BIOLOGICAL OVERVIEW

Skittles is a type of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) recently identified in Drosophila (Knirr, 1997a). Phosphoinositol lipids have been postulated to play important roles in various cellular processes including growth, differentiation, and vesicular secretion (for more information, see Drosophila Phosphotidylinositol 3 kinase 92E). The phosphatidylinositol pathway consists of a series of conversions of phosphatidylinositol (a membrane lipid bearing a sugar moiety attached to the lipid via an intermidiate phosphate residue) into singly, doubly, and triply phosphorylated products (Carpenter, 1996). An important branching point in the pathway occurs when phosphatidylinositol 4-phosphate (PtdIns[4]P) is phosphorylated to become phosphatidylinositol 4,5-bis-phosphate (PtdIns[4,5]P2 or PIP2), a step catalyzed by phosphatidylinositol 4-phosphate 5-kinase (PIP5K; Boronenkov, 1995 and Ishihara, 1996). There are two types of PIP5Ks (PIP5KI and PIP5KII) with distinct biochemical and immunohistochemical properties, but they both catalyze the conversion of PtdIns[4]P into PIP2 (reviewed in Loijens, 1996b). Skittles corresponds to a PIP5KI (Knirr, 1997a). The hydrolysis of PIP2 by phospholipase C (PLC) produces the second messengers diacylglycerol (DAG) and inositol tris-phosphate (IP3). DAG is an activator of protein kinase C (PKC: see Drosophila Protein kinase C), and IP3 plays an important role in the release of intracellular calcium. In addition, PIP2 is converted into phosphatidylinositol 3,4,5-tris-phosphate, which activates some PKC isoforms (Hassan, 1998 and references).

PIP2 is itself a second messenger that has been implicated in the modulation of the function of cytoskeletal regulatory proteins such as profilin, cofilin, fascin, and gelsolin (Janmey, 1994). There is also evidence that phosphoinositide metabolism is involved in signal transduction and cytoskeleton regulation via the interaction with the Rho family of small G proteins (Chong, 1994 and Ren, 1996). Rho and Rac small GTPases associate with type-I phosphatidylinositol 4-phosphate 5-kinase to regulate the production of phosphatidylinositol 4,5-bisphosphate. This lipid appears to mediate some of the effects of Rho and Rac on the actin cytoskeleton. The genes for several type-I phosphatidylinositol 4-phosphate 5-kinases have been cloned recently but it is not known which ones interact with Rho and/or Rac. Rho family GTPases also interact with phosphatidylinositol 3-kinase (see Drosophila Pi3K92E), though this kinase can be either upstream or downstream of the GTPases depending upon the system (Ren, 1998). Other work has suggested an interaction between phosphoinositides and receptor tyrosine kinases. It has also been suggested that PIP5K function may be associated with, or required for, DNA synthesis and cell proliferation. Finally, PIP5KI has been shown to be required for vesicular secretion in PC12 cells (Hay, 1995 ), while PIP5KII appears to be involved in vesicular trafficking in the budding yeast (Yamamoto, 1995). Most of the understanding of how PIP5Ks function to regulate cellular processes is derived from in vitro data. Whether the various, and apparently distinct, functions in which PIP5K is thought to be involved are related remains unknown. It also remains to be established whether these postulated roles for PIP2 are relevant in vivo and how the modulation of PIP5K levels affects development in animals (Hassan, 1998 and references).

The cloning of Drosophila skittles (sktl) makes the genetic and developmental analysis of the in vivo requirements of this gene possible and facilitates understanding the role(s) played by phosphoinositides in various tissues and cell types. sktl has been shown to be essential for cell and organism viability and is required for cytoskeletal regulation during sensory structure development. sktl is also required for germline development. This analysis resolves an issue pertinent to the function of another gene, inscuteable (insc). sktl maps to the first intron of insc, whose function is required for cell fate determination during neuronal and myogenic lineage development (Kraut, 1996a; Knirr, 1997b; Ruiz-Gomez, 1997 and Carmena, 1998). To date, all studies on neuronal insc function have been carried out using deletion alleles that remove or affect both genes, allowing for the possibility that the described phenotypes may be in part due to the loss of sktl or from the combined loss of sktl and insc. Hassan (1998) showed that the loss of sktl is not responsible for the insc phenotype.

