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Endocytosis promotes polarity and growth in eukaryotes. In Schizosaccharomyces pombe fission yeast, endocytosis occurs at the polarized cell ends and division site and at the non-polarized cell sides. Our characterization of endocytic actin patches show that they are differentially regulated. The patches at the cell ends and division site internalize successfully while those at the sides are weak and erratic. The major regulator of cell polarity, Cdc42, and its target Pak1 kinase only localize to the cell ends and division site. We find that these proteins regulate assembly and internalization of patches at these sites but not at the cell sides. Moreover, Cdc42 specifically activated by the GEF Gef1 promotes proper patch dynamics. Endocytosis requires phosphorylation of the Type I Myosin Myo1 by the Pak1 kinase. Myo1 localizes to the cell ends, division site, and the cell sides. We find that unlike Cdc42 and Pak1, Myo1 also promotes patch assembly at the cell sides. Our data indicate that while Myo1 can globally promote branched actin assembly, successful endocytic patch dynamics and internalization at polarized sites require Cdc42 and Pak1 kinase. 

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.