Spatiotemporal Regulation of Cdc42 Activity Directs Specific Membrane Trafficking Events at Distinct Cell Sites

Abstract

Polarization allows cells to form and maintain morphologies necessary for their diverse functions during processes such as growth, division, differentiation, and migration. Signaling proteins such as the family of small Rho GTPases promote polarization by spatiotemporally regulating cytoskeleton dynamics and coordinating membrane trafficking. Here, we investigate and define roles of the Rho GTPase Cdc42 in promoting polarization in S. pombe. As fission yeast, S. pombe cells grow from their cell ends during interphase and divide by medial fission to produce two new daughter cells. As cell-walled organisms, growth and division require intricate remodeling and expansion of the cell wall via incorporation of new membrane and proteins at these polarized sites. Thus, growth and division require specific sequences of membrane trafficking events to deliver and remove cargo at appropriate times and locations. During cytokinesis, fission yeast cells divide by synthesizing new cell wall called the septum to medially bisect the cell. The septum is synthesized behind the actomyosin ring to aid its constriction. Once ring constriction completes and the septum matures, the septum is partially digested to physically separate the daughter cells. Previous work has shown that Cdc42 promotes the delivery of specific but not all septum-synthesizing enzymes as well as septum-digesting enzymes, but it was not known how Cdc42 activation is regulated at the division site to temporally coordinate these processes. Here, we show that the Cdc42 GAPs Rga4 and Rga6 promote proper septum synthesis and timely cell separation by locally decreasing Cdc42 activation during late cytokinesis. This work also reveals a role for Cdc42 in regulating clathrin-mediated endocytosis, both at the division site as well as at growing cell ends. To further explore this role, we systematically examined the behaviors of endocytic actin patches in mutants of Cdc42 regulators and compared these dynamics to wild-type controls. This characterization led to the observation that endocytic patches are best formed to induce successful patch internalization at sites of polarization where Cdc42 is active. In this work, we show that Cdc42 activation promotes proper endocytic patch behavior in a dose-dependent manner and that Cdc42 regulates endocytosis via its downstream effector, the Pak1 kinase. We also demonstrate that Cdc42 and Pak1 activity promote endocytosis through at least two pathways which regulate branched actin formation. First, we show that Cdc42 and Pak1 promote proper endocytic actin patch formation. Secondly, we show that Pak1-mediated phosphorylation of the endocytic Type I myosin promotes timely internalization of endocytic actin patches.