Tuning the Contractility and Deformation Modes of Active Actin-Based Assemblies <em>In Vitro</em>: From Two-Dimensional Active Networks to Liquid Crystal Drops

Actin cytoskeleton-based materials are widely investigated as model cellular materials to elucidate physical mechanisms of cell mechanics, such as shape regulation and force production, as well as intriguing soft polymeric materials. In this method, we detail creating actin-based assemblies in vitro using purified protein for fluorescence microscopy studies. We polymerize long actin filaments in a sample chamber and use a polymer depletant to crowd filaments into a two-dimensional (2D)-entangled network against a surface passivated with a surfactant layer. Adding skeletal muscle myosin II filaments in the presence of adenosine triphosphate (ATP) induces contraction of the actin network. By bundling actin filaments with a crosslinker, we tune the contractility of the assembly, transitioning from a material that buckles to a material that slides at the microscale. By reducing the length of the actin filaments through co-polymerizing actin in the presence of capping protein, we tune the material from being a 2D network to a liquid crystal. Cross-linking of dispersed short actin filaments results in three-dimensional (3D) liquid crystal droplet formation.