InteractiveFly: GeneBrief

four wheel drive: Biological Overview | References

Gene name - four wheel drive

Synonyms -

Cytological map position - 61C1-61C1

Function - enzyme

Keywords - a Golgi-localized lipid kinase, the homologue of phosphatidylinositol 4-kinase IIIβ which mediates the phosphorylation of phosphatidylinositol to generate phosphatidylinositol 4-phosphate, involved in spermatocyte cytokinesis and male fertility, mutants exhibit profound locomotor deficits and shortened lifespan

Symbol - fwd

FlyBase ID: FBgn0004373

Genetic map position - chr3L:305,393-319,884

Classification - PKc_like: Protein Kinases, catalytic domain

Cellular location - cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein

fwd orthologs: Biolitmine
Recent literature
O'Brien, C. E., Younger, S. H., Jan, L. Y. and Jan, Y. N. (2023). The GARP complex prevents sterol accumulation at the trans-Golgi network during dendrite remodeling. J Cell Biol 222(1). PubMed ID: 36239632
Membrane trafficking is essential for sculpting neuronal morphology. The GARP and EARP complexes are conserved tethers that regulate vesicle trafficking in the secretory and endolysosomal pathways, respectively. Both complexes contain the Vps51, Vps52, and Vps53 proteins, and a complex-specific protein: Vps54 in GARP and Vps50 in EARP. In Drosophila, both complexes were found to be required for dendrite morphogenesis during developmental remodeling of multidendritic class IV da (c4da) neurons. Having found that sterol accumulates at the trans-Golgi network (TGN) in Vps54KO/KO neurons, genes that regulate sterols and related lipids at the TGN were investigated. Overexpression of oxysterol binding protein (Osbp) or knockdown of the PI4K four wheel drive (fwd) exacerbates the Vps54KO/KO phenotype, whereas eliminating one allele of Osbp rescues it, suggesting that excess sterol accumulation at the TGN is, in part, responsible for inhibiting dendrite regrowth. These findings distinguish the GARP and EARP complexes in neurodevelopment and implicate vesicle trafficking and lipid transfer pathways in dendrite morphogenesis.
O'Brien, C. E., Younger, S. H., Jan, L. Y. and Jan, Y. N. (2023). The GARP complex prevents sterol accumulation at the trans-Golgi network during dendrite remodeling. J Cell Biol 222(1). PubMed ID: 36239632
Membrane trafficking is essential for sculpting neuronal morphology. The GARP and EARP complexes are conserved tethers that regulate vesicle trafficking in the secretory and endolysosomal pathways, respectively. Both complexes contain the Vps51, Vps52, and Vps53 proteins, and a complex-specific protein: Vps54 in GARP and Vps50 in EARP. In Drosophila, both complexes were found to be required for dendrite morphogenesis during developmental remodeling of multidendritic class IV da (c4da) neurons. Having found that sterol accumulates at the trans-Golgi network (TGN) in Vps54KO/KO neurons, genes that regulate sterols and related lipids at the TGN were investigated. Overexpression of oxysterol binding protein (Osbp) or knockdown of the PI4K four wheel drive (fwd) exacerbates the Vps54KO/KO phenotype, whereas eliminating one allele of Osbp rescues it, suggesting that excess sterol accumulation at the TGN is, in part, responsible for inhibiting dendrite regrowth. These findings distinguish the GARP and EARP complexes in neurodevelopment and implicate vesicle trafficking and lipid transfer pathways in dendrite morphogenesis.

Balanced mitochondrial fission and fusion play an important role in shaping and distributing mitochondria, as well as contributing to mitochondrial homeostasis and adaptation to stress. In particular, mitochondrial fission is required to facilitate degradation of damaged or dysfunctional units via mitophagy. Two Parkinson's disease factors, PINK1 and Parkin, are considered key mediators of damage-induced mitophagy, and promoting mitochondrial fission is sufficient to suppress the pathological phenotypes in Drosophila Pink1/parkin mutants. Additional factors were sought that impinge on mitochondrial dynamics and which may also suppress Pink1/parkinphenotypes. The Drosophila phosphatidylinositol 4-kinase IIIβ homologue, Four wheel drive (Fwd), promotes mitochondrial fission downstream of the pro-fission factor Drp1. Previously described only as male sterile, this study identified several new phenotypes in fwd mutants, including locomotor deficits and shortened lifespan, which are accompanied by mitochondrial dysfunction. Finally, fwd overexpression can suppress locomotor deficits and mitochondrial disruption in Pink1/parkin mutants, consistent with its function in promoting mitochondrial fission. Together these results shed light on the complex mechanisms of mitochondrial fission and further underscore the potential of modulating mitochondrial fission/fusion dynamics in the context of neurodegeneration (Terriente-Felix, 2020).

Mitochondria are dynamic organelles that are transported to the extremities of the cell and frequently undergo fusion and fission events that influence their size, branching and degradation. Many of the core components of the mitochondrial fission and fusion machineries have been well characterised. There include the pro-fusion factors Mfn1/2 and Opa1, and pro-fission factors Drp1 and Mff. Maintaining an appropriate balance of fission and fusion, as well as transport dynamics, is crucial for cellular health and survival as mutations in many of the core components cause severe neurological conditions in humans and model organisms. Recently, a role for phosphatidylinositol 4-phosphate [PI(4)P] in mitochondrial fission has been elucidated in cultured cells (Nagashima, 2020), but the in vivo consequences have not yet been described (Terriente-Felix, 2020).

The mitochondrial fission/fusion cycle has been linked to the selective removal of damaged mitochondria through the process of autophagy (termed mitophagy), in which defective mitochondria are engulfed into autophagosomes and degraded by lysosomes. Two genes that have been firmly linked to the mitophagy process are PINK1 and PRKN. Mutations in these genes cause autosomal-recessive juvenile parkinsonism, associated with degeneration of midbrain dopaminergic neurons and motor impairments, among other symptoms and pathologies. Studies from a wide variety of model systems have shown various degrees of mitochondrial dysfunction associated with mutation of PINK1/PRKN homologues including disrupted fission/fusion. Drosophila have proven to be a fruitful model for investigating the function of the conserved homologues Pink1 and parkin, with these mutants exhibiting robust mitochondrial disruption and neuromuscular phenotypes. Importantly, several studies have shown that the pathological consequences of loss of Pink1 or parkin can be largely suppressed by genetic manipulations that increase mitochondrial fission or reduce fusion (Terriente-Felix, 2020).

To identify genes involved in mitochondrial quality control and homeostasis, an RNAi screen was performed in Drosophila S2 cells to identify kinases and phosphatases that phenocopy or suppress hyperfused mitochondria caused by loss of Pink1 (Pogson, 2014). This study identified the phosphatidylinositol 4-kinase IIIβ homologue, four wheel drive (fwd), whose knockdown phenocopied Pink1 RNAi, resulting in excess mitochondrial fusion. Drosophila mutant for fwd have been reported to be viable but male sterile due to incomplete cytokinesis during spermatogenesis. While muscle-specific knockdown has shown to impact neuromuscular junction formation (Forrest, 2013), no other organismal phenotypes or mitochondrial involvement have been described to date. Thus, this study sought to better understand the role of Fwd in mitochondrial homeostasis (Terriente-Felix, 2020).

