puc and the MAPKs seems to stablish a narrow range of activity thresholds within which normal number of synapses is determined (Jordan-Alvarez, 2017).

The concept of signaling thresholds is also unveiled in this study by the identification of another signaling pathway that opposes synapse formation. The pro- and anti-synaptogenesis pathways have similar constituents, including small GTPases, MAPKs and transcriptional effectors, Mad/Smad, which are canonical for RSTK receptors. The RSTK type II receptor Put, which can mediate diverse signaling pathways according to the co-receptor bound can be discarded in either the pro- or the anti-synaptogenesis pathways. Thus, the main receptor for the anti-synaptogenesis pathway remains to be identified (Jordan-Alvarez, 2017).

Concerning small GTPases, the pro-synaptogenesis pathway uses Ras85D while its counterpart uses the poorly studied Ras64B. The anti-synaptogenesis pathway includes an additional member of this family of enzymes, Rala. This small GTPase plays a role in the exocyst-mediated growth of the muscle membrane specialization that surrounds the synaptic bouton as a consequence of synapse activity. That is, Rala can influence synapse physiology acting from the postsynaptic side. The experimental expression of a constitutively active form of Rala in the neuron does not seem to affect the overall synaptic terminal branching. However, the null ral mutant shows reduced synapse branching and its vertebrate homolog is expressed in the central nervous system. This study found that Rala under-expression in neurons yields an elevated number of synapses. Thus, it is likely that this small GTPase acts as a break to synaptogenesis, hence its inclusion in the antagonistic pathway (Jordan-Alvarez, 2017).

Synaptogenesis and neuritogenesis are distinct processes since each one can be differentially affected by the same mutant (e.g.: Hiw). Both features, however, share some signals (e.g., Wnd, Hep). This signaling overlap is akin to the case of axon specification versus spine formation for constituents of the apico/basal polarity complex Par3-6/aPKC [127]. These and other examples illustrated in this study underscore the need to discriminate between synapses and boutons. This study is focused on the cell autonomous signaling that takes place in the neuron. Non-cell autonomous signals (e.g., originated in the glia or hemolymph circulating) have not been considered. The active role of glia in axon pruning and bouton number has been the subject of other studies. Considering the reported role of Hiw through the midline glia in the remodeling of the giant fiber interneuron it is not unlikely that the glia-to-neuron signaling may share components with the neuron autonomous signaling addressed here (Jordan-Alvarez, 2017).

The summary scheme (see Summary diagram of antagonistic signaling pathways for synaptogenesis and their interactions) describes the scenario where two signaling pathways mutually regulate each other. Epistasis assays are the only experimental approach for in vivo studies of more than one signaling component, albeit this type of assay is only feasible in Drosophila Thus, it is plausible that vertebrate synaptogenesis will be regulated by a similar antagonistic signaling (Jordan-Alvarez, 2017).

The regulatory equilibrium as a mechanism to determine a biological parameter is the most relevant feature in this scenario for several reasons. First, because this type of mechanism can respond very fast to changes in the physiological status of the cell, and, second because it provides remarkable precision to the trait to be regulated, synapse number in this case. Although bi-stable regulatory mechanisms are known in other contexts, the case of synapse number may seem unexpected because the highly dynamic nature of synapse number has been recognized only recently. Consequently, a molecular signaling mechanism endowed with proper precision and time resolution must sustain this dynamic process. The balanced equilibrium uncovered in this study, although most likely still incomplete in terms of its components, offers such a mechanism (Jordan-Alvarez, 2017).

A postsynaptic PI3K-cII dependent signaling controller for presynaptic homeostatic plasticity

Presynaptic homeostatic plasticity stabilizes information transfer at synaptic connections in organisms ranging from insect to human. By analogy with principles of engineering and control theory, the molecular implementation of PHP is thought to require postsynaptic signaling modules that encode homeostatic sensors, a set point, and a controller that regulates transsynaptic negative feedback. The molecular basis for these postsynaptic, homeostatic signaling elements remains unknown. In this study, an electrophysiology-based screen of the Drosophila kinome and phosphatome defines a postsynaptic signaling platform that includes a required function for PI3K-cII, PI3K-cIII and the small GTPase Rab11 during the rapid and sustained expression of PHP. Evidence is presented that PI3K-cII localizes to Golgi-derived, clathrin-positive vesicles and is necessary to generate an endosomal pool of PI(3)P that recruits Rab11 to recycling endosomal membranes. A morphologically distinct subdivision of this platform concentrates postsynaptically where it is proposed to functions as a homeostatic controller for retrograde, trans-synaptic signaling (Hauswirth, 2018).

Homeostatic signaling systems stabilize the functional properties of individual neurons and neural circuits through life. Despite widespread documentation of neuronal homeostatic signaling, many fundamental questions remain unanswered. For example, given the potent action of homeostatic signaling systems, how can neural circuitry be modified during neural development, learning, and memory? Although seemingly contradictory, the homeostatic signaling systems that stabilize neural function throughout life may actually enable learning-related plasticity by creating a stable, predictable background upon which learning-related plasticity is layered. Therefore, defining the underlying molecular mechanisms of homeostatic plasticity may not only be informative about the mechanisms of neurological disease, these advances may be informative regarding how complex neural circuitry is able to accomplish an incredible diversity of behaviorally relevant tasks and, yet, retain the capacity for life-long, learning-related plasticity (Hauswirth, 2018).

Neuronal homeostatic plasticity encompasses a range of compensatory signaling that can be sub-categorized based upon the cellular processes that are controlled, including ion channel gene expression, neuronal firing rate, postsynaptic neurotransmitter receptor abundance and presynaptic vesicle release. Presynaptic homeostatic potentiation (PHP) is an evolutionarily conserved form of neuronal homeostatic control that is expressed at the insect, rodent and human neuromuscular junctions (NMJ) and has been documented at mammalian central synapses. PHP is initiated by the pharmacological inhibition of postsynaptic neurotransmitter receptors. The homeostatic enhancement of presynaptic vesicle release can be detected in a time frame of seconds to minutes, at both the insect and mouse NMJ. This implies the existence of postsynaptic signaling systems that can rapidly detect the disruption of neurotransmitter receptor function and convert this into retrograde, trans-synaptic signals that accurately adjust presynaptic neurotransmitter release. Notably, the rapid induction of PHP is transcription and translation independent, and does not include a change in nerve terminal growth or active zone number (Hauswirth, 2018).

There has been considerable progress identifying presynaptic effector molecules responsible for the expression of PHP. There has also been progress identifying postsynaptic signaling molecules that control synaptic growth at the Drosophila NMJ as well as the long-term, translation-dependent maintenance of PHP. However, to date, nothing is known about the postsynaptic signaling systems that initiate and control the rapid induction and expression of PHP (Hauswirth, 2018).

This paper reports the completion of an unbiased, forward genetic screen of the Drosophila kinome and phosphatome, and the identification of a postsynaptic signaling system for the rapid expression of PHP that is based on the activity of postsynaptic Phosphoinoside-3-Kinase (PI3K) signaling. There are three classes of PI3-Kinases, all of which phosphorylate the 3 position of phosphatidylinositol (PtdsIns). Class I PI3K catalyzes the conversion of PI(4,5)P₂ to PI(3,4,5)P₃ (PIP3) at the plasma membrane, enabling Akt-dependent control of cell growth and proliferation, and participating in the mechanisms of long-term potentiation. Class II and III PI3Ks (PI3K-cII and PI3K-cIII, respectively) both catalyze the conversion of PI to PI(3)P, which is a major constituent of endosomal membranes. PI(3)P itself may be a signaling molecule with switch like properties, functioning in the endosomal system as a signaling integrator. The majority of PI(3)P is synthesized by PI3K-cIII, which is involved in diverse cellular processes. By contrast, the cellular functions of PI3K-cII remain less well defined. PI3K-cII has been linked to the release of catecholamines, immune mediators, insulin, surface expression and recycling of integrins, and GLUT4 translocation to the plasma membrane, a mediator of metabolic homeostasis in muscle cells. This study demonstrates that Class II and Class III PI3K-dependent signaling are necessary for the rapid expression of PHP, controlling signaling from Rab11-dependent, recycling endosomes. By doing so, this study defines a postsynaptic signaling platform for the rapid expression of PHP and defines a novel action of PI3K-cII during neuronal homeostatic plasticity. This is the first established postsynaptic function for PI3K-cII at a synapse in any organism (Hauswirth, 2018).

Recently, it has become clear that the endosomal system has a profound influence on intracellular signaling and neural development. There is evidence that early and recycling endosomes can serve as sites of signaling intersection and may serve as signaling integrators and processors. Furthermore, protein sorting within recycling endosomes, and novel routes of protein delivery to the plasma membrane, may specify the concentration of key signaling molecules at the cell surface. The essential role of endosomal protein trafficking is underscored by links to synapse development and neurodegeneration. Yet, connections to homeostatic plasticity remain to be established. Based upon the data presented in this study and building upon prior work on endosomal signaling in other systems, it is speculated that postsynatpic PI3K-cII and Rab11-dependent recycling endosomes serve as as a postsynaptic 'homeostatic controller' that is essential for the specificity of retrograde, transsynaptic signaling (Hauswirth, 2018).

The Drosophila kinome and phosphatome were screened for genes that control the rapid expression of PHP. This screen identified three components of a conserved, postsynaptic lipid signaling pathway that is essential for the robust expression of PHP including: (1) class II PI3K, (2) class III PI3K (Vps34) and a gene encoding the Drosophila orthologue of PI4K (not examined in detail in this study). Pi3K68D was shown to be is essential, postsynaptically for PHP. Pi3K68D resides on a Clathrin-positive membrane compartment that is positioned directly adjacent to Golgi membranes, throughout muscle and concentrated at the postsynaptic side of the synapse. Pi3K68D is necessary for the maintenance of postsynaptic PI(3)P levels and the recruitment of Rab11 to intracellular membranes, likely PI(3)P-positive recycling endosomes. Postsynaptic Rab11 and Vps34 knockdown block PHP in an unusual, calcium-dependent manner that phenocopies Pi3K68D. Thus, this study has identified a postsynaptic signaling platform, centered upon the formation of PI(3)P and Rab11-positive recycling endosomes, that is essential for PHP (Hauswirth, 2018).

First it is considered whether postsynaptic Pi3K68D, Vps34 and Rab11 might alter PHP through modulation of postsynaptic glutamate receptor abundance. There is no consistent change in mEPSP amplitude in Pi3K68D mutants or following muscle-specific knockdown of Rab11 or Vps34 that could account for altered PHP. Therefore, functionally, there is no evidence for a change in glutamate receptor abundance at the postsynaptic membrane that could drive the phenotypic effects that were observe. Anatomically, data is presented examining GluR staining levels. In the Pi3K68D mutants, no change was found in GluRIIA levels. GluRIIA subunit containing receptors are the primary mediator of PhTx-dependent PHP. This study also reports a very modest (16%), though statistically significant, increase in GluRIIB levels. Based on these combined data, it seems unlikely that a change in GluR trafficking is a causal event leading to altered expression of PHP. It is noted that previous work showed limited GluRIIA receptor mobility within the PSD at the Drosophila NMJ. Thus, it is speculated that the function of Pi3K68D, Vps34 and Rab11 during PHP is not directly linked to postsynaptic GluR trafficking (Hauswirth, 2018).

