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

The actin cytoskeleton forms a mesh-like network that drives cellular deformations. The network property is defined by the network density and the species of actin-binding proteins. However, the relationship between the network density, the penetration ability of actin-binding proteins into the network, and resulting network dynamics remains elusive. Here, we report an in vitro optogenetic system, named OptoVCA, which induces Arp2/3 complex-mediated actin network assembly on a lipid membrane. By changing the illumination power, duration, and pattern, the OptoVCA flexibly manipulates the density, thickness, and shape of the actin network. Taking these advantages, we examine the effects of the network density on two representative actin-binding proteins, myosin and ADF/cofilin. We find that the penetration of myosin filaments into the network is strictly inhibited by only a several-fold increase in network density due to the steric hindrance. Furthermore, penetrated myosin filaments induce directional actin flow when the network has a density gradient. On the other hand, ADF/cofilin penetrates into the network regardless of network density. However, network disassembly is dramatically inhibited by only a several-fold increase in network density. Thus, the OptoVCA contributes to understanding cell mechanics by examining the network density-dependent effects on actin-binding proteins.