This study has characterised fwd mutants for organismal phenotypes associated with Pink1/parkindysfunction and analysed the impact on mitochondrial form and function. Genetic interactions were investigated between fwd and Pink1/parkin, as well as with mitochondrial fission/fusion factors. It was found that loss of fwd inhibited mitochondrial function, causing increased mitochondrial length and branching, and decreased respiratory capacity. These effects were associated with shortened lifespan and dramatically reduced locomotor ability, similar to Pink1 and parkin mutants. Furthermore, fwd overexpression was sufficient to significantly suppress Pink1/parkin mutant locomotor deficits and mitochondrial phenotypes. Interestingly, it was found that the mitochondrial and locomotion phenotypes in fwd mutants can be rescued by loss of pro-fusion factors Marf and Opa1, but the pro-fission activity of Drp1 appears to require fwd. These results support a role for fwd in regulating mitochondrial morphology, specifically in facilitating mitochondrial fission, and further substantiate the important contribution of aberrant mitochondrial fission/fusion dynamics in Pink1/parkinphenotypes (Terriente-Felix, 2020).

Previous work identified fwd as a gene whose knockdown induces mitochondrial hyperfusion in cultured cells, similar to loss of Pink1 (Pogson, 2014). This study has validated that the genetic loss or knockdown of fwd also causes excess mitochondrial fusion in neuronal cells in vivo, leading to increased mitochondrial length and branching. As mitochondrial fission/fusion dynamics have been shown to be important for mitochondrial homeostasis, it is not surprising that this also has an impact on respiration at the organismal level and on organismal fitness and vitality. While fwd mutants have mainly been characterised for their male sterility phenotype, this study describes new organismal phenotypes associated with loss of fwd: profound locomotor deficits and shortened lifespan. Interestingly, while the data reveal a stronger requirement for fwd in the nervous system compared to the musculature to maintain normal motor behaviour, fwd is required in muscle for neuromuscular junction formation (Brill, 200). Furthermore, consistent with the observations on lifespan, Fwd overexpression has previously been shown to confer increased lifespan (Landis, 2003). Thus, Fwd clearly has a more widespread role in organismal vitality than previously appreciated (Terriente-Felix, 2020).

The robust locomotor phenotype allowed a test of the genetic relationship between fwd and core components of the mitochondrial fission/fusion machinery. Given the excess mitochondrial fusion upon loss of fwd, suppression of the organismal phenotypes by reduction of fusion factors Marf and Opa1 was expected. However, it was surprising that overexpression of the fission factor Drp1 was unable to ameliorate organismal phenotypes or even the increased mitochondrial length, though it was able to revert the increased branching caused by loss of fwd. These results suggested that Drp1 requires Fwd to drive mitochondrial fission. Consistent with this, Drp1 overexpression was no longer able to rescue Pink1/parkin mutant phenotypes in the absence of fwd. These genetic experiments strongly hint at a functional link between Drp1 and Fwd but do not illuminate the molecular mechanism underpinning it. Fwd is the Drosophila homologue of phosphatidylinositol 4-kinase IIIβ [PI(4)KB], which mediates the phosphorylation of phosphatidylinositol to generate phosphatidylinositol 4-phosphate [PI(4)P] (Godi, 1999). PI(4)P is one of the most abundant phosphoinositides, which is usually concentrated in the trans-Golgi network (Di Paolo, 2006); thus, the mechanism by which PI(4)P may influence mitochondrial dynamics is not immediately obvious. However, while this manuscript was in preparation, Godi, 1999 reported that Golgi-derived PI(4)P-containing vesicles were required for the final stages of mitochondrial fission (Nagashima, 2020). In that study, the authors found that loss of PI(4)KIIIβ led to hyperfusion and increased branching of the mitochondrial network, consistent with what was observed in this study. Moreover, Nagashima described that while Drp1 was still recruited, it was unable to fully execute the scission event, although the reason is unclear, leading to extended mitochondrial constriction sites. Genetic evidence that the action of Drp1 requires Fwd is consistent with these findings, and provides an in vivo validation of Nagashima's results. Currently, it is unclear why Drp1 overexpression was able to revert the increased branching caused by loss of fwd but the mechanisms of branch formations are not well understood. It is interesting to note that while Nagashima suggest a universal role for PI(4)P in mitochondrial fission, the current in vivo analysis reveals that while fwd affected mitochondrial morphology in the nervous system, it appeared to have a much more limited role in the musculature. These tissue-specific requirements were borne out in the strong locomotor deficits caused by neuronal loss of fwd but much less so by knockdown in muscles. Clearly, further work is required to better understand the complexities of regulated fission/fusion events in different cell contexts in vivo (Terriente-Felix, 2020).

A key role of mitochondrial fission/fusion dynamics is in contributing to a quality control mechanism of mitochondrial sorting to eliminate dysfunctional units via mitophagy. A substantial body of evidence from cellular models indicates that mammalian PINK1 and Parkin act to promote damage-induced mitophagy, and some in vivo evidence from Drosophila also supports this. However, the precise nature of PINK1/Parkin-mediated mitochondrial turnover in vivo is debated with contradictory results emerging. Nevertheless, interventions to combat the decline in mitochondrial homeostasis remain a key challenge to combatting PINK1/PRKN related pathologies. One mechanism that seems to provide substantial benefit in physiological contexts is through augmenting mitochondrial fission, which presumably facilitates the flux of damaged mitochondrial components towards turnover. This study, provide further evidence that augmenting a pro-fission pathway is beneficial against Pink1 and parkin dysfunction. As phosphoinositides can be interconverted by the action of multiple enzymes that may be druggable, these findings suggest another potential route towards a therapeutic intervention (Terriente-Felix, 2020).

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

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

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

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

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

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

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

The Drosophila genome harbors one gene each for the three families of PI4 kinases (PI4Ks). The three families of PI4 kinases produce PI4P using PI as a substrate at distinct intracellular membranes. Among the three genes, viz. fwd, Pi4KIIα and PI4KIIIα, phenotypes were observed only upon knockdown of fwd and PI4KIIIα. As mentioned earlier, loss of PI4KIIIα resulted in a complete loss of knockdown clones in the mosaic screen and led to pupal lethality when it was downregulated in the entire eye tissue. As a result, it appears likely that PI4KIIIα is essential for cellular viability. On the other hand, smaller knockdown clones were observed when fwd was downregulated in the mosaic CoinFLP Gal4 screen. However, like PI3K68D and Vps34, downregulation of fwd across the entire developing eye failed to show any significant phenotypes, again suggesting cell-cell interactions between fwd-deficient and neighboring wild-type cells to be the likely reason for reduced size of fwdRNAi clones. fwd knockout flies are viable and female fertile. fwd knockout male flies are sterile due to defects in cytokinesis during male meiosis (Brill, 2000). Both fly and mammalian fwd (PI4Kβ) bind and recruit Rab11 to the Golgi and are required for the maintenance of Golgi integrity and secretion (de Graaf, 2004; Giansanti, 2007; Polevoy, 2009). These reports suggest that fwd may have pleiotropic cellular roles, causing the phenotypes to vary depending on the tissues in which its levels are manipulated (Janardan, 2019).

