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).

Molecular networks linked by Moesin drive remodeling of the cell cortex during mitosis

The cortical mechanisms that drive the series of mitotic cell shape transformations remain elusive. This paper identifies two novel networks that collectively control the dynamic reorganization of the mitotic cortex. Moesin, an actin/membrane linker, integrates these two networks to synergize the cortical forces that drive mitotic cell shape transformations. The Pp1-87B/Slik phosphatase restricts high Moesin activity to early mitosis and down-regulates Moesin at the polar cortex, after anaphase onset. Overactivation of Moesin at the polar cortex impairs cell elongation and thus cytokinesis, whereas a transient recruitment of Moesin is required to retract polar blebs that allow cortical relaxation and dissipation of intracellular pressure. This fine balance of Moesin activity is further adjusted by Skittles and Pten, two enzymes that locally produce phosphoinositol 4,5-bisphosphate and thereby, regulate Moesin cortical association. These complementary pathways provide a spatiotemporal framework to explain how the cell cortex is remodeled throughout cell division (Roubinet, 2011).

These findings unravel how, by integrating two regulatory networks, Moe activity provides a spatiotemporal framework to control cell shape transformations during division (see Model of the spatiotemporal regulation of Moe activity throughout the successive steps of the cell cycle). The increase in cortical rigidity that drives cell shape remodeling at the interphase/mitosis transition involves a Pp1-87B/Slik molecular switch that timely regulates Moe phosphorylation (Roubinet, 2011).

PI(4,5)P2 was further identified as a spatial cue that controls Moe distribution at the cortex. This latter aspect coordinates the spatial balance in cortical stiffness/contractility that is required for anaphase cell elongation and cytokinesis. It is proposed that the concerted action of these two regulatory networks ensures the proper series of mitotic cell shape transformations required for the fidelity of cell division (Roubinet, 2011).

A global increase in cortical actomyosin forces generate cell rounding at mitosis entry. These forces are transmitted to the plasma membrane through the activation of ERM proteins. At mitosis exit, both cortical contractions and ERM activity must be down-regulated to allow cells to go back to their interphase shape. In Drosophila cells, the Slik kinase was shown to activate Moe at mitosis entry (Carreno, 2008; Kunda, 2008). This study identifies the Pp1-87B phosphatase as essential for Moe inactivation after cytokinesis and in interphase (Roubinet, 2011).

Although Slik homogenously associates with the cell cortex in both interphase and early mitosis, Pp1-87B is cytoplasmic in interphase and relocalizes to the spindle in pro/metaphase. An attractive model would be that together with a 'constitutive' cortical association of the Slik activator in interphase and pro/metaphase, intracellular redistribution of the Pp1-87B inhibitor represents an efficient way to restrict high levels of Moe phosphorylation to mitosis entry. During anaphase, Pp1-87B concentrates near the chromosomes migrating toward the polar cortex, whereas Slik accumulates at the cleavage furrow. In this model, redistribution of both Pp1-87B and Slik after the anaphase onset contributes to enrich Moe at the equator and to decrease it at poles. Finally, relocalization of Pp1-87B in the cytoplasm after cytokinesis would contribute to relax the cortex for the next interphase by maintaining low Moe activity. A growing number of evidence supports that Pp1 phosphatases play important roles in the temporal control of cell division. Pp1-87B being required for mitotic spindle morphogenesis, this phosphatase could contribute to synchronize cell shape control operated through Moe regulation to chromosome segregation. Although additional investigations will be required to unravel how the activity and distribution of Pp1-87B and Slik are regulated, these results indicate that the Slik/Pp1-87B switch represents an important control of Moe activity during the cell cycle (Roubinet, 2011).

The results show that local levels of PI(4,5)P2 provide an additional mechanism to regulate Moe function at the cortex of dividing cells. Several studies have established a role of PI(4,5)P2 in the localization of ERM proteins in polarized processes of differentiated cells. This study provides evidence that during mitosis, PI(4,5)P2-rich membrane domains act as a spatial cue that regulates both Moe distribution and activation at the cortex (Roubinet, 2011).

The distribution of PI(4,5)P2 at the plasma membrane is tightly regulated during mitosis. As in mammalian cells, it was found that PI(4,5)P2 is actively enriched at the equator of anaphase Drosophila S2 cells, suggesting that equatorial accumulation of PI(4,5)P2 is a feature shared by most animal cells. Although a previous study did not detect PI(4,5)P2 enrichment at the cleavage furrow of Drosophila spermatocytes, whether this is caused by an intrinsic difference between mitosis and meiosis or by experimental limitations in vivo remains to be established. However, how this dynamic localization is regulated remained unknown. This study shows that the equatorial enrichment of PI(4,5)P2 relies, at least in part, on the enzymatic activity of Skittles and Pten. During cytokinesis, the equatorial accumulation of PI(4,5)P2 plays a role in cleavage furrow formation and ingression, through controlling the activity and/or recruitment of several components of the contractile ring. PI(4,5)P2 hydrolysis is also necessary for maintaining cleavage furrow stability and efficient cytokinesis. The current findings extend the functional repertoire of PI(4,5)P2 during mitosis to the control of local properties of the mitotic cortex, which are required for polar relaxation and cell elongation. Through functional screenings, novel regulators of cell division were identified among the entire set of enzymes implicated in phosphoinositide biosynthesis. Two main pathways regulate PI(4,5)P2 levels in mitotic cells, and their alterations provoke similar cortical disorganization. The first pathway involves the Pten tumor suppressor, a PI(3,4,5)P3 3-phosphatase. Pten was shown to accumulate at the septum of dividing yeast cells, as well as at the cleavage furrow in Dictyostelium discoideum. The results of living Drosophila cells show a progressive delocalization of Pten from the polar cortex to the equator after anaphase onset, suggesting that Pten dynamics rely on mechanisms conserved throughout evolution. Furthermore, depletion of Pten leads to a significant enrichment of PI(3,4,5)P3 at the cortex, especially at the cleavage furrow. These results show that Pten uses PI(3,4,5)P3 to spatially control PI(4,5)P2 levels at the mitotic cortex (Roubinet, 2011).

