Cdc42 prevents precocious Rho1 activation during cytokinesis in a Pak1-dependent manner

Abstract

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.

The Arp2/3 complex promotes periodic removal of Pak1-mediated negative feedback to facilitate anticorrelated Cdc42 oscillations

“…Further, Pak1 plays multiple roles at the membrane in addition to Scd1 phosphorylation such as activating Myo1 which promotes Pak1 removal [32][33][34]70]. Thus, the inhibitory role of increasing Pak1 activity causes a gradual decrease in its own recruitment until, eventually, Pak1 removal rate exceeds recruitment rate.…”

“…Next, it is likely that, Pak1 phosphorylates Scd1 to hinder Scd1-mediated Cd42 activation and dampen positive-feedback similar to budding yeast [35, 36]. Further, Pak1 plays multiple roles at the membrane in addition to Scd1 phosphorylation such as activating Myo1 which promotes Pak1 removal [32–34, 70]. Thus, the inhibitory role of increasing Pak1 activity causes a gradual decrease in its own recruitment until, eventually, Pak1 removal rate exceeds recruitment rate.…”

“…The Pak1 kinase is activated and recruited to the growing cell ends when it binds active Cdc42 [29,30]. When active, Pak1 phosphorylates effector proteins to modulate a host of biological pathways [11,[29][30][31][32][33][34]. In the absence of Pak1 activity, Scd1 and Scd2 accumulate at the cell ends leading to increased Cdc42 activity [11].…”