In summary, this screen identified several components of the phosphoinositide metabolism toolkit as regulators of cell growth. Using the power of mosaic analysis in the Drosophila eye, it was possible to classify these into those exerting their effect in a cell-autonomous manner and those likely acting via cell-cell interactions in a plane of developing cells. The screen identified three genes that may regulate growth via cell-cell interactions. These include Pi3K68D, Vps34 and fwd. Interestingly, Pi3K68D is found only in a subset of metazoans, Bilateria. The observation that Pi3K68D is not present in single cell eukaryotes but are only found in multicellular eukaryotes further supports the findings that Pi3K68D may have a role in cell-cell interactions. The products of the three identified enzymes, PI(3,4)P2, PI3P and PI4P, have so far not been directly linked to cell competition. The identification of these genes as regulators of growth has thus opened up new links between phosphoinositide metabolizing enzymes and cell growth that invites further studies to explore underlying mechanisms (Janardan, 2019).

The current screen included phosphoinositide kinases, phosphatases and a few other phosphoinositide metabolizing enzymes. However, signaling events downstream of their generation are dependent on the ability of these lipids to bind target proteins and modulate their activities. There are about 70 phosphoinositide binding proteins annotated in Drosophila. Extending the CoinFLP screen to these phosphoinositide binding proteins in the future would further understanding of the mechanisms by which phosphoinositides regulate growth (Janardan, 2019).

Increased levels of phosphoinositides cause neurodegeneration in a Drosophila model of amyotrophic lateral sclerosis

The Vesicle-associated membrane protein (VAMP)-Associated Protein B (VAPB) is the causative gene of amyotrophic lateral sclerosis 8 (ALS8) in humans. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective death of motor neurons leading to spasticity, muscle atrophy and paralysis. VAP proteins have been implicated in various cellular processes, including intercellular signalling, synaptic remodelling, lipid transport and membrane trafficking and yet, the molecular mechanisms underlying ALS8 pathogenesis remain poorly understood. This study has identified the conserved phosphoinositide phosphatase Sac1 as a Drosophila VAP (DVAP)-binding partner and showed that DVAP is required to maintain normal levels of phosphoinositides. Downregulating either Sac1 or DVAP disrupts axonal transport, synaptic growth, synaptic microtubule integrity and the localization of several postsynaptic components. Expression of the disease-causing allele (DVAP-P58S) in a fly model for ALS8 induces neurodegeneration, elicits synaptic defects similar to those of DVAP or Sac1 downregulation and increases phosphoinositide levels. Consistent with a role for Sac1-mediated increase of phosphoinositide levels in ALS8 pathogenesis, this study found that Sac1 downregulation induces neurodegeneration in a dosage-dependent manner. In addition, this study reports that Sac1 is sequestered into the DVAP-P58S-induced aggregates and that reducing phosphoinositide levels rescues the neurodegeneration and suppresses the synaptic phenotypes associated with DVAP-P58S transgenic expression. These data underscore the importance of DVAP-Sac1 interaction in controlling phosphoinositide metabolism and provide mechanistic evidence for a crucial role of phosphoinositide levels in VAP-induced ALS (Forrest, 2013).

Amyotrophic lateral sclerosis (ALS) is a progressive, degenerative disorder characterized by the selective loss of motor neurons in the brain and spinal cord leading to paralysis, muscle atrophy and eventually, death. Two missense mutations in the gene encoding the human Vesicle-associated membrane protein (VAMP)-Associated Protein B (hVAPB) causes a range of dominantly inherited motor neuron diseases including ALS8. VAP family proteins are characterized by an N-terminal major sperm protein (MSP) domain, a coiled-coil (CC) motif and a transmembrane (TM)-spanning region. They are implicated in several biological processes, including regulation of lipid transport, endoplasmic reticulum (ER) morphology and membrane trafficking. Drosophila Vap-33-1 (hereafter, DVAP) regulates synaptic structure, synaptic microtubule (MT) stability and the composition of postsynaptic glutamate receptors. MSP domains in DVAP are cleaved and secreted into the extracellular space where they bind Ephrin receptors. MSPs also bind postsynaptic Roundabout and Lar-like receptors to control muscle mitochondria morphology, localization and function. Transgenic expression of the disease-linked alleles (DVAP-P58S and DVAP-T48I) in the larval motor system recapitulates major hallmarks of the human disease, including aggregate formation, locomotion defects and chaperone upregulation. Several studies have also implicated the ALS mutant allele in abnormal unfolded protein response (UPR) and in the disruption of the anterograde axonal transport of mitochondria. However, it is unclear how these diverse VAP functions are achieved and which mechanisms underlie the disease pathogenesis in humans. One way to address these questions is to search for DVAP-interacting proteins. This study identified Sac1 (Suppressor of Actin 1), an evolutionarily conserved phosphoinositide phosphatase, as a DVAP-binding protein. Phosphoinositides are low-abundance lipids that localize to the membrane-cytoplasm interface and function by binding various effector proteins. The inositol group can be reversibly phosphorylated at the 3', 4' and 5' positions to generate seven possible phosphoinositide derivatives, each with a specific intracellular dynamic distribution. Sac1 predominantly dephosphorylates PtdIns4P pools, although PtdIns3P and PtdIns(3,5)P2 can also function as substrates. In yeast, Sac1 has been linked to several processes, including actin organization, vacuole morphology and sphingomyelin synthesis. Drosophila Sac1 mutants die as embryos and exhibit defects in dorsal closure and axonal pathfinding. Mouse lines deficient for Sac1 are cell lethal, whereas Sac1 downregulation in mammalian cell cultures results in disorganization of Golgi membranes and mitotic spindles. Interestingly, SAC3 (also known as FIG4), another member of the Sac phosphatase family, is mutated in familial and sporadic cases of ALS. Inactivation of SAC3 in mice also results in extensive degeneration and neuronal vacuolization in the brain, most relevantly in the motor cortex. This study identified Sac1 and DVAP as binding partners and shows that DVAP is required to maintain normal levels of PtdIns4P. Loss of either Sac1 or DVAP function disrupts axonal transport, MT stability, synaptic growth and the localization of a number of postsynaptic markers. Rhe disease-causing mutation (DVAP-P58S) induces neurodegeneration and displays synaptic phenotypes similar to those of either Sac1 or DVAP loss-of-function, including an increase in PtdIns4P levels. Importantly, reducing PtdIns4P levels rescues the neurodegeneration associated with DVAP-P58S and suppresses the synaptic phenotypes associated with DVAP-P58S and DVAP loss-of-function alleles. Consistent with these observations, Sac1 is sequestered into DVAP-P58S-mediated aggregates and downregulation of Sac1 in neurons induces increased PtdIns4P levels and degeneration. These data highlight the crucial role of DVAP and Sac1 in regulating phosphoinositides and support a causative role for PtdIns4P levels in ALS8 pathogenesis (Forrest, 2013).