Knirr (1997a) reported that sktl is expressed in germ cells during oogenesis. At stage 6, sktl expression is restricted to the future oocyte. By stage 9, expression is initiated in the nurse cells. At the end of oogenesis, large amounts of sktl transcript are present in the mature egg. To examine the function of sktl in germline development, sktl germline clones were generated using one of the excision alleles (sktl^Delta15). Negative control crosses in which no recombination was induced result in 100% female sterility. The ovaries of these females, carrying the dominant sterile ovo^D1 marker, are severely atrophic and show very early arrest of egg chamber development. Positive control crosses showed that 47% of the females were fertile. In contrast, all sktl recombinant females are sterile. Ovaries were dissected and stained with DAPI to reveal the nuclei. Oogenesis in these females is arrested after stage 10, and very few eggs are fully developed. Arrested egg chambers show defects in nurse cell nuclei at and after stage 10, but no defects in nuclear morphology are seen before that stage. The affected nuclei appear very small and fragmented, suggesting that sktl is required for nurse cell viability, and therefore proper egg chamber development. In contrast, the oocyte nucleus does not appear to be affected. The few eggs that did develop were smaller than the eggs produced by control flies and show defects in their dorsal appendages. Generally, the dorsal appendages of sktl mutant eggs are short and thick. The sterility associated with the partial loss of sktl in the female germline precludes the determination of the consequences of the loss of sktl in embryos (Hassan, 1998).

sktl is expressed widely in the wing disc. To examine the function of sktl in wing disc development sktl mutant clones were generated using the sktl^Delta5 and sktl^Delta15 alleles. The absence of the yellow marker was used to identify the clones. Approximately 76% of the control flies had yellow bristle clones on their notum, legs, and wing margin. These bristles showed no defects. In contrast, only 1–2% of the recombinant flies had yellow bristles on the notum. In addition the size of these clones was markedly reduced in comparison to control flies: each clone consisted of either a single bristle, or rarely, two bristles. The few mutant bristles recovered showed structural abnormalities ranging from a wavy shape (in most cases) to sharp bends (in a few cases), suggesting cytoskeletal defects. No mutant bristles were observed on the wing margin or the legs. The very small number of clones obtained combined with the small size of each clone suggest that during wing disc development sktl is required for either cell viability, proliferation, or both (Hassan, 1998).

To determine if sktl is required in the eye disc, where it is abundantly expressed, sktl mutant clones were generated. In control experiments, 45% of the recombinant flies had white eye clones of variable sizes. In contrast, no sktl mutant clones were observed, supporting the conclusion that sktl function is required for cell viability or cell division in imaginal discs. It should be noted that third instar larvae transheterozygous for the sktl^Delta15 and fam^k07505 alleles show no defects in the sizes of the imaginal discs and the brain. Therefore it is more likely that the absence of sktl clones results from an effect on cell viability. While this hypothesis is favored, the possibility cannot be excluded that the sktl^Delta15/fam^k07505 combination, while being lethal, is not severe enough to reveal a role for sktl in cell proliferation (Hassan, 1998).

