Cell Cycle Regulation of Membrane Trafficking

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 control 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.
We will use biochemical and genetic approaches to identify substrates or binding partners of separase on
vesicles to define the mechanism by which it promotes exocytosis. The dynamic localization of separase is
regulated during cell division and separase only localizes to vesicles during anaphase. We 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
embryo lethality of separase mutants. 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. The functions of PPH-5 and HSP-90 will be characterized to
determine whether they directly regulate separase during the meiotic divisions. 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.

Grant Number: 5R01GM114471-11
NIH Institute/Center: NIH
Principal Investigator: Joshua Bembenek