Any model to explain the role of PI3K, Vps34 and Rab11-dependent endosomal signaling during homeostatic plasticity must account for the phenotypic observation that PHP is only blocked at low extracellular concentrations. More specifically, in animals deficient for Pi3K68D, Rab11 or Vps34, PHP is fully expressed at elevated calcium, following PhTX application or in the GluRIIA mutant. However, PHP completely fails when extracellular calcium is acutely decreased (following induction) below 0.7 mM [Ca2+]e. Clearly, the PHP induction mechanisms remain fully intact. Instead, the presynaptic expression of PHP has been rendered calcium-dependent. It is important to note that PHP can be fully induced in the absence of extracellular calcium, so the concentration of calcium itself is not the defect. In addition, this study documents trans-heterozygous interactions of Pi3K68D with presynaptic rim and dmp, arguing for the loss of trans-synaptic signaling and a specific function of Pi3K68D in the mechanisms of PHP. In very general terms, it is concluded that PI3K and Rab11-dependent endosomal signaling platform is necessary to enable the normal expression of PHP. Ultimately, some form of retrograde signaling must be defective due to either: 1) the absence of a retrograde signal that should have normally participated in PHP or 2) the presence of an aberrant or inappropriate signal that dominantly obstructs normal PHP expression. Both of these ideas in greater depth (Hauswirth, 2018).

First, the possibility is considered that the absence of postsynaptic PI3K and Rab11 signaling could alter the molecular composition or development of the presynaptic terminal due to the persistent absence of a retrograde signal that controls generalized synapse development or growth. Several observations demonstrate that impaired PHP is not a secondary consequence of a general defect in synapse development. Three independent postsynaptic manipulations are reported (postsynaptic expression of kinase dead Pi3K68D, postsynaptic knockdown of Rab11, and postsynaptic knockdown of Vps34) that have no effect on presynaptic release at any [Ca2+]e, yet block PHP at low [Ca2+]e. In addition, no obvious defect was found in anatomical synapse development (Hauswirth, 2018).

Next, the possibility is considered that postsynaptic PI3K and Rab11 signaling eliminate a retrograde signal that is specific for PHP. It was recently demonstrated that Semaphorin2b (Sema2b) and PlexinB (PlexB) define a retrograde signal at the Drosophila NMJ that is necessary for PHP. However, both Sema2b and PlexB are essential for the rapid induction of PHP, inclusive of experiments at low and elevated extracellular calcium. Further, acute application of recombinant Sema2b is sufficient to fully induce PHP. Since the induction of PHP remains fully intact in the Pi3K68D mutant, and since PHP is rendered calcium sensitive, it suggests that altered Sema2b secretion is not the cause of impaired PHP in the Pi3K68D mutant. Nevertheless, this possibility will be directly tested in the future (Hauswirth, 2018).

Next, the possibility is considered that the loss of PI3K and Rab11 signaling causes aberrant or inappropriate retrograde signaling, thereby impairing the expression of PHP. This is a plausible scenario because the induction of presynaptic homeostatic plasticity suffers from a common problem inherent to many intra-cellular signaling systems: two incompatible outcomes (1. presynaptic homeostatic potentiation and 2. presynaptic homeostatic depression -- PHD) are produced from a common input, and it remains unclear how signaling specificity is achieved. The topic of signaling specificity has been studied in several systems. One system, budding yeast, is a good example. Different pheromone concentrations can induce several distinct behaviors in budding yeast despite having a common input (pheromone concentration) and underlying signaling systems. Signaling specificity degrades in the background of mutations that affect Map Kinase scaffolding proteins. In a similar fashion, presynaptic homeostatic plasticity is induced by a change in mEPSP amplitude. A decrease in mEPSP amplitude causes the induction of PHP, whereas an increase in mEPSP amplitude causes the induction of presynaptic homeostatic depression (PHD). If a common sensor is employed to detect deviations in average mEPSP amplitude, how is this converted into the specific induction of either PHP or PHD? It has been shown that PHD and PHP can be sequentially induced. But, it remains unknown what would happen if the mechanisms of PHP and PHD were simultaneously induced. Under normal conditions this would never occur because mEPSP amplitudes cannot be simultaneously increased and decreased. But, if signaling specificity were degraded in animals lacking postsynaptic PI3K or Rab11, then the expression of PHP and PHD might coincide and create a mechanistic clash within the presynaptic terminal (Hauswirth, 2018).

Signaling and recycling endosomes are, in many respects, ideally suited to achieve signaling specificity during homeostatic plasticity. Signaling specificity can be achieved by mechanisms including sub-cellular compartmentalization of pathways, physically separating signaling elements with protein scaffolds, or through mechanisms of cross-pathway inhibition. Well-established mechanisms of protein sorting within recycling endosomes could physically compartmentalize signaling underlying PHP versus PHD. Alternatively, recycling endosomes can serve as a focal point for signal digitization, integration, and, perhaps, cross-pathway inhibition. Thus, it is proposed that the loss of postsynaptic PI3K and Rab11 compromises the function of the postsynaptic endosomal platform that this study has identified, thereby degrading homeostatic signaling specificity. As such, this platform could be considered a 'homeostatic controller' that converts homeostatic error signaling into specific, homeostatic, retrograde signaling for either PHP or PHD (Hauswirth, 2018).

Other models are considered, but are not favored. It remains formally possible that the calcium-sensitivity of PHP expression could be explained by a partially functioning PHP signaling system. This seems unlikely given that the same phenotype is observed in four independent genetic manipulations including a null mutation in Pi3K68D, postsynaptic expression of kinase dead Pi3K68D, postsynaptic knockdown of Rab11, and postsynaptic knockdown of Vps34. Furthermore, prior experiments examining hypomorphic and trans-heterozygous genetic interactions among essential PHP genes suggest that PHP is either diminished across the entire calcium spectrum or fully functional. So, there is no evidence that partial disruption of PHP could account for calcium-sensitive expression of PHP. Finally, the experiments argue against the possibility that compensatory changes in Vps34 expression partially rescue the Pi3K68D mutant phenotype (Hauswirth, 2018).

Another common signaling module that emerged from the genetic screen is considered. Both CamKII and CamKK were identified as potential hits. The identification of CamKII is supported by prior work showing the expression of dominant negative CamKII transgenes disrupt the long-term maintenance of PHP in the GluRIIA mutant background (Haghighi, 2003). It has been assumed that postsynaptic calcium is used to detect the PhTX or GluRIIA-dependent perturbation. But, the logic remains unclear. PHP is induced by diminished GluR function and, therefore, diminished postsynaptic calcium influx. This should diminish activation of CamKII and yet, loss of CamKII blocks PHP. An interesting alternative model is that calcium and calmodulin-dependent kinase activity facilitate the function of the postsynaptic endosomal membrane system. Both calcium and calmodulin are necessary for endosomal membrane fusion. In this manner, the action of CamKK and CamKII would be entirely consistent with the identification of Class II/III PI3K and Rab11 as homeostatic plasticity genes (Hauswirth, 2018).

This study has uncovered novel postsynaptic mechanisms that drive homeostatic plasticity. Eventually, continued progress in this direction may make it possible to not only reveal how stable neural function is achieved throughout life, but to uncover new rules that are essential for the processing of information throughout the nervous system. In particular, PHP has a very large dynamic range, whether one considers data from Drosophila or human NMJ or mammalian central synapses. The homeostatic control of presynaptic release can achieve a 7-fold change in synaptic gain, and yet retains the ability to offset even small changes in postsynaptic neurotransmitter receptor function. Thus, it is expected that the regulatory systems that achieve PHP will be complex and have a profound impact on brain function. This study has defined a postsynaptic signaling system responsible for the rapid expression of PHP and a novel, albeit speculative, model is proposed for the postsynaptic control of PHP, taking into account the need for signaling specificity. The identification of this pathway paves the way for future advances in understanding how homeostatic signaling is designed and implemented at a cellular and molecular level (Hauswirth, 2018).

Janardan, V., Sharma, S., Basu, U. and Raghu, P. (2019). A genetic screen in Drosophila to identify novel regulation of cell growth by phosphoinositide signaling. G3 (Bethesda). PubMed ID: 31704710

A genetic screen in Drosophila to identify novel regulation of cell growth by phosphoinositide signaling

Phosphoinositides are lipid signaling molecules that regulate several conserved sub-cellular processes in eukaryotes, including cell growth. Phosphoinositides are generated by the enzymatic activity of highly specific lipid kinases and phosphatases. For example, the lipid PIP3, the Class I PI3 kinase that generates it and the phosphatase PTEN that metabolizes it are all established regulators of growth control in metazoans. To identify additional functions for phosphoinositides in growth control, a genetic screen was performed to identify proteins which when depleted result in altered tissue growth. By using RNA-interference mediated depletion coupled with mosaic analysis in developing eyes, additional candidates were identified and classified in the developing Drosophila melanogaster eye that regulate growth either cell autonomously or via cell-cell interactions. This study reports three genes, Pi3K68D, Vps34 and fwd, that are important for growth regulation and suggest that these are likely to act via cell-cell interactions in the developing eye. These findings define new avenues for the understanding of growth regulation in metazoan tissue development by phosphoinositide metabolizing proteins (Janardan, 2019).

As part of the two-step screen a small set of genes were identified where the RNAi-mediated knockdown clones for these genes were smaller than the wild-type clones in the mosaic CoinFLP screen. However, whole-eye knockdown of the same set of genes failed to show any effect upon the adult eyes, which remained similar in size when compared to control flies. This indicated that such genes might support cell growth and/or survival through cell-cell signaling, including mechanisms that involve cell-cell competition. Pi3K68D, Vps34 and one of the PI4Ks (i.e., four wheel drive (fwd)) fell in this category (Janardan, 2019).

Pi3K68D codes for a Class II PI3K enzyme that has been shown to localize to the plasma membrane and endo-lysosomal structures. It utilizes PI or PI4P as substrates to synthesize PI3P or PI(3,4)P2, respectively (see Phosphoinositide metabolism in eukaryotic cells). Pi3K68D has been previously shown to regulate patterning in Drosophila wing imaginal discs but did not affect eye imaginal discs under the conditions tested. Genetic interactions of PI3K68D with EGF receptor and Notch signaling pathways were seen to be important for this regulation of patterning. No study directly links Class II PI3K to cell growth or survival in Drosophila. In HeLa cells and CHO cells, downregulation of PI3K-C2a, one of the three mammalian Class II PI3K isoforms, results in increased apoptosis. However, contrary to this, downregulation of PI3K-C2a in human muscle cells, human lung epithelial fibroblasts and rat insulinoma cells shows no effect on proliferation. While an initial mosaic screen suggested that loss of PI3K68D may lead to apoptosis as seen in HeLa or CHO cells, this was unlikely as knocking down PI3K68D had no effect in whole eyes. The screen therefore implicates PI3K68D as an important regulator of cell-cell interaction and the underlying mechanism, if investigated, may reveal novel modes of growth regulation (Janardan, 2019).