The Drosophila adult peripheral nervous system has cells that extend sensory bristles rich in actin, providing an excellent system for the study of the defects in cytoskeleton assembly. Mutations in the Drosophila actin interacting proteins profilin (chickadee in Drosophila) and fascin (singed in Drosophila) show severe defects in bristle morphology. Loss of function of either protein results in bristles that lack actin filament integrity, causing bending and branching during extension. The analysis of Skittles indicates that the alteration of PIP5KI levels results in structural defects in sensory bristles, providing genetic evidence for the involvement of PIP5KIs in cytoskeletal regulation. Ectopic expression of Skittles causes ectopic bristle formation on the notum and wing blade. The appearance of ectopic bristles on the wing blade was seen in ~20% of the flies, but the majority of the flies (70%) showed ectopic bristles on the notum. These results prompted an examination of the effects of generalized overproduction of sktl in the wing disc. Using the 32B-Gal4 driver, which is expressed ubiquitously at high levels in the third instar wing disc, overexpression of sktl results in ectopic bristles (macrochaetae and microchaetae) on the notum and the wing blade. On the notum, both the ectopic and normal bristles showed severe structural defects. The structural defects of the bristles are consistent with a role for sktl in regulating cytoskeletal components. All ectopic bristles were associated with socket cells, suggesting that the production of ectopic bristles may be the result of the specification of extra sense organ precursors rather than the transformation of a socket cell into a bristle cell. The phenotypes observed in sktl clones are similar but not identical to those observed in singed and chickadee mutants. Specifically, while the sktl loss- and gain-of-function mutations resulted in bent and wavy bristles, no branching bristles were observed. Finally, it is interesting to note that mutations in all three genes (chickadee, singed, and sktl) result in female sterility. Therefore, both germline and bristle development present model systems in which to study the interactions between PIP5K and the actin cytoskeleton (Hassan, 1998).

The appearance of extra bristles associated with socket cells as a result of the overexpression of sktl in the wing disc can be explained in one of two ways: extra cell division, or specification of an extra precursor cell. The sterility resulting from the removal of the maternal component and the failure of somatic clones to survive does not allow for a correlation of the ectopic production of bristles with a loss-of-function phenotype. However, it is interesting to note that cytoskeleton-associated proteins like Inscuteable (Kraut, 1996a and Kraut, 1996b) and sanpodo (a Drosophila tropomodulin homologue; Dye, 1998) play a significant role in cell fate specification in the nervous system. This result suggests that sktl too may play a role in cell fate specification in the peripheral nervous system (Hassan, 1998).

Phosphatidylinositol 4,5-bisphosphate regulates cilium transition zone maturation in Drosophila melanogaster

Cilia are cellular antennae that are essential for human development and physiology. A large number of genetic disorders linked to cilium dysfunction are associated with proteins that localize to the ciliary transition zone (TZ), a structure at the base of cilia that regulates trafficking in and out of the cilium. Despite substantial effort to identify TZ proteins and their roles in cilium assembly and function, processes underlying maturation of TZs are not well understood. This study reports a role for the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) in TZ maturation in the Drosophila melanogaster male germline. Reduction of cellular PIP2 levels by ectopic expression of a phosphoinositide phosphatase or mutation of the type I phosphatidylinositol phosphate kinase Skittles induces formation of longer than normal TZs. These hyperelongated TZs exhibit functional defects, including loss of plasma membrane tethering. It is also reported that the onion rings (onr) allele of Drosophila exo84 decouples TZ hyperelongation from loss of cilium-plasma membrane tethering. These results reveal a requirement for PIP2 in supporting ciliogenesis by promoting proper TZ maturation (Gupta, 2018).

Cilia are sensory organelles important for signalling in response to extracellular cues, and for cellular and extracellular fluid motility. Consistent with their importance, defects in cilium formation (i.e. ciliogenesis) are associated with genetic disorders known as ciliopathies, which can display neurological, skeletal and fertility defects, in addition to other phenotypes. Many ciliopathies are associated with mutations in proteins that localize to the transition zone (TZ), the proximal-most region of the cilium that functions as a diffusion barrier and regulates the bidirectional transport of protein cargo at the cilium base. For example, the conserved TZ protein CEP290 is mutated in at least six different ciliopathies and is important for cilium formation and function in humans and Drosophila (Basiri, 2014). Although the protein composition of TZs has been investigated in various studies, the process of TZ maturation, through which it is converted from an immature form to one competent at supporting cilium assembly, is relatively understudied (Gupta, 2018).

