Inorganic phosphate in Arp2/3 complex acts as a rapid switch for the stability of actin filament branches
The rate at which actin filaments turn over modulates actin network architecture and mechanosensitivity. Actin filaments often appear as branches, nucleated by the Arp2/3 complex. Within the Arp2/3 complex, Arp2 and Arp3 are ATPases, which, similar to actin, may adopt slightly different conformations depending on their bound nucleotide. We investigate the impact of the nucleotide state of Arp2/3 complexes on branch stability, by mechanically pulling on them. Remarkably, we find that branches with mammalian ADP-Pi and ADP-Arp2/3 complexes detach with the same exponential increase as a function of force, but ADP-Pi-Arp2/3 complex branch junctions are 20 times more stable at all forces. We observe that inorganic phosphate (Pi) is in rapid equilibrium with ADP-Arp2/3 at the branch junction, and is released at a rate greater than 1 s-1. Upon branch dissociation, the surviving Arp2/3 complex, remaining attached to the mother filament, is a thousand times more stable in the ADP-Pi than in the ADP state. Surprisingly, branch regrowth from surviving ADP-Pi-Arp2/3 complex does not require reloading ATP, indicating that the Arp2/3 complex remained in an active state upon debranching. We reveal that GMF destabilizes ADP-Arp2/3 complex branch junctions by accelerating first the dissociation of the daughter filament, and then the dissociation of the surviving Arp2/3 complex from the mother filament. We also report that cortactin stabilizes branches in a force dependent manner, and enhances branch renucleation. GMF and cortactin do not bind to Arp2/3 in the ADP-Pi state. Overall, Our results give a new importance to cytoplasmic Pi, as a possible regulator of branched actin network stability.