The second pathway relies on Skittles, a PI(4)P 5-kinase that plays a major role in regulating the levels and localization of PI(4,5)P2 during mitosis. Skittles switches from an isotropic cortical distribution in pro/metaphase to equatorial enrichment after the anaphase onset. Depletion of Skittles results in a phenotype similar to the mitotic cortical defects observed after inducible PI(4,5)P2 hydrolysis. It was also found that CG10260, a phosphoinositide 4-kinase, contributes to the organization of the mitotic cortex. Genetics screens have identified a role for phosphoinositide 4-kinases in the division of budding and fission yeast as well as for cytokinesis of male spermatocytes in flies. CG10260 is involved in PI(4)P synthesis, the major substrate of Skittles to produce PI(4,5)P2. Together, these data show that Skittles acts as a key regulator of PI(4,5)P2 levels and Moe activation at the mitotic cortex. Interestingly, Skittles is required for Moe activation in Drosophila oocytes, suggesting that this enzyme plays a broad role in the regulation of ERM proteins (Roubinet, 2011).

An important question is how Skittles and Pten are enriched at the equator in anaphase. It has been reported that activated RhoA stimulates a PI(4)P 5-kinase activity and promotes PI(4,5)P2 synthesis in mammalian cells. During anaphase, activated RhoA localizes at the equatorial cortex, where it could recruit and/or activates Skittles to promote PI(4,5)P2 production. This anisotropy in PI(4,5)P2 distribution might be in turn reinforced by the localized activity of Pten, whose membrane association is itself dependent on PI(4,5)P2. Together, the activity of Skittles and Pten could therefore provide a feed-forward regulatory loop of local PI(4,5)P2 levels at the cortex of dividing cells (Roubinet, 2011).

The metaphase/anaphase transition is characterized by a break in cortical symmetry, with concomitant relaxation of the polar cortex and contraction of the equator. The anisotropic distribution of Moe participates in coordinating this differential in cortical tension. Overactivation of Moe impairs cell elongation and causes cytokinesis failure, suggesting that the polar cortex is too rigid for cell division. Accumulation of F-actin at the cleavage furrow can be attributed, at least in part, to a cortical flow of F-actin filaments from polar regions to the equator. Overactivation of Moe at the poles could block this actin cortical flow, through an excessive bridging of the actin cytoskeleton with the plasma membrane, leading to an abnormal stiffness of the polar cortex. Therefore, redistribution of activated Moe from the polar cortex to the equator participates in polar relaxation, anaphase cell elongation, and cytokinesis fidelity (Roubinet, 2011).

Contraction of the equatorial actomyosin ring increases the cytoplasmic pressure exerted on the plasma membrane. Relaxation of the polar cortex is thus required to dissipate this extra pressure by increasing the cellular volume, a process that was proposed to involve short-lived polar blebs. These polar blebs were recently found to play important roles during cell division. Perturbation of their dynamics triggers anaphase spindle rocking and destabilization of cleavage furrow positioning. Although recent studies have addressed how cortical blebs are regulated in interphase, understanding of the signalization that controls dynamics of cortical blebs in mitosis has poorly progressed since pioneering studies. The results show that a transient recruitment of Moe at the mitotic bleb membrane is required for efficient polar bleb retraction, as are the functions of the Moe positive regulators Slik, Skittles, and Pten. Active Moe contributes to cortical bleb organization because alteration of Moe function (or regulation) disrupts actin organization and efficient bleb retraction. This leads to disorganization of the mitotic cortex, characterized by giant blebs that continue growing in an unregulated manner. Therefore, although a global decrease in Moe activity at the polar cortex contributes to cell elongation and cytokinesis, transient and local association of Moe at the rim of polar blebs is important for their retraction. If the binding of Moe to PI(4,5)P2 is required at both the equator and bleb membrane, the influence of the Slik kinase on Moe activation appears different between these two regions of the anaphase cortex. Although Slik depletion abolishes Moe recruitment to polar blebs, remnants of cortical Moe are still visible at the equator, likely as a result of high PI(4,5)P2 levels at the furrow (Roubinet, 2011).

Although these mechanisms synergistically contribute to the cortical contractility at the equator, they also allow cortical relaxation at the polar cortex through control of transient anaphase blebs. It is proposed that this dual mechanism of Moe regulation is exploited by animal cells to ensure proper cell division (Roubinet, 2011).

A dPIP5K dependent pool of Phosphatidylinositol 4,5 Bisphosphate (PIP2) is required for G-Protein coupled signal transduction in Drosophila photoreceptors

Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity occur at the neuronal plasma membranes, yet levels of this lipid at the plasma membrane are remarkably stable. Although the existence of unique pools of PIP2 supporting these events has been proposed, the mechanism by which they are generated is unclear. In Drosophila photoreceptors, the hydrolysis of PIP2 by G-protein coupled phospholipase C activity is essential for sensory transduction of photons. This study identified dPIP5K as an enzyme essential for PIP2 re-synthesis in photoreceptors. Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane. Overexpression of dPIP5K was able to accelerate the rate of PIP2 synthesis following light induced PIP2 depletion. Other PIP2 dependent processes such as endocytosis and cytoskeletal function were unaffected in photoreceptors lacking dPIP5K function, and are probably carried out by Skittles. These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction. These results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes (Chakrabarti, 2015).

The detection and conversion of external stimuli into physiological outputs is a fundamental property of neurons and depends on intracellular signal transduction pathways. Phosphoinositides, the seven phosphorylated derivatives of phosphatidylinositol are key signalling molecules and of these the most abundant PIP2 has multiple roles in neurons. Several neuronal receptors (such as the metabotropic glutamate, growth factor and sensory receptors) transduce stimuli into cellular information using the hydrolysis of PIP2 by phospholipase C enzymes. Additionally, within the context of neuronal cell biology PIP2 has several roles including cytoskeletal function and several ion channels and transporters require PIP2 for their activity. At the pre-synaptic terminal, a regulated cycle of PIP2 turnover is essential to regulate synaptic vesicle cycling. Thus PIP2 plays multiple roles at the plasma membrane of neurons; hence not surprisingly, changes in phosphoinositide metabolism have been linked to several inherited diseases of the human nervous system. Finally, one of the molecular targets of lithium, used in the treatment of bipolar disorders, is inositol monophosphatase a key regulator of PIP2 turnover in neurons (Chakrabarti, 2015).