This study has identified Sac1 as a DVAP-binding protein and uncovered a hitherto unknown function of Sac1 in postembryonic synaptic maturation and neurodegeneration. Presynaptic reduction of either DVAP or Sac1 levels induces structural changes, disruption of the synaptic MT cytoskeleton and accumulation of clusters of proteins and vesicles along the axons. In addition, muscle down-regulation of either Sac1 or DVAP leads to a strikingly aberrant synaptic morphology and abnormal localization and distribution of several postsynaptic markers, including adducin and β-spectrin. Depletion of DVAP as well as Sac1 expression induces an increase in PtdIns4P levels. Sac1 downregulation in the adult nervous system was shown to cause early death and neurodegeneration in a dosage-dependent manner, a phenotype similar to that of DVAP-P58S transgenic expression. This analysis indicates that the DVAP-P58S allele has a dominant negative effect, as its transgenic expression leads to an upregulation of PtdIns4P and its mutant phenotypes are similar to those associated with either DVAP or Sac1 loss-of-function. In agreement with the hypothesis that neurodegeneration in the DVAP-P58S context is due to a loss-of-function of both DVAP and Sac1, it is reported that both wild-type DVAP and Sac1 are depleted from their normal localization and are sequestered into DVAP-P58S-mediated aggregates. Altogether, these data are consistent with a model in which DVAP is required for Sac1 activity and for the regulation of intracellular PtdIns4P levels. Loss-of-function of DVAP and Sac1 by a DVAP-P58S-mediated dominant-negative mechanism induces cell degeneration by an upregulation of PtdIns4P, which is also responsible for the observed disruption of fundamental biological processes at the NMJs . It has been previously shown that transgenic expression of DVAP proteins carrying the equivalent ALS8 mutations in Drosophila mimic the human disease. Notably, expression of hVAPB in flies rescues the lethality and the phenotypes associated with DVAP mutants, indicating an evolutionarily conserved function for VAP proteins. Collectively, these data indicate that DVAP-mediated molecular pathways are likely to be important for understanding of the disease pathogenesis in humans (Forrest, 2013).

There is evidence supporting that DVAP functions to maintain normal cellular levels of PtdIns4P by interacting with Sac1. First, Sac1 and DVAP bind to each other and colocalize in many different tissues. It has been reported that phosphoinositol transfer proteins/phosphoinositide-binding proteins associate directly with phosphatases and kinases to control their activities. Specifically, VAP has been shown to bind PtdIns4P in vitro and to be required for Sac1 activity in yeast. Second, PtdIns4P levels are upregulated in DVAPRNAi mutants, suggesting that DVAP function is required for normal PtdIns4P levels. Similarly, in yeast, inactivation of Scs2/Scs22 VAP genes induces an increase in the levels of PtdIns4P. Third, the phenotypic similarity associated with either DVAP or Sac1 loss-of-function mutations supports the idea that the pool of PtdIns4P that is upregulated in DVAP mutants is the same as the one dephosphorylated by Sac1. Previous studies attributed a prominent functional role to the N-terminal MSP domain of DVAP. The MSP domain is cleaved and secreted and binds to the extracellular domain of Ephrin receptors. Secreted MSP also binds to Robo and Lar-like receptors to control mitochondria morphology, localization and function in muscles. A new DVAP-binding activity was identified that is MSP-independent and involves a C-terminal fragment encompassing the TM domain. Interestingly, a new hVAPB mutation replacing valine at position 234 with an isoleucine in the conserved TM domain of hVAPB has been shown to cause ALS8 in humans. These data may provide direct evidence of a role of hVAPB-Sac1 interaction in the disease pathogenesis in humans (Forrest, 2013).

In yeast and mammalian cells, Sac1 is an integral membrane protein localized to the ER and the Golgi. This study reports a similar localization for the Drosophila homologue of Sac1. In yeast and mammalian cells, Sac1 localization appears to be very dynamic, as this protein shuttles between ER and Golgi upon nutrient conditions. Specifically, glucose starvation in yeast or growth factor deprivation in mammalian cells causes relocalization of Sac1 from the ER to the Golgi complex, where it reduces PtdIns4P levels and slows protein trafficking. The ER-Golgi shuttling ability of Sac1 is reversed when nutrients or growth factors are added back to the growth medium. The growth factor-induced translocation of Sac1 from the Golgi to the ER requires p38 MAPK (mitogen-activated protein kinase) activity. These data suggest that Sac1 trafficking may be regulated by stressors that activate p38 MAPK. Some of these stressors such as oxidative damage and ER stress are triggers of neurodegeneration. This raises the intriguing possibility that a p38 MAPK-activated mechanism of PtdIns4P spatial regulation may be implicated in neurodegenerative processes (Forrest, 2013).

Scs2/Scs22 VAP proteins in yeast play a pivotal role in tethering the ER to the PM to form ER/PM contact sites. Studies have highlighted the role of membrane junctions between organelles as important sites for lipid metabolism and intracellular signalling controlled by PtdIns4P. Depletion of Scs2/Scs22 VAP proteins located to the ER/PM contact sites leads to a retraction of the ER into internal structures, elevated levels of PtdIns4Ps at the PM and induction of the UPR. At the ER/PM contact sites, Sac1 dephosphorylates PtdIns4P on the PM in trans from the ER. This reaction requires the Scs2/Sc22p VAP proteins and the oxysterol-binding homology proteins that act as PtdIns4P sensors and activates Sac1 phosphatase activity. ER/PM junctions have been described in many organisms and cell types, including neurons and Drosophila photoreceptors. In addition, VAP proteins have been implicated in ER-Golgi, ER-endosomes and ER-mitochondria contacts in mammalian cells, suggesting that they may function as a tether for several organelle/membrane contact sites. In conclusion, emerging evidence suggests that VAP proteins may be a crucial component of a hub controlling PtdIns4P metabolism in yeast and possibly, in higher eukaryotes as well (Forrest, 2013).

The ability of either PI4KIIIα or four wheel drive fwd, a Golgi-localized lipid kinase that synthesizes phosphatidylinositol 4-phosphate from phosphatidylinositol, to suppress the synaptic and neurodegenerative phenotypes associated with transgenic expression of DVAP-P58S is somewhat surprising, as their yeast homologues (Stt4 and Pik1, respectively) are supposed to play non-redundant functions and to control spatially separate pools of PtdIns4P. This is based on previously published data showing that, in yeast, Stt4 and Pik1 are both essential for cell viability but control different cellular processes. Pik1 is essential for anterograde vesicular trafficking, whereas Stt4 plays a role in actin cytoskeleton organization and protein kinase C signalling. Both Pik1 and Stt4 play distinct roles in regulating MAPK signalling. Localization studies further suggest that Pik1p is primarily present in the nucleus and in the Golgi, whereas Stt4p is mainly cytoplasmic and is recruited to the PM for localized synthesis of PtdIns4P (Forrest, 2013).

However, at present, the precise degree to which Stt4 and Pik1 functions have been conserved and apportioned among their homologues in flies remains unclear. In Drosophila, the Stt4 homologue PI4KIIIα is required for oocyte polarization and its intracellular localization has not been determined. On the other hand, previous studies revealed that the fly Pik1 homologue Fwd is required for male germ-line cytokinesis. In spermatocytes, Fwd localizes to the Golgi and it is required for the accumulation of PtdIns4P on this organelle, implying that its function in providing PtdIns4P in the Golgi is evolutionarily conserved with yeast. However, whereas Pik1 is required for cell viability, Fwd appears to be dispensable for normal development, suggesting that it is redundant with similar genes in carrying out its function (Forrest, 2013).

