Fig. 5. Mechanism of cortactin coactivation of Arp2/3 complex with NPFs and branch stabilization.

Actin-loaded monomeric (top) or dimeric (bottom) NPFs bind inactive Arp2/3 complex through the high-affinity site 1 on Arp2-ArpC1. Cortactin competes with NPF binding to site 2 on Arp3, but not to site 1. NPF binding to Arp2-ArpC1 shifts the equilibrium of Arp2/3 complex toward the active, short-pitch conformation and delivers actin at the barbed end of Arp2, whereas cortactin recruits this “primed” complex to the mother filament. In the case of monomeric NPFs, the binding of actin at the barbed end of Arp3 is diffusion limited. For dimeric NPFs, however, one arm of the dimer is bound to Arp2-ArpC1, whereas the other arm is close enough to deliver actin to Arp3. Binding of the primed complex to the side of the mother filament shifts the equilibrium further in the direction of the active, short-pitch conformation. The actin subunits that begin adding at the barbed end of the Arps compete with NPF binding to sites 1 and 2, causing NPF release. Upon NPF release, the short-pitch conformation is fully stabilized through interactions of Arp2/3 complex with actin subunits of the mother and daughter filaments. Cortactin remains bound at the branch junction, interacting with either the mother or daughter filament, which increases the stability of the branch.