Given the multiple functions of PIP2 at the plasma membrane, it is unclear if a common pool of PIP2 supports all these functions. Alternatively, if there are distinct pools, it is unclear how these are generated and sequestered on the nanoscale structure of the membrane. In principle, PIP2 can be generated by the activity of two classes of phosphatidylinositol phosphate kinase (PIPK) enzymes, designated PIP5K and PIP4K; PIP5K phosphorylates PI4-P at position 5 of the inositol ring, whereas PIP4K phosphorylates PI5-P at position 4. Although PIP4K and PIP5K synthesize the same end product, they are not functionally redundant and studies of the mammalian enzymes has defined the molecular basis of substrate specificity. Genes encoding PIP5K are present in all sequenced eukaryotes; however PIP4K appears to be a feature of metazoans; mammalian genomes contain three distinct genes for each of these two activities. However, the functional importance of these two classes of enzymes in generating plasma membrane PIP2 has remained unclear (Chakrabarti, 2015).

Drosophila photoreceptors are a well-established model for analyzing phosophoinositide signaling in-vivo. In these cells, the absorption of photons is transduced into neuronal activity by G-protein coupled, phospholipase Cβ (PLCβ) mediated PIP2 hydrolysis (NorpA). Thus, during phototransduction, PIP2 needs to be resynthesized to match consumption by ongoing PLCβ activity. PIP2 turnover is tightly regulated in photoreceptors; mutants in molecules that regulate PIP2 turnover show defects in phototransduction. However the role of PIPK enzymes in regulating PIP2 synthesis during phototransduction is unknown. This study analyzed each of the three PIPK encoded in the Drosophila genome that could generate PIP2 in the context of phototransduction. This analysis defines three pools of PIP2 supporting distinct molecular processes in photoreceptors (Chakrabarti, 2015).

The hydrolysis of PIP2 by PLC in response to receptor activation is a widespread mechanism of signalling at the plasma membrane. In some cells such as neurons, activation of cell surface receptors by neurotransmitter ligands (e.g glutamate, Ach) or sensory stimuli triggers high rates of PLC activation and rapid consumption of PIP2. Under these conditions, it is essential that levels of PIP2, the substrate for PLC are maintained as failure to do so would likely result in desensitization. In mammalian cells, multiple classes of PIPK, the enzymes that resynthesize PIP2 have been described; yet the contribution of these enzymes to PIP2 resynthesis following PLC activation during cell signalling in vivo remains unclear. Broadly two classes of PIPK can synthesize PIP2 have been described; PIP5K that phosphorylates PI4P at position 5 or PIP4K that can phosphorylate PI5P at position 4. This study has analyzed the consequence of loss of each of these two classes of PIPK to resynthesis following PLC mediated PIP2 depletion during Drosophila phototransduction (Chakrabarti, 2015).

Loss of dPIP5K function results in profound defects in the light activated electrical response as well as slower recovery of plasma membrane PIP2 levels. Conversely overexpression of dPIP5K was able to substantially accelerate the recovery of PIP2 levels following stimulation with a bright flash of light. dPIP5K is localized to the microvillar plasma membrane, the site at which PIP2 needs to be produced to support ongoing light induced PLC activity. Finally, this study found that loss of dPIP5K enhances the ERG defect in a hypomorphic allele of rdgB, a gene with a well-established defect in the response to light. Collectively these observations strongly suggest that dPIP5K activity underlies the conversion of PI4P to PIP2 at the microvillar membrane where it is then available as a substrate for light induced PLCβ activity. By contrast loss of the only PIP4K enzyme in the Drosophila genome has minimal effects on phototransduction and this enzyme is not targeted to the microvillar plasma membrane. These findings also imply that dPIP4K activity (and hence the conversion of PI5P into PIP2) is dispensable for maintaining PIP2 levels during Drosophila phototransduction. This is consistent with a previous study which found no reduction in the levels of PIP2 in flies lacking dPIP4K function. These observations validate the conclusion from biochemical studies in mammalian cells that the levels of PI5P are substantially lower than those of PIP2 and hence it is unlikely to be the source of the majority of PIP2 in cells. The identity of the PI4K isoform that generates the substrate, PI4P used by dPIP5K remains unknown although a recent study in mammalian systems suggests that PI4KIIIα is likely to be the relevant isoform (Chakrabarti, 2015).

Although the ERG response is severely affected in the dPIP5K18 variant, it is not abolished as seen in null mutants of PLCβ (norpA) that are not able to hydrolyse PIP2. Additionally, the resting levels of PIP2 as detected by the PIP2 biosensor are comparable to wild type and following a bright flash of light that depletes PIP2, its levels do recover albeit at a slower rate than in wild type photoreceptors. Given that dPIP5K18 is a protein null allele, these observations imply that there must be a second pool of PIP2 in dPIP5K18 cells that is able to support phototransduction and microvillar PIP2 re-synthesis albeit with lower efficiency. This second pool of PIP2 is likely available with low efficiency for PLC activity in the absence of the dPIP5K dependent pool thus accounting for the residual light response and observed PIP2 dynamics in dPIP5K18 photoreceptors (Chakrabarti, 2015).

