Self-organized patterns of actin filaments in cell-sized confinement

Cells use actin filaments to define and maintain their shape and to exert forces on the surrounding tissue. Accessory proteins like crosslinkers and motors organize these filaments into functional structures. However, physical effects also influence filament organization: steric interactions impose packing constraints at high filament density and spatially confine the filaments within the cell boundaries. Here we investigate the combined effects of packing constraints and spatial confinement by growing dense actin networks in cell-sized microchambers with nonadhesive walls. We show that the filaments spontaneously form dense, bundle-like structures above a threshold concentration of 1 mg ml⁻¹, in contrast to unconfined networks, which are homogeneous and undergo a bulk isotropic-to-nematic phase transition above 5 mg ml⁻¹. Bundling requires quasi-2D confinement in chambers with a depth comparable to the mean filament length (6 μm). The bundles curve along the walls and central bundles align along the chamber diagonal or, in elongated chambers, along the long axis. We propose that bundling is a result of the polydisperse length distribution of the filaments: filaments shorter than the chamber depth introduce an entropic depletion attraction between the longest filaments, which are confined in-plane. Bundle alignment reflects a competition between bulk liquid-crystalline ordering and alignment along the boundaries. This physical mechanism may influence intracellular organization of actin in combination with biochemical regulation and actin–membrane adhesion.