Vps34 is a Class III PI3K that converts PI to PI3P on endosomes. In mammalian cells, signaling via Vps34 is important for the transduction of amino acid and glucose signals into mTORC1 output which further regulates cell growth. In such a scenario, Vps34 would be expected to autonomously regulate cell growth via mTORC1 signaling. In Drosophila, while the requirement of mTOR activity to mediate amino acid sensing into growth is conserved, Vps34 has been reported to be dispensable for normal mTOR signaling in fat body cells (Janardan, 2019).

Vps34 also plays an important role in the regulation of autophagy. Autophagy is shown to be both pro-survival and pro-death in a context dependent manner. Reduction of autophagy reduces cell death in larval salivary glands. Similarly, knockdown of many genes involved in autophagy, including Vps34, delays the programmed cell death of obsolete Drosophila larval midgut. In contrast to these, the mosaic clones of Vps34 were smaller than controls suggesting that Vps34 has a pro-survival role in the developing eye tissue. It is likely that an interplay between mTORC1-dependent regulation of cell growth and mTOR-independent regulation of autophagy decides the fate of Vps34 knockdown cells (Janardan, 2019).

In addition, the results suggest that Vps34 has a role in cell competition as whole-eye knockdown of Vps34 did not result in a reduction in the size of the eye and despite an under representation of clones in the CoinFLP screen. Epithelial cells with disrupted apicobasal polarity are known to be eliminated by neighboring wild-type cells by the process of cell competition during which JNK activation is seen in 'loser cells'. Loss of Vps34 results in activation of JNK pathway, leading to disruption of epithelial organization. Taken together, these studies hint toward the possibility that Vps34 knockdown leads to JNK activation mediated disruption of apicobasal polarity and loss of cells (Janardan, 2019).

The Drosophila genome harbors one gene each for the three families of PI4 kinases (PI4Ks). The three families of PI4 kinases produce PI4P using PI as a substrate at distinct intracellular membranes. Among the three genes, viz. fwd, Pi4KIIα and PI4KIIIα, phenotypes were observed only upon knockdown of fwd and PI4KIIIα. As mentioned earlier, loss of PI4KIIIα resulted in a complete loss of knockdown clones in the mosaic screen and led to pupal lethality when it was downregulated in the entire eye tissue. As a result, it appears likely that PI4KIIIα is essential for cellular viability. On the other hand, smaller knockdown clones were observed when fwd was downregulated in the mosaic CoinFLP Gal4 screen. However, like PI3K68D and Vps34, downregulation of fwd across the entire developing eye failed to show any significant phenotypes, again suggesting cell-cell interactions between fwd-deficient and neighboring wild-type cells to be the likely reason for reduced size of fwdRNAi clones. fwd knockout flies are viable and female fertile. fwd knockout male flies are sterile due to defects in cytokinesis during male meiosis. Both fly and mammalian fwd (PI4Kβ) bind and recruit Rab11 to the Golgi and are required for the maintenance of Golgi integrity and secretion. These reports suggest that fwd may have pleiotropic cellular roles, causing the phenotypes to vary depending on the tissues in which its levels are manipulated (Janardan, 2019).

Toll receptors remodel epithelia by directing planar-polarized Src and PI3K activity

Toll-like receptors are essential for animal development and survival, with conserved roles in innate immunity, tissue patterning, and cell behavior. The mechanisms by which Toll receptors signal to the nucleus are well characterized, but how Toll receptors generate rapid, localized signals at the cell membrane to produce acute changes in cell polarity and behavior is not known. This study shows that Drosophila Toll receptors direct epithelial convergent extension by inducing planar-polarized patterns of Src and PI3-kinase (PI3K) activity. Toll receptors target Src activity to specific sites at the membrane, and Src recruits PI3K to the Toll-2 complex through tyrosine phosphorylation of the Toll-2 cytoplasmic domain. Reducing Src or PI3K activity disrupts planar-polarized myosin assembly, cell intercalation, and convergent extension, whereas constitutive Src activity promotes ectopic PI3K and myosin cortical localization. These results demonstrate that Toll receptors direct cell polarity and behavior by locally mobilizing Src and PI3K activity (Tamada, 2021).

Cell-surface receptors convert extracellular signals into changes in cell behavior that are essential for the formation, remodeling, and repair of multicellular tissues. Toll-like receptors (TLRs) are a conserved family of receptors with roles in dorsal-ventral patterning and cell-pathogen recognition in the innate immune system. More recently, members of the Toll-receptor family have also been shown to regulate cell behavior during the development of epithelial tissues and the nervous system. Toll receptors organize cell movement, direct nervous system wiring, repair wounds, and eliminate less fit cells through cell competition, defining new functions for this receptor family. In the immune system, Toll-like receptor (TLR) signaling activates the expression of immune-response genes by promoting the nuclear translocation of transcriptional regulators in the NF-κB, AP-1, and IRF families. However, how Toll receptors elicit fast-acting and localized responses in cells to generate spatially regulated changes in cell behavior is not understood (Tamada, 2021).

Tyrosine phosphorylation is a rapid and reversible mechanism that transduces extracellular information into molecular changes within cells. Cell-surface receptors such as growth-factor receptors initiate signaling cascades through their intrinsic tyrosine-kinase activity, which is activated by ligand binding and receptor dimerization. In addition, receptors that lack kinase activity can also participate in tyrosine-kinase signaling by recruiting nonreceptor tyrosine kinases. Src-family nonreceptor tyrosine kinases are essential for signaling by growth-factor receptors, T and B cell receptors, integrins, and cadherins, and have diverse roles in cell-cell adhesion, and cytoskeletal organization. A critical event in receptor-initiated tyrosine kinase signaling is often phosphorylation of the receptor itself, which generates binding sites that recruit downstream effector proteins. Although canonical TLR signaling involves a serine/threonine kinase cascade triggered by the recruitment of TLR-associated adaptor proteins, tyrosine kinases in the Src family have also been shown to facilitate the response to TLR signaling in cultured cells. However, the effects of Src-family kinases on Toll-receptor signaling in vivo, and whether tyrosine-kinase pathways mediate the rapid and localized effects of Toll receptors on cell behavior, are not known (Tamada, 2021).

Convergent extension is a conserved morphogenetic process that shapes developing organs and elongates the body axis of multicellular organisms. In Drosophila, three Toll-family receptors expressed in distinct striped patterns provide critical spatial cues that orient cell rearrangements and elongate the embryo along the head-to-tail axis. Toll receptors guide cell movements by directing the planar-polarized localization of proteins involved in actomyosin contractility and cell adhesion. However, the signaling pathways that generate spatially regulated changes in the organization and activity of the force-generating machinery downstream of Toll receptors are unknown. This study shows that a Src-mediated tyrosine-kinase pathway is essential for planar polarity and Toll-receptor signaling during convergent extension in Drosophila. Toll receptors generate a planar-polarized pattern of Src kinase activity, and Src in turn phosphorylates the Toll-2 C-terminal domain, promoting the association of Toll-2 with PI3-kinase (PI3K). Drosophila Src-family kinases, the regulatory and catalytic subunits of the PI3K complex, and tyrosine phosphorylation of the Toll-2 cytoplasmic domain are all necessary for planar polarity, cell intercalation, and convergent extension. These results identify a localized signaling mechanism by which Toll receptors induce rapid changes in cell polarity and behavior during development (Tamada, 2021).

Toll-family receptors are best known for their roles in signaling to the nucleus to activate the expression of immune-response genes. This study shows that Toll receptors also direct cell polarity and behavior during convergent extension by promoting the localized activities of Src and PI3K at the cell membrane. Toll receptors recruit active Src kinases to planar-polarized membrane domains, and localized Src activity generates spatially regulated actomyosin contractility by promoting the phosphorylation of two clusters of tyrosines in the Toll-2 cytoplasmic domain, creating docking sites for PI3K-reg. Src and PI3K are critical effectors of Toll-receptor signaling, as the loss of Src or PI3K recapitulates the effects of Toll-receptor mutants on planar polarity, cell intercalation, and convergent extension. Moreover, disruption of a single cluster of tyrosines in the Toll-2 C-terminal domain, which is phosphorylated in a Src-dependent fashion, inhibits Toll-2/PI3K complex formation and abolishes Toll-2 function during convergent extension. Taken together, these results identify Src and PI3K-reg as critical effectors of Toll-receptor signaling during convergent extension and reveal a noncanonical mechanism by which Toll receptors induce spatially regulated cell behavior by mobilizing two potent signaling proteins to specific subcellular domains (Tamada, 2021).

These results demonstrate that a critical symmetry-breaking event during convergent extension in Drosophila is the formation of localized Toll-2/PI3K complexes that generate tissue-wide patterns of actomyosin contractility. The upstream signals that activate Toll receptors in specific cellular domains are not known but could involve a localized extracellular ligand that increases Toll-2 binding to Src or enhances Src activity. Alternatively, mechanisms that promote Toll-receptor clustering could amplify the activity of associated Src kinases by enhancing their proximity to multiple substrates. Toll-2 and Toll-6 both interact with PI3K-reg and are tyrosine phosphorylated by Src kinases in vitro, suggesting that a Src/PI3K-signaling mechanism could link both Toll-2 and Toll-6 to PI3K. By contrast, Toll-8 is not phosphorylated by Src kinases, although it does associate weakly with PI3K-reg. Toll-8 could signal through a distinct mechanism, or it could communicate with Src and PI3K indirectly through heterodimerization with receptors such as Toll-6, which is expressed in many of the same cells. The defects in embryos lacking Src or PI3K appear to be more severe than the defects in Toll-2,6,8 mutants, suggesting that Src and PI3K may represent a common mechanism that mediates signaling downstream of multiple receptors during convergent extension (Tamada, 2021).

Tyrosine phosphorylation is a widely used signaling mechanism in multicellular organisms, with a particular enrichment of tyrosine phosphorylation at sites of contact between cells and with the extracellular matrix. Identifying the substrates required for the diverse roles of tyrosine-kinase signaling in vivo remains a formidable challenge. This study demonstrates that Src-dependent phosphorylation of a single cluster of tyrosines in the Toll-2 C-terminal domain is sufficient to eliminate Toll-2 function during convergent extension. In addition to Toll-2, Src kinases may also promote the phosphorylation of other proteins involved in Toll-receptor signaling. For example, Src-family kinases can phosphorylate Rho-kinase and recruit Rho-kinase and myosin II to focal adhesions and adherens junctions in fibroblasts and epithelial cells, and can phosphorylate and activate the Abl tyrosine kinase, which regulates planar polarized junctional dynamics during convergent extension. In addition, Src kinases can stabilize cell adhesion, enhance E-cadherin dynamics, influence cytoskeletal organization, and modulate cell polarity. The recruitment of active Src kinases to Toll-receptor complexes in Drosophila demonstrates an essential role for tyrosine-kinase pathways in signaling by Toll receptors and suggests a mechanism by which Toll receptors could trigger a wide range of cellular events that contribute to epithelial remodeling (Tamada, 2021).

