skittles

REGULATION

Interplay between Rab5 and PtdIns(4,5)P₂ controls early endocytosis in the Drosophila germline

Phosphoinositides have emerged as key regulators of membrane traffic through their control of the localization and activity of several effector proteins. Both Rab5 and phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P₂] are involved in the early steps of the clathrin-dependent endocytic pathway, but little is known about how their functions are coordinated. This study investigated the role of PtdIns(4,5)P₂ and Rab5 in the Drosophila germline during oogenesis. Rab5 was found to be required for the maturation of early endocytic vesicles. PtdIns(4,5)P₂ is required for endocytic-vesicle formation, for Rab5 recruitment to endosomes and, consistently, for endocytosis. Furthermore, a previously undescribed role of Rab5 was revealed in releasing PtdIns(4,5)P₂, PtdIns(4,5)P₂-binding budding factors and F-actin from early endocytic vesicles. Finally, overexpressing the PtdIns(4,5)P₂-synthesizing enzyme Skittles leads to an endocytic defect that is similar to that seen in rab5 loss-of-function mutants. Hence, these results argue strongly in favor of the hypothesis that the Rab5-dependant release of PtdIns(4,5)P₂ from endosomes that was discovered in this study is crucial for endocytosis to proceed (Compagnon, 2009).

Regulation of the level of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P₂] within a cell and recruitment of the small GTPase Rab5 to clathrin-coated vesicles (CCVs) are required to promote the formation and maturation of these vesicles. Although these two mechanisms appear to participate in the same step of clathrin-dependant endocytosis, the potential interdependency between them is not known. The existence in Drosophila of a well-characterized endocytic route in the oocyte makes its oogenesis a very attractive model in which to address this issue in a physiological context. The ovarian follicle is composed of a 16-cell germline cyst -- one cell of which is determined as the oocyte -- that is surrounded by follicle cells. The most-studied endocytic process in the oocyte is the uptake of yolk proteins (Yp1-Yp3) via clathrin-mediated endocytosis. From stages 8 to 11, the oocyte intakes, via endocytosis, a large quantity of vitellogenins, which are yolk-protein precursors that are synthesized in the fat body and the follicle cells. After their entry into the oocyte, yolk proteins are stored inside large late endocytic compartments, yolk granules, which will later provide the reserves necessary for embryonic development. This specific accumulation of embryonic reserves inside the oocyte is achieved by the localization of the vitellogenin receptor Yolkless (Yl) at the plasma membrane (PM) of the oocyte during stages 8 to 11. The morphology of the endocytic intermediates in insect oocytes is well described at the ultrastructural level. For instance, it is inside the ovary of Aedes aegypti that coated vesicles were observed for the first time. Furthermore, the succession of these intermediates has also been well described by means of a thermosensitive dominant negative allele of shibire, the Drosophila homolog of dynamin. The vitellogenins are first found in clathrin-coated pits (CCPs) at the oocyte PM; after fission, these CCPs form CCVs. These vesicles then lose their coat and form tubular intermediates that fuse with the forming yolk-protein storage granules, thus filling the lumen of these granules with yolk proteins to form mature storage granules (Compagnon, 2009).

The small GTPase Rab5 is a well-known regulator of early endocytosis in mammals. Rab5 is involved in the budding of CCVs in vitro and also regulates their subsequent maturation by promoting the fusion of early endocytic vesicles (EEVs) with sorting endosomes. This contribution to endocytic-vesicle maturation relies on the recruitment of several effector proteins that trigger a local enrichment in the endosomal membrane of phosphatidylinositol 3-phosphate [PtdIns(3)P] (Christoforidis, 1999; Erdmann, 2007; Hyvola, 2006; Shin, 2005). The Rab5-dependent formation of the PtdIns(3)P-positive endosomal domain on early endosomes participates in the recruitment of endosomal factors regulating various aspects of early-endosome function, such as tethering, fusion and mobility. Another aspect of endocytosis-related phosphatidylinositol (PtdIns) regulation is the control of PtdIns(4,5)P₂ levels during early endocytic steps. PtdIns(4,5)P₂ plays a crucial role in the selective recruitment of endocytic proteins to the PM for CCV formation. It binds to endocytic clathrin adaptor complexes such as AP2 to initiate the assembly of the coat and also to dynamin, which controls the fission reaction. Consistent with this, lower levels of PtdIns(4,5)P₂ impair endocytosis. There is yet another requirement in the regulation of PtdIns(4,5)P₂ after CCV formation. The PtdIns(4,5)P₂ 5-phosphatase activity of Synaptojanin (Synj) is necessary for the hydrolysis of PtdIns(4,5)P₂ from EEVs, thereby triggering coat-component shedding (Compagnon, 2009 and references therein).

