The regulation and mechanics of actin-based motility

During cell motility, biochemical processes have mechanical consequences. By using the intracellular pathogen, Listeria monocytogenes , as a model of cell motility, I probed the mechanics and mechanisms of actin-based protrusion. In my first two studies, I examined the role of Enabled/Vasodilator Stimulated Protein or Ena/VASP in enhancing filopodial protrusions and its role in promoting Listeria infectivity. For promoting filopodial protrusions, it was proposed that Ena/VASP could directly stimulate existing actin nucleating protein Arp2/3 or sequentially convert existing lamellar networks into filopodial bundles. Using biochemical complementation of functional mutants that can only bind to Arp2/3 or Ena/VASP in a reconstituted bead motility assay, I examined the kinetics of actin tail formation. The kinetics and stoichiometry of actin protrusion (tails) from these particles disprove the notion that Ena/VASP directly regulates existing nucleators. My data supports the alternative, sequential conversion model.; For promoting Listeria infectivity, Ena/VASP both accelerates motility and stabilizes "steering" by making motility straighter. Although steering correlated with infectivity more closely than with speed, all the biochemical activities attributed to Ena/VASP could enhance both speed and steering in many ways. To distinguish the multiple activities, I used a mechanical approach and grouped them into three hypotheses. Using high-resolution laser-tracking and comparing the Ena/VASP mutant to wildtype, I show that Ena/VASP improved steering by increasing the sheer size of the actin tail. This find discounted alternative hypotheses that predicted the mechanical consequences of Ena/VASP biochemistry were not important for steering Listeria. Hence, stability of motility is largely due to hydrodynamic drag.; For my subsequent studies, I use only purified proteins to reconstitute aspects of motility. My initial experiments showed that prior methods were inappropriate for evaluating these new reconstituted systems, necessitating the development of new and more promising approaches. When using the full set of purified proteins, Listeria motility generated forces at least an order of magnitude stronger than in crude extracts. Very regular 3 nm steps were detectable in this system. The minimal subset that generated transient motility requires capping protein (CP). CP plays a paradoxical role and mechanical measurements were used to distinguish hypotheses. Although we attempted dynamic mechanical measurements, a promising steady-state mechanical approach is described.