Another way to explain the rescue data would be to admit that upregulation of PtdIns(4,5P)2 and not PtdIns4P is responsible for DVAP-P58S mutant phenotypesPtdIns4P formed by the PM-associated STT4 can function as a substrate of PI4P 5-kinase to generate PtdIns(4,5)P2 at the cell cortex. It is also possible that PtdIns4P that is phosphorylated by plasmalemmal PI4P 5-kinase originates from intracellular sources. In the Golgi, PtdIns4P levels play a central role in the formation of vesicles delivered from the trans-Golgi network to the PM and their lipid cargo could be the substrate for the plasmalemmal PtdIns(4,5)P2 synthesis (676767). As PtdIns4P in the Golgi is mainly produced by Pik1 is therefore possible that PtdIns(4,5)P2 associated with the PM and its effector proteins are downstream of both Stt4 and Pik1. Moreover, upregulation of PtdIns(4,5)P2 would explain the MT phenotypes, the mislocalization of post-synaptic markers and the axonal transport defects. Indeed, PtdIns(4,5)P2-enriched microdomains in the PM have been shown to participate in the regulation of MT plus-end capture and stabilization during polarized mobility. In Caenorhabditis elegans, the microtubular motor UNC-4 gene was shown to be anchored to synaptic vesicles, using a pleckstrin homology domain, thus implicating PtdIns(4,5)P2 in MT-based intracellular motility. Finally, spectrin proteins and adducin require PtdIns(4,5)P2 for their correct localization to the cell cortex (Forrest, 2013).

However, if this were true, an increase in PtdIns(4,5)P2 levels whould be observed wherever an upregulation of PtdIns4P is observed. By using an antibody specific for PtdIns(4,5)P2, PtdIns(4,5)P2 levels were quantified in tissues in which either Sac1 or DVAP were downregulated as well as in tissues expressing the DVAP-P58S transgene. Surprisingly, PtdIns(4,5)P2 levels were not affected by the dramatic upregulation of PtdIns4P in any of the genotypes described earlier. Consistent with these data, it was previously reported that, in Drosophila eye imaginal discs, depletion of Sac1 exhibits a dramatic increase in PtdIns4P levels, whereas PtdIns(4,5)P2 and PtdIns3P levels remain similar to wild-type. In addition, loss of PM PtdIns4P by downregulation of PI4KIIIα was not matched by a decrease in PtdIns(4,5)P2 levels. It was shown that the major function of PtdIns4P is not to generate the pool of PtdIns(4,5)P2 on the PM but rather to contribute to the generation of a polyanionic lipid environment in the inner leaflet of the PM. PtdIns4P would then function in recruiting soluble proteins to the PM by electrostatic interaction with their polycationic surface. It was also shown that PtdIns4P contributes to processes such as modulation of ion channel activity that have been traditionally associated with changes in PtdIns(4,5)P2. Finally, upregulation of PtdIns(4,5)P2 by inactivation of the Drosophila PI(4,5)P2 5-phosphatase synaptojanin leads to a distinct endocytotic phenotype due to defects in synaptic vesicle recycling. In synaptojanin mutants, synaptic vesicles are severely depleted and those remaining are clearly clathrin-coated. Intracellular recordings revealed enhanced synaptic depression during prolonged high-frequency stimulation. Ultrastructural and electrophysiological analysis of DVAP mutants do not exhibit a synaptojanin-like phenotype, indicating that upregulation of PtdIns(4,5)P2 does not mimic the phenotype associated with increased levels of PtdIns4P. Taken together, these considerations suggest that increased levels of PtdIns4P could be the main factor determining the observed synaptic and neurodegenerative phenotypes. Further studies using fluorescent phosphoinositide probes and genetic analyses will be needed to fully clarify the contribution of PtdIns4P versus PtdIns(4,5)P2 pools to NMJ physiology and neurodegeneration (Forrest, 2013).

Emerging evidence indicates that VAP and Sac1 may also play an important and specific role in membrane homeostasis. Biogenesis of sphingolipids, sterols and phosphoinositides that together determine the structural and functional properties of cell membranes must be closely coordinated. VAP interacts with both oxysterol-binding protein (OSBP) and ceramide transfer protein (CERT), recruiting them to contact sites between the ER and the Golgi complex. CERT has a FFAT (diphenylalanine in an acidic tract) motif that mediates its binding to ER-localized VAP and a PH domain that recognizes the PtdIns4P-enriched Golgi membrane. It has been proposed that CERT, because of its dual-binding ability, shuttles ceramide from the ER to the Golgi, where it is converted into sphingomyelin. Sphingomyelin continues to move through the secretory pathway to the PM, where it is most abundant. OSBP has an analogous function to CERT but instead mediates inter-membrane sterol transfer. This functional similarity is also reflected in OSBP's domain architecture: like CERT, it contains a PtdIns4P-binding PH domain and a VAP-binding FFAT motif. It has been shown that sterols regulate sphingolipid metabolism by inducing a significant increase in SM synthesis that is dependent on OSBP, CERT and their shared binding partner VAP. The precise mechanism is not yet known but OSBP appears to activate CERT by promoting its recruitment to membranes and its binding to VAP. It is likely that disruption of the VAP-Sac1 interaction may have profound effects on the lipid composition of the PM, affecting its curvature and thickness and by consequence, vesicle budding and membrane remodelling. Interestingly, synaptic growth requires membrane remodelling and, at the Drosophila NMJs, occurs mainly by the budding of new boutons from pre-existing ones (Forrest, 2013).

VAPB is involved in the IRE1/XBP1 signalling pathway of the UPR, an ER reaction inhibiting the accumulation of unfolded/misfolded proteins. In the disease-context, the hVAPB-P56S protein recruits its wild-type counterpart into the aggregates and it attenuates its ability to induce the UPR. This together with the observation that, in yeast, depletion of VAP proteins from the ER/PM contact sites induces a constitutive activation of the UPR suggests that motor neurons in ALS8 could be particularly vulnerable to cell death-induced ER stress. In addition, VAP proteins have been shown to be involved in lipid transfer and metabolism and accumulation of lipids and intermediates of lipid biosynthetic pathways are potent inducers of apoptosis. Finally, recent studies in yeast have shown that defects in the PtdIns4K Pik1 activity lead to a blockage of autophagy, a process controlling the degradation of long-lived proteins, damaged organelles and bulk cytoplasm in response to various types of stress. Many questions remain to be explored concerning the precise molecular mechanism underlying neurodegeneration in ALS. However, over the last few years, an increasing number of experimental models have been generated and they represent an excellent tool for identifying molecular pathways in ALS and for evaluating their contribution to the disease pathogenesis (Forrest, 2013).

