Structural and biochemical characterization of the mechanism of actin filament branching mediated by Arp2/3 complex in Schizosaccharomyces pombe

My research focused on answering the mechanistic question about the mechanism of actin filament nucleation by Arp2/3 complex and how the process contributes to clathrin-mediated endocytosis in fission yeast Schizosaccharomyces pombe. I used biochemical and biophysical experiments to dissect two aspects of this question. First, I performed extensive biochemical investigations on the thermodynamics and kinetics of the interactions involved in the assembly of actin filament branches. Isothermal titration calorimetry showed that two VCA regions from the C-terminus of the nucleation promoting factor WASp bind one Arp2/3 complex with significantly different affinities. Fission yeast Wsp1p-VCA binds site 1 on fission yeast Arp2/3 complex with Kd of ∼ 0.1 microM, and binds site 2 with Kd of ∼ 1 microM. VCA bound to site 1 inhibits binding of Arp2/3 complex to the sides of preexisting actin filaments, but the association of Arp2/3 complex with actin filaments increases the affinity of site 2 for VCA about 20-fold. Arp2/3 complex bound to an actin filament binds actin-VCA crosslinked product with ∼ 100-fold higher affinity than free Arp2/3 complex. My colleague Christopher Jurgenson also provided the first high-resolution crystal structure of part of VCA bound to site 2 Arp2/3 complex, and my mutagenesis of this site confirmed that VCA binding to site 2 is essential for the viability of fission yeast. I proposed a possible mechanism of the formation of branched actin filaments that is consistent with all the evidence from biochemistry, chemical crosslinking and protein structures.;Second, I purified and characterized active S. pombe actin. Although fission yeast S. pombe is a popular model organism for studying endocytosis and cytokinesis, most of the previous biochemical experiments on fission yeast actin-binding proteins were done with skeletal muscle actin, because the purified, active S. pombe actin was not available. I developed a protocol to purify active S. pombe actin, and compared several functional properties of S. pombe and muscle actins. S. pombe and skeletal muscle actins have the same critical concentration, but ATP dissociates 40-fold faster from monomers, S. pombe actin form trimers 5-fold faster and the melting temperature of S. pombe actin is 10 °C lower than skeletal muscle actin monomers. Phosphate dissociates 20- to 100-fold faster from filaments of S. pombe ADP-P i-actin filaments than muscle actin filaments, and the affinity of S. pombe Arp2/3 complex binds S. pombe actin filaments with a higher affinity than muscle actin filaments. These biochemical parameters explain the published discrepancies between computer simulations of actin patch dynamics in S. pombe and the biochemical parameters measured with skeletal muscle actin.