Optical Control of Actin Network Assembly on the Supported Lipid Bilayer.

The spatiotemporal dynamics and density of actin networks are key determinants of actin cytoskeleton-mediated cellular functions. In vitro reconstitution systems have been widely used to study actin cytoskeletal dynamics; however, many existing approaches offer limited flexibility in controlling the geometry, thickness, and density of the assembled actin networks. Here, we present an in vitro optogenetic protocol that enables precise control of actin network assembly on supported lipid bilayers using an improved light-induced dimer (iLID)-SspB-based light-inducible dimerization system. In this system, His-mEGFP-iLID is anchored to a Ni-NTA-containing lipid bilayer, while SspB-mScarlet-I-VCA, a nucleation-promoting factor fused with SspB, together with other actin cytoskeletal proteins, is supplied in bulk solution. Upon blue light illumination, SspB-mScarlet-I-VCA is recruited to the membrane in a spatially and temporally defined manner, inducing localized actin polymerization. By tuning illumination patterns and duration, actin networks with defined density, thickness, and geometry can be generated, and polymerization can be rapidly halted by stopping illumination. This protocol provides a versatile platform for reconstructing actin networks with controlled spatial organization and density, enabling quantitative analysis of density-dependent interactions between actin networks and actin-binding proteins. Key features • Actin networks with varying densities and arbitrary shapes can be formed on the same supported lipid bilayer by controlling blue light illumination through the objective lens. • Actin polymerization can be stopped simply by turning off blue light illumination, enabling the formation of actin networks with defined thicknesses. • This protocol requires purified actin and actin-binding proteins.