Julie A. Brill

PIP2 Hydrolysis and Calcium Release Are Required for Cytokinesis in Drosophila Spermatocytes

The role of calcium (Ca(2+)) in cytokinesis is controversial, and the precise pathways that lead to its release during cleavage are not well understood. Ca(2+) is released from intracellular stores by binding of inositol trisphosphate (IP3) to the IP3 receptor (IP3R), yet no clear role in cytokinesis has been established for the precursor of IP3, phosphatidylinositol 4,5-bisphosphate (PIP2). Here, using transgenic flies expressing PLCdelta-PH-GFP, which specifically binds PIP2, we identify PIP2 in the plasma membrane and cleavage furrows of dividing Drosophila melanogaster spermatocytes, and we establish that this phospholipid is required for continued ingression but not for initiation of cytokinesis. In addition, by inhibiting phospholipase C, we show that PIP2 must be hydrolyzed to maintain cleavage furrow stability. Using an IP3R antagonist and a Ca(2+) chelator to examine the roles of IP3R and Ca(2+) in cytokinesis, we demonstrate that both of these factors are required for cleavage furrow stability, although Ca(2+) is dispensable for cleavage plane specification and initiation of furrowing. Strikingly, providing cells with Ca(2+) obviates the need to hydrolyze PIP2. Thus, PIP2, PIP2 hydrolysis, and Ca(2+) are required for the normal progression of cytokinesis in these cells.

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

Fwd shuttles Rab11 to the cleavage furrow by both kinase-dependent and -independent mechanisms.

Drosophila spermiogenesis: Big things come from little packages.

Drosophila melanogaster spermatids undergo dramatic morphological changes as they differentiate from small round cells approximately 12 μm in diameter into highly polarized, 1.8 mm long, motile sperm capable of participating in fertilization. During spermiogenesis, syncytial cysts of 64 haploid spermatids undergo synchronous differentiation. Numerous changes occur at a subcellular level, including remodeling of existing organelles (mitochondria, nuclei), formation of new organelles (flagellar axonemes, acrosomes), polarization of elongating cysts and plasma membrane addition. At the end of spermatid morphogenesis, organelles, mitochondrial DNA and cytoplasmic components not needed in mature sperm are stripped away in a caspase-dependent process called individualization that results in formation of individual sperm. Here, we review the stages of Drosophila spermiogenesis and examine our current understanding of the cellular and molecular mechanisms involved in shaping male germ cell-specific organelles and forming mature, fertile sperm.

Two phosphatidylinositol 4-kinases control lysosomal delivery of the Gaucher disease enzyme, β-glucocerebrosidase

Trafficking of glucocerebrosidase (GBA) enzyme from the endoplasmic reticulum to the lysosome requires lysosomal integral membrane protein type 2 (LIMP-2), which is a receptor for GBA. This study shows that phosphatidylinositol 4-kinase (PI4K) type IIIβ controls the exit of LIMP-2/GBA complex from the Golgi, while PI4KIIα is required for the post-Golgi trafficking of the complex via the late endosomes.

Stabilization of the Actomyosin Ring Enables Spermatocyte Cytokinesis in Drosophila

The scaffolding protein anillin recruits septins to the cleavage furrow and constrains actomyosin contractility. Expression of E-cadherin suppresses the cytokinesis defects caused by anillin knockdown and stabilizes F-actin in the furrow, thereby providing an alternate means of coupling the actomyosin ring to the plasma membrane during cytokinesis.

Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

BackgroundPhosphatidylinositol 4,5-bisphosphate (PIP2) is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn induces calcium (Ca2+) release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated.ResultsHere we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH3 (edelfosine) on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3.ConclusionWe have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring.

Depletion of plasma membrane PtdIns(4,5)P2 reveals essential roles for phosphoinositides in flagellar biogenesis

Axonemes are microtubule-based organelles of crucial importance in the structure and function of eukaryotic cilia and flagella. Despite great progress in understanding how axonemes are assembled, the signals that initiate axoneme outgrowth remain unknown. Here, we identified phosphatidylinositol phosphates (phosphoinositides) as key regulators of early stages of axoneme outgrowth in Drosophila melanogaster spermatogenesis. In a study of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] function in developing Drosophila male germ cells, we depleted PtdIns(4,5)P2 by expression of a potent phosphoinositide phosphatase. Phosphatase expression dramatically inhibited sperm tail formation and perturbed microtubule organization in a manner reversible by co-expression of a PtdIns 4-phosphate 5-kinase. Depletion of PtdIns(4,5)P2 caused increased levels of basal body γ-tubulin and altered the distribution of proteins known to be required for axoneme assembly. Examination of PtdIns(4,5)P2-depleted spermatids by transmission electron microscopy revealed defects in basal body docking to the nuclear envelope, and in axoneme architecture and integrity of the developing flagellar axoneme and axial sheath. Our results provide the first evidence that phosphoinositides act at several steps during flagellar biogenesis, coordinately regulating microtubule and membrane organization. They further suggest that phosphoinositides play evolutionarily conserved roles in flagella and cilia, across phyla and in structurally diverse cell types.

Continuous phosphatidylinositol metabolism is required for cleavage of crane fly spermatocytes

Successful cleavage of animal cells requires co-ordinated regulation of the actomyosin contractile ring and cleavage furrow ingression. Data from a variety of systems implicate phosphoinositol lipids and calcium release as potential regulators of this fundamental process. Here we examine the requirement for various steps of the phosphatidylinositol (PtdIns) cycle in dividing crane fly (Nephrotoma suturalis) spermatocytes. PtdIns cycle inhibitors were added to living cells after cleavage furrows formed and began to ingress. Inhibitors known to block PtdIns recycling (lithium), PtdIns phosphorylation (wortmannin, LY294002) or phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] hydrolysis [U73122 (U7)] all stopped or slowed furrowing. The effect of these drugs on cytokinesis was quite rapid (within 0-4 minutes), so continuous metabolism of PtdIns appears to be required for continued cleavage furrow ingression. U7 caused cleavage furrow regression concomitant with depletion of F-actin from the contractile ring, whereas the other inhibitors caused neither regression nor depletion of F-actin. That U7 depletes furrow-associated actin seems counterintuitive, as inhibition of phospholipase C would be expected to increase cellular levels of PtdIns(4,5)P2 and hence increase actin polymerization. Our confocal images suggest, however, that F-actin might accumulate at the poles of U7-treated cells, consistent with the idea that PtdIns(4,5)P2 hydrolysis may be required for actin filaments formed at the poles to participate in contractile ring assembly at the furrow.

AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

Clathrin and AP-1 are required for the formation of mucin-type secretory granules in Drosophila larval salivary gland cells. Clathrin and AP-1 colocalize with secretory cargo at the trans-Golgi network (TGN) and on immature granules. Moreover, clathrin recruitment to the TGN requires AP-1. Strikingly, loss of AP-1 or clathrin profoundly blocks granule biogenesis.

Phosphatidylinositol 4,5-bisphosphate Directs Spermatid Cell Polarity and Exocyst Localization in Drosophila

This study identifies phosphoinositides as key regulators of spermatid cell polarity. Polarization and elongation of spermatids in Drosophila are regulated through local synthesis of PIP2 by Sktl, which drives polarized localization of the exocyst complex to promote targeted membrane delivery and polarization of the elongating spermatid cysts.