PI3K is a conserved lipid kinase with widespread roles in cell growth, proliferation, survival, and polarity. This study demonstrates that PI3K directs planar polarity in the Drosophila embryo by promoting the localized assembly of contractile myosin networks in epithelial cells. These results are consistent with the altered cell topology and deregulated myosin activity observed in epithelial cells lacking the PI3K-antagonizing enzyme PTEN. The mechanisms by which PI3K influences tissue structure are not well understood. PI3K promotes the activity of the Rac GTPase in a number of cell types, but PI3K can also influence epithelial organization by activating PDK1, and PI3K can regulate the localization and activity of RhoA and Rho-kinase in fibroblasts and endothelial cells. PI3K could influence planar polarity by regulating Rho GTPase signaling, which has been linked to Toll-2 and is essential for convergent extension. The effectors that link Toll-2/Src/PI3K signaling to changes in actomyosin organization remain to be identified. These could include classical PI3K effectors that signal to the actin cytoskeleton or other proteins such as RhoGEFs that interact with the phospholipid product of PI3K, phosphatidylinositol (3,4,5)-trisphosphate (PIP3) (Tamada, 2021).

TLRs initiate signaling by recruiting adaptor proteins that assemble large multiprotein signaling complexes. The Toll-receptor signaling complexes involved in morphogenesis have not been defined. This study demonstrated that the C-terminal-most tyrosine cluster of Toll-2 is targeted by Src kinases and recruits Src and PI3K to the Toll-2 complex. This tyrosine cluster bears homology to immunoreceptor tyrosine-based activation motifs (ITAMs and hemITAMs). These motifs are present in several classes of immunoreceptors and their associated adaptor proteins and, when phosphorylated, create binding sites for downstream effector proteins. Toll-2 therefore displays features of multiple receptors in the immune system: an extracellular domain and intracellular TIR domain characteristic of TLRs and a C-terminal extension that couples to effectors through Src-family kinases and PI3K, reminiscent of other immunoreceptors such as C-type lectin-like receptors and the T cell receptor/CD3 complex. Although mammalian TLRs do not display this bipartite structure, tyrosine-rich adaptor proteins that associate with mammalian TLRs, such as BCAP and SCIMP, could create docking sites for the assembly of downstream signaling components. Toll-family receptors have conserved roles in regulating spatially regulated cell behaviors in flies and mammals, including axon growth, synapse formation, epithelial remodeling, and wound healing. An intriguing possibility is that Src and PI3K could represent a core module of a spatially regulated branch of Toll-receptor signaling that promotes the rapid and directional mobilization of cells during tissue development and immune defense (Tamada, 2021).

This study demonstrates a functional connection between Toll receptors and localized Src and PI3K activity during convergent extension in Drosophila. However, several limitations remain. First, phosphospecific antibodies to the C2 tyrosine cluster of Toll-2 could not be generated, and the other phosphospecific Toll-2 antibodies that were generated in this study worked only for western blots, but not for immunostaining. Therefore, it was not possible to analyze the spatiotemporal pattern of Toll-2 tyrosine phosphorylation in the embryo. Second, embryos were analyzed that had only a partial loss of Src42, PI3K-reg, and PI3K-cat function, due to essential roles of these proteins at earlier stages of development. Therefore, these proteins may have additional functions in this tissue that were not detected in this study. Third, focus was placed on protein localization and cell behavior in the adherens junction domain, and the results do not exclude the possibility that defects in basolateral regions of these cells or in other parts of the embryo could contribute to the phenotypes observed (Tamada, 2021).

Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis

Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. This study shows that autophagy controls DNA fragmentation during late oogenesis in Drosophila. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. These results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death (Nezis, 2010).

Dying nurse cells exhibit several markers of apoptosis during late oogenesis in Drosophila such as caspase activation, chromatin condensation, and DNA fragmentation. To address the role of autophagy in nurse cell death, transgenic flies were generated carrying a UASp-GFP-mCherry-DrAtg8a transgene. The double-tagged Atg8a protein emits yellow (green merged with red) fluorescence in nonacidic structures such as autophagosomes, and is red only in the autolysosomes due to quenching of GFP in these acidic structures. Upon expression of GFP-mCherry-DrAtg8a in the germline, several GFP-mCherry-DrAtg8a yellow puncta were detected in the cytoplasm of nurse cells during early stage 12. After the completion of transport of the majority of the nurse cell cytoplasm to the growing oocyte during late stage 12, GFP-mCherry-DrAtg8a yellow puncta remained in nurse cell cytoplasm in close proximity to the nurse cell nuclei. Ultrastructural analysis of the nurse cells at the same developmental stage also revealed the presence of autophagosomes in the remaining nurse cell cytoplasm. Interestingly, during late stage 13 when the majority of nurse cells have degenerated, a large number of red structures were observed, indicating that the majority of the autophagosomes became autolysosomes. This was confirmed by ultrastructural analysis through detection of large autolysosomes associated with the condensed and fragmented nurse cell nucleus. These autolysosomes often contained condensed material resembling the material of the fragmented nurse cell nucleus, suggesting that the nurse cell nuclear remnants are removed by autophagy. Indeed, nurse cells of late stage 13 egg chambers expressing UASp-mCherry-DrAtg8a exhibited mCherry-DrAtg8a puncta that are located either adjacent to or attached to the fragmented nucleus, indicative of nuclear autophagy. To further examine the presence of autophagy during late oogenesis in Drosophila, protein trap lines were used that express GFP-tagged Atg5 and Atg8a. Atg5-GFP and Atg8a-GFP were detected as punctae around the nurse cell nuclei during late oogenesis, revealing the presence of autophagic compartments. These findings indicate that autophagy occurs during nurse cell death and degradation in late oogenesis in Drosophila (Nezis, 2010).

To explore the potential role of autophagy in nurse cell death during late oogenesis, germline mutant cells were generated for the core Drosophila autophagy genes atg1 and atg13 and cell death was examined using the TUNEL assay to detect fragmented DNA. Interestingly, in either atg1 or atg13 germline mutants, a significant increase was observed in the number of stage 14 egg chambers that had persisting TUNEL-negative nurse cell nuclei. This phenotype differs from wild-type stage 14 egg chambers, in which nurse cell nuclei can rarely be detected, and those few that remain are exclusively TUNEL positive. TUNEL-positive nurse cell nuclei can be detected in the wild-type egg chambers in earlier developmental stages but not in autophagy germline mutants. To further examine the role of autophagy in nurse cell degeneration, germline mutants were generated for vps34, a member of the class III PI3-kinase complex that is responsible for the production of phosphatidylinositol 3-phosphate, a phosphoinositide required for autophagy. Like the other autophagy mutants, the vps34 germline mutant egg chambers displayed significant increase in the number of egg chambers that had persisting TUNEL-negative nurse cell nuclei during late oogenesis. All autophagy germline mutants exhibited accumulation of Ref(2)P, a marker for autophagic flux, in the nurse cell cytoplasm compared with the wild type, further confirming that autophagy was inhibited. Interestingly, in all the autophagy germline mutants, the persisting nurse cell nuclei exhibited condensed nuclear staining. To examine whether proteolytic processing of caspase-3 was affected by inhibition of autophagy, immunolabeling for cleaved caspase-3 was performed in the atg1, atg13, and vps34 germline mutant egg chambers. Cleaved caspase-3 levels were markedly attenuated in autophagy germline mutants compared with the wild type, with 92% cleaved caspase-3 labeling in w¹¹¹⁸ late stage 12-14 egg chambers, 38% in atg13^−/− GLCs, and 33% in vps34^−/− GLCs late stage 12-14 egg chambers. Together, these data demonstrate that autophagy functions upstream of caspase processing and DNA fragmentation during late oogenesis in Drosophila (Nezis, 2010).

How can autophagy promote caspase activity, DNA fragmentation, and cell death in the same cell? It was hypothesized that proteins crucial for cell survival could be degraded by autophagy, thus promoting cell death. To test this hypothesis, the localization of Drosophila IAPs in the nurse cells was investigated during late oogenesis along with their relationship to the autophagic marker GFP-Atg8a. Three of four known Drosophila IAPs, DIAP1, DIAP2, and dBruce were investigated. DIAP1 and DIAP2 exhibit a rather diffuse cytoplasmic staining that did not colocalize with GFP-Atg8a. In contrast, dBruce exhibited an interesting localization pattern. dBruce could not be detected in stage 10B egg chambers. Interestingly, during early stage 12, colocalization of dBruce and Atg8a-GFP was observed in structures 0.5-1.5 µm in diameter resembling autophagosomes. A similar pattern of colocalization was observed during late stage 12. In contrast, in later stages when nurse cell cytoplasm was completely transferred to the oocyte, dBruce exhibited a diffuse localization pattern mainly in the follicle cells surrounding the nurse cells remnants. These data suggest that dBruce might be degraded by autophagy. To test this hypothesis, the localization of dBruce was investigated in atg1, atg13, and vps34 germline mutants. Significantly, dBruce accumulated in the remaining cytoplasm of the nurse cells of all of these autophagy mutants and formed large aggregates 5-10 µm in diameter. Western blot analyses showed that autophagy germline mutant egg chambers contain higher levels of dBruce protein than wild-type egg chambers. These observations support the hypothesis and indicate that dBruce is degraded by autophagy in the nurse cells during late oogenesis (Nezis, 2010).

It was next asked how dBruce might be targeted for autophagy. p62 is a known adaptor protein that targets substrates for autophagic degradation (Pankiv, 2007). It was asked whether the Drosophila orthologue of p62, Ref(2)P (Nezis, 2008) may target dBruce for autophagy. Immunofluorescence analysis demonstrated that Ref(2)P staining in the nurse cells of late stage egg chambers has no correlation with the autophagic marker Atg8a-GFP. Additionally, Ref(2)P mutant egg chambers exhibited a normal pattern of DNA fragmentation, cell death, and degradation in the nurse cells during late oogenesis, which suggests that targeting dBruce for autophagy does not depend on Ref(2)P function (Nezis, 2010).

dBruce belongs to the IAP protein family. It contains both BIR (baculoviral IAP repeat, which is responsible for caspase inhibition) and UBC (responsible for ubiquitin conjugation) domains in the N and C termini, respectively. The function was tested of three different dBruce mutant alleles that result in truncated proteins with deletions either in the BIR or UBC domains. Two of them (dBruce^E16 and dBruce^e00984) have a deletion in the UBC domain, and one of them (dBruce^E81) has a deletion in the BIR domain. All dBruce mutant alleles displayed a significant increase in the number of degenerating egg chambers during mid-oogenesis compared with the wild type. To further investigate the role of autophagic degradation of dBruce in nurse cell death, double mutants were constructed for either atg1 and dBruce^E81 or vps34 and dBruce^E81. Both double mutant egg chambers contained persistent nurse cell nuclei that were TUNEL positive. These data indicate that autophagic degradation of dBruce controls DNA fragmentation in the nurse cells during oogenesis in Drosophila (Nezis, 2010).

