InteractiveFly: GeneBrief

Phosphotidylinositol 3 kinase 59F: Biological Overview | References

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 class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila

Degradation of cytoplasmic components by autophagy requires the class III phosphatidylinositol 3 [PI(3)]-kinase Vps34, but the mechanisms by which this kinase and its lipid product PI(3) phosphate (PI(3)P) promote autophagy are unclear. In mammalian cells, Vps34, with the proautophagic tumor suppressors Beclin1/Atg6, Bif-1, and UVRAG, forms a multiprotein complex that initiates autophagosome formation. Distinct Vps34 complexes also regulate endocytic processes that are critical for late-stage autophagosome-lysosome fusion. In contrast, Vps34 may also transduce activating nutrient signals to mammalian target of rapamycin (TOR), a negative regulator of autophagy. To determine potential in vivo functions of Vps34, mutations were generated in the single Drosophila Vps34 orthologue (Phosphotidylinositol 3 kinase 59F), causing cell-autonomous disruption of autophagosome/autolysosome formation in larval fat body cells. Endocytosis is also disrupted in Vps34^-/- animals, but this does not account for their autophagy defect. Unexpectedly, TOR signaling is unaffected in Vps34 mutants, indicating that Vps34 does not act upstream of TOR in this system. Instead, TOR/Atg1 signaling regulates the starvation-induced recruitment of PI(3)P to nascent autophagosomes. These results suggest that Vps34 is regulated by TOR-dependent nutrient signals directly at sites of autophagosome formation (Juhász, 2008).

Engulfment of cytoplasmic material into specialized double-membrane vesicles known as autophagosomes is the defining feature of a process referred to as macroautophagy or simply autophagy. Subsequent fusion of autophagosomes with the endolysosomal network leads to hydrolytic degradation of the sequestered material. This process provides eukaryotic cells with a mechanism for cytoplasmic renewal by which they can rid themselves of defective organelles and protein complexes. In addition, nonselective autophagy can be induced to high levels by starvation, providing an internal source of nutrients on which cells can survive extended periods of nutrient deprivation. Conversely, under some circumstances autophagy may be used as a killing mechanism, acting as an alternative or augmentation to apoptotic cell death. As autophagy has been implicated in several physiological and pathological conditions, including neurodegeneration, tumorigenesis, and aging, better understanding of the molecular mechanisms controlling autophagy and identification of pharmacological regulators of this process are important goals (Juhász, 2008).

Wortmannin and 3-methyladenine are well established inhibitors of autophagy. These compounds are broad-spectrum phosphatidylinositol 3 [PI(3)]-kinase inhibitors that disrupt autophagy by inhibiting Vps34 (Petiot, 2000), the enzymatic component of a multiprotein complex which also includes Vps15, Beclin1/Atg6, UVRAG, and Bif-1 in mammals and Vps15, Atg6, and Atg14 in yeast (Mari, 2007). Localized production of PI(3) phosphate (PI(3)P) by Vps34 can act to recruit proteins containing FYVE and PX domains to specific membrane compartments (Lindmo, 2006). In yeast, this Vps34 complex is critical for recruiting autophagy-related (Atg) proteins to the preautophagosomal structure, the yeast-specific site of autophagosome formation. The role of PI(3)P in autophagosome biogenesis is less well understood in higher eukaryotes, and whether it functions at the autophagosomal, the donor, or another membrane has not been determined (Juhász, 2008).

Vps34 is also required more broadly for several vesicular trafficking processes that may have indirect impacts on autophagy. These include sorting of hydrolytic enzymes to the lysosome/vacuole and early steps in the endocytic pathway (Lindmo, 2006). In mammalian cells, autophagosomes have been shown to fuse with early or late endosomes before fusion with lysosomes, resulting in intermediate structures known as amphisomes. Recently, mutations in components of the endosomal sorting complex required for transport (ESCRT) complex, which is required for the transition from early to late (multivesicular) endosomes, have been shown to block autophagy by inhibiting autophagosome-endosome fusion. Thus, the effect of PI(3)-kinase inhibitors on autophagy may be due, in part, to these more general trafficking functions of Vps34 (Juhász, 2008).

Recent work has shown that Vps34 can also function in a nutrient-sensing pathway upstream of the target of rapamycin (TOR) in several mammalian cell lines (Byfield, 2005; Nobukuni, 2005). Disruption of Vps34 activity with blocking antibodies or siRNA was found to inhibit activation of TOR by insulin, amino acids, and glucose. As TOR signaling inhibits autophagy, these findings are at odds with the conserved role of Vps34 in promoting autophagy under starvation conditions, suggesting that distinct complexes or pools of Vps34 may be subject to different modes of regulation (Juhász, 2008).

This paper addresses how these multiple potential roles of Vps34 are coordinated to regulate autophagy in Drosophila. The findings suggest that despite a critical role for Vps34 in endocytic uptake and recycling, its primary function in autophagy in vivo is limited to its direct role at the nascent autophagosome. Vps34 is not required for TOR activity in this system, and starvation results in a TOR/Atg1-dependent recruitment of Vps34 activity to the autophagosomal membrane (Juhász, 2008).

