Mechanism of Actin Nucleation by Arp2/3 Complex Visualized by Single Molecule Fluorescence

Abstract

Nucleation of actin filaments by the Arp2/3 complex is a critical process in cell motility and endocytosis. It is well established that activity of Arp2/3 complex is stimulated by interactions with the VCA domain of WASP family proteins and pre-existing (mother) filaments. However the kinetic mechanism by which these proteins, together with monomeric actin, cooperate to rapidly assemble a critical nucleus for new (daughter) filament growth is not fully understood. We used multi-wavelength single molecule fluorescence microscopy to directly visualize in vitro the dynamic interactions of individual fluorescently labeled S. cerevisiae Arp2/3 complexes with actin filaments and to follow the pathway of new filament nucleation. We observed that Arp2/3 complex associated with mother filament sides, not ends. The majority of Arp2/3 complexes rapidly dissociated from filament sides after a characteristic distribution of lifetimes, while the remaining sub-population nucleated daughter filaments. Pointed ends of daughter filaments remained stably associated with the mother filament for durations estimated to be orders of magnitude longer than the dissociation times of Arp2/3 complexes that did not generate daughter filaments. Arp2/3 complexes nucleated some daughter filaments even in the absence of VCA, consistent with a mechanism in which VCA shifts an equilibrium between nucleation-inactive and nucleation-primed states of filament-bound Arp2/3 complexes. Taken together, these novel measurements reveal key features of the kinetic mechanism of actin nucleation by Arp2/3 complex and will help to more clearly define the VCA-regulated steps in the pathway.