Reconstitution of actin-based motility of Listeria and Shigella using pure proteins

Abstract

Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (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 rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, alpha-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

Profilin-1 is a negative regulator of mammary carcinoma aggressiveness

“…In the case of MDA-MB-231 cells, although we noticed a somewhat decrease in the overall flare of protrusion after Pfn1 depletion, polarised protrusion at the leading edge was still evident. It was previously implied that Pfn1 facilitates, but is not absolutely critical for the canonical WASP-Arp2/3-mediated, actinbased protrusions in migrating cells (Loisel et al, 1999). Since WASP-family proteins, Arp-2/3 as well as their upstream regulators (Rac1, Cdc42) are known to be overexpressed in several invasive cancers including breast cancer (Sahai, 2005), protrusion of tumour cells may be much less sensitive to loss of Pfn1 expression compared to vascular endothelial cells.…”

Theory from the Oster Laboratory Leaps Ahead of Experiment in Understanding Actin-Based Cellular Motility

“…Mogilner and Oster showed that short stiff filaments are favorable for the elastic Brownian ratchet. Loisel et al (10) also showed that cofilin is required for robust comet tails. Cofilin promotes the turnover of the filaments so comet tails maintain a constant length as the bacterium moves forward.…”

“…Selective pressures during evolution of the system~1 billion years ago arrived at the physically optimal angle of the branches. Reconstitution experiments (10) showed that movement of bacteria by actin comets depends on capping the filaments so their lengths are limited. Mogilner and Oster showed that short stiff filaments are favorable for the elastic Brownian ratchet.…”

“…Actin polymerization next to the bacterium assembles a comet tail of actin filaments that propels the bacterium through the cytoplasm (8,9). This work culminated in reconstitution of bacterial motility from purified actin and a few other proteins (10).…”

Spatial and temporal regulation of actin polymerization for cytoskeleton formation through Arp2/3 complex and WASP/WAVE proteins

“…Actin comets can be reconstituted in vitro with five proteins and N-WASP-, or Listeria protein ActA-coated plastic beads [Cameron et al, 1999;Loisel et al, 1999;Pantaloni et al, 2001;Suetsugu et al, 2001c]. The five proteins are ADF/cofilin, profilin, Arp2/3 complex, barbed-end capping protein, and actin (Fig.…”