The actin cytoskeleton participates in a range of cellular behaviors involving motility. A number of bacterial pathogens hijack the host cell cytoskeleton as part of their infectious cycle. Listeria monocytogenes, for example, polymerizes actin to form comet-like tails that propels it through the cytoplasm. Because these pathogens bypass many of the normal cytoskeletal controls and because their movement has been recapitulated in cell-free extracts, they have been used as model systems to study actin polymerization. Here, we describe the use of Listeria to identify novel factors involved in actin dynamics and force generation. We have found that Listeria move at different rates in different cells extracts. Listeria move at high rates in human platelet and frog egg extracts, but at low rates in mouse or bovine brain extracts. Addition of high-motility extracts to the brain extracts confers fast movement to brain extract. We propose to use this activity to purify components present in platelet and egg extracts that are involved in fast bacterial motility. Specifically, we plan to: (1) further characterize Listeria motility in different cell extracts, (2) fractionate extracts and follow high-motility activity to purify factors that enhance bacterial movement, and (3) identify and characterize the purified proteins. We believe this approach will isolate novel factors involved in actin dynamics and force generation.