The ultrastructure of dPIP5K18 photoreceptors was essentially normal. This was particularly surprising given that in addition to phototransduction, PIP2 at the microvillar membrane is also expected to regulate multiple processes required to maintain normal microvillar structure including dynamin dependent endocytosis as well as cytoskeletal function. However, using multiple readout, molecular readouts of endocytosis and cytoskeletal function were found to be unaffected in dPIP5K18 photoreceptors. These observations imply that the PIP2 required for these processes is not dependent on dPIP5K activity; rather PIP2 generated by a separate PIPK supports these processes. Thus far, dPIP4K has not been detected on the microvillar plasma membrane, dPIP4K29 photoreceptors show normal ultrastructure on eclosion and do not undergo light dependent microvillar degeneration; thus dPIP4K is unlikely to be the critical enzyme that generates the PIP2 required to support dynamin dependent endocytosis, p-Moesin localization, or phototransduction. The Drosophila genome encodes an additional PIP5K activity, sktl that is expressed at low levels in the adult retina but is localized to both the microvillar and basolateral membrane and hence could synthesize PIP2 at both these locations. Complete loss of sktl function is cell lethal and overexpression of sktl in developing photoreceptors results in a severe block in rhabdomere biogenesis whereas overexpression of sktl results in light dependent retinal degeneration in post-development photoreceptors. These findings presumably reflect an essential and non-redundant role for SKTL in supporting fundamental PIP2 dependent cellular processes such as endocytosis and cytoskeletal function that are not dependent on PIP2 hydrolysis by PLC. This model is consistent with the cell-lethal phenotype of photoreceptors that are null for sktl and previous studies showing a role for sktl in supporting cytoskeletal function and endocytosis in other Drosophila tissues and processes such as spermiogenesis and oogenesis (Chakrabarti, 2015).

Collectively, these observations imply that there are at least two pools of PIP2 in photoreceptors; one generated by dPIP5K that is required to support a normal electrical response to light but is dispensable for non-PLC dependent functions of PIP2 in photoreceptors and another that is generated by enzymes other than dPIP5K (most likely SKTL) that is also capable of supporting PIP2 synthesis during the light response albeit with reduced efficiency. In summary the PIP2 pool synthesized by dPIP5K is unique in that it is required for a normal light response and apparently dispensable for other PIP2 dependent functions/processes. It also reflects the existence of distinct/segregated pools of PIP2 on the same microvillar plasma membrane that are maintained by distinct kinases (Chakrabarti, 2015).

A number of previous studies have shown that in multiple eukaryotic cell types, plasma membrane PIP2 levels are remarkably stable, undergoing transient fluctuations despite ongoing PLC mediated PIP2 hydrolysis. However the reasons for this remarkable finding have remained unclear although pharmacological studies have suggested the importance of PIP2 resynthesis in this process. One potential explanation for this idea is the existence at the plasma membrane of two pools of PIP2, a larger but less dynamic pool of that is not normally accessed by PLC and supporting non-PLC dependent functions of this lipid and a second, quantitatively smaller but more dynamic pool that is the substrate for PLC activity. What underpins such pools of PIP2? The existence of separate enzymes that generate unique pools of PIP2 has been previously suggested but there have been limited experimental studies to support this model. In murine platelets where thrombin induced PIP2 hydrolysis appears to be dependent on PIP5K1β but not PIP5Kγ; since both these enzymes are expressed in platelets this implies the existence of two pools of PIP2 in these cells of which the PIP5K1β dependent pool is available for thrombin dependent PIP2 turnover (Chakrabarti, 2015).

This finding together with this study in Drosophila photoreceptors implies that the plasma membrane in general may contain a specific pool of PIP2 dedicated for the use of receptor dependent PLC signalling and synthesized by a specific PIPK. It is possible that given the high rates of PLC activated PIP2 turnover at the plasma membrane (such as the microvillar membrane in photoreceptors) eukaryotic cells have evolved a mechanism to generate distinct PIP2 pool for this purpose so that other PIP2 dependent functions at the plasma membrane remain unaffected by ongoing receptor activated PIP2 hydrolysis. It is likely that dPIP5K and mammalian PIP5K1β represent PIP5K enzymatic activities required to support such a pool of PIP2 at the plasma membrane (Chakrabarti, 2015).

It is presently unclear what properties might make dPIP5K more suitable for generating PIP2 in the context of receptor triggered PLC activity. One possibility is that the kinetic properties of the enzyme encoded by dPIP5K is distinct from that encoded by sktl allowing it to function in the context of high rates of PIP2 turnover. Alternatively (or additionally) within the nanoscale organization of the microvillar plasma membrane, it is possible that dPIP5K is segregated such that PIP2 generated by this enzyme is available within molecular distances of the phototransduction machinery. Interestingly, Drosophila photoreceptors contain within their microvillar membrane a macromolecular signalling complex organized by the PDZ domain protein INAD. It is presently not known if dPIP5K is part of a similar complex but the existence of such mechanisms has been previously shown for mammalian PIP5K1γ in the context of focal adhesion function. Interestingly, it has been reported that the INAD protein complex that includes PLCβ is recruited to detergent resistant membranes during light stimulation which themselves have been previously implicated in the formation of PIP2 microdomains and receptor activated PIP2 turnover. It is possible that the two PIPKs, SKTL and dPIP5K show differential localization to such domains thus generating and segregating such pools of PIP2 and further studies in this direction are likely to provide insight into this issue. Nevertheless this study has provided evidence for the concept of distinct PIPK enzymes as the basis for functionally distinct pools of PIP2 at the plasma membrane. Further analysis in this system is likely to reveal the molecular basis for the organization of PIP2 pools at cellular membranes (Chakrabarti, 2015).



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).


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).


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. sktlDelta20 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).

