Optogenetic actin network assembly on lipid bilayer uncovers the network density-dependent functions of actin-binding proteins.

The actin cytoskeleton forms a meshwork that drives cellular deformation. Network properties, determined by density and actin-binding proteins, are crucial, yet how density governs protein penetration and dynamics remains unclear. Here, we report an in vitro optogenetic system, named OptoVCA, enabling Arp2/3 complex-mediated actin assembly on lipid membranes. By tuning illumination power, duration, and pattern, OptoVCA flexibly manipulates the density, thickness, and shape of the actin network. Taking these advantages, we examine how network density affects two actin-binding proteins: myosin and ADF/cofilin. We find that even modest increases in density strictly inhibit myosin filament penetration by steric hindrance. Penetrated myosin filaments generate directional actin flow in networks with density gradients. In contrast, ADF/cofilin accesses networks regardless of density, yet network disassembly is markedly reduced by increased density. Thus, OptoVCA reveals that network density differentially regulates actin-binding protein penetration and activity. These findings advance understanding of cell mechanics through precise, light-based manipulation of cytoskeletal structure.