Force generation by actin comet tails: Physical influence of the moving object and its surroundings

Polymerizing actin filaments in comet tails generate force for movement of the bacterial pathogen, Listeria monocytogenes. Coexisting populations of actin filaments in the tail exert pushing forces and retarding forces on the bacterial surface, while hydrodynamic drag forces oppose motion. In order to understand the magnitude, distribution, and balance of these forces we have designed experiments to perturb the surface properties of moving objects on actin-based movement. To investigate the influence of hydrodynamic drag force on bacterial movement, we compared the speeds of moving Listeria monocytogenes in normal infected fibroblasts to those in cells lacking intermediate filaments. Although the diffusion coefficient of bacteria increases two-fold in cells lacking intermediate filaments, the speed of bacteria in both cell types is identical, suggesting that force generated by actin comet tails is insensitive to a two-fold change in cytoplasmic viscosity. To probe the magnitude and distribution of forces by actin comet tails, we developed a model system where lipid vesicles coated with the ActA protein from L. monocytogenes are propelled by comet tails in cytoplasmic extract. Motile vesicles are deformed due to nanoNewton compression forces exerted by the actin comet tail. Net pushing and retarding forces in the comet tail spatially segregate, such that pushing forces predominate along the sides of the vesicle while retarding forces predominate at the rear. ActA is polarized on vesicles, with the highest density of protein coinciding with the maximal retarding force. To examine how force depends on shape and/or the polar expression of ActA, we examined the motility of ActA-coated ellipsoidal beads in cytoplasmic extracts. At steady-state, beads move either parallel or perpendicular to their major axis at identical speeds. During symmetry-breaking, the beads initiate movement parallel to their major axis, suggesting that a compression-based mechanism for force production depends on the regime of actin-based movement. Lipid-coated ellipsoidal beads move parallel to the major axis and a subset of these beads move in tight persistent circles. These data suggest that fluid surfaces aid in localizing pushing and retarding forces and influence force distribution and force balance in the actin-comet tail.