Phosphatidylinositol 4,5-bisphosphate directs spermatid cell polarity and exocyst localization in Drosophila

During spermiogenesis, Drosophila spermatids coordinate their elongation in interconnected cysts that become highly polarized, with nuclei localizing to one end and sperm tail growth occurring at the other. Remarkably little is known about the signals that drive spermatid polarity and elongation. This study identified phosphoinositides as critical regulators of these processes. Reduction of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) by low-level expression of the bacterial PIP2 phosphatase SigD or mutation of the PIP2 biosynthetic enzyme Skittles (Sktl) results in dramatic defects in spermatid cysts, which become bipolar and fail to fully elongate. Defects in polarity are evident from the earliest stages of elongation, indicating that phosphoinositides are required for establishment of polarity. Sktl and PIP2 localize to the growing end of the cysts together with the exocyst complex. Strikingly, the exocyst becomes completely delocalized when PIP2 levels are reduced, and overexpression of Sktl restores exocyst localization and spermatid cyst polarity. Moreover, the exocyst is required for polarity, as partial loss of function of the exocyst subunit Sec8 results in bipolar cysts. These data are consistent with a mechanism in which localized synthesis of PIP2 recruits the exocyst to promote targeted membrane delivery and polarization of the elongating cysts (Fabian, 2010).

Evidence is provided for a critical role for PIP2 as a regulator of spermatid cyst polarization during spermiogenesis. Reduction of PIP2 levels by ectopic expression of the bacterial phosphoinositide phosphatase SigD or by mutation of the PIP5K Sktl resulted in formation of bipolar spermatid cysts. These cysts lack plasma membranes separating the elongating sperm tails, suggesting an additional defect in membrane addition during elongation. The observed effects on cyst polarity are likely due to reduction of PIP2 or to an imbalance between PIP2 and PI4P because studies examining genetic interactions between SigD-low and two PI4Ks as well as a PI4P phosphatase indicate that elevated levels of PI4P are not responsible for the polarity defects observed in SigD-low cysts (Fabian, 2010).

These results suggest that PIP2 localization in developing sperm primarily results from asymmetric distribution of the PIP5K Sktl rather than from localized action of the PIP3 phosphatase PTEN (phosphatase and tensin homolog), as germ cell clones homozygous for a mutation in PTEN have normal polarity. Subcellular localization of PIP5Ks likely represents a conserved mechanism for ensuring local synthesis of PIP2. Localized distribution of PIP5Ks has recently been implicated in establishing cell polarity in early C. elegans embryos, as well as in root hair and pollen tube elongation in plants (Fabian, 2010).

The factors that regulate localization of PIP5Ks are not well understood. In C. elegans, concentration of the Sktl homolog PPK-1 at the posterior pole of early embryos is dependent on casein kinase 1, which negatively regulates its distribution. In mammalian tissue culture cells, plasma membrane association of PIP5KIα is regulated by Rho and Rac. In neurons, mammalian PIP5KIγ661 is recruited and activated by binding to the focal adhesion protein talin and the clathrin adaptor AP-2. The finding that Sktl localization is abnormal in SigD-low cysts suggests the existence of a positive feedback loop, whereby locally high concentrations of PIP2 retain the PIP5K, which in turn stimulates local PIP2 synthesis. Such a feedback loop could operate by maintaining locally activated Rho family G proteins, which—together with their activators—bind PIP2 and which in turn could recruit PIP5K. Alternatively, the feedback mechanism could be more direct, because PIP5Ks were recently shown to localize to the plasma membrane via positively charged amino acids that bind phosphoinositides. In either case, mislocalization of YFP-Sktl in SigD-low cysts likely reflects a failure to retain sufficiently high levels of PIP2 at the plasma membrane in the presence of SigD. The mechanism by which Sktl concentrates at the growing ends of spermatid cysts remains an area for future study (Fabian, 2010).

This study shows that PIP2 levels affect polarized exocyst distribution in a developmental context. In a survey of proteins that were candidates to be regulated by PIP2 during spermatid cyst polarization, it was discovered that actin cytoskeletal proteins (anillin, P-moesin, and spectrin) associate with the growing ends of the flagellar axonemes in the middle of the bipolar SigD-low cysts. These cytoskeletal proteins do not seem to be involved in establishing or maintaining spermatid polarity in Drosophila. For example, anillin is dispensable for spermatid cyst polarity, and PIP2 binding by β-spectrin is not required for viability or male fertility. In contrast, the exocyst was completely delocalized upon PIP2 reduction, and exocyst mutants show defects in spermatid cyst polarity (Fabian, 2010).

Based on previous studies from yeast and mammalian cells, regulation of the exocyst by PIP2 is likely to be direct. Indeed, binding of the exocyst subunits Sec3 and Exo70 to PIP2 appears critical for exocyst function. Consistent with this idea, it was found that the exocyst subunit Sec8 localizes immediately adjacent to PIP2 at the growing end of spermatid cysts and that localization of Sec8 and Sec6 is strongly influenced by PIP2 levels: reduction of PIP2 caused complete delocalization of the exocyst, and rescue of PIP2 by Sktl expression restored colocalization. Furthermore, the exocyst is required for polarity of Drosophila spermatid cysts. A hypomorphic mutation in fun, which encodes Sec8, caused defects in cyst polarity, whereas loss of function of onr, which encodes Exo84, caused formation of apolar cysts. These defects in polarity, common to exocyst mutants and SigD transgenic flies, were not due to failure of cytokinesis, because cytokinesis was normal in SigD-low flies. Instead, the data suggest that establishment of spermatid cyst polarity relies on localized recruitment of the exocyst, which drives targeted membrane delivery to the growing end. Alternatively, or in addition, PIP2 and the exocyst may help establish the axis of polarization by linking the spermatid nuclear envelope to the plasma membrane, as suggested from analysis of sktl2.3 mutant clones during oogenesis or by regulating formation of the flagellar membrane that surrounds the growing end of the axoneme. Indeed, given the structural similarity between flagella and cilia and the requirement for the exocyst in ciliogenesis, PIP2 could play a conserved role in regulating this critical process (Fabian, 2010).

Rhabdomere biogenesis in Drosophila photoreceptors is acutely sensitive to phosphatidic acid levels

Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. This study shows that before a critical time period during rhabdomere biogenesis in Drosophila photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. It is proposed that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis (Raghu, 2009).

