Reconstitution of Actin Based Motility in Cell Extracts

Although the ActA protein was identified rather rapidly following the initial discovery of L. monocytogenes actin-based motility, it took longer to identify the essential host factors that participate in actin polymerization. The delay was due to a lack of genetic tools in host organisms and a lack of biochemical tools for dissecting motility in vitro. A breakthrough that set the stage for the identification of host factors was the reconstitution of L. monocytogenes motility in vitro in concentrated cytoplasmic extracts made from X. laevis eggs (Theriot et al. 1994). Reconstitution was later achieved in human platelet cytoplasmic and

L. monocytogenes capping protein

Figure 10.4. Cartoon diagram depicting the location and function of proteins known to participate in actin nucleation (Arp2/3 complex, yellow), elongation (VASP, dark blue; profilin, light blue), organization (Arp2/3 complex; capping protein, green), and depoly-merization (ADF/cofilin, orange) during L. monocytogenes motility. (A color version of this figure appears between pages 196 and 197.)

L. monocytogenes capping protein

Figure 10.4. Cartoon diagram depicting the location and function of proteins known to participate in actin nucleation (Arp2/3 complex, yellow), elongation (VASP, dark blue; profilin, light blue), organization (Arp2/3 complex; capping protein, green), and depoly-merization (ADF/cofilin, orange) during L. monocytogenes motility. (A color version of this figure appears between pages 196 and 197.)

cytoskeletal extracts, which are highly enriched in cytoskeletal proteins (Welch et al. 1997b). Actin-based motility in vitro requires the use of a bacterial strain (Leimeister-Wachter and Chakraborty 1989) that overexpresses ActA and other virulence factors, indicating that optimal levels of expression differ for motility in different environments. Nevertheless, the motility parameters in Xenopus egg and human platelet cytoplasmic extracts are remarkably similar to those in infected cells—bacteria move at an average speed of ~6 ^m/ min (slightly slower than in infected cells) and assemble characteristic actin comet tails. The experimental advantage of these reconstitution systems is that the function of known host proteins can be assessed by addition or depletion, and the identification of unknown proteins can proceed by biochemical fractionation. Using this and other systems, a core set of host proteins that are involved in actin polymerization and motility has been identified (Figure 10.4.), as is described in the following sections.

The host factor that nucleates actin assembly with ActA was purified from human platelet extracts by fractionating and assaying for factors that could promote actin assembly at the L. monocytogenes surface (Welch et al. 1997b). This factor was identified as the Arp2/3 complex (Welch et al. 1997b), an evolutionarily conserved protein complex consisting of the actin-related proteins Arp2 and Arp3 and five additional subunits that was originally isolated from Acanthamoeba castellanii (Machesky et al. 1994). The Arp2/3 complex normally functions in host cells to nucleate actin assembly at the cortex during processes such as membrane protrusion, phagocytosis, and endocytosis (Welch and Mullins 2002). Additionally, Arp2/3 complex is necessary for bacterial actin-based motility as depletion of or interference with its activity in cell extracts (May

Was this article helpful?

0 0

Post a comment