In Drosophila, Rab5 has been found to localize on PtdIns(3)P-containing early endosomes at the neuromuscular junction, where it is required for synaptic-vesicle recycling. It has also been demonstrated that Rab5 function is required for the formation of PtdIns(3)P-containing early endosomes. Thus, Rab5 is, in Drosophila, a fundamental regulator of the early endocytic pathway, similar to its mammalian homologs (Compagnon, 2009).

This study used complete loss of rab5 function in the germline cyst to study the consequences on the endocytic pathway. Rab5 was found to be required for maturation of the EEV and yolk-protein endocytosis in the oocyte. Using loss of function of skittles (sktl), coding for a type I phosphatidylinositol 4-phosphate 5-kinase, it was shown that PtdIns(4,5)P₂ is required for endocytic-vesicle formation, for Rab5 recruitment and accordingly for yolk-protein endocytosis. Furthermore, a previously undescribed role for Rab5 was revealed in controlling the release of PtdIns(4,5)P₂, PtdIns(4,5)P₂-binding budding factors and F-actin from EEVs. Finally, it was shown that overexpressing the PtdIns(4,5)P₂-synthesizing enzyme Sktl first leads to the formation of an abnormal early endocytic compartment containing Rab5, PtdIns(4,5)P₂-binding coat component and F-actin, and, second, affects yolk-protein endocytosis. Hence, these results argue strongly in favor of the hypothesis that Rab5-dependent release of PtdIns(4,5)P₂ from EEVs is crucial for endocytosis to proceed (Compagnon, 2009).

The first stage of endocytosis, CCV formation, has previously been shown to rely on the presence of PtdIns(4,5)P₂ at the PM for coat-component recruitment and fission in mammals (for review see, Di Paolo and De Camilli, 2006). In Drosophila, the situation has been less clear; early studies attempting to address the requirement of PtdIns(4,5)P₂ for endocytosis were inconclusive. The current results in sktl mutant oocytes indicate that, when PtdIns(4,5)P₂ level is lowered, endocytosis is impaired. Moreover, analysis of endocytic-compartment morphology at the ultrastructural level in this context revealed a depletion of all intracytoplasmic endocytic intermediates. This strongly suggests a conserved requirement of PtdIns(4,5)P₂ for the formation of endocytic vesicles in the oocyte. Interestingly, the build-up of coated pits along invaginations of the PM that was observed in sktl mutant oocytes is very similar to what is observed with a dominant-negative allele of dynamin, encoding an essential PtdIns(4,5)P₂-binding regulator of fission. Moreover, in sktl mutant oocytes, the subcortical recruitment of Rab5 during vitellogenic stages was affected. These observations suggest that PtdIns(4,5)P₂-dependent CCV formation is necessary for Rab5 recruitment (Compagnon, 2009).

Consistent with a role of Rab5 following EEV formation, analysis of endocytic-compartment morphology at the ultrastructural level in the absence of Rab5 showed an accumulation of EEVs, which was associated with a depletion of later endocytic structures. This suggests a conserved requirement of Rab5 for EEV maturation, but does not exclude that Rab5 might be also required for other steps along the endocytic pathway. Surprisingly, loss of function of rab5 impairs the removal of PtdIns(4,5)P₂ from the endosomal membrane (Compagnon, 2009).

The importance of PtdIns(4,5)P₂ turnover for endocytic-vesicle maturation was demonstrated by the study of Synj, a PtdIns 5-phosphatase, in synaptic termini. In synj knock-out mice or Drosophila mutants, synaptic-vesicle recycling is impaired and CCVs accumulate in thecytoplasm (Cremona, 2001; Verstreken, 2003). This study observed, in mutants with strong loss of synj function, that neither yolk-protein endocytosis nor actin restriction at the cortex were impaired in the oocyte. Nevertheless, it was found that an excess in the PtdIns(4,5)P₂-producing enzyme Sktl led to the formation of an abnormal endocytic compartment containing PtdIns(4,5)P₂ and, accordingly, a reduction of yolk-protein endocytosis in this context was also observed. The results indicate that, in vivo, besides its known requirement in neuronal cells, PtdIns(4,5)P₂ removal from endosomal membranes is also essential for endocytosis to proceed in other cell types. Furthermore, this study found a situation different from that in neurons; removing PtdIns(4,5)P₂ from endosomal membrane of oocytes does not require Synj. The finding also echoes the recent observation that Synj is not required for endocytosis in S2 cells (Korolchuk, 2007). Altogether, this suggests that different enzymes could fulfill this function in various cell types. Interestingly, the finding that Rab5 is present on the abnormal endocytic structures induced by Sktl overexpression suggests that, in this context, endocytosis is blocked at the stage when Rab5 is required to proceed further along the endocytic path. Altogether, these observations make the finding of a role of Rab5 in the removal of PtdIns(4,5)P₂ from endosomal membrane all the more relevant (Compagnon, 2009).

