Dynamics of Branched Actin Filaments in Schizosaccharomyces Pombe : Structural and biochemical characterization of the mechanism of actin filament branching mediated by Arp2/3 complex

De novo formation of actin filaments from the side of mother filaments plays crucial roles at the leading edge of motile cells and in endocytosis. The seven-subunit Arp2/3 complex initiates these actin filament branches. Fission yeast S. pombe has been used as a genetic model organism on studying the mechanism of endocytosis and cytokinesis. However, there is a gap between microscopic observations in pombe cells and in vitro biochemistry because most of the biochemical and biophyscical properties of the fission yeast actin binding proteins have been done with muscle skeletal actin for matters of convenience. I filled the gap with the newly developed protocols for the purification and characterization of active pombe actin by affinity chromatography with C-terminal half of mouse gelsolin (G4-6). I also showed one Arp2/3 complex binds two VCAs and described the intriguing interactions of Arp2/3 complex's two VCA binding sites with VCAs and actin filaments. Our crystal structure showed the N-terminal tryptophan of CA located at Arp3, and we surmised this is the low affinity site 2. My fission yeast genetic results verified the importance of this binding site on cell viability.