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

Abstract

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.

Mutations inCog7affect Golgi structure, meiotic cytokinesis and sperm development duringDrosophilaspermatogenesis

“…However, axonemal of PIP2-depleted flies also exhibited alterations not observed in Cog7 flies, such as triplets as well as doublets among outer microtubules, suggesting a role for PIP2 or a downstream second messenger in controlling the transition from basal body to axonemal microtubule arrays (Wei et al, 2008). The defects in axoneme architecture are not merely the consequence of cytokinesis failure, as other mutants defective in cytokinesis form regular axonemes (Wei et al, 2008).…”

“…Spermatid elongation, which involves a 100-fold increase in cell surface area, may necessitate the addition of new membrane derived from Golgi vesicles and so may be especially sensitive to defects in Golgi structure and/or function (Fabian et al, 2010;Farkas et al, 2003;Fuller, 1993;Tates, 1971;Wei et al, 2008;Zhou et al, 2011). In addition, loss of Cog7 affected architecture of the developing flagellar axonemes and resulted in dissociation of the basal body from the nuclear envelope, suggesting defects in the docking to the nuclear envelope.…”

“…Recent studies demonstrated that depletion of the phosphoinositide phosphatidylinositol 4,5 biphosphate (PIP2) results in defects in both axoneme architecture and in basal docking to the nuclear envelope (Wei et al, 2008). However, axonemal of PIP2-depleted flies also exhibited alterations not observed in Cog7 flies, such as triplets as well as doublets among outer microtubules, suggesting a role for PIP2 or a downstream second messenger in controlling the transition from basal body to axonemal microtubule arrays (Wei et al, 2008).…”

“…During Drosophila spermiogenesis, round spermatids, ,12 mm in diameter, undergo drastic morphological changes in cell surface before reaching the impressive 1.8 mm in length of sperm tails (Fuller, 1993;Tates, 1971;Zhou et al, 2011). The 100-fold increase in cell length that characterises spermatid elongation is particularly dependent on membrane biosinthesis and membrane remodelling (Fabian et al, 2010;Farkas et al, 2003, Wei et al, 2008Zhou et al, 2011). Among the COG subunits, the Drosophila Cog5 homologue Four way stop (Fws) has been implicated in both male meiotic cytokinesis and spermatid elongation .…”

PI(4,5)P2 regulates myoblast fusion through Arp2/3 regulator localization at the fusion site

“…High levels of the reporter are shown to sequester PI(4,5)P2 moieties in the inner leaflet of the plasma membrane and therefore block PI(4,5)P2 signaling (Kwik et al, 2003;Raucher et al, 2000;Wei et al, 2008). To titrate PI(4,5)P2 levels during myoblast fusion, we manipulated both the number of transgenes and temperature.…”