Dual roles for the Drosophila PI 4-kinase four wheel drive in localizing Rab11 during cytokinesis

Successful completion of cytokinesis relies on addition of new membrane, and requires the recycling endosome regulator Rab11, which localizes to the midzone. Despite the critical role of Rab11 in this process, little is known about the formation and composition of Rab11-containing organelles. This study identified the phosphatidylinositol (PI) 4-kinase III beta Four wheel drive (Fwd) as a key regulator of Rab11 during cytokinesis in Drosophila spermatocytes. Fwd is required for synthesis of PI 4-phosphate (PI4P) on Golgi membranes and for formation of PI4P-containing secretory organelles that localize to the midzone. Fwd binds and colocalizes with Rab11 on Golgi membranes, and is required for localization of Rab11 in dividing cells. A kinase-dead version of Fwd also binds Rab11 and partially restores cytokinesis to fwd mutant flies. Moreover, activated Rab11 partially suppresses loss of fwd. These data suggest Fwd plays catalytic and noncatalytic roles in regulating Rab11 during cytokinesis (Polevoy, 2009).

The discovery that Drosophila PI4Kβ Fwd and fission yeast PI4P 5-kinase Its3 are required for cytokinesis provided the first genetic evidence that phosphoinositides play a critical role in this process. Consistent with this, the phosphatidylinositol transfer proteins Gio and Nir2 are also required for cytokinesis, and may serve in part to provide the PI precursor for PI4P. In addition, the pool of PI4P synthesized by PI4Kβ may serve as a precursor to PIP2, which is also required for cytokinesis. Nonetheless, individual phosphoinositides and their regulatory enzymes likely play unique roles, regulating distinct steps of the process. Importantly, a role for PI4Kβ -- and therefore PI4P -- in cytokinesis appears conserved (Polevoy, 2009).

Experiments reveal that Fwd is required for synthesis of PI4P on Golgi membranes and for formation of PI4P- and Rab11-associated secretory organelles at the midzone. On the surface, this result appears at odds with previous observations suggesting that Fwd and Gio function at a later step to promote fusion of Lva-containing Golgi-derived vesicles with the cleavage furrow. However, because Lva serves as a Golgi scaffold, accumulation of Lva at the midzone in fwd and gio mutant cells may reveal a defect in segregation of a subset of Golgi membranes to the poles of the cell rather than a defect in vesicle fusion (Polevoy, 2009).

Although Rab11 has been shown to traffic to the midzone during cytokinesis, the membrane composition of Rab11-containing organelles was previously unknown. The current finding that PI4P is present on these organelles is consistent with proteomic analyses demonstrating an enrichment of Rab11 and PI4Kβ on PI4P-containing liposomes. Interestingly, these liposomes were also enriched in actin regulatory factors such as Rac1 and Wave/Scar. As actin is transported on vesicles to the midzone in Drosophila embryos, and the Rab11 effector Nuf promotes actin polymerization at the furrow, PI4P-dependent organelles may concentrate or recruit factors such as Nuf that contribute to maintenance of F-actin in the contractile ring. Consistent with this idea, mutations in fwd, like mutations in nuf and rab11, are associated with failure to maintain proper actin organization during cytokinesis (Polevoy, 2009).

The regulatory relationship between Fwd and Rab11 is evolutionarily conserved. In budding yeast, the Rab11 homologues Ypt31/32 act downstream of Pik1 to regulate post-Golgi trafficking. The two Arabidopsis thaliana PI4Kβs, PI-4Kβ1 and PI-4Kβ2, show genetic interactions with the Rab11 homologue Rab4Ab in root hair development and colocalize with RabA4b on root hair tip-associated membranes, and PI-4Kβ1 binds GTP-bound RabA4b in vitro. Moreover, RabA4b-containing membranes exhibit altered morphology in PI-4Kβ1/β2 double mutants, suggesting RabA4b may act downstream of PI4Kβs in this process. Mammalian PI4Kβ binds activated Rab11, and is thought to recruit Rab11 to Golgi membranes to promote post-Golgi secretory trafficking. The results of this study demonstrate that Fwd acts upstream of Rab11 during cytokinesis, and that bovine and human PI4Kβ can fully substitute for Fwd in vivo (Polevoy, 2009).

PI4Kβ and PI4P participate in vesicular and nonvesicular trafficking of cellular membranes and their lipid constituents, suggesting that, in addition to its role in formation of secretory organelles, Fwd may direct other trafficking pathways. For example, several conserved lipid transport proteins bind PI4P and depend on PI4Kβ for their localization and function in yeast and mammalian cells. PI4P is also found at ER exit sites (also called transitional ER, or tER). Intriguingly, tER was recently shown to accumulate at the midzone of dividing S. pombe cells, and normal ER morphology in dividing Caenorhabditis elegans embryos was found to require Rab11. Future experiments will be required to determine if Fwd-dependent tER or nonvesicular trafficking pathways actively participate in cytokinesis (Polevoy, 2009).

Despite strong parallels between cytokinesis in mammalian cells and in Drosophila, the mechanism by which Rab11 affects completion of cytokinesis is not entirely conserved. In mammalian cells, Rab11 associates indirectly with the plasma membrane regulator Arf6 via FIP3, a Rab11-binding protein with homology to Nuf. Both Rab11 and Arf6 bind members of the exocyst complex, which in turn mediates targeting of endosomes to the midzone. In contrast, in Drosophila, Arf6 and Rab11 appear to function in separate pathways. Nuf binds and colocalizes with Rab11, yet fails to bind Arf6. Consistent with this, Rab11 is essential and has specific functions at multiple stages of development, whereas Arf6 is required only for spermatocyte cytokinesis. Even in spermatocytes, Arf6 promotes trafficking of Rab4-positive but not Rab11-positive vesicles. Thus, in spermatocytes, Arf6/Rab4 and Fwd/Rab11 appear to constitute nonredundant membrane trafficking pathways required for completion of meiotic cytokinesis (Polevoy, 2009).

Despite its vital role in spermatocyte cytokinesis, Fwd is dispensable for normal development and female fertility. Drosophila tissue culture cells show only a weak requirement for fwd during cytokinesis, with knockdown of fwd by RNAi resulting in a small increase in binucleate cells. This is particularly surprising given that yeast PIK1 is required for post-Golgi secretory trafficking and endocytosis. As secretion and endocytosis are essential processes, it is hypothesized that fwd is redundant with other genes for carrying out these functions outside of the male germline. Future investigations will determine the identity of these fwd-interacting genes (Polevoy, 2009).

Rab11 is required for membrane trafficking and actomyosin ring constriction in meiotic cytokinesis of Drosophila males

Rab11 is a small GTPase that regulates several aspects of vesicular trafficking. This study shows that Rab11 accumulates at the cleavage furrow of Drosophila spermatocytes and that it is essential for cytokinesis. Mutant spermatocytes form regular actomyosin rings, but these rings fail to constrict to completion, leading to cytokinesis failures. rab11 spermatocytes also exhibit an abnormal accumulation of Golgi-derived vesicles at the telophase equator, suggesting a defect in membrane-vesicle fusion. These cytokinesis phenotypes are identical to those elicited by mutations in giotto (gio) and four wheel drive (fwd) that encode a phosphatidylinositol transfer protein and a phosphatidylinositol 4-kinase, respectively. Double mutant analysis and immunostaining for Gio and Rab11 indicated that gio, fwd, and rab11 function in the same cytokinetic pathway, with Gio and Fwd acting upstream of Rab11. It is proposed that Gio and Fwd mediate Rab11 recruitment at the cleavage furrow and that Rab11 facilitates targeted membrane delivery to the advancing furrow (Giansanti, 2007; full text of article).

