Reconstitution of actin-based motility of Listeria and Shigella using pureproteins

Actin polymerization is essential for cell locomotion and is thought to gen erate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to si gnalling(1-6). The bacteria Listeria and Shigella bypass the signalling pat hway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells(7-10). However, the Arp2/3 complex alone is insu fficient to promote movement. Here we have used pure components of the acti n cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by A TP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing fac tor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rat e of unidirectional growth of actin filaments at the surface of the bacteri um. The movement is more effective when profilin, alpha-actinin and VASP (f or Listeria) are also included. These results have implications for our und erstanding of the mechanism of actin-based motility in cells.