Alignment of nematic and bundled semiflexible polymers in cell-sized confinement

The finite size of cells poses severe spatial constraints on the network of semiflexible filaments called the cytoskeleton, a main determinant of cell shape. At the same time, the high packing density of cytoskeletal filaments poses mutual packing constraints. Here we investigate the competition between excluded volume interactions in the bulk and surface packing constraints on the orientational ordering of confined actin filaments as a function of filament density and the presence of crosslinks. We grow fluorescently labeled actin filaments in shallow (thickness d_z 3 μm), rectangular microchambers with a systematically varied length (d_y between 5 and 100 μm) and in-plane aspect ratio (d_x/d_y between 1 and 10). We determine the nematic director field by image analysis of fluorescence confocal images. We find that high-density (nematic) solutions respond sensitively to changes in the size and aspect ratio of the chambers. In small chambers (d_y ≤ 20 μm), filaments align parallel to the long walls as soon as the aspect ratio is ≥1.5, indicating that surface-induced ordering dominates. In larger chambers, the filaments instead align along the chamber diagonal, indicating that bulk packing constraints dominate. The nematic order parameter is maximal in small and highly anisometric chambers. In contrast to the nematic solutions, low-density (isotropic) solutions are rather insensitive to confinement. Bundled actin solutions behave similarly to nematic solutions, but are less well-ordered. Our observations imply that the orientational order of actin filaments in flat confining geometries is primarily determined by a balance between bulk and surface packing constraints with a minimal effect of the enthalpic cost of filament bending. Our assay provides an interesting platform for the future reconstitution of more complex, active cytoskeletal systems with actively treadmilling filaments or molecular motors.