Engineering and Characterization of a Fluorescent Fission Yeast Arp2/3 Complex for Single Molecule Mechanistic Investigations.

Abstract

Arp2/3 complex is a seven-component protein complex that facilitates the assembly of branched actin filament networks by binding to pre-existing "mother" actin filaments and initiating the nucleation of new branched "daughter" filaments. Arp2/3 complex must be activated by a nucleation promoting factor such as the WASP/Scar protein family. The direct visualization of Arp2/3 complex during the branching pathway is critical to elucidate the underlying molecular mechanisms under diverse assembly conditions. Here we have successfully purified and fluorescently labeled a fission yeast Arp2/3 complex that is activated by different classes of fission yeast nucleation promoting factors. We visualized Arp2/3 complex actin filament binding and branch formation in two-color TIRF microscopy assays, revealing that a single complex of Arp2/3 complex nucleates a branched actin filament ~2 s after binding. We have therefore generated an important reagent that can help elucidate a clearer understanding of the pathway and regulation of Arp2/3 complex-mediated actin filament branch formation.