Previous work in mammalian and yeast systems has identified a wide range of vesicle trafficking processes regulated by Vps34, including autophagy, endocytosis, endosome maturation, and both anterograde and retrograde trafficking between the Golgi and lysosome (Lindmo, 2006). The current findings indicate that despite these activities, the in vivo role of Vps34 in autophagy is largely limited to its function at the autophagosome. Although fluid-phase endocytosis, endocytic recycling of Notch, and trafficking of lysosomal proteins are disrupted by mutation of Vps34, the results suggest that events subsequent to autophagosome formation, including fusion between autophagosomes and endosomes or lysosomes and subsequent lysosomal degradation, are not rate limiting in the absence of Vps34. Why does endocytic disruption lead to autophagosome fusion defects in ESCRT mutants but not in Vps34 mutants? Accumulation of endocytic tracer at the periphery of Vps34 mutant cells suggests that Vps34 functions at an early step of endocytosis, and apparently this event, as well as normal endocytic flux is not essential for fusion of autophagosomes with elements of the endosomal-lysosomal compartment. Interestingly, the accumulation of autophagosomes in ESCRT/Vps34 double mutants indicates that loss of Vps34 does not completely prevent autophagosome formation. Similarly, the lack of autophagosome accumulation in Vps34 single mutants indicates that ESCRT complexes are at least partially functional in the absence of Vps34. Thus, PI(3)P may not be absolutely essential for these processes, or perhaps sufficient levels of PI(3)P are generated independently of Vps34 by the class II PI(3) kinase or by PI(3,4)P or PI(3,5)P phosphatases. Since ESCRT components are required for multivesicular body formation but not autophagy in yeast, it will be interesting to determine whether the requirement for ESCRT complexes in autophagy in higher eukaryotes reflects their role in multivesicular body formation or an alternate function (Juhász, 2008).

The cellular compartment in which Vps34 acts to promote autophagy and the mechanisms by which it is regulated by nutrient signals have remained unresolved. In mammalian cells, Beclin1/Atg6 has been reported to localize to the trans-Golgi network, ER, and mitochondria. It was recently shown that Beclin1-Vps34 complexes can be inhibited by the antiapoptotic factor Bcl-2 in a nutrient-dependent manner (Pattingre, 2005). Bcl-2 mutants that are targeted to the ER, but not to mitochondria, retain their capacity to inhibit starvation-induced autophagy, suggesting that the ER is an important site of Beclin1-Vps34 regulation. However, it is unknown how these organelles contribute to the formation of autophagosomes, and recent studies suggest that rather than budding off a preexisting compartment, the autophagosomal membrane is likely to form de novo from small lipid transport vesicles or lipoprotein complexes. The finding that myc-2xFYVE is recruited to GFP-Atg8a-positive structures under starvation conditions indicates that Vps34 activity is targeted directly to autophagosomes in a TOR/Atg1-dependent manner. Although these results do not distinguish between a role for TOR/Atg1 signaling in regulating Vps34 activity versus providing a platform on which Vps34 complexes can assemble, together with these previous studies they indicate that Vps34 is likely to promote autophagy by different mechanisms from multiple cellular locations (Juhász, 2008).

How the TOR signaling pathway senses intracellular levels of nutrients, such as amino acids, has been poorly understood despite considerable work in yeast, mammalian, and other model systems. The recent identification of Vps34 as a transducer of this signal in mammalian cells thus represents a significant new insight into this issue (Byfield, 2005; Nobukuni, 2005). However, further work is necessary to determine the extent to which this mechanism is generally conserved, since starvation appears to have opposing effects on Vps34 activity in different cell types. The results presented in this study fail to support this model in Drosophila; mutation of Vps34 does not appear to influence TOR-dependent phenotypes nor to disrupt TOR-dependent signaling. This may reflect a fundamental difference in signaling mechanisms between the fly and mammalian systems. The makeup of Vps34 complexes has diverged significantly between yeast and metazoans, and perhaps components of this complex, such as Ambra1 (Fimia, 2007), that appear to be unique to vertebrates may confer functions not found in flies. It is also possible that production of PI(3)P by Vps34-independent mechanisms is more efficient in D. melanogaster than in mammalian cells and, thus, a role for Vps34 in TOR signaling may be obscured by these other sources. The continued ESCRT function and basal level of autophagy in Vps34 null mutants are consistent with this possibility. Alternatively, the current findings may reflect important differences between the roles of TOR and Vps34 in vivo versus in cultured cells as well as the experimental paradigms of these systems. For example, although complete starvation is commonly used to inactivate TOR in cell culture studies, such experiments may not accurately mimic physiologically relevant events, given the inherent capacity of intact organisms to buffer changes in nutrient levels. Additional studies in an in vivo mammalian system will be helpful to clarify these issues (Juhász, 2008).

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).

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: 5 December 2010

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