Inhibition of Rac by the GAP activity of centralspindlin is essential for cytokinesis

Abstract

During cytokinesis, the guanosine triphosphatase (GTPase) RhoA orchestrates contractile ring assembly and constriction. RhoA signaling is controlled by the central spindle, a set of microtubule bundles that forms between the separating chromosomes. Centralspindlin, a protein complex consisting of the kinesin-6 ZEN-4 and the Rho family GTPase activating protein (GAP) CYK-4, is required for central spindle assembly and cytokinesis in Caenorhabditis elegans. However, the importance of the CYK-4 GAP activity and whether it regulates RhoA remain unclear. We found that two separation-of-function mutations in the GAP domain of CYK-4 lead to cytokinesis defects that mimic centralspindlin loss of function. These defects could be rescued by depletion of the GTPase Rac or its effectors, but not by depletion of RhoA. Thus, inactivation of Rac by centralspindlin functions in parallel with RhoA activation to drive contractile ring constriction during cytokinesis.

Figure 1. CYK-4 GAP domain mutants phenocopy Centralspindlin loss-of function

A) Residues changed in CYK-4 and ZEN-4 mutant proteins. B) The E448K and T546I substitutions destabilize CYK-4 GAP domain structure. C) Plot of mean furrow diameter versus time. Error bars=SEM. D) Time-lapse montage of the furrow region in embryos expressing a GFP::plasma membrane marker. Series begin at anaphase onset. Scale bar, 20μm.

Figure 2. Central spindle assembly is not disrupted in the GAP mutants

A) Immunofluorescence staining for tubulin, ZEN-4, and DNA. B) Images of GFP::AuroraB^AIR-2 40s post-anaphase onset. C) Plot of mean centrosome separation verses time. Error bars=SEM. Scale bars, 10 μm.

Figure 3. Rac depletion suppresses the cytokinesis defect in CYK-4^GAP(E448K) embryos

Time-lapse montage of the furrow region in embryos expressing a GFP::plasma membrane marker. Series begin at anaphase onset. A) CYK-4^GAP(E448K) embryos exhibit partial furrow ingression followed by regression. B) Partial depletion of RhoA^RHO-1 enhances the CYK-4^GAP(E448K) cytokinesis defect. C) RNAi of other Rho family members does not disrupt cytokinesis. D) Depletion of Rac^CED-10 or Rac^RAC-2 rescues cytokinesis success in CYK-4^GAP(E448K) embryos. Scale bar, 20 μm.

Figure 4. Negative regulation of Rac, WASP/WAVE, and the Arp2/3 complex by CYK-4 is essential for cytokinesis

Cytokinesis in CYK-4^GAP(E448K) embryos is rescued by co-depletion of A) the Rac effectors WASP^WSP-1/WAVE^WVE-1 or B) of the downstream target Arp2^ARX-2. Scale bar, 20 μm. C) Model for signaling during cytokinesis.