A Conserved RhoGAP Limits M Phase Contractility and Coordinates with Microtubule Asters to Confine RhoA during Cytokinesis

Abstract

SUMMARY During animal cell cytokinesis, the spindle directs contractile ring assembly by activating RhoA in a narrow equatorial zone. Rapid GTPase activating protein (GAP)-mediated inactivation (RhoA flux) is proposed to limit RhoA zone dimensions. Testing the significance of RhoA flux has been hampered by the fact that the GAP targeting RhoA is not known. Here, we identify M-phase GAP (MP-GAP) as the primary GAP targeting RhoA during mitosis/cytokinesis. MP-GAP inhibition caused excessive RhoA activation in M-phase leading to the uncontrolled formation of large cortical protrusions and late cytokinesis failure. RhoA zone width was broadened by attenuation of the centrosomal asters but was not affected by MP-GAP inhibition alone. Simultaneous aster attenuation and MP-GAP inhibition led to RhoA accumulation around the entire cell periphery. These results identify the major GAP restraining RhoA during cell division and delineate the relative contributions of RhoA flux and centrosomal asters in controlling RhoA zone dimensions.

Cytokinesis in Animal Cells

“…3) (Miller and Bement 2009). However, similar findings have not been reported in other systems and at least two other GAPs, p190RhoGAP and ARHGAP11A, have also been reported to inhibit RhoA during cell division (Mikawa et al 2008;Zanin et al 2013). Therefore, it is currently unclear whether this role of RacGAP1 is conserved.…”

Anillin interacts with microtubules and is part of the astral pathway that defines cortical domains

“…In the absence of microtubules (e.g. in nocodazoletreated cells), other cortical regulators, such as MP-GAP, could modulate anillin localization (Zanin et al, 2013).…”

“…These data suggest that both sets of microtubules independently contribute to the localization of contractile proteins. Alternative mechanisms, including chromatin-based and MP-GAP (also known as ARHGAP11A), likely restrict anillin localization in the absence of microtubules (Kiyomitsu and Cheeseman, 2013;Zanin et al, 2013).…”

“…S1; RNAi efficiency is shown in supplementary material Fig. S2; Rankin and Wordeman, 2010;Zanin et al, 2013). These observations were quantified by measuring anillin fluorescence intensity across the central plane of the cell to include the equatorial cortex (the line scan was drawn away from the DNA in metaphase cells) and averaging cells with identical line lengths.…”

“…The concentration of nocodazole (30 ng/ml) used for this experiment preferentially removed polar astral microtubules but left some of the central spindle microtubules intact (supplementary material Fig. S3A; Zanin et al, 2013). Anillin fluorescence was measured across the central plane of cells at various mitotic stages ( Fig.…”