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bioRxiv · 10.1101/2025.07.18.665541

Inorganic phosphate in Arp2/3 complex acts as a rapid switch for the stability of actin filament branches

Xiao, J.·Pages, R.·Romet-Lemonne, G.·Jegou, A.
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Abstract

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.

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Xiao, J., Pages, R., Romet-Lemonne, G., Jegou, A.. 2025-07-20. Inorganic phosphate in Arp2/3 complex acts as a rapid switch for the stability of actin filament branches. https://doi.org/10.1101/2025.07.18.665541

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