Syndapin Functions in Orchestrated Remodelling of the Membrane and Actin Cytoskeleton

Abstract

The syncytial nuclear division cycles during early Drosophila embryo development involves orchestrated remodelling of the membrane and cytoskeleton. We have shown that the F-BAR domain protein Syndapin, that is known to have roles in membrane tubulation, endocytosis and cytoskeletal remodelling, is increasingly enriched at the tip of syncytial pseudocleavage furrows with each nuclear cycle. The dynamics of pseudocleavage furrow progression depends on Syndapin function. We showed that Syndapin function was important for recruitment of septin Peanut and organization of the formin Diaphanous at the furrow tip downstream of the furrow initiation protein RhoGEF2. Syndapin-depleted embryos showed disorganized actin on the furrow, also seen in RhoGEF2 mutants. Interstingly, Syndapin overexpression in RhoGEF2-depleted background, restored actin organization and extent of furrow ingression. Addtionally, we showed that Syndapin or RhoGEF2 depletion causes reduction in the number of Rab5 puncta; however, Syndapin overexpression in RhoGEF2- depleted background cannot restore the number of Rab5 puncta. Therefore, we conclude that Syndapin can rescue the furrow extension phenotype by actin organization alone and not because of its role in early endocytosis. Actin turnover is also crucial for dynamic expansion of the apical caps prior to furrow formation. Arp2/3 function is important for expansion of the apical cap. We find that syndapin mutants limit cap expansion and show abnormally large actin caps. We are able to rescue this cap expansion phenotype by combining mutants of a component of the Arp2/3 complex, ArpC1 with the syndapin mutant. Using TIRF microscopy, we find that remodelling of apical actin structures is defective in these mutants giving rise to the observed phenotypes. Therefore, Syndapin and Arp2/3 complex oppose each other to regulate dynamics of actin cap expansion in the syncytial embryo. Apical cap expansion defects also correlate with defects in furrow extension and formation of edges of polygonal compartments where the expanding caps meet. Therefore, we also studied effects of Syndapin depletion and also of other actin regulatory proteins on the distribution of polygon classes that are conserved across organized epithelial sheets in various tissues with a maximum of hexagons. The result was a frequency distribution skewed towards pentagons rather than hexagons. Interestingly, however, the recruitment of junctional polarity proteins was not affected in embryos depleted of Syndapin.