The role of autophagy in cell death has been controversial. Previous studies have shown that autophagy promotes cell death in Drosophila larval salivary glands, midgut, and embryonic serosal membrane. However, the precise mechanism by which autophagy executes the death of these cells is not clear. This study has shown that autophagic degradation of the IAP dBruce controls DNA fragmentation in nurse cells during Drosophila late oogenesis. The data also demonstrate that autophagy acts genetically upstream of caspase activation and DNA fragmentation in this developmental context and indicate that autophagy directly contributes to the activation of cell death. This agrees with recent evidence from cultured mammalian cells in which autophagy appears to act upstream of caspase-3 activation under specific experimental settings (Laane, 2009; Zalckvar, 2010; Nezis, 2010 and references therein).

dBruce has been previously shown to suppress cell death in the Drosophila eye and also has a crucial function in nuclear degeneration during sperm differentiation in Drosophila. Interestingly, dBruce was recently shown to regulate autophagy and cell death during early and mid-oogenesis in Drosophila. In this earlier study, dBruce and caspase activity were shown to influence autophagy. In contrast, this study provides the first evidence for a mechanism by which autophagy regulates dBruce and cell death. This study provides genetic evidence that dBruce is degraded by autophagy in the degenerating nurse cells during late oogenesis and that it regulates DNA fragmentation. The fact that chromatin condensation is not affected in autophagy mutants indicates that this process is regulated independently from DNA fragmentation (Nezis, 2010),

Degradation of proteins that are crucial for cell survival is one of the mechanisms by which a cell can trigger its own death. For instance, selective depletion of catalase by autophagy has been shown to promote cell death in mammalian cells in vitro. Furthermore, it was recently shown that chaperone-mediated autophagy modulates the neuronal survival machinery by regulating the neuronal survival factor MEF2D, and dysregulation of this pathway is associated with Parkinson's disease. In a recent study, it was also demonstrated that autophagy promotes synaptogenesis in Drosophila neuromuscular junction by degrading Highwire, an E3 ubiquitin ligase which limits neuromuscular junction growth. The current in vivo data further support the idea that autophagic degradation of survival factors can promote cell death and indicate that IAPs can be degraded by autophagy, thereby causing cell death. Autophagy not only functions during late oogenesis as the cause of cell death, but can also function to efficiently degrade the nurse cell nuclei remnants, as previously shown in salivary glands. It was recently reported that dying nurse cells during late oogenesis exhibit characteristics of programmed necrosis and that the lysosomal genes dor, spinster, and cathepsin D are required for this process (Bass, 2009), showing that autophagy and necrosis participate in nurse cell death and degradation during late oogenesis. In conclusion, these findings indicate that autophagy plays an important role in nurse cell death during late oogenesis in Drosophila, first by acting upstream of DNA fragmentation, thereby causing cell death, and then by scavenging nurse cell remnants (Nezis, 2010).

Shaping development of autophagy inhibitors with the structure of the lipid kinase Vps34

Phosphoinositide 3-kinases (PI3Ks) have diverse and profound roles in health and disease. The primordial PI3K, Vps34, is present in all eukaryotes and has essential roles in autophagy, endosomal sorting, phagocytosis and signalling upstream of mTOR in nutrient sensing. The crystal structure of Drosophila Vps34 reveals a constricted adenine-binding pocket, shedding light on why specific inhibitors of this class of PI3K have proven elusive. Both the phosphoinositide-binding loop and the C-terminal helix of Vps34 have dual roles: they are essential for catalysis on membranes and they suppress futile ATPase cycles. Vps34 appears to alternate between a closed form in the cytosol and an open form on the membrane. Structures of Vps34 complexes with a series of inhibitors show why the autophagy inhibitor 3-methyladenine preferentially inhibits Vps34 and lay a foundation for generating new potent and specific Vps34 inhibitors (Miller, 2010: Full text of article).

Drosophila Mtm and class II PI3K coregulate a PI(3)P pool with cortical and endolysosomal functions

Reversible phosphoinositide phosphorylation provides a dynamic membrane code that balances opposing cell functions. However, in vivo regulatory relationships between specific kinases, phosphatases, and phosphoinositide subpools are not clear. This study identified Myotubularin (Mtm), a Drosophila melanogaster MTM1/MTMR2 phosphoinositide phosphatase, as necessary and sufficient for immune cell protrusion formation and recruitment to wounds. Mtm-mediated turnover of endosomal phosphatidylinositol 3-phosphate (PI(3)P) pools generated by both class II and III phosphatidylinositol 3-kinases (Pi3K68D and Vps34, respectively) is needed to down-regulate membrane influx, promote efflux, and maintain endolysosomal homeostasis. Endocytosis, but not endolysosomal size, contributes to cortical remodeling by mtm function. It is proposed that Mtm-dependent regulation of an endosomal PI(3)P pool has separable consequences for endolysosomal homeostasis and cortical remodeling. Pi3K68D depletion (but not Vps34) rescues protrusion and distribution defects in mtm-deficient immune cells and restores functions in other tissues essential for viability. The broad interactions between mtm and class II Pi3K68D suggest a novel strategy for rebalancing PI(3)P-mediated cell functions in MTM-related human disease (Velichkova, 2010).

If Mtm dephosphorylates a distinct PI(3)P pool, mtm function could antagonize the PI(3)P production by a specific PI3-kinase. As in mammals, Drosophila encodes three classes of PI3-kinases, with one member per class capable of PI(3)P synthesis in vitro (class I, Pi3K92E; class II, Pi3K68D; and class III, Vps34). Vps34 was a likely candidate for production of an Mtm-functional substrate given known roles for PI(3)P synthesis on early endosomes and for autophagy (Lindmo, 2006). In testing all three PI3-kinases, it was found that knockdown or expression of kinase-dead form of Vps34 and, surprisingly to an even greater extent, knockdown of Pi3K68D each individually resulted in dispersion of localized 2xFYVE biosensors, demonstrating that both class II and III PI3-kinases are required for significant PI(3)P pools in immune cells. A recovery of the 2xFYVE-localized distribution was obtained from codisruption of mtm with Pi3K68D or Vps34, indicating that interference with either PI3-kinase was sufficient to restore the mtm-dependent PI(3)P imbalance. Given that Pi3K68D contribution to PI(3)P synthesis has not been characterized in vivo, its role was further investigated. The effects on PI(3)P total cellular levels were confirmed from myo-inositol radiolabeled Kc₁₆₇ cell lysates. Altered levels of PI(3)P were observed upon knockdown of mtm phosphatase (1.7-fold increase) and Pi3K68D kinase (2.8-fold decrease), respectively, that returned nearer to normal levels upon their codepletion (1.6-fold decrease), mirroring the genetic interaction seen with 2xFYVE distribution. Overexpression of Pi3K68D cDNA phenocopied mtm depletion effects of expanded 2xFYVE distribution, which is consistent with Pi3K68D synthesis of PI(3)P (Velichkova, 2010).

If Mtm roles are mediated through down-regulation of a distinct PI(3)P pool, then mtm could antagonize the function of a specific PI3-kinase. As observed for PI(3)P, codepletion of Pi3K68D with mtm suppressed the giant endolysosome size. Conversely, it was found that overexpression of Pi3K68D cDNA in WT hemocytes resulted in greatly enlarged LysoTracker-positive organelles. This condition phenocopyied mtm depletion and is consistent with Pi3K68D coregulation of a PI(3)P pool important for endolysosomal size. Although both Vps34 and Pi3K68D disruption reduced 2xFYVE-detected PI(3)P distribution to a similar degree, knockdown of Vps34 alone or in combination with mtm exhibited minor effects on LysoTracker-positive organelles in Kc₁₆₇ cells. Disruption of Vps34 with either a null mutant allele or targeted expression of a kinase-dead form in hemocytes, however, resulted in diffuse LysoTracker staining throughout the cells, suggesting disruption of lysosomal H⁺-ATPase trafficking or of the integrity or size of acidified organelles. Codepletion or double mutants of Vps34 and mtm suppressed both individual endolysosomal defects in hemocytes. Unlike Pi3K68D, overexpression of Vps34 WT cDNA in hemocytes had little to no effect on LysoTracker-positive organelles. These results suggest that mtm is antagonistic to both Pi3K68D and Vps34 functions but that each kinase exhibits distinct roles for normal acidified endolysosomes and differential requirements in Kc₁₆₇ cells and hemocytes (Velichkova, 2010).

This study identified a class II Pi3K68D-dependent PI(3)P pool as a functional and likely direct Mtm substrate. Pi3K68D and mtm play major roles in the coregulation of a hemocyte PI(3)P pool, and both were necessary and sufficient for PI(3)P-mediated endolysosomal homeostasis. Alternatively, mtm and Pi3K68D could interact through interconverted PIP pools, e.g., if class II PI3K synthesis of PI(3,4)P₂ led to inositol polyphosphate 4-phosphatase generation of endosomal PI(3)P (Velichkova, 2010).

Importantly, Pi3K68D loss of function suppressed multiple mtm-dependent hemocyte functions and essential roles in multiple tissues. The results suggest that a conserved pathway linking MTM1/MTMR2 and class II PI3-kinases could also be important for similar roles in mammals. Expression of human MTMR2 in flies rescued the lethality associated with mtm depletion in different tissues, highlighting potential significance from use of the fly to better understand MTMR2-related human disease. Recent studies in T cells identified roles for PI3KC2α and MTMR6 in PI(3)P-mediated regulation of a calcium-activated K⁺ channel, indicating that class II PI3-kinases may play broad and dedicated roles in conjunction with different MTM family members (Velichkova, 2010).

A subset of mtm functions also shared interactions with class III PI3-kinase, Vps34. The genetic interactions observed between mtm and Vps34 in PI(3)P and endolysosomal homeostasis, but not cell remodeling or essential functions in different tissues, suggest several possibilities. Vps34 function may regulate a Pi3K68D function or be partially redundant with Pi3K68D for certain mtm functions; Vps34 may indirectly interact with mtm through converging PI(3)P membrane pools, and/or there may be additional essential consequences of Vps34 functions, e.g., that antagonize different MTM family member functions. Similar partial and redundant interactions have been observed in Caenorhabditis elegans, where reduction of mtm-1 rescued endocytosis defects but not lethality of vps-34 mutants (Xue, 2003), and increased apoptotic cell corpse engulfment upon mtm-1 depletion was found to be dependent on both vps-34 and the class II PIKI-1 functions (Zou, 2009; Velichkova, 2010 and references therein).

mtm was not only required for but could also promote cortical remodeling, specifically modulating cell protrusion formation. MTMs have not previously been ascribed specific roles in cellular remodeling, although MTM1/MTMR2 and PI3KC2 isoforms have been associated with the cortex, and MTM1 overexpression led to cell protrusions. Cortical F-actin organization and dynamics are under control of competing Rho GTPase activities, namely roles for Rac, Rho, and Cdc42 in lamellipodia versus protrusion formation. A mutant form of MTM1 was detected at the plasma membrane upon constitutive Rac1 GTPase activation, mtm-1 was identified as a negative regulator of Rac-mediated engulfment (Zou, 2009), and Rho1 pathway hyperactivation resulted from combined essential function of ymr1 (MTM), sjl2, and sjl3 lipid phosphatases in yeast PI(3)P regulation (Parrish, 2005). However, PI3KC2β-expressing cell lysates exhibit increased levels of activated Cdc42, and PI3KC2α depletion interfers with Rho-mediated smooth muscle contraction. Interestingly, endocytic trafficking of Rac was shown important for its spatially regulated activity. Thus, one consequence of opposing Pi3K68D/mtm functions in hemocytes may be in the cortical balance of specific Rho GTPases, either through PI(3)P-mediated membrane trafficking or recruitment of PI(3)P-binding regulatory proteins to discrete membrane domains (Velichkova, 2010 and references therein).

