Cell Cycle Regulation of Membrane Trafficking

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Project Summary Cell division requires a carefully coordinated series of essential events that must be precisely regulated. A central feature of cell division is the accurate segregation of chromosomes into daughter cells. Other compartments of the cell must also be carefully packaged into daughter cells, and coordinated with chromosome segregation. Membrane trafficking pathways are essential for the completion of cytokinesis at the end of cell division. How cells trigger membrane trafficking during cytokinesis is not well understood. The large protease separase is a central player in chromosome segregation due to its role in cohesin cleavage, which allows chromosome separation at the onset of anaphase. After chromosome segregation, separase promotes several events during anaphase. This proposal aims to understand a novel role of separase in the exocytosis of RAB-11 vesicles required for cytokinesis. Separase also regulates exocytosis of large cortical granules during anaphase of meiosis I to block polyspermy, which is an ideal context to analyze this regulatory pathway. This proposal describes research aimed at understanding how separase promotes exocytosis in collaboration with other closely related cell cycle regulators that have well characterized roles in chromosome segregation. The dynamic localization of separase is regulated during cell division and only localizes to vesicles during anaphase. Proposed research will investigate how chromosome segregation regulators control separase activity and localization to vesicles. Overexpression of non-degradable securin will be used to determine how this inhibitory chaperone controls the exocytic function of separase. Mutations of the PPH-5 phosphatase and its activator HSP-90 were identified as suppressors of separation of function alleles of separase. The functions of PPH-5 and HSP-90 will be characterized to determine whether they directly regulate separase during the meiotic divisions. Separase phosphorylation sites will be mapped and phosphorylation mutants will be studied to determine how they affect separase function. PPH-5 will be tested to determine if it directly dephosphorylates separase in vitro. Finally, biochemical and genetic approaches will be employed to identify substrates or targets of separase on vesicles to define the mechanism by which it promotes exocytosis. These studies will be performed using the genetically tractable C. elegans embryo. This work will provide new insight into how cells coordinate the essential process of chromosome segregation with exocytosis during cytokinesis, which is relevant to understanding normal development and diseases such as infertility and cancer.