Force Regulation of Capping and Arp2/3 Nucleation of Branched Actin Networks

Abstract

The assembly of branched actin networks provides the driving force for numerous cell motilities of all eukaryotic cells. While pushing the cytoplasm membrane moving forward, the networks are also polymerizing under a counter force. At the leading edge of the cell motilities, such as Lamellipodium, the pushing force and network formation are dynamically regulated by nucleation, elongation, and capping of individual growing filaments in the branched actin networks. Although it has been shown that the motor activity and mechanical properties of growing networks adapt to the counter forces, the force dependence of many other biochemical events and their molecular mechanisms are still not clear. Here we show how the counter force regulates capping and nucleation at molecular level. We found that the capping of filament polymerizing ends (free barbed ends) is force-regulated in the same way as for filament elongation. We also discovered that counter forces slow the rate of Arp2/3-mediated filament nucleation via a previously unsuspected mechanism, which involves force-dependent balance of free barbed ends, nucleation promoting factor protein, and Arp2/3 complex. This work not only uncovers a previously unknown and functionally significant effects of force in branched actin network assembly, but also reveals the responsible molecular mechanisms. Based on the newly discovered force-dependent capping and nucleation mechanisms, we have successfully explained the previously observed force-insensitive filament length and force-induced increase of number of free barbed ends. This work provides the very fundamental molecular mechanisms for cell motilities and we anticipate our assay to be a start point for more systematic studies on the influence of physical perturbations in actin branched network biochemistry.