In the absence of Rab5, PtdIns(4,5)P₂, found in the ectopic endosomal compartments, is associated with the PtdIns(4,5)P₂-binding factors necessary for coat recruitment and fission, and with F-actin aggregates, hence suggesting that the defective PtdIns(4,5)P₂ regulation impairs the dynamics of budding factors and F-actin organization. Although the involvement of Rab5 in these processes independently from its effect on PtdIns(4,5)P₂ distribution cannot be ruled out, the interpretation that these phenotypes are a direct consequence of altered PtdIns(4,5)P₂ removal is favored for several reasons. First, recent studies using live-cell imaging have shown that there is an intimate connection between the regulation of PtdIns(4,5)P₂ levels and coat assembly and/or disassembly (Sun, 2007; Zoncu, 2007). Second, it has been established that the PtdIns(4,5)P₂-dependent shut-down of actin polymerization is required for endocytosis to proceed in yeast (Sun, 2007). Third, the phenotypes are reminiscent of those observed when the PtdIns(4,5)P₂-synthesizing enzyme Sktl was overexpressed (Compagnon, 2009).

These observations raise the issue of a possible link that could exist between Rab5 and the spatial restriction of PtdIns(4,5)P₂. Among the Rab5 effectors known to be involved in PtdIns metabolism, three could directly regulate PtdIns(4,5)P₂ levels: the PtdIns 3-kinase p110 is able to use PtdIns(4,5)P₂ as a substrate to produce PtdIns(3,4,5)P₃, and the PtdIns 5-phosphatases INPP5B and ORCL are able to use both PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ to produce PtdIns(4)P and PtdIns(3,4)P₂, respectively. This study found that PtdIns(3,4,5)P₃ accumulates on endosomes along with PtdIns(4,5)P₂ in rab5² mutant oocytes. This leads to favoring of the assumption that the accumulation of PtdIns(4,5)P₂ that was revealed in this study is more likely to arise from defective PtdIns 5-phosphatase recruitment than defective PtdIns 3-kinase recruitment. Another hypothesis, compatible with the previous assumption, is that Rab5 can also restrict PtdIns(4,5)P₂ synthesis by negatively regulating Sktl activity from the endosomes. Two observations are in line with this scenario: (1) Sktl overexpression led to defects that were similar to those observed in rab5 loss-of-function mutants, and (2) Sktl is found on abnormally maturing endosomes in rab5² mutant oocytes. It may thus prove fruitful in the future to search for a Rab5 effector that is able to restrict Sktl localization from endosomes, and to explore the influence of Rab5-recruited PtdIns 5-phosphatase on the regulation of PtdIns(4,5)P₂ levels along the endocytic pathway (Compagnon, 2009).

DEVELOPMENTAL BIOLOGY

Embryonic

In situ hybridization shows that during embryogenesis sktl is expressed at all stages, but there is a very dynamic pattern of regulation in various developing tissues. At all stages there is a basal level of expression in all cells. At stage 5, strong expression is seen in the procephalic neuroectoderm. During gastrulation, expression is elevated in the invaginating cells of the ventral and cephalic furrows. At stage 11 all central nervous system and peripheral nervous system precursor cells express high levels of sktl. At stage 13 most developing tissues (heart, gut, muscles, CNS, and PNS) express high levels of skittles. By the end of embryogenesis (stage 17) expression is prominent in a few CNS cells and the gut. This expression pattern, particularly in the nervous system, is remarkably similar, if not identical, to that of insc (Kraut, 1996a and Knirr, 1997b). This suggests that the two genes may share common regulatory elements and raises the question of whether they interact during nervous system development. Alternatively, sktl may be under the control of insc enhancers and may serve an unrelated function (Hassan, 1998).

Larval

During third instar larval development sktl is expressed widely in all imaginal discs. In the leg disc expression is ubiquitous and uniform. In the wing disc, expression is elevated in the precursors of the anterior wing margin sensory organs and along the anterior-posterior axis. Expression is very low or absent along the dorso-ventral axis. In the eye disc, expression is elevated in the row of cells anterior to the morphogenetic furrow from which the R8 photoreceptors will differentiate. In the third instar larval brain, sktl is expressed widely but not ubiquitously. Areas of expression include the outer proliferation center of the optic lobes, several patches of cells in the midbrain, and subsets of cells in the ventral ganglion (Hassan, 1998).

Adult

During oogenesis, sktl expression is first observed in region 2 of the germarium. At this stage, sktl appears to be expressed in a subset of cells building the different cysts. In egg chambers of stagee S1-S14 strong expression is observed in the oocyte. Only weak expression is detected in the nurse cells from stage S1 to S8. At stage S8, expression in the nurse cells becomes more profound and increases during subsequent stages. No expression is observed in the follicle cells surrounding the egg chambers. Sktl transcripts are uniformly distributed in the unfertilized egg. skit is also expressed in the male germline. Sktl transcripts first occur in primary spermatocytes and later during meiotic prophase, whereas in early germ cells, such as the stem cells and the spermatogonia, as well as in postmeiotic stages, Sktl transcripts are absent (Knirr, 1997a).