During development, eukaryotic cells undergo morphogenetic changes to suit ongoing physiological needs. Effecting cell shape changes involves complex cell biological processes, including changes in both the cell membrane and the cytoskeletal. An essential element of membrane biogenesis is the need to achieve regulated vesicular transport such that membranes can be delivered to the desired domain of the cell. This process is thought to involve a complex interplay of the physical properties of the lipid constituents in membranes as well as the activities of proteins that can affect membrane curvature. Conceptually, the lipid constituents of the cell membranes could be those with essentially structural roles (such as phosphatidylcholine [PC], phosphatidylethanolamine, phosphatidylserine (PS), and cholesterol) and signaling lipids whose levels change in a regulated manner. These signaling lipids include DAG, its phosphorylated derivative phosphatidic acid (PA), and several phosphorylated species of phosphatidylinositol (PI) (Raghu, 2009).

In the simple eukaryote Saccharomyces cerevisiae that recapitulates most basal transport pathways conserved in higher eukaryotes, genetic analysis has implicated several lipids in regulating membrane traffic. Evidence showing that DAG and PA can affect membrane transport comes from yeast through analysis of SEC14, a gene that encodes a PI/PC transfer protein essential for viability and transport from the Golgi. The sec14 phenotype can be suppressed/bypassed by mutants in several genes that control biosynthesis of PI and PC. However, the ability of such mutants to bypass sec14 has an obligate requirement for SPO14 that encodes phospholipase D (PLD), an enzyme that generates PA from PC. Although Spo14p is not required for vegetative growth, it is required to form the prospore membrane (Rudge et al., 1998) and for PA synthesis during sporulation; loss of Spo14p leads to accumulation of undocked prospore membrane precursors vesicles on the spindle pole body. Thus, in yeast, PA generated by Spo14p activity plays a key role in this membrane trafficking event. Although the analysis of spo14 has implicated PA and its downstream lipid metabolites in membrane transport, to date there is little direct evidence to suggest that PA can function as a regulator of membrane traffic in metazoans. The idea that PA can function in a signaling capacity during membrane transport has been fueled by the observations that (1) in vitro ADP ribosylation factor (Arf) proteins, key mediators of membrane transport, can regulate the activity of PLD, (2) overexpression of PLD in several different cell types affects processes likely to require exocytosis, and (3) overexpression of mammalian PLD1 is reported to promote generation of β-amyloid precursor protein-containing vesicles from the TGN. However, the role of PA in regulating secretion in these settings remains unclear, and currently, there is little evidence linking demonstrable changes in PA levels with the molecular machinery that regulates membrane traffic in vivo (Raghu, 2009).

This study used Drosophila melanogaster photoreceptors as a model system to test the effect of altered PA levels on membrane traffic. It was shown that elevated levels of PA, achieved by manipulation of three genes (CDP diglyceride synthetase, Phospholipase D and retinal degeneration A), disrupt membrane transport to the apical domain of photoreceptors with defects in the endomembrane system (Raghu, 2009).

Work in mammalian cell culture models has suggested that the activity of a PI4K enzyme generating a Golgi localized pool of PI(4)P is important for regulating TGN exit. To test whether the activity of a PI(4)P-generating enzyme might be critical for rhabdomere biogenesis, the effect of down-regulating PI4K activity in developing photoreceptors was tested.The effect of down-regulating two genes that could encode PI4K activity: CG2929 (PI4KIIβ) and CG10260 (PI4KIIIα). This analysis revealed that down-regulation of CG2929 using RNAi phenocopied key aspects of the phenotype of Pld overexpression: (1) down-regulation in the levels of Rh1 protein, (2) formation of small and deformed rhabdomere, and (3) accumulation of abnormal endomembranes within the cell body. These findings suggest that the activity of PI4K is important for membrane transport (Raghu, 2009).

This study elevated PA levels using either cds1 and Pld or rdgA overexpression in each of which the only common and immediate biochemical outcome is the accumulation of PA. Using EM to directly visualize photoreceptor membranes, it was demonstrate that all three genetic manipulations cause defects in endomembrane organization characterized by a reduction in the size of the apical rhabdomere membrane and/or the accumulation of expanded membranous structures in the cell body. These observations, which are consistent with a defect in membrane transport to the apical domain, are highly reminiscent of defects seen in photoreceptors from Drosophila p47 and Rab11 mutants. Importantly, it was also demonstrated that in all three genotypes used to modulate PA levels, the abundance of a single molecular species of PA (16:0/18:2) was elevated without changes in the mass of structural lipids such as PC or of signaling lipids such as PI and DAG. Because this species of PA accounts for <10% of the total PA in photoreceptors, it is hypothesized that it represents a quantitatively minor phospholipid that functions in a signaling capacity to modulate membrane transport. The importance of PA for the described phenotypes is supported by the observation that overexpression of a type II PA phosphatase is able to partially revert the defects in rhabdomere biogenesis and endomembrane structure. Together, these findings provide compelling evidence that PA can affect the transport and organization of endomembranes in metazoan cells (Raghu, 2009).

Interestingly, although cds1, Pld, and rdgA overexpression all caused endomembrane defects in photoreceptors, the ultrastructural features of the abnormal transport intermediates were variable. All three genotypes showed variable degrees of defect in rhabdomere biogenesis. In addition, in the case of cds1, the accumulated endomembranes in the cell body resembled ER-like structures; with Pld overexpression, there were concentric and sheetlike tubular membranes, whereas with rdgA overexpression, in addition to tubular membranes, there were several vesicular intermediates that accumulated. It is likely that these differences reflect the distinct subcellular locations at which PA accumulates in each genotype. In cds1, PA probably accumulates in the ER site at which CDP-DAG synthase activity is normally present; PLD localization is limited to a compartment at the base of the rhabdomeres, and when overexpressed, DGK is distributed in punctate fashion throughout the ER. The generation of a suitable probe to visualize PA levels in a spatial dimension will be required to address this issue (Raghu, 2009).

