Phase separation determines treadmilling-like movement of actin bundles

Cellular motility relies on the dynamic turnover of actin filaments, which treadmill through continuous polymerization at the barbed end and disassembly at the pointed end. Yet, the underlying physical principles remain poorly understood. A long-standing challenge has been experimentally reconstituting stable, persistent treadmilling leading to higher-order actin organization. Here, we reconstitute a minimal in vitro system, in which liquid-liquid phase-separated condensates of zyxin and VASP balance cofilin-driven disassembly to enable persistent treadmilling-like movement of actin bundles. The condensates crosslink filaments into dynamic bundles while promoting barbed-end polymerization. The localized stabilization based on condensate-mediated bundling competes against the activity of cofilin and CAP1, enabling selective disassembly at pointed ends and recycling of monomers. To elucidate the physical basis underlying this emergent behavior, we complement our experiments with agent-based simulations that quantitatively recapitulate key experimental findings. This combined approach demonstrates that an intermediate range of zyxin-VASP self-affinity, driving the phase separation, enables robust treadmilling-like movement. A weakened zyxin-VASP self-affinity fails to stabilize the bundles, whereas an excessive affinity impedes filament dynamics. Experimental reconstitution and theoretical modeling together reveal a physical mechanism by which the material properties of multivalent protein condensates govern cytoskeletal turnover, and suggest a general design principle by which biomolecular condensates can spatiotemporally organize cytoskeletal structures.