These experiments show that Mtm has a role in policing traffic at the late endosome, which is consistent with a normal function to down-regulate membrane influx and promote efflux. mtm is important to maintain the balance, but not ability, for membrane influx from endocytic and autophagic routes. It was found through genetic interactions, marker analysis, and time-lapse microscopy that mtm function antagonizes PI(3)P-mediated membrane flux consistent with known roles in transport, tethering, and fusion of endosomes with lysosomes and of autophagosomes with late endosomes. Importantly, mtm-dependent functions for endolysosomal size and cortical remodeling are separable, as indicated by mtm interactions with Atg1 or Vps34 that rescues endolysosome size but not hemocyte protrusions. Live cell imaging also revealed lack of dynamic tubulation, indicative of exiting membrane, in mtm-depleted hemocytes, suggesting that mtm function promotes undetermined routes of membrane efflux from PI(3)P-containing compartments. In addition, several results point to a role for mtm in autophagy: the increased number of double-membrane-bound structures and autophagolysosomes in mtm-depleted cells, reversion of enlarged endolysosomal size with mtm and Atg1 codepletion, and Mtm localization to small rings associated with LysoTracker-positive organelles and within Rab7 compartments, suggestive of autophagosomes. Given the PI(3)P dependence and intersection with endolysosomes, there are likely roles for MTM phosphatase regulation in autophagy (Velichkova, 2010 and references therein).

Collectively, a model is favored that a PI(3)P pool directly coregulated by Pi3K68D-mediated synthesis and Mtm-mediated turnover is involved in membrane delivery and exit, respectively, at an endosomal compartment that maintains homeostasis of both cortical dynamics and endolysosome size. Pi3K68D localization and motility suggest interaction at the level of dynamic PI(3)P pools synthesized at the cortex or on internal membranes. The lack of cell protrusions upon mtm disruption could result from elevated Pi3K68D-dependent PI(3)P that inhibits membrane efflux to undefined recycling endosomes and, thus, blocks redelivery of a cortical regulator that promotes cell protrusions. Pi3K68D overexpression did not phenocopy the lack of protrusions, which may indicate that Pi3K68D requires a limiting cofactor or scaffold protein or that levels of Mtm are sufficient to override ectopic activity. Conversely, ectopic cell protrusions that form upon Mtm overexpression could result from inappropriate depletion of a Pi3K68D-synthesized PI(3)P pool that leads to excessive efflux, and, thus, persistent recycling of the same cortical regulator. Consistent with this, an endosomal-tethered form of MTM1 was able to induce membrane tubulation. In turn, mtm function down-regulates PI(3)P-mediated endosome transport, tethering, and fusion, restricting endolysosome size (Velichkova, 2010).

The genetic analysis uncovered critical requirements for mtm- and phosphoinositide-dependent muscle and immune cell functions in Drosophila. Defects in remodeling cell shape upon either knockdown or overexpression of mtm both corresponded with defects in hemocyte dispersion and recruitment to wound sites. These results indicate the significance of mtm-dependent cellular regulation to immune cell behaviors in the animal, analogous to those performed by mammalian macrophages in response to wounding and infection. The identification of Pi3K68D-generated PI(3)P pools as a likely in vivo substrate of Mtm, and the specific cellular roles modulated by the balance of this pool in animals, has significance in better understanding roles for conserved MTM1/MTMR2 and PI3KC2 in mammals. The results highlight the potential that class II PI3K-activating mutations could underlie unassigned MTM-related human diseases. Furthermore, class II PI3K could serve as a therapeutic target to oppose deleterious effects of MTM mutations associated with human disease (Velichkova, 2010).

The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates

Autophagy is involved in cellular clearance of aggregate-prone proteins, thereby having a cytoprotective function. Studies in yeast have shown that the PI 3-kinase Vps34 and its regulatory protein kinase Vps15 are important for autophagy, but the possible involvement of these proteins in autophagy in a multicellular animal has not been addressed genetically. This study created a Drosophila deletion mutant of vps15 and investigated its role in autophagy and aggregate clearance. Homozygous δvps15 Drosophila died at the early L3 larval stage. Using GFP-Atg8a as an autophagic marker, fluorescence microscopy was employed to demonstrate that fat bodies of wild type Drosophila larvae accumulated autophagic structures upon starvation whereas δvps15 fat bodies showed no such response. Likewise, electron microscopy revealed starvation-induced autophagy in gut cells from wild type but not δvps15 larvae. Fluorescence microscopy showed that δvps15 mutant tissues accumulated profiles that were positive for ubiquitin and Ref(2)P, the Drosophila homolog of the sequestosome marker SQSTM1/p62. Biochemical fractionation and Western blotting showed that these structures were partially detergent insoluble, and immuno-electron microscopy further demonstrated the presence of Ref(2)P positive membrane free protein aggregates. These results provide the first genetic evidence for a function of Vps15 in autophagy in multicellular organisms and suggest that the Vps15- containing PI 3-kinase complex may play an important role in clearance of protein aggregates (Lindmo, 2008).

Studies of the involvement of specific PI3Ks in autophagy in higher organisms such as Drosophila and mammals by pharmacological PI3K inhibitors have been complicated by the fact that these animals express multiple classes of PI3Ks that may have opposing roles. It has been found that class I PI3K represses autophagy during the early larval stages in Drosophila, and that its downregulation in response to ecdysone signaling triggers developmental autophagy. The present study sought to clarify the possible involvement of class III PI3K in autophagy and aggregate clearance by generating a Drosophila mutant in which the gene for the regulatory Vps15 subunit was deleted. The δvps15 mutant larvae turned out to be defective for starvation induced autophagy. Importantly, vps15 mutant animals accumulated detergent-soluble and -insoluble structures that are likely to represent endosomes and sequestosomes, respectively. This provides evidence for the involvement of Vps15 in autophagy and aggregate clearance in metazoans (Lindmo, 2008).

The only PI3K in S. cerevisae, Vps34, can participate in two distinct protein complexes; one consisting of Vps34, Vps15, Vps30 and Vps38 that functions in vacuolar protein sorting and one consisting of Vps34, Vps15, Vps30 and Atg14 that functions in autophagy (Kihara, 2001). So far, no metazoan homolog of Atg14 has been reported, whereas metazoan homologs of Vps34, Vps15 and Vps30 are known. Of these, the Vps30 homolog, Beclin-1, an interactor of the antiapoptotic proteins Bcl-2 and Bcl-XL, has been most studied for its role in autophagy in metazoans. Overexpression of Beclin-1 in MCF7 breast carcinoma cells promotes autophagy and inhibits cell proliferation, whereas its depletion promotes apoptosis. The possible role of Beclin-1 in aggregate clearance has not been investigated, nor have metazoan Vps34 and Vps15 been studied in this context. It was therefore considered important to study whether the metazoan Vps34-Vps15 subcomplex is required for autophagy and aggregate clearance. Because of the availability of appropriate Drosophila FRT strains, a specific deletion of the vps15 gene was generated in Drosophila. The inhibition of starvation-induced autophagy in gut and fat body tissues of δvps15 larvae demonstrates the importance of the Vps15 for autophagy in metazoans. Most importantly, the accumulation of protein aggregates in the δvps15 mutants shows that this complex is critically required for normal clearance of such aggregates (Lindmo, 2008).

The polyubiquitin binding p62 protein accumulates strongly on ubiquitin-positive protein aggregates and serves as a reporter for such structures. Protein aggregates are not formed in p62/Ref(2)P mutants and the fact that p62 binds directly to the mammalian Atg8 homolog LC3 and recruits it to ubiquitin-positive aggregates suggests that p62 may serve to mark the protein aggregates for autophagic degradation. The present report used antibodies against conjugated ubiquitin and the Drosophila homolog of p62, Ref(2)P, as a marker for protein aggregates. Although Ref(2)P was originally identified as a factor involved in male fertility and sigma virus replication, it contains all the structural hallmarks of a p62 ortholog, including the PB1, ZZ and UBA domains. Interestingly, δvps15 Drosophila larvae accumulated numerous Ref(2)P-positive structures, indicative of impaired metabolism of protein aggregates. Consistent with this, the δvps15 mutants also accumulated ubiquitin-positive structures. Because depletion of certain proteins involved in endocytic trafficking causes the accumulation of ubiquitinated membrane proteins in early endosomes, some of the ubiquitin- and Ref(2)P positive profiles might correspond to endosomes. This is supported by the finding that a fraction of the ubiquitin- and Ref(2)P-positive structures could be solubilized in Tx. Because confocal and electron microscopy indicated that Ref(2)P is preferentially found in membrane-free structures in δvps15 mutants, an alternative explanation for the partial Tx solubility of ubiquitin- and Ref(2)P-positive structures may be that smaller accumulations of aggregating proteins are Tx soluble. In any case, a substantial fraction of the ubiquitin- and Ref(2)P positive structures that accumulated in δvps15 mutants were Tx insoluble, strongly suggesting that protein aggregates accumulate in the absence of Vps15. The ultrastructural appearance of these aggregates has striking resemblance to Ref(2)P positive structures found in neuronal tissue of atg8 mutant flies. In both cases, accumulation of vesicular structures surrounding a densely labeled matrix was observed. This might indicate that either the recruitment of autophagic membranes onto or their functional elongation around protein aggregates is dependent on both Atg8 function and PI3K class III activity (Lindmo, 2008).

In conclusion, this study has shown that the PI3K class III co-activator, Vps15, is required for autophagy in Drosophila. δvps15 mutant tissues accumulate Tx-insoluble ubiquitin and Ref(2)P positive structures, indicating a role of Vps15 in autophagic clearance of aggregate-prone proteins. Given that enhanced autophagy can inhibit aggregate-induced neurodegeneration in Huntington models, neuronal-specific stimulation of the Vps34-Vps15 complex might provide a prospective strategy for developing drugs against neurodegenerative diseases (Lindmo, 2008).