Effects of Mutation or Deletion

sktl mutants are lethal in early first instar larvae. Because sktl is expressed at high levels in most if not all nervous system precursors, the consequences of the loss of sktl on nervous system development were investigated. sktl^Delta20 mutant embryos were used to determine if sktl is required for nervous system development. Nervous system development was examined using anti-ELAV and Mab 22C10 antibodies to detect neurons and anti-PROS to detect neuronal precursors during early neurogenesis and glial cells during late neurogenesis. No detectable defects are seen by stage 16 with these markers. The absence of an insc phenotype in sktl mutants demonstrates that the phenotypes reported are indeed caused by the absence of insc and not by the loss of function of either sktl alone or both sktl and insc, as for example, the loss of nervous system cells due to mislocalization of Numb and Prospero during neuronal lineage development (Hassan, 1998).

PIP5KI is required for Ca²⁺-dependent neuropeptide secretion from PC12 cells (Hay, 1995). In addition, several lines of evidence suggest that PIP5Ks play a crucial role in regulating membrane trafficking. Furthermore, sktl is expressed in many cells in the ventral ganglion of third instar larvae. Some of these cells may correspond to motor neurons innervating the larval body wall muscles. The Drosophila third instar larval neuromuscular junction is an excellent system for measuring neurotransmitter release from motor neurons. To test the requirement of sktl for neurotransmitter release, third instar larvae transheterozygous for sktl alleles were used. One combination results in late second instar lethality with a few third instar escapers. Electrophysiological recordings at the neuromuscular junction were done to examine spontaneous release as well as evoked release. The size, shape, and frequency of evoked and spontaneous responses were examined in control larvae (heterozygous for either allele alone) and in transheterozygous mutant larvae. Evoked response was measured after either single or repetitive stimulation. Tests were perfomed for nerve fatigue by repetitive stimulation. No detectable defects were observed in any of the above measurements, suggesting that sktl is not required for glutamate neurotransmitter release, at least at the larval neuromuscular junction. It is therefore unlikely to play a role in regulating vesicular trafficking at that junction. The lack of a vesicular secretion phenotype may be due to the activity of other PIP5Ks in Drosophila. Drosophila has a PIP5K type II that maps to the tip of chromosome 4 and that appears, from preliminary expression analysis, to be expressed specifically in the late embryonic CNS (B. Hassan, unpublished results cited in Hassan, 1998). It remains to be established whether this form of PIP5K functions in neuronal secretion (Hassan, 1998).

Zhong (1995) showed in Drosophila that repetitive stimulation at the neuromuscular junction at 20 Hz or higher results not only in a fast evoked response but also in a slow, neuropeptide-dependent, depolarization. Neuropeptides are released by dense core vesicles from the nerve terminal. Hay (1995) showed that PIP5KI is required for dense core vesicle secretion from PC12 cells. To test the requirement of sktl for dense core vesicle secretion, motor nerves were stimulated at 30 and 50 Hz. The resulting slow depolarization profile in sktl mutants is indistinguishable from that of heterozygous controls or wild-type larvae. Therefore sktl does not appear to play a role in regulating dense core vesicle secretion, at least at the larval neuromuscular junction. It is not possible to exclude the possibility that the transheterozygous combination used in these experiments, while being strong enough to cause larval lethality, is not strong enough to have an effect on peptide release. However, this is unlikely because even viable hypomorphic mutations of numerous proteins involved in neurotransmission, such as synaptotagmin and RAS opposite (ROP), show severe electrophysiological defects (Hassan, 1998).

To characterize the function of sktl, overexpression studies were carried out. Flies with a UAS-sktl construct were used to overexpress sktl using a variety of Gal4 drivers. The neuronal-specific elav-Gal4 driver resulted in no detectable phenotypes and gave rise to fertile adults. Overexpression using the ubiquitous daughterless-Gal4 driver and the heat shock-Gal4 driver resulted in no obvious phenotypes during embryogenesis but caused early larval lethality. Expression with the ubiquitous imaginal disc driver T80-Gal4 resulted in third instar larval lethality. The lethality associated with the ubiquitous expression of sktl, which is itself very widely expressed, precluded the use of the UAS-sktl construct to rescue sktl mutants. Overexpression with the dpp-Gal4 driver, expressed in the morphogenetic furrow of the eye disc and along the anterior-posterior boundary in the wing disc, showed no detectable phenotypes in the eye (Hassan, 1998).

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date revised: 25 June 2009

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