Cytokinesis consists of a series of coordinated multi-step events that partition a dividing cell. Accurate regulation of cytokinesis is essential for proliferation and genome integrity. In fission yeast, these coordinated events ensure that the actomyosin ring and septum start ingressing only after chromosome segregation. How cytokinetic events are coordinated is unclear. The GTPase Cdc42 is required for the delivery of certain cell wall-building enzymes while the GTPase Rho1 is required for activation of these enzymes. Here we show that Cdc42 prevents early Rho1 activation during cytokinesis. Using a Rho-probe, we report that Rho1 is activated in late anaphase, just before the onset of ring constriction, even though, the primary Rho1 activators Rgf1 and Rgf3 localize to the division site in early anaphase. We find that loss of Cdc42 activation enables precocious Rho1 activation in early anaphase. Furthermore, this inhibition of Rho1 activation is dependent on the downstream Cdc42 effector Pak1 kinase. Disrupting pak1 function resulted in early Rho1 activation accompanied by precocious septum deposition and ring constriction. We provide functional and genetic evidence which indicates that Pak1 regulates Rho1 activation likely via the regulation of its GEF Rgf1. Our work proposes a mechanism of regulation of Rho1 by active Cdc42 to coordinate timely septum formation and cytokinesis fidelity.

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During cytokinesis a series of coordinated events partition a dividing cell. Accurate regulation of cytokinesis is essential for proliferation and genome integrity. In fission yeast, these coordinated events ensure that the actomyosin ring and septum start ingressing only after chromosome segregation. How cytokinetic events are coordinated remains unclear. The GTPase Cdc42 promotes recruitment of certain cell wall-building enzymes while the GTPase Rho1 activates these enzymes. We show that Cdc42 prevents early Rho1 activation during cytokinesis. Using an active Rho-probe, we find that although the Rho1 activators Rgf1 and Rgf3 localize to the division site in early anaphase, Rho1 is not activated until late anaphase, just before the onset of ring constriction. We find that loss of Cdc42 activation enables precocious Rho1 activation in early anaphase. Furthermore, we provide functional and genetic evidence that Cdc42-dependent Rho1 inhibition is mediated by the Cdc42 target Pak1 kinase. Our work proposes a mechanism of Rho1 regulation by active Cdc42 to coordinate timely septum formation and cytokinesis fidelity.