PI3K signaling and Stat92E converge to modulate glial responsiveness to axonal injury

Glial cells are exquisitely sensitive to neuronal injury but mechanisms by which glia establish competence to respond to injury, continuously gauge neuronal health, and rapidly activate reactive responses remain poorly defined. This study shows glial PI3K signaling in the uninjured brain regulates baseline levels of Draper, a receptor essential for Drosophila glia to sense and respond to axonal injury. After injury, Draper levels are up-regulated through a Stat92E-modulated, injury-responsive enhancer element within the draper gene. Surprisingly, canonical JAK/STAT signaling does not regulate draper expression. Rather, injury-induced draper activation is downstream of the Draper/Src42a/Shark/Rac1 engulfment signaling pathway. Thus, PI3K signaling and Stat92E are critical in vivo regulators of glial responsiveness to axonal injury. Evidence is provided for a positive auto-regulatory mechanism whereby signaling through the injury-responsive Draper receptor leads to Stat92E-dependent, transcriptional activation of the draper gene. It is proposed that Drosophila glia use this auto-regulatory loop as a mechanism to adjust their reactive state following injury (Doherty, 2014: PubMed).

ird1 is a Vps15 homologue important for antibacterial immune responses in Drosophila

The immune response-deficient 1 (ird1, also known as VPS15, the regulatory subunit of Pi3k59F) gene was identified in a forward genetic screen as a novel regulator for the activation of Imd NFkappaB immune signalling pathway in Drosophila. ird1 animals are also more susceptible to Escherichia coli and Micrococcus luteus bacterial infection. ird1 encodes the Drosophila homologue of the Vps15/p150 serine/threonine kinase that regulates a class III phosphoinositide 3-kinase and is necessary for phagosome maturation and starvation-induced autophagy in yeast and mammalian cells. To gain insight into the role of ird1 in the immune response, how amino acid starvation affects the immune signalling pathways in Drosophila was examined. Starvation, in the absence of infection, leads to expression of antimicrobial peptide (AMP) genes and this response is dependent on ird1 and the Imd immune signalling pathway. Starvation, in addition to bacterial infection, suppresses the AMP response in wild-type animals and reduces the ability to survive M. luteus infection. These results suggest that starvation and innate immune signalling may be intimately linked processes (Wu, 2007).

The unexpected discovery that ird1, a Vps15 kinase, is important for the innate immune response is a good example of how forward genetic screens can uncover new functions for known genes. This kinase has been primarily studied for its roles in endocytosis and autophagy (Petiot, 2000; Fratti, 2001; Futter, 2001; Vieira, 2001) because the yeast mutant phenotype indicated its importance in cellular trafficking events (Herman, 1991; Stack, 1995). An examination of the mutant phenotype in a multicellular organism indicates that this kinase also plays a key role in regulating specific NFkappaB signalling pathways in the Drosophila immune response. In ird1 mutants, the Imd pathway is not activated in response to bacterial infection, whereas the Toll pathway appears to be constitutively activated. This indicates that ird1 may act at a nexus point for the regulation of both pathways (Wu, 2007).

The Imd and Toll signalling pathways are often presented as distinct, independent pathways but this is an oversimplified view. Examinations of mutations in the pathways indicate that the two pathways interact. Mutations in the Toll pathway (spätzle) show higher levels of Diptericin induction; this suggests that the Toll pathway is negatively regulating the Imd pathway and that loss of Toll signalling may result in a compensatory, higher activation of the Imd pathway. ird1 mutants show a complementary effect with loss of Imd pathway and constitutive activation of the Toll pathway. This may be unique to ird1, as other Imd pathway mutants do not show constitutive activation of the Toll pathway. The results of this study suggest that despite the appearance of constitutive signalling via the Toll pathway in ird1 mutants, or during starvation, this is actually deleterious for the fly's immune response to bacterial infection. Recent papers also indicate that a constitutive Toll response is harmful and causes flies to be more susceptible to Drosophila X virus or Listeria monocytogenes infection. Not much is known about how the immune signalling pathways are shut off, but this appears to be as important as activation, for the animal's overall immune competence (Wu, 2007).

Activation and maintenance of an immune response involves a metabolic cost to the organism. This phenomenon is most apparent when the immune response is always on. In Arabidopsis, mutations that constitutively activate systemic acquired resistance (SAR) result in much smaller plants that have reduced fitness. In humans, a chronic inflammatory response results in metabolic adaptations to produce acute-phase proteins at the expense of skeletal muscle. Hence, organisms need to find a balance between using available energy resources and mounting an adequate immune response. In Drosophila, the fat body serves both as the primary nutrient responsive tissue and as the primary site for AMP production. The requirement for ird1 for starvation-induced AMP responses and the functional studies of its yeast and mammalian homologues indicate that it is positioned in a nutrient-sensing pathway. The finding that ird1 is also necessary to activate the Imd pathway and to keep the Toll pathway in check indicates that ird1 can affect both the known AMP immune signalling pathways. Having a gene, ird1, necessary for both nutrient sensing and immune signalling would provide a means for the organism to quickly integrate signals from these pathways and modulate the strength of its immune response relative to the available energy sources. In the future, it will be important to determine if other components of nutrient sensing pathways can also influence the immune signalling pathways (Wu, 2007).

PtdIns(3)P controls mammalian cell cytokinesis through KIF13A-mediated recruitment of FYVE-CENT to the midbody

Several subunits of the class III phosphatidylinositol-3-OH kinase (PI(3)K-III) complex are known as tumour suppressors. This study uncovered a function for this complex and its catalytic product phosphatidylinositol-3-phosphate (PtdIns(3)P) in cultured mammalian cell cytokinesis. PtdIns(3)P localizes to the midbody during cytokinesis and recruits a centrosomal protein, FYVE-CENT (ZFYVE26), and its binding partner TTC19, which in turn interacts with CHMP4B, an endosomal sorting complex required for transport (ESCRT)-III subunit implicated in the abscission step of cytokinesis. Translocation of FYVE-CENT and TTC19 from the centrosome to the midbody requires another FYVE-CENT-interacting protein, the microtubule motor KIF13A. Depletion of the VPS34 or Beclin 1 subunits of PI(3)K-III causes cytokinesis arrest and an increased number of binucleate and multinucleate cells, in a similar manner to the depletion of FYVE-CENT, KIF13A or TTC19. These results provide a mechanism for the translocation and docking of a cytokinesis regulatory machinery at the midbody (Sagona, 2010).

Cytokinesis, the final step of mitosis in which the two daughter cells separate, is defined by the constriction of the cytoplasm between the two re-forming nuclei through the assembly of an actomyosin contractile ring. This constriction results in the formation of a narrow intercellular bridge, the midbody, filled with central spindle microtubules. Completion of cytokinesis requires cleavage of the midbody in a process called abscission. Failure to complete cytokinesis has been shown to promote aneuploidy and has been associated with tumorigenesis. Recent studies have identified certain proteins that localize to centrosomes early in mitosis and later to the midbody and are required for the completion of abscission. How centrosomal proteins move to the midbody and are retained there remains largely unknown (Sagona, 2010).

One mechanism for anchoring cytosolic proteins to membranes is by virtue of their binding to phosphorylated derivatives of the membrane lipid phosphatidylinositol (PtdIns), known as phosphoinositides. These lipids regulate cytoskeletal functions, membrane trafficking and receptor signalling through the reversible recruitment of protein complexes to specific membranes. A well-studied example is PtdIns(3)P, which is enriched on endosomal membranes. The first known functions of PtdIns(3)P were in the regulation of vesicular trafficking in the endolysosomal system. However, PtdIns(3)P has also been implicated in other signalling processes, including nutrient sensing, receptor signalling and autophagy. Even though endosomes have been implicated in cytokinesis, it is not known whether PtdIns(3)P and its effectors are involved in this process (Sagona, 2010).

This study investigated the possible function of PtdIns(3)P in cytokinesis. PtdIns(3)P was shown to be required for proper cytokinesis and a PtdIns(3)P-binding protein, FYVE-CENT, and its interacting partners TTC19 and KIF13A were identified as components of a machinery that regulates cytokinesis. KIF13A-dependent recruitment of FYVE-CENT and TTC19 to the midbody controls cytokinesis (Sagona, 2010).

To study the localization pattern of PtdIns(3)P during cytokinesis, a green fluorescent protein (GFP)-tagged tandem FYVE domain (GFP-2×FYVE), which is a widely used probe for monitoring PtdIns(3)P distribution, was used. HeLa cells transfected with the GFP-2×FYVE probe show a punctuate pattern of GFP-positive spots that mostly represent endosomes. In cells undergoing early and late cytokinesis, the 2×FYVE probe, but not a point mutant defective in PtdIns(3)P binding, stained the Aurora B-positive midbody. PtdIns(3)P-containing vesicles were frequently observed close to the midbody ring, presumably in transit to the ring. These vesicles were found to co-localize with the recycling endosomal marker transferrin (Sagona, 2010).

To examine the possible requirement for PtdIns(3)P during cytokinesis, small interfering RNA (siRNA) was used to knock down the class II and III phosphatidylinositol-3-OH kinases (PI(3)K-II and PI(3)K-III) known to mediate its formation. Depletion of one of these, the PI(3)K-III VPS34, did indeed cause an increased number of cells in cytokinesis and also led to increased numbers of binucleate and multinucleate cells. The same was true when an accessory subunit of the PI(3)K-III complex, the tumour suppressor Beclin 1, was knocked down. Back-transfection of the VPS34 and Beclin 1 transgenes reverted the phenotype. Consistent with a role for PI(3)K-III in cytokinesis was the observation that endogenous VPS34 localized to the midbody. To test the importance of VPS34 for cell division in vivo, clones of vps34 mutant cells were studied in the follicular epithelium of the Drosophila egg chamber. This analysis showed that cells mutant for vps34 showed a fivefold increase in the binucleate phenotype compared with the wild-type neighbouring cells. Taken together, these data demonstrate that PtdIns(3)P is localized to the midbody during cytokinesis and is required for its normal completion in cell culture and in a multicellular model organism (Sagona, 2010).

To identify possible effectors of PtdIns(3)P in cytokinesis, HeLa cells were screened with a siRNA library targeting proteins with known PtdIns(3)P-binding domains, the FYVE and PX domains. The strongest positive hit with this strategy was ZFYVE26, a roughly 285-kDa FYVE domain protein whose depletion caused the highest proportion of cells arrested in cytokinesis. To study the expression and intracellular localization of ZFYVE26, a rabbit antiserum was raised against two specific peptides located in its amino-terminal and carboxy-terminal parts, respectively. Fluorescence microscopy with anti-ZFYVE26 showed that the major localization of this protein was on one or two punctate structures in each cell, and co-localization with the centrosomal marker γ-tubulin revealed that ZFYVE26 localizes on the centrosome. The protein product of the ZFYVE26 gene was therefore named FYVE-CENT (FYVE-domain-containing centrosomal protein) (Sagona, 2010).

To establish the localization of FYVE-CENT during the cell cycle the subcellular localization of FYVE-CENT was examined in HeLa cells that were synchronized and fixed in G1, S or M phase. This analysis showed that FYVE-CENT is localized to the centrosome at all stages of the cell cycle. The protein also localized to the midbody during cytokinesis. Immunoelectron microscopy of cells in G1 phase revealed the presence of FYVE-CENT on the centrioles. FYVE-CENT did not co-localize with various endosomal (EEA1, CD63 or endocytosed transferrin) or autophagic (GFP-LC3) markers. A previous study reported localization of the overexpressed ZFYVE26 gene product to early endosomes in COS-7 cells. However, in the current study, overexpressed FYVE-CENT localized to the centrosome in HeLa cells, which is consistent with the localization of endogenous FYVE-CENT. Deletion mutagenesis showed that the C-terminal part of FYVE-CENT was sufficient for localization to the centrosome and midbody. However, the N-terminal part also showed a weak localization to the centrosome. These results suggest that the subcellular targeting of FYVE-CENT is primarily mediated by its C terminus, and that the N terminus possibly also contributes by binding to centrosomal proteins (Sagona, 2010).

