Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila

Abstract

Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression.

Phosphatidylinositol 4,5-bisphosphate regulates cilium transition zone maturation in Drosophila melanogaster

“…Stocks expressing GFP-tagged Cby and Mks1 were provided by Bénédicte Durand (Institut NeuroMyoGéne, Université Claude Bernard Lyon-1, France) (Enjolras et al, 2012;Vieillard et al, 2016). The Exo84 onr mutant was described previously (Giansanti et al, 2015). Stocks for generating sktl 2.3 clones were originally provided by Antoine Guichet (Institut Jacques Monod, Université Paris-Diderot, France) (Gervais et al, 2008).…”

Phosphoinositide signaling in sperm development

“…For example, subunits of the exocyst, an octameric complex that mediates targeted plasma membrane delivery, bind PIP2 [90,91]. Two exocyst subunits, Sec8, which binds PIP2, and Exo84, were recently shown to be required for spermatocyte cytokinesis [86]. Dividing spermatocytes from exocyst mutants show defects in plasma membrane expansion, consistent with a role for the exocyst in membrane delivery.…”

“…In male germ cells, cytokinesis is incomplete, resulting in formation of stable intercellular bridges that interconnect clonally related cells within a cyst [59] (reviewed in [60]). Because PIPs regulate actin assembly and membrane trafficking, they are ideally suited to orchestrate cytokinesis (reviewed in [61,62] [80,84,86,88] and others remain to be tested.…”

Peripheral axonal ensheathment is regulated by Ral GTPase and the exocyst complex

“…We identified Ral and exocyst as factors required for wrapping glia membrane growth. This pathway has been implicated in membrane addition in other cellular contexts, including postsynaptic growth (Teodoro et al 2013; Patrício-Rodrigues & Teodoro 2018), nanotube formation (Hase et al 2009) and in growth phases during cell division (Murthy et al 2010; Holly et al 2015; Giansanti et al 2015). While the Ral/Exocyst pathway is clearly a mechanism widely used for growth regulation, our results indicate that it is not simply a default growth signal, since subperineurial glia size was unchanged in the mutants and this glia subtype also depends on hypertrophic growth during larval development.…”