Ciliogenesis begins with assembly of a nascent TZ at the tip of the basal body (BB). During TZ maturation, its structure and protein constituents change, allowing for establishment of a compartmentalized space, bounded by the ciliary membrane and the TZ, where assembly of the axoneme, a microtubule-based structure that forms the ciliary core, and signalling can occur. In Drosophila, nascent TZs first assemble on BBs during early G2 phase in primary spermatocytes. This occurs concomitantly with anchoring of cilia to the plasma membrane (PM), microtubule remodelling within the TZ, and establishment of a ciliary membrane that will persist through meiosis (Gupta, 2018).

TZ maturation has been described in Paramecium, Caenorhabditis elegans and Drosophila (Gottardo, 2013), and is most readily observed in the Drosophila male germline by an increase in TZ length. Previous work has shown that the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) is essential for proper axoneme structure in the Drosophila male germline. PIP2, which is one of seven different phosphoinositides (PIPs) present in eukaryotes, localizes primarily to the PM, where it is required for vesicle trafficking among other processes. PIP2 has recently been linked to cilium function. Although the ciliary membrane contains very little PIP2 due to action of the cilium resident PIP phosphatase INPP5E, the cilium base is enriched in PIP2. Inactivation of INPP5E causes a buildup of intraciliary PIP2, which disrupts transport of Hedgehog signalling proteins in vertebrates and ion channels involved in mechanotransduction in Drosophila (Park, 2015). In light of current understanding of PIP2 as a modulator of cilium function, this study sought to investigate the cause of defects observed in axoneme assembly in Drosophila male germ cells with reduced levels of PIP2 (Gupta, 2018).

To investigate how reduction of cellular PIP2 affects ciliogenesis in the Drosophila male germline, transgenic flies were used expressing the Salmonella PIP phosphatase SigD under control of the spermatocyte-specific β2-tubulin promoter (hereafter β2t-SigD). To examine whether axoneme defects in β2t-SigD were caused by aberrant TZ function, localization of fluorescently-tagged versions of the core centriolar/BB protein Ana1 (CEP295 homolog) and the conserved TZ protein Cep290 was analyzed during early steps of cilium assembly. Cep290 distribution appeared similar in control and β2t-SigD in early G2 phase, when TZs are still immature. In contrast, Cep290-labelled TZs were significantly longer in β2t-SigD compared to controls by late G2, following TZ maturation. Unlike Drosophila cep290 mutants, which contain longer than normal BBs, Ana1 length was not affected in β2t-SigD, and no strong correlation was observed between Cep290 and Ana1 lengths. Consistent with this result, the ultrastructure of BBs in β2t-SigD is normal, and localization of the centriolar marker GFP-PACT is similar in controls and β2t-SigD. In contrast, TZ proteins Chibby (Cby) and Mks1 exhibited hyperelongation in β2t-SigD, indicating that this phenotype is not unique to Cep290. TZ hyperelongation was highly penetrant (>70%, n >200) and showed high correlation (>0.95) within syncytial germ cell cysts, suggesting a dosage-based response to a shared cellular factor, presumably SigD. Despite persistence of hyperelongated TZs through meiosis, axonemes were able to elongate in post-meiotic cells. Nonetheless, the ultrastructure of these axonemes is frequently aberrant, either lacking nine-fold symmetry or containing triplet microtubules in addition to the usual doublets (Gupta, 2018).