Because the siRNA screen was performed with pools of four siRNAs, the screening result needed to be verified with deconvoluted single siRNAs. Both the siRNA pool and the individual siRNAs against FYVE-CENT caused a significant decrease in the 285-kDa FYVE-CENT-reactive band compared with cells treated with control RNA duplex. To ascertain whether knockdown of FYVE-CENT with single siRNAs affects cytokinesis, HeLa cells were transfected with FYVE-CENT siRNAs and observed by confocal fluorescence microscopy, using Aurora B as a marker for the midbody and Hoechst staining to reveal nuclei. Quantification of multiple micrographs showed that the percentages of binucleate and multinucleate cells and cells undergoing cytokinesis were markedly increased after FYVE-CENT knock-down even with single siRNAs. Back-transfection of the FYVE-CENT transgene reverted the phenotype. Together, these results indicate that FYVE-CENT, like PI(3)K-III, is required for proper cytokinesis (Sagona, 2010).

To examine whether the FYVE domain of FYVE-CENT binds to PtdIns(3)P, the recombinant FYVE domain was purified as a glutathione S-transferase (GST) fusion protein and incubated with liposomes containing various phosphoinositides [PtdIns(3)P, PtdIns(4,5)P₂, PtdIns(3,5)P₂ and PtdIns(3,4,5)P₃]. The GST-tagged FYVE domain of FYVE-CENT bound strongly only to PtdIns(3)P, as determined by western blotting with an anti-GST antibody. As expected, mutation of a conserved arginine residue (R1835A) in the FYVE domain predicted to bind directly to PtdIns(3)P, significantly decreased the ability to bind PtdIns(3)P. It is concluded that the FYVE domain of FYVE-CENT, similarly to other FYVE domains tested, binds specifically to PtdIns(3)P (Sagona, 2010).

To study the importance of the FYVE domain in the subcellular targeting of FYVE-CENT, the R1835A mutant version of the C terminus of FYVE-CENT was next expressed in HeLa cells and its intracellular localization was determined. In contrast to the wild-type FYVE-CENT C terminus, which localized to the midbody during cytokinesis, the R1835A mutant did not localize to the midbody but had a cytosolic and nuclear distribution. The same occurred with a construct in which the FYVE domain had been deleted. In interphase cells the R1835A construct localized to the centrosome similarly to the wild-type C terminus. These findings indicate that the FYVE domain is critical for the targeting of FYVE-CENT to the midbody but not for targeting to the centrosome (Sagona, 2010).

FYVE-CENT is the first PtdIns(3)P-binding protein that has been shown to translocate from the centrosome to the midbody during cytokinesis; whether its interacting partners could shed light on its mechanisms of localization and function was examined. To identify interacting partners for FYVE-CENT, a yeast two-hybrid screen was performed, using the C-terminal part of FYVE-CENT as bait. The two strongest hits in this screen were KIF13A and TTC19. KIF13A is a plus-end-directed microtubule-dependent motor protein involved in mannose-6-phosphate receptor transport to the plasma membrane. Its interaction with FYVE-CENT did not map to its canonical cargo-binding domain but was instead located to a region containing a forkhead-homology-associated (FHA) domain, a domain found in all members of the kinesin-3 family. TTC19, a previously uncharacterized protein, contains five tetratricopeptide repeats, first described as a protein-protein interaction domain in yeast cell cycle proteins. To verify the interactions biochemically, cell lysates containing Myc-epitope-tagged KIF13A or TTC19 were incubated with beads containing the C terminus of FYVE-CENT fused to GST. This assay showed that Myc-KIF13A and Myc-TTC19 were pulled down by the C terminus of GST-FYVE-CENT by not by GST alone, thereby confirming the results from the two-hybrid screen. Finally, endogenous KIF13A and FYVE-CENT were co-immunoprecipitated with an antibody against endogenous TTC19, indicating that the three proteins can exist as a physical complex in vivo (Sagona, 2010).

The interactions of FYVE-CENT with KIF13A and TTC19 raised the possibility that these proteins share the subcellular distribution of FYVE-CENT. Immunofluorescence microscopy showed that endogenous KIF13A co-localizes with γ-tubulin on the centrosome and with Aurora B on the midbody. Overexpressed Myc-epitope-tagged KIF13A also co-localized with CEP55, a known marker for the midbody and the centrosome. Similarly, endogenous TTC19 co-localized with γ-tubulin and Aurora B on the centrosome and the midbody. TTC19 did not localize to the centrosomes during cytokinesis, suggesting that it translocates from the centrosome to the midbody during this phase of cell division. siRNA-mediate depletion of KIF13A or TTC19 resulted in arrest in cytokinesis and in increased numbers of binuclear and multinuclear cells. Back-transfection of the KIF13A and TTC19 transgenes rescued the phenotype. Thus, similarly to their interacting partner FYVE-CENT, KIF13A and TTC19 are regulators of cytokinesis (Sagona, 2010).

Having established a role for PI(3)K-III complex, FYVE-CENT, KIF13A and TTC19 in cytokinesis, attempts were made to identify at which stage in cytokinesis these proteins come into action. For this purpose, each of these proteins was knocked down by siRNA, the cells were stained for Aurora B, α-tubulin and DNA, and analyses of cell division profiles were recorded from a large number of cells with an automated fluorescence microscope. Using this strategy it was observed that cells depleted of VPS34, FYVE-CENT and KIF13A were mainly arrested in early cytokinesis (defined by Aurora-B-positive midbodies and incompletely decondensed chromatin), although to some extent also in late cytokinesis (defined by Aurora-B-positive midbodies and fully decondensed nuclei). However, TTC19-depleted cells were mainly arrested in late cytokinesis, similarly to TSG101, which is thought to function in the final abscission step together with other components of the endosomal sorting complex required for transport (ESCRT) machinery (Morita, 2007; Carlton, 2007). This suggests that TTC19 functions after PtdIns(3)P, FYVE-CENT and KIF13A in cytokinesis (Sagona, 2010).

TTC19 has previously been identified as a possible interacting partner of the ESCRT-III subunit CHMP4A in a yeast two-hybrid screen, which is of interest because ESCRT-III components have been proposed to mediate midbody abscission through their interaction with centrosomal proteins and midbody membranes. Because it was found that endogenous CHMP4B, which is closely related to CHMP4A, localizes strongly to the midbody during cytokinesis, whether TTC19 interacts with this protein was examined. TTC19 was found to interact with CHMP4B in a pull-down assay and endogenous TTC19 could be co-immunoprecipitated with endogenous CHMP4B from a HeLa cell lysate. When cells were depleted of CHMP4B, the localization of TTC19 on the midbody was markedly decreased. This suggests that CHMP4B and TTC19 interact functionally at the midbody, and is consistent with the possibility that TTC19 could be a regulator of CHMP4B (Sagona, 2010).

Given the capability of KIF13A to translocate vesicles and cargo molecules towards the cell periphery, it was speculated that this microtubule motor protein might mediate the localization of FYVE-CENT and TTC19 to the midbody. To test this idea, KIF13A in HeLa cells was knocked down with siRNA and the cells were stained for FYVE-CENT and TTC19. Depletion of KIF13A did indeed abolish FYVE-CENT and TTC19 localization from the midbody. Furthermore, depletion of FYVE-CENT also abolished the localization of TTC19 from the midbody. Localization of FYVE-CENT and TTC19 to the midbody was significantly decreased when VPS34 and Beclin 1 were depleted, but midbody localization of KIF13A was not affected. In contrast, TTC19 knockdown had no effect on FYVE-CENT or KIF13A localization to the midbody. Taken together, these data suggest that KIF13A transports FYVE-CENT and TTC19 to the midbody, where FYVE-CENT can dock to PtdIns(3)P and TTC19 to CHMP4B. It is possible that the specific docking at the midbody is mediated by a simultaneous detection of PtdIns(3)P and TTC19, although it is also possible that transport to the midbody is required for exposure of the FYVE domain of FYVE-CENT. On the basis of these data and high-content microscopy, it is proposed that PtdIns(3)P controls the KIF13A-dependent recruitment of FYVE-CENT and TTC19 to the midbody, and that TTC19 is the most downstream effector of the three, possibly controlling the function of CHMP4B (Sagona, 2010).

These data have demonstrated that PtdIns(3)P production is essential for proper cytokinesis and suggest that the PtdIns(3)P-binding centrosomal protein FYVE-CENT and its interacting protein TTC19 control cytokinesis through their translocation from the centrosome to the midbody mediated by the kinesin protein KIF13A. This model explains the significant increase in cells arrested in cytokinesis and also binucleate and multinucleate cells when FYVE-CENT, TTC19 and KIF13A are depleted. Even though such depletions only affected a minor proportion of the cell population (a similar penetrance to that of depletion of the ESCRT protein TSG101), they are likely to reflect an important regulatory function of FYVE-CENT, TTC19 and KIF13A in cytokinesis. In terms of oncogenesis, the loss of protein functions that affect only a minor proportion of cells could well be more severe than the loss of more central factors, because the latter is more likely to cause cell death than tumourigenesis (Sagona, 2010).

Even though multiple proteins are known to mediate (or regulate) cytokinesis, the exact biochemistry of the abscission step remains unknown. The recent identification of the ESCRT-III complex, a well-known machinery for inward membrane budding, in cytokinesis is conceptually interesting because it suggests a model for the final membrane abscission step. ESCRT-III components have been proposed to mediate midbody abscission through their constriction of midbody membranes. The interaction of the centrosomal protein TTC19 with the ESCRT-III subunit CHMP4B uncovers an interplay between this regulator of late cytokinesis and the ESCRT machinery. It is proposed that TTC19, through its direct interaction with FYVE-CENT and CHMP4B, could regulate the function of ESCRT-III components at the midbody, possibly by controlling the oligomerization of CHMP4B, and could in this way control the abscission process (Sagona, 2010).

It was recently shown that the ZFYVE26 gene encoding FYVE-CENT is mutated in patients with hereditary spastic paraplegia. Spastin, a protein that is often found mutated in patients with hereditary spastic paraplegia, has also been shown to localize to the midbody and to control cytokinesis. This raises the possibility that there could be a correlation between aberrant cytokinesis and the aetiology of some forms of hereditary spastic paraplegia (Sagona, 2010).

Three subunits of the PI(3)K-III complex, namely Beclin 1, Bif-1 (BAX-interacting factor 1) and UVRAG (UV radiation resistance associated), have previously been identified as tumour suppressors. Their tumour suppressor activities have been attributed to their involvement in autophagy, a catabolic process that is thought to promote genome stability by scavenging damaged organelles. The present data suggest an additional potential mechanism for the tumour suppressor function of the PI(3)K-III complex. Failure to complete cytokinesis has been suggested to promote tumorigenesis, and it is interesting to note that the ZFYVE26 gene encoding FYVE-CENT has been found mutated in breast cancer samples with a frequency of more than 10%. Taken together, these findings reveal a regulatory role for the PI(3)K-III complex and its catalytic product, and it will be interesting to investigate whether this is relevant to carcinogenesis (Sagona, 2010).

Search PubMed for articles about Drosophila Pi3k59F

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date revised: 19 June 2024

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