The actin cytoskeleton underlies many critical processes including morphogenesis, trafficking, and motility. The organization of actin filaments into higher order networks or bundles is required to sustain the forces involved in many of the mechanical functions of the cytoskeleton. Actin bundling is controlled by numerous actin regulators. Recently, many proteins that comprise this network have been found to undergo liquid-liquid phase separation. How might these liquid-like phases contribute to actin organization and filament bundling? Here we show that the processive actin polymerase, VASP, forms liquid-like droplets under physiological conditions. We find that these droplets catalyze actin polymerization and bundling. Specifically, VASP droplets mediate filament bundling through a mechanism that balances the mechanical properties of droplets against those of the actin filaments. As actin polymerizes within VASP droplets, elongating filaments gather around the edges of the droplet to minimize curvature, forming a ring-like bundle of actin within the droplet. Here the rigidity of the bundle is balanced by the surface tension of the droplet, in agreement with computational modeling. However, as the bundle grows thicker, its rigidity increases and eventually overcomes the surface tension of the droplet, deforming it into a linear bundle. These results reveal a novel mechanism of filament bundling that is mediated by protein droplets. The liquid-like nature of these droplets is important because it supports strong localization of actin, while permitting the dynamic rearrangement necessary for polymerization and bundling. We anticipate that this dynamic assembly mechanism may be relevant in the context of multiple structures rich in actin bundles, such as filopodia and focal adhesions.

Polymerization of actin inside liquid-like protein droplets drives spontaneous actin bundling

ACS Spring 2022

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