Title: Genetic and biochemical studies of cytokinesis
Description: Cytokinesis is the last step in the cell cycle, in which the cell physically separates into two daughters. It is a fundamental biological process of medical importance since successful cytokinesis is critical for the maintenance of genome stability. Cytokinesis requires coordinated activities of the cytoskeleton, membrane trafficking systems and cell cycle engine that are precisely controlled in space and time. We need to understand the mechanisms underlying each of these diverse processes and how they are coordinated. I used three approaches to try to understand cytokinesis mechanism. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila tissue culture cells, looking for genes encoding proteins required for cytokinesis and small molecules that target them. We identified 50 small molecule inhibitors and 214 important genes. Most proteins whose knockdown caused strong inhibition were already known, but we did identify a new subunit of the Aurora B kinase complex. Our weaker hits include important cytokinesis proteins likely required at other times in the cell cycle, including 12 genes encoding vesicle transport proteins. We also identified 54 genes predicted to encode proteins of unknown function.

I characterized a series of mutations in the anillin gene, a strong hit in our RNAi screen. I found defects in cytokinesis, cellularization and pole cell formation. Mutations that result in amino acid changes in Anillin's C-terminal PH domain caused defects in septin recruitment to the cellularization front and contractile ring during zygotic cell divisions, perturbing both processes. Our data indicate an important role for Anillin in scaffolding cleavage furrow proteins, directly stabilizing intracellular bridges, and indirectly stabilizing newly deposited plasma membrane during cellularization. Cell-free systems allow researchers to analyze biological processes in isolation, setting the stage for inhibition and fractionation of the underlying biochemistry. I explored whether cell cycle-regulated Xenopus extracts might provide a cell-free system to study aspects of cytokinesis. Upon comparing mitotic and interphase extracts, I found they exhibited very different actomyosin dynamics. My exploration on a molecular level revealed cell cycle differences in actin filament nucleation. This has led me to propose novel hypotheses concerning regulation of actin nucleation during the cell cycle.