During development, the precursor cells of the Drosophila eye undergo a substantial increase in size with the concomitant requirement for generating new plasma membrane. During the last 30% of pupal development, photoreceptors show an approximately fourfold increase in plasma membrane surface area, a process that requires a massive surge in polarized membrane transport capacity starting at ~70% pupal development. This study has defined a critical time window ~70% pupal development before which elevation of PA levels by overexpressing Pld results in the endomembrane defects. As this window precedes the onset of rapid membrane transport accompanying rhabdomere biogenesis, it is postulated that PA regulates the activity of a component of the molecular machinery that mediates polarized membrane transport during this period. Conceptually, in this respect the current findings are reminiscent of observations in the yeast Spo14 mutant, in which membrane transport defects are evident only during the generation of the prospore membrane. These findings are the first report of regulation of polarized membrane transport by PA in metazoans (Raghu, 2009).

During this study, it was observed that the effects of elevated PA (through both cds1 and Pld overexpression) were sensitive to the activation state of Arf1. In the cds1 mutant, in which PA is likely to be elevated in the ER, overexpression of the Arf1-GEF garz resulted in significantly less developed apical rhabdomere membrane but was not associated with enhanced accumulation of membranes in the cell body, which is consistent with the known effects of expressing constitutively active Arf1 in cells. In contrast, overexpression of dArf1-GAP resulted in an enhancement of defective rhabdomere biogenesis as well as a massive accumulation of ER membrane-like intermediates in the cell body. This observation suggests that the PA accumulating at the ER in cds1 influences the Arf1 cycle in this setting, resulting in the transport defects described. Previous biochemical analysis has shown that the activity of Arf1-GAP proteins can be regulated by at least three different lipids relevant to this study, namely PC, DAG, and PA. In the lipidomic analysis of cds1 retinae, it was found that the levels of 34:2 DAG and 34:2 PC were no different from wild type, whereas levels of 34:2 PA were elevated. On the basis of these findings, it is likely that the 34:2 PA that accumulates in cds1 photoreceptors causes the transport defects that were described by down-regulating the activity of Arf1 via dArf1-GAP (Raghu, 2009).

The development and maintenance of apical membranes in polarized cells requires both sorting at the TGN with exocytic transport as well as endocytosis. Thus, the phenotypes resulting from PLD overexpression could be a result of (1) altered membrane transport along one of the steps in the secretory pathway from the ER to the developing rhabdomere or (2) the consequence of enhanced endocytosis from the rhabdomere into the cell body (Raghu, 2009).

Experimental evidence presented in this study shows that in photoreceptors overexpressing Pld, the defect in rhabdomere biogenesis was dependent on the levels of active Arf1. In contrast, it was found that (1) altering the activity of Arf6, (2) down-regulation of α-adaptin, and (3) a reduction in the function of dynamin (shi) did not suppress the effects of overexpressing Pld. Collectively, these three observations strongly suggest that excessive clathrin-mediated endocytosis of rhabdomeral plasma membrane does not underlie the endomembrane defects resulting from Pld overexpression. A recent study has suggested a role for Arf1 in regulating a dynamin-independent endocytic pathway in Drosophila cells. The role of this pathway in the effects of Pld overexpression remains unknown (Raghu, 2009).

Arf1 also exerts several effects on distinct steps of the exocytic pathway, including bidirectional transport between the ER and Golgi between Golgi cisternae and the regulation of exit from the late Golgi. In photoreceptors overexpressing Pld, the current analysis suggests that ER to trans-Golgi transport was normal, implying that the observed phenotypes are likely to involve a transport step between the TGN and plasma membrane, although observed phenotypes do not phenocopy exocyst loss of function. In Drosophila photoreceptors, PLD localizes to a restricted subcompartment at the base of the rhabdomeres. Although the molecular identity of this compartment has not been established, its subcellular localization is consistent with the ability of PA produced by PLD to regulate transport between the rhabdomeres and cell body. In TEMs of photoreceptors overexpressing Pld, the endomembranes that were observed in the cell body showed a tubulovesicular morphology extending throughout the cytoplasm. These membranes resemble large pleiomorphic carriers, transport intermediates that derive from the TGN destined for acceptor compartments like the plasma membrane. Furthermore, vesicles containing proteins destined for and normally restricted to the apical rhabdomere membrane (such as Rh1) are found in the cell body of photoreceptors overexpressing Pld. These observations are particularly interesting in the light of previous studies suggesting that PA generated by PLD can regulate the release of vesicles from the Golgi in an Arf1-dependent manner. However, in the absence of a clear identification of the accumulated membranes, the precise definition of the affected transport intermediates that were observed remains elusive (Raghu, 2009).

Arf1 can influence several events at the TGN, including the recruitment and activation of phospholipid-metabolizing enzymes. These include the recruitment and activation of PI4KIIIβ, generating PI(4)P, as well a direct role in activating the type I PIPkin on Golgi membranes in vitro. During this study, it was found that (1) down-regulating the levels of a PI4K expressed during photoreceptor development phenocopies key aspects of that seen with Pld overexpression, and (2) a strong hypomorph of the type I PIPkin (sktl) was able to substantially suppress the effects of Pld overexpression on rhabdomere biogenesis. These observations reflect the importance of tightly regulating type I PIPkin activity by PA for normal transport to the apical domain in polarized cells. They suggest that the regulation of PI(4)P levels is critical for rhabdomere biogenesis. In the context of interpreting the effects of Pld overexpression, it is possible that raised PA levels lead to enhanced activity of type I PIPkin consuming PI(4)P at the TGN, resulting in consequent transport defects to the apical membrane. Although it has not been possible to demonstrate reduced PI(4)P or increased PI(4,5)P2 levels at the Golgi in photoreceptors overexpressing Pld, the observation that overexpression of sktl in developing photoreceptors before the critical time window (but not a kinase-dead version) results in a massive defect in rhabdomere biogenesis underscores the importance of tight regulation of type I PIPkin activity during this process. Thus, a tight regulation of the balance of PI(4)P and PI(4,5)P2 levels through Arf1 activity may underlie the effects of PA in this system (Raghu, 2009).

Given the large number of effectors that can be regulated by PA, in the future, it will be important to identify and understand the functions of those that play a role in the biogenesis of rhabdomeres during photoreceptor development (Raghu, 2009).

