Inorganic phosphate rapidly switches the stability of Arp2/3-induced actin branches

Actin filaments often appear as branches, nucleated by the Arp2/3 complex. Arp2 and Arp3 are ATPases, which adopt different nucleotide-dependent conformations. We investigated how the nucleotide state of mammalian Arp2/3 complexes affects branch stability, by applying mechanical load. Branch junctions are 30-fold more stable when Arp2/3 is in the ADP-inorganic phosphate (Pi) rather than the ADP state. Pi is in rapid equilibrium with the ADP-Arp2/3 complex at the branch junction (release rate 0.2 s-1). Upon branch dissociation, Arp2/3 complexes remaining attached to the mother filament in the ADP-Pi state are 100-fold more stable, release their phosphate slowly (0.05 s-1), and can regrow branches without reloading ATP. Glia maturation factor (GMF) accelerates the dissociation of surviving ADP-Arp2/3 complexes, but does not prevent branch regrowth at physiological ATP concentration. Cortactin stabilizes branches and enhances renucleation. Neither GMF nor cortactin affects branch stability and renucleation of ADP-Pi-Arp2/3. Overall, these results identify Pi in the Arp2/3 complex as a critical regulator of branched actin network stability.