The Gio PITP is enriched at the furrow membrane and that it is required for Drosophila cytokinesis (Giansanti, 2006). This study shows that the furrow membrane is also enriched in Rab11 and that Rab11 localization at the equatorial membrane requires the wild-type activity of both giotto (gio) and four wheel drive (fwd). In addition, the wild-type functions of gio, fwd, and rab11 are all required for membrane-vesicle fusion during cytokinesis, because mutations in these genes result in an abnormal accumulation of Golgi-derived vesicles at the equator of telophase cells (Giansanti, 2006). Finally, the results strongly suggest that gio, fwd, and rab11 function in the same cytokinesis pathway. These observations suggest a model for the mechanisms underlying membrane addition to the cleavage furrow during spermatocyte cytokinesis. It is proposed that Gio mediates transfer of PtdIns monomers to the furrow membrane, causing a local enrichment in PtdIns molecules. The association of Gio with this membrane domain may facilitate recruitment of the PtdIns-4-kinase encoded by fwd, which would mediate phosphorylation of PtdIns to PtdIns(4)P, allowing their further phosphorylation to PtdIns(4,5)P2. Fwd may also mediate Rab11 recruitment at the cleavage furrow, allowing targeted Rab11-dependent vesicle fusion events necessary for completion of cytokinesis. It is realized that this is a rather speculative model. Its major drawback is that the subcellular localization and the molecular interactions of the Drosophila Fwd protein are currently unknown. However, studies in S. pombe have shown that one of the PtdIns-4-kinases present in this organism interacts with Cdc4p, a contractile ring protein essential for cytokinesis. This finding indicates that, at least in fission yeast, one of the PtdIns-4-kinases is associated with the cleavage furrow. In addition, a recent study has shown that one of the mammalian PtdIns-4-kinases interacts physically with Rab11 and is required for Rab11 localization in the Golgi complex. The same study has also shown that recruitment of this kinase to the Golgi does not require Rab11 (de Graaf, 2004). These results are consistent with the current findings, and they lead to the belief that Gio, Fwd, and Rab11 are all enriched at cleavage furrow, where they work in concert to ensure proper vesicle docking and fusion (Giansanti, 2007).

Mutations in rab11 cause frequent failures in meiotic cytokinesis of males without affecting cytokinesis of larval brain neuroblasts. The mutations analyzed are obviously hypomorphic since they cause lethality at the larval and pupal stages, whereas rab11 null alleles result in embryonic lethality. Thus, it is possible that the rab11 mutants analyzed retain a residual Rab11 activity that is sufficient for neuroblast cytokinesis but not meiotic cytokinesis. Alternatively, Rab11 may not be required for mitotic cytokinesis. A strong support for a specific involvement of Rab11 in meiotic cytokinesis comes from recent RNAi screens that have shown that Rab11 has little or no role in S2 cell cytokinesis (Giansanti, 2007).

Previous studies have shown that null mutations in fwd and fws disrupt spermatocyte cytokinesis but that they have no observable effects on larval neuroblast mitosis (Brill, 2000; Farkas, 2003; Giansanti, 2004). Thus, at least three proteins involved in membrane traffic, Rab11, Cog5 (encoded by four way stop or fws), and a PtdIns-4-kinase, seem to be specifically required for meiotic cytokinesis. This specificity is unlikely to depend on the peculiar features of the final steps of spermatocyte cytokinesis. In male meiotic cells, the cytoplasmic bridges generated by ring constriction are not severed by a canonical abscission process, as occurs in larval neuroblasts; they instead persist and are stabilized by the formation of a specialized structure called ring canal. Mutations in rab11, fws and fwd inhibit ring constriction and furrow ingression during early telophase and block cytokinesis well before the formation of a cytoplasmic bridge. These observations rule out the possibility that the spermatocyte-specific effects of these mutations reflect problems in the final step of cytokinesis when ring canals are assembled (Giansanti, 2007).

The specific role of Rab11, Cog5, and Fwd in spermatocyte cytokinesis may reflect a specifically high requirement for formation of new membrane at the advancing cleavage furrow. To fulfill this requirement, male meiotic cells may exploit all the extant pathways for membrane addition. These pathways would be redundant in mitotic cell where the requirements for membrane expansion at the advancing furrow are relatively low. Alternatively, the specific requirement of membrane trafficking functions for spermatocyte cytokinesis may reflect the organization of membrane stores within these cells. Spermatocytes contain a large ER that includes astral and parafusorial membranes, and they do not possess a detectable pericentriolar RE. Larval neuroblasts do exhibit a spindle envelope, but, in contrast to spermatocytes, they are devoid of astral membranes and possess pericentriolar REs (Giansanti, 2006). Thus, formation of new membrane during spermatocyte cytokinesis might utilize membrane trafficking activities that are at least in part distinct from those used by mitotic cells, depending on the organization of membrane stores within the two cell types (Giansanti, 2007).

Whatever the reason for their specific sensitivity to mutations that disrupt membrane-related functions, Drosophila spermatocytes are emerging as an extremely useful model system for studying membrane traffic during animal cell cytokinesis. There is indeed growing evidence that the analysis of mutations that disrupt spermatocyte cytokinesis can reveal membrane-trafficking genes that play redundant cytokinetic roles in other animal cell systems (Giansanti, 2007).

Genetic dissection of meiotic cytokinesis in Drosophila males

This study has used Drosophila male meiosis as a model system for genetic dissection of the cytokinesis mechanism. Drosophila mutants defective in meiotic cytokinesis can be easily identified by their multinucleate spermatids. Moreover, the large size of meiotic spindles allows characterization of mutant phenotypes with exquisite cytological resolution. This study has screened a collection of 1955 homozygous mutant male sterile lines for those with multinucleate spermatids, and thereby identified mutations in 19 genes required for cytokinesis. These include 16 novel loci and three genes, diaphanous, four wheel drive, and pebble, already known to be involved in Drosophila cytokinesis. To define the primary defects leading to failure of cytokinesis, meiotic divisions were analyzed in male mutants for each of these 19 genes. Examination of preparations stained for tubulin, anillin, KLP3A, and F-actin revealed discrete defects in the components of the cytokinetic apparatus, suggesting that these genes act at four major points in a stepwise pathway for cytokinesis. The results also indicated that the central spindle and the contractile ring are interdependent structures that interact throughout cytokinesis. Moreover, genetic and cytological analyses provide further evidence for a cell type-specific control of Drosophila cytokinesis, suggesting that several genes required for meiotic cytokinesis in males are not required for mitotic cytokinesis (Giansanti, 2004).

