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

Abstract

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.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of <em>Drosophila</em> Testes

“…In Figure 4, microtubules in a fixed testes preparation were visualized via the intrinsic fluorescence of GFPtagged beta1-tubulin (expressed from a transgene under control of a globally expressed ubiquitin promoter). Alternatively, transgenic flies in which expression is under the control of a tissue-specific promoter; for example, the promoter of the beta2-tubulin gene have been used for testes-specific expression 35 . Alternatively, the expression of a protein of interest can be restricted to specific male germline cells by using the UAS-Gal4 system 36 .…”

Cytokinesis in Animal Cells

“…Consistent with this, the PI4-kinase Four-wheel drive (Fwd) and the PI(4)P binding protein GOLPH3 are both required for cytokinesis in Drosophila spermatocytes (Brill et al 2000;Polevoy et al 2009;Sechi et al 2014). Some evidence also indicates that the phosphoinositide phosphatidylinositol 4,5-biphosphate (PI(4,5)P2) is enriched at the cleavage furrows of dividing tissue culture cells and Drosophila spermatocytes, in which it regulates formation and stability of the cytokinetic ring (Field et al 2005b;Wong et al 2005;Echard 2012). PI(4,5)P2 interacts with the contractile ring components anillin and septins and regulates F-actin polymerization by modulating the activity of the actin-binding proteins profilin and cofilin (Yin and Janmey 2003;Bertin et al 2010;Liu et al 2012).…”

Rho GTPase activity zones and transient contractile arrays

“…(64,65) The PIP2 could then act as a GDF to liberate Rho from the GDI-Rho-GDP complex (see above). Accumulated PIP2 could also promote recruitment of ERM proteins such as radixin, which localize to cytokinetic furrows (66) and act as GDFs (see above). Another potential mechanism for selfamplification could come from Rho-dependent stimulation of microtubule stabilization in the zone region, (67) assuming that such stable microtubules promote Rho activation.…”