Biophysical parameters influence actin-based movement, trajectory, and initiation in a cell-free system

Using a biochemically complex cytoplasmic extract to reconstitute actin-based motility of Listeria monocytogenes and polystyrene beads coated with the bacterial protein ActA, we have systematically varied a series of biophysical parameters and examined their effects on initiation of motility, particle speed, speed variability, and path trajectory. Bead size had a profound effect on all aspects of motility, with increasing size causing slower, straighter movement and inhibiting symmetry-breaking. Speed also was reduced by extract dilution, by addition of methylcellulose, and paradoxically by addition of excess skeletal muscle actin, but it was enhanced by addition of nonmuscle (platelet) actin. Large, persistent individual variations in speed were observed for all conditions and their relative magnitude increased with extract dilution, indicating that persistent alterations in particle surface properties may be responsible for intrinsic speed variations. Trajectory curvature was increased for smaller beads and also for particles moving in the presence of methylcellulose or excess skeletal muscle actin. Symmetry breaking and movement initiation occurred by two distinct modes: either stochastic amplification of local variation for small beads in concentrated extracts, or gradual accumulation of strain in the actin gel for large beads in dilute extracts. Neither mode was sufficient to enable spherical particles to break symmetry in the cytoplasm of living cells.