Kinetic trapping organizes actin filaments within liquid-like protein droplets

Abstract

Actin is essential for various cellular functions such as growth, migration, and endocytosis. Recent evidence suggests that several actin-binding proteins phase separate to form condensates and that actin networks have different architectures in these droplets. In this study, we use computational modeling to investigate the conditions under which actin forms different network organizations in VASP droplets. Our simulations reveal that the binding and unbinding rates of actin and VASP determine the probability of formation of shells and rings, with shells being more probable than rings. The different actin networks are highly dependent on the kinetics of VASP-actin interactions, suggesting that they arise from kinetic trapping. Specifically, we showed that reducing the residence time of VASP on actin filaments promotes assembly of shells rather than rings, where rings require a greater degree of actin bundling. These predictions were tested experimentally using a mutant of VASP, which has decreased bundling capability. Experiments reveal an increase in the abundance of shells in VASP droplets, consistent with our predictions. Finally, we investigated the arrangements of filaments within deformed droplets and found that the filament length largely determines whether a droplet will straighten into a bundle or remain kinetically trapped in a ring-like architecture. The sphere-to-ellipsoid transition is favored under a wide range of conditions while the ellipse-to-rod transition is only permitted when filaments have a specific range of lengths. Our findings have implications for understanding how the interactions between phase-separated actin binding proteins and actin filaments can give rise to different actin network architectures.

Liquid-like condensates that bind actin drive filament polymerization and bundling

“…This phenomenon has been reported whenever filaments grow inside spherical containers with diameters below the persistence length of actin, 10-20 μm. 13,18,58,59 This partitioning results in the assembly of an actin shell or ring at the inner surfaces of the droplet. As more filaments join these structures, their rigidity eventually overcomes the surface tension of the droplet.…”

“…When the rigidity of the actin ring overcame the surface tension of the VASP condensate, the filaments within the ring began to straighten, deforming the initially spherical VASP condensates into elliptical and rod-like structures filled with parallel bundles of actin filaments. 13,18…”

Liquid-like condensates mediate competition between actin branching and bundling

“…The simulations also accounted for Brownian dynamics of the molecules considered. While kinetic parameters for Arp2/3 were chosen from experimental data (Supplementary Table S3), kinetic parameters for VASP were chosen based on simulations of actin polymerization within VASP droplets that lacked Arp2/3 (35). The present simulations are described in detail in the Supplementary Methods section, Supplementary Figure S2, and Supplementary Table S3.…”