Characterization of constitutively active Arp2/3

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My goal is a quantitative understanding of the activation of Arp2/3 complex, a protein assembly instrumental in cellular motility. I propose three lines of experiments to understand how activators change the conformation of Arp2/3 complex and how this conformational change influences actin filament nucleation and branching in vivo and in vitro. . Use disulfide crosslinking between cysteine residues engineered into fission yeast Arp2/3 complex to create constitutively active and inactive Arp2/3 complex. I will compare native Arp2/3 complex and these two frozen conformations in spectroscopic and single particle fluorescence microscopy assays to analyze the kinetics and thermodynamics of Arp2/3 complex activation by nucleation promoting factors such as the VGA regions of WASp and Scar, actin monomers, and pre-existing actin filaments. . Use high-resolution 3D fluorescence microscopy to characterize the effects of a panel of mutations on the ability of Arp2/3 complex to support actin filament nucleation in live fission yeast. Use simulations to study Arp2/3 subunit mechanisms involved in nucleotide binding and activation.