Rab11 Is Required for Membrane Trafficking and Actomyosin Ring Constriction in Meiotic Cytokinesis ofDrosophilaMales

Abstract

Rab11 is a small GTPase that regulates several aspects of vesicular trafficking. Here, we show 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. We propose that Gio and Fwd mediate Rab11 recruitment at the cleavage furrow and that Rab11 facilitates targeted membrane delivery to the advancing furrow.

Mutations inCog7affect Golgi structure, meiotic cytokinesis and sperm development duringDrosophilaspermatogenesis

“…In fixed dividing spermatocytes coexpressing Gio-RFP with Rab11-GFP, Gio colocalized with Rab11, consistent with previous results with anti-Gio and antiRab11 antibodies (supplementary material Fig. S5) (Giansanti et al, 2007). During telophase Gio and Rab11 concentrated at the cleavage furrow ( Fig.…”

“…Fluorescent WGA also stains the equatorial plasma membrane of Drosophila wild-type spermatocytes during late anaphase/telophase indicating an enrichment of sialic or N-acetylglucosamine glycoconjugates ( Fig. 5B) (Giansanti et al, 2007) but failed to detect an accumulation of WGA receptors in all the Cog7 cells examined (Fig. 5B, n580 for wild type, n584 for Cog7).…”

“…Previous data showed that the phosphatidylinositol transfer protein (PITP) Giotto/Vib (Gio/Vib) and the small GTPase Rab11 accumulate at the cleavage site and are both required for spermatocyte cytokinesis (Gatt and Glover, 2006;Giansanti et al, 2006;Giansanti et al, 2007;Polevoy et al, 2009). It was suggested that Rab11, Gio and the Phosphatidylinositol 4-kinase b (PI4Kb) Fwd function in the same pathway during cytokinesis, with Gio and Fwd acting upstream of Rab11 (Giansanti et al, 2007;Polevoy et al, 2009). Our immunofluorescence analysis revealed that loss of Cog7 impaired the recruitment of Gio at the cell equator in 89% of telophase spermatocytes ( Fig.…”

“…S4). Since localisation of Rab11 to the cleavage site is dependent on both Gio (Giansanti et al, 2007) and Cog7 (this study) we performed Co-IP experiments using Drosophila testis protein extracts aimed at verifying interactions among these proteins. Protein extracts from testes expressing either GFP-Cog7 or Rab11-GFP were immunoprecipitated using the GFP-Trap (see Materials and Methods).…”

“…For example, Drosophila spermatocytes are quite large cells (more than 20 mm in diameter), that complete two meiotic divisions in less than two hours (Fuller, 1993;Giansanti et al, 2012). Spermatocyte cytokinesis proved to be sensitive to mutations affecting vesicle traffic components (Brill et al, 2000;Farkas et al, 2003;Dyer et al, 2007;Gatt and Glover, 2006;Giansanti et al, 2006;Giansanti et al, 2007;Polevoy et al, 2009;Robinett et al, 2009;Xu et al, 2002a;Zhou et al, 2011). 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).…”