Although PIP2 is its major substrate in eukaryotic cells in vivo, SigD can dephosphorylate multiple PIPs in vitro. To address whether TZ hyperelongation observed in β2t-SigD represented a physiologically relevant phenotype due to decreased PIP2, attempts were made to rescue this phenotype by co-expressing β2t-SigD with fluorescently-tagged Skittles (Sktl) under control of the β2- tubulin promoter. Sktl expression was able to suppress TZ hyperelongation to various degrees in a cilium-autonomous manner. Furthermore, the BB/TZ protein Unc-GFP exhibited TZ hyperelongation at a low penetrance in sktl2.3 mutant clones, indicating that Sktl is important for TZ maturation. Vertebrate type I PIP kinase PIPKIγ is important for cilium formation in cultured cells. The Drosophila PIPKIs, Sktl and PIP5K59B, arose from recent duplication of the ancestral PIPKI gene, and are not orthologous to specific vertebrate PIPKI isoforms. Sktl has diverged more than its paralog PIP5K59B and seems to be functionally related to PIPKIγ and the C. elegans PPK-1 in having roles at cilia. However, unlike the human PIPKIγ, which licenses TZ assembly by promoting CP110 removal from BBs, the current results suggest that Sktl functions in regulating TZ length but not TZ assembly. Consistent with this, neither inactivation nor overexpression of cp110 affects cilium formation in Drosophila, and Cp110 is removed from BBs in early primary spermatocytes (Gupta, 2018).

Attempts were made to examine whether TZ hyperelongation due to SigD expression affected TZ function. Following meiosis in the Drosophila male germline, TZs detach from BBs and migrate along growing axonemes, maintaining a ciliary compartment at the distal-most ~2μm, where tubulin is incorporated into the axoneme. As shown by Unc and Cep290 localization, TZs in β2t-SigD were frequently incapable of detaching from BBs and migrating along axonemes despite axoneme and cell elongation. Indeed, the previously reported 'comet-shaped' Unc-GFP localization in β2t-SigD persists during cell elongation after meiosis despite elongation of the axoneme (Gupta, 2018).

In Drosophila and humans, BBs consist of microtubule triplets, whereas axonemes contain microtubule doublets due to termination of C-tubules at the TZ (Gottardo, 2013). Consistent with a defect in this transition and the presence of microtubule triplets in axonemes in β2t-SigD, a subset of cilia (<5%) in β2t-SigD contained puncta of Ana1 at the distal tips of TZs. Treatment of germ cells with the microtubule-stabilizing drug Taxol increased penetrance of this phenotype from <5% in untreated cells to >25% in cells treated with 4 μM Taxolwithout significantly affecting Cep290 length. Taxol-treated controls did not exhibit TZ- distal Ana1 puncta. Fluorescently-tagged Asterless (CEP152 homolog), a pericentriolar protein, did not localize to TZ-distal puncta in β2t-SigD suggesting these TZ-distal sites are not fully centriolar in protein composition. Taxol has been hypothesized to disrupt TZ maturation by inhibiting microtubule remodelling in the Drosophila male germline (Riparbelli, 2013). Indeed, similar to β2t-SigD, Taxol-treated male germ cells assemble long axonemes that contain triplet microtubules (Riparbelli, 2013), further supporting a functional relationship between PIP2 and microtubule reorganization in TZ maturation (Gupta, 2018).

Male flies homozygous for the onion rings (onr) mutant of Drosophila exo84 are sterile and exhibit defects in cell elongation and polarity similar to β2t-SigD. Exo84 is a component of the octameric exocyst complex, which binds PIP2 at the PM. To investigate whether defects in TZ hyperelongation could be explained by defective Exo84 function, TZs were examined in onr mutants. Unlike β2t-SigD, onr did not display hyperelongated TZs, suggesting Exo84 is dispensable for TZ maturation. Due to involvement of the exocyst in membrane trafficking, whether cilium- associated membranes were affected in β2t-SigD or onr mutants in a manner similar to dilatory; cby mutants (Vieillard, 2016) was examined. Dilatory (Dila), a conserved TZ protein, cooperates with Cby to assemble TZs in the Drosophila male germline (Vieillard, 2016). TZs in β2t-SigD and onr cells were able to dock at the PM initially, but were unable to maintain membrane connections, and were rendered cytoplasmic, similar to TZs in dila; cby mutants. In addition, fluorescently-tagged Exo70, a PIP2-binding exocyst subunit, localized to BBs. The current results suggest that the exocyst, and Exo84 in particular, regulates cilium-PM association, similar to PIP2, and that TZ hyperelongation and loss of cilium-PM association are genetically separable phenotypes (Gupta, 2018).

