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ABSTRACT 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 whereas the GTPase Rho1 activates these enzymes. We show that Cdc42 prevents early Rho1 activation during fission yeast 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. 

Abstract Fission yeast cytokinesis is driven by simultaneous septum synthesis, membrane furrowing and actomyosin ring constriction. The septum consists of a primary septum flanked by secondary septa. First, delivery of the glucan synthase Bgs1 and membrane vesicles initiate primary septum synthesis and furrowing. Next, Bgs4 is delivered for secondary septum formation. It is unclear how septum synthesis is coordinated with membrane furrowing. Cdc42 promotes delivery of Bgs1 but not Bgs4. We find that after primary septum initiation, Cdc42 inactivators Rga4 and Rga6 localize to the division site. Inrga4Δrga6Δmutants, Cdc42 activity is enhanced during late cytokinesis and cells take longer to separate. Electron micrographs of the division site in these mutants exhibit malformed septum with irregular membrane structures. These mutants have a larger division plane with enhanced Bgs1 delivery but fail to enhance accumulation of Bgs4 and several exocytic proteins. Additionally, these mutants show endocytic defects at the division site. This suggests that Cdc42 regulates primary septum formation and only certain membrane trafficking events. As cytokinesis progresses Rga4 and Rga6 localize to the division site to decrease Cdc42 activity to allow coupling of Cdc42‐independent membrane trafficking events with septum formation for proper septum morphology.