A search for doxycycline-dependent mutations that increase Drosophila melanogaster life span identifies the VhaSFD, Sugar baby, filamin, fwd and Cctl genes

A P-type transposable element called PdL has been engineered with a doxycycline-inducible promoter directed out through the 3' end of the element. Insertion of PdL near the 5' end of a gene often yields doxycycline-dependent overexpression of that gene and a mutant phenotype. This functional genomics strategy allows for efficient screening of large numbers of genes for overexpression phenotypes. PdL was mobilized to around 10,000 new locations in the Drosophila melanogaster genome and used to search for genes that would extend life span when overexpressed. Six lines were identified in which there was a 5%-17% increase in life span in the presence of doxyxcycline. The mutations were molecularly characterized and in each case a gene was found to be overexpressed using northern blots. Two genes did not have previously known phenotypes and are implicated in membrane transport: VhaSFD encodes a regulatory subunit of the vacuolar ATPase proton pump (H+-ATPase), whereas Sugar baby (Sug) is related to a maltose permease from Bacillus. Three PdL mutations identified previously characterized genes: filamin encodes the homolog of an actin-polymerizing protein that interacts with presenilins. four wheel drive (fwd) encodes a phosphatidylinositol-4-kinase (PI 4-kinase) and CTP:phosphocholine cytidylyltransferase-l (Cctl) encodes the rate-limiting enzyme in phosphatidylcholine synthesis. Finally, an apparently novel gene (Red herring, Rdh) was found in the first intron of the encore gene. It is concluded that screening for conditional mutations that increase Drosophila life span has identified genes implicated in membrane transport, phospholipid metabolism and signaling, and actin cytoskeleton organization (Landis, 2003).

A phospholipid kinase regulates actin organization and intercellular bridge formation during germline cytokinesis

The endgame of cytokinesis can follow one of two pathways depending on developmental context: resolution into separate cells or formation of a stable intercellular bridge. This study shows that the four wheel drive (fwd) gene of Drosophila melanogaster is required for intercellular bridge formation during cytokinesis in male meiosis. In fwd mutant males, contractile rings form and constrict in dividing spermatocytes, but cleavage furrows are unstable and daughter cells fuse together, producing multinucleate spermatids. fwd is shown to encode a phosphatidylinositol 4-kinase (PI 4-kinase), a member of a family of proteins that perform the first step in the synthesis of the key regulatory membrane phospholipid PIP2. Wild-type activity of the fwd PI 4-kinase is required for tyrosine phosphorylation in the cleavage furrow and for normal organization of actin filaments in the constricting contractile ring. These results suggest a critical role for PI 4-kinases and phosphatidylinositol derivatives during the final stages of cytokinesis (Brill, 2000).

Functions of four wheel drive orthologs in other species

Golgi-derived PI(4)P-containing vesicles drive late steps of mitochondrial division

Mitochondrial plasticity is a key regulator of cell fate decisions. Mitochondrial division involves Dynamin-related protein-1 (Drp1) oligomerization, which constricts membranes at endoplasmic reticulum (ER) contact sites. The mechanisms driving the final steps of mitochondrial division are still unclear. This study found that microdomains of phosphatidylinositol 4-phosphate [PI(4)P] on trans-Golgi network (TGN) vesicles were recruited to mitochondria-ER contact sites and could drive mitochondrial division downstream of Drp1. The loss of the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) or its effector, phosphatidylinositol 4-kinase IIIbeta [PI(4)KIIIbeta], in different mammalian cell lines prevented PI(4)P generation and led to a hyperfused and branched mitochondrial network marked with extended mitochondrial constriction sites. Thus, recruitment of TGN-PI(4)P-containing vesicles at mitochondria-ER contact sites may trigger final events leading to mitochondrial scission (Nagashima, 2020).


Search PubMed for articles about Drosophila four wheel drive

Brill, J. A., Hime, G. R., Scharer-Schuksz, M. and Fuller, M. T. (2000). A phospholipid kinase regulates actin organization and intercellular bridge formation during germline cytokinesis. Development 127(17): 3855-3864. PubMed ID: 10934029

de Graaf, P., Zwart, W. T., van Dijken, R. A., Deneka, M., Schulz, T. K., Geijsen, N., Coffer, P. J., Gadella, B. M., Verkleij, A. J., van der Sluijs, P. and van Bergen en Henegouwen, P. M. (2004). Phosphatidylinositol 4-kinasebeta is critical for functional association of rab11 with the Golgi complex. Mol Biol Cell 15(4): 2038-2047. PubMed ID: 14767056

Di Paolo, G. and De Camilli, P. (2006). Phosphoinositides in cell regulation and membrane dynamics. Nature 443(7112): 651-657. PubMed ID: 17035995

Forrest, S., Chai, A., Sanhueza, M., Marescotti, M., Parry, K., Georgiev, A., Sahota, V., Mendez-Castro, R. and Pennetta, G. (2013). Increased levels of phosphoinositides cause neurodegeneration in a Drosophila model of amyotrophic lateral sclerosis. Hum Mol Genet 22(13): 2689-2704. PubMed ID: 23492670

Giansanti, M. G., Farkas, R. M., Bonaccorsi, S., Lindsley, D. L., Wakimoto, B. T., Fuller, M. T. and Gatti, M. (2004). Genetic dissection of meiotic cytokinesis in Drosophila males. Mol Biol Cell 15(5): 2509-2522. PubMed ID: 15004238

Giansanti, M. G., Belloni, G. and Gatti, M. (2007). Rab11 is required for membrane trafficking and actomyosin ring constriction in meiotic cytokinesis of Drosophila males. Mol Biol Cell 18(12): 5034-5047. PubMed ID: 17914057

Godi, A., Pertile, P., Meyers, R., Marra, P., Di Tullio, G., Iurisci, C., Luini, A., Corda, D. and De Matteis, M. A. (1999). ARF mediates recruitment of PtdIns-4-OH kinase-beta and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex. Nat Cell Biol 1(5): 280-287. PubMed ID: 10559940

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

Landis, G.N., Bhole, D. and Tower, J. (2003). A search for doxycycline-dependent mutations that increase Drosophila melanogaster life span identifies the VhaSFD, Sugar baby, filamin, fwd and Cctl genes. Genome Biol. 4(2):R8. PubMed ID: 12620118

Nagashima, S., Tabara, L. C., Tilokani, L., Paupe, V., Anand, H., Pogson, J. H., Zunino, R., McBride, H. M. and Prudent, J. (2020). Golgi-derived PI(4)P-containing vesicles drive late steps of mitochondrial division. Science 367(6484): 1366-1371. PubMed ID: 32193326

Pogson, J. H., Ivatt, R. M., Sanchez-Martinez, A., Tufi, R., Wilson, E., Mortiboys, H. and Whitworth, A. J. (2014). The complex I subunit NDUFA10 selectively rescues Drosophila pink1 mutants through a mechanism independent of mitophagy. PLoS Genet 10(11): e1004815. PubMed ID: 25412178

Polevoy, G., Wei, H. C., Wong, R., Szentpetery, Z., Kim, Y. J., Goldbach, P., Steinbach, S. K., Balla, T. and Brill, J. A. (2009). Dual roles for the Drosophila PI 4-kinase four wheel drive in localizing Rab11 during cytokinesis. J Cell Biol 187(6): 847-858. PubMed ID: 19995935

Terriente-Felix, A., Wilson, E. L. and Whitworth, A. J. (2020). Drosophila phosphatidylinositol-4 kinase fwd promotes mitochondrial fission and can suppress Pink1/parkin phenotypes. PLoS Genet 16(10): e1008844. PubMed ID: 33085661

Biological Overview

date revised: 22 January 2021

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