Maturation of a TZ from a nascent to a fully functional state, leading ultimately to axoneme assembly and ciliary signalling, requires orchestration of various proteins and cellular pathways. The current results indicate that normal execution of this process requires PIP2, and that depletion of PIP2 induces TZs to grow longer than normal. Similar to β2t-SigD, Drosophila dila; cby and cby mutants display hyperelongated TZs, whereas mks1 mutants have shorter TZs. Because both Cby and Mks1 are hyperelongated in β2t-SigD cells, PIP2 regulates TZ length independently of an effect on Cby or Mks1 recruitment. This study shows that hyperelongated TZs are dysfunctional. Similar to dila; cby (Vieillard, 2016) and cep290 (Basiri, 2014) mutants, axonemes can assemble in β2t-SigD, albeit with aberrant ultrastructure, despite lack of functional TZs or membrane association. The presence of TZ-distal Ana1 puncta in β2t-SigD, without the increase in BB length seen in cep290 mutants lacking a functional TZ barrier, suggests that β2t-SigD selectively disrupts the ability of TZs to restrict C-tubules and Ana1 without abolishing the TZ barrier entirely. CEP295, the human Ana1 ortholog, regulates post-translational modification of centriolar microtubules, which might explain the presence of TZ-distal Ana1 along with supernumerary microtubules in β2t-SigD cells. Asterless (Asl), a pericentriolar protein important for centrosome formation and centriole duplication, did not exhibit this TZ-distal localization, possibly due to differences in dynamics of Ana1 and Asl loading onto centrioles or the more peripheral nature of Asl distribution within the centriole (Gupta, 2018).

The majority of PIP2 at the PM is produced by PIPKIs. Mutation of the PIPKI Sktl induced hyperelongated TZs, and expression of Sktl could suppress TZ hyperelongation in β2t-SigD, suggesting Sktl might function in situ to regulate TZ length. In humans, PIPKIC is linked to lethal congenital contractural syndrome type 3 (LCCS3), which has been suggested to represent a ciliopathy. The recent discovery of a role for another LCCS-associated protein in cilium function corroborates this hypothesis. The current data support the idea that PIPKIs might represent ciliopathy-associated genes or genetic modifiers of disease. Members of the exocyst complex are important for cilium formation in cultured cell lines and zebrafish, but their precise roles in ciliogenesis are not well understood. The subunits Sec3 and Exo70 regulate exocyst targeting to the PM through a direct interaction with PIP2. Previous work has shown that the onr allele of Drosophila exo84 phenocopies defects in cell polarity and elongation observed in β2t-SigD. This study showed that the onr mutation phenocopies loss of cilium-membrane contacts in β2t-SigD but not TZ hyperelongation. Thus, TZ hyperelongation is not a prerequisite for failure of cilium-PM association in male germ cells, and Exo84 uniquely regulates the latter process, potentially by supplying membrane required to maintain cilium-PM tethering. That the TZ is dispensable for this function is supported by the Drosophila cep290 mutant, which lacks a functional TZ but retains cilium-PM association. Notably, EXOC8, which encodes the human Exo84, has been linked to the ciliopathy Joubert syndrome, and a similar defect in ciliogenesis might be present in humans with mutations in EXOC8 (Gupta, 2018).

GENE STRUCTURE

sktl is nested in the first intron (10 kb) of inscuteable (Knirr, 1997b).

PROTEIN STRUCTURE

Amino Acids - 462

Structural Domains

Skittles bears a high degree of similarity to the human STM-7.1 gene. STM-7 has been assigned to the neurodegenerative disorder Friedrichs ataxia and codes for a PI4P5-kinase (Carvajal, 1996). Skittles shows similarity to a human placental PI4P5-kinase, two murine pancreatic PI4P5-kinases and two yeast genes: FAB1 and MSS1. Sktl shows 84% identity to the human STM-7 and the murine PI4P5K-1alpha protein and 60% identity to the yeast MSS4 protein (Knirr, 1997a).

date revised: 11 June 2022

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