PI(4,5)P2 produced by the PI4P5K SKTL controls apical size by tethering PAR-3 in Drosophila epithelial cells

The control of apical-basal polarity in epithelial layers is a fundamental event in many processes, ranging from embryonic development to tumor formation. A key feature of polarized epithelial cells is their ability to maintain an asymmetric distribution of specific molecular complexes, including the phosphoinositides PI(4,5)P2 and PI(3,4,5)P3. The spatiotemporal regulation of these phosphoinositides is controlled by the concerted action of phosphoinositide kinases and phosphatases. Using the Drosophila follicular epithelium as a model system in vivo, this study shows that PI(4,5)P2 is crucial to maintain apical-basal polarity. PI(4,5)P2 is essentially regulated by the PI4P5 kinase Skittles (SKTL), whereas neither the phosphatase PTEN nor the PI(4,5)P3 kinase DP110 lead to loss of apical-basal polarity. By inactivating SKTL and thereby strongly reducing PI(4,5)P2 levels in a single cell of the epithelium, the disassembly was observed of adherens junctions, actin cytoskeleton reorganization, and apical constriction leading to delamination, a process similar to that observed during epithelial-mesenchymal transition. Evidence is provided that PI(4,5)P2 controls the apical targeting of PAR-3/Bazooka to the plasma membrane and that the loss of this polarized distribution is sufficient to induce a similar cell shape change. Finally, it was shown that PI(4,5)P2 is excluded from the cell apex and that PAR-3 diffuses laterally just prior to the apical constriction in a context of endogenous invagination. All together, these results indicate that the PIP5 kinase SKTL, by controlling PI(4,5)P2 polarity, regulates PAR-3 localization and thus the size of the apical domain (Claret, 2014).

The results indicate that PI(4,5)P2 distribution is critical for BAZ tethering and AJ apical localization control in order to regulate apical constriction in the follicular epithelium. As PI(4,5)P2 is the major source of PI(3,4,5)P3, a joint implication of PI(3,4,5)P3 in this process cannot be ruled out. However, the results indicate that PI(3,4,5)P3 alone is not implicated during the constriction in anterodorsal cells (ADCs). The apical levels of PI(3,4,5)P3 increase contrary to what is expected if it was jointly implicated together with PI(4,5)P2. Furthermore, the overexpression of SKTL modulates PI(4,5)P2 levels in ADCs without changing the level of PI(3,4,5)P3. Hence, altogether and in accordance with previous observations in other Drosophila tissues, the results suggest that PI(4,5)P2 is the important phosphoinositide during the regulation of apical constriction. Interestingly, the results further reveal an efficient regulatory process that tightly limits the apical amount of SKTL, especially during tubulogenesis (Claret, 2014).

Drosophila or mammalian PAR-3/BAZ has been shown to bind to a broad spectrum of phosphoinositides in vitro. This paper reports that in vivo, PI(4,5)P2 is crucial for BAZ membranous localization, but that PI(3,4,5)P3 is dispensable. AJs are also affected by PI(4,5)P2 depletion, though potentially indirectly. In fact, it has been shown that BAZ and the actin cytoskeleton are two major contributors to AJ stability (Claret, 2014).

Previously work has shown that in the Drosophila oocyte, SKTL is required for BAZ and PAR-1 localization to the cortex. However, the mutual exclusion between PAR-1 and BAZ prevented assessing which one is delocalized first. In FCs at stage 9/10, the apical-basal polarity is very stable, perhaps because of AJs. In these cells, evidence is provided that PI(4,5)P2 specifically affects BAZ, but not PAR-1, and that BAZ localization relies mostly on the PI4P5K SKTL, rather than the PI3 phosphatase PTEN. The results, together with previous biochemical studies, suggest that PI(4,5)P2 is crucial for BAZ apical localization (Claret, 2014).

In Drosophila, the function of MOE with regards to AJs seems to vary depending on the tissue. In the wing disc, moe mutant cells can be extruded basally from the pseudoepithelium, suggesting than in this tissue MOE is important for epithelium integrity. However, in photoreceptor cells, MOE is dispensable for AJ maintenance and is rather important for apical morphogenesis. In FCs, MOE inactivation upon PI(4,5)P2 removal or MOE depletion has no effect on apical domain size and epithelium integrity. Therefore, the delamination of PI(4,5)P2 depleted cells seems to be independent of MOE (Claret, 2014).

It is proposed that the apical downregulation of PI(4,5)P2 during dorsal appendage morphogenesis may be crucial for the onset of tubulogenesis, when cells should constrict their apices in order to allow epithelial invagination. Indeed, a more drastic downregulation of PI(4,5)P2 by inactivation of SKTL triggers columnar epithelial cells to adopt a bottle shape before delamination, resembling an EMT. It should be noted that an active participation of wild-type neighboring cells in the delamination of sktl mutant cells cannot be excluded. Surprisingly, during the first steps of tubulogenesis, phospholipid polarity is totally reversed, with PI(3,4,5)P3 apical and PI(4,5)P2 lateral. This inversion does not appear to be the simple outcome of DP110 activation and production of PI(3,4,5)P3 from PI(4,5)P2 at the apex. Therefore, another process must contribute to the decrease in PI(4,5)P2 at the apical membrane. Interestingly, the PI(4,5)P2 phosphatase OCRL interacts with clathrin-coated pits and the GTPase RAB5, which is particularly enriched in the subapical region in ADCs. Thus, it is possible that endocytosis of PI(4,5)P2 leads to its apical exclusion. Likewise, this may involve a concomitant downregulation of the activity of the PI4P5K SKTL (Claret, 2014).

Finally, evidence is provided that the level of PI(4,5)P2 required for polarity maintenance in Drosophila epithelia is controlled by the PI4P5K SKTL, rather than by the tumor-suppressor gene PTEN. These results suggest that the control of PI4P5K activity is important during normal developmental but could also be implicated in pathological processes such as metastasis (Claret, 2014).


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skittles: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 25 March 2015

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