Novel Roles of Actin Binding Proteins in Listeria monocytogenes Actin-Based Motility Revealed Within a Cellular Context

Novel Roles of Actin Binding Proteins in Listeria monocytogenes Actin-Based Motility Revealed Within a Cellular ContextBy Raphael H. Buencamino Actin-based motility is essential in many biological processes from fighting harmful organisms to the development of our bodies in the correct configuration. It has been dissected to its basic parts, finding the minimal set of actin binding proteins needed to produce movement. In contrast, actin-based motility in cells allows movement at a fast pace with a diverse set of activities and structures based on the cell's immediate function within the body. We dissected the mechanism of actin-based motility in vivo and found a set of proteins whose depletion drastically changes the character and efficiency of actin based movement. We accomplished this by using Listeria monocytogenes, a pathogen that expresses one bacterial protein, ActA, that allows Listeria to hijack the host proteins necessary to move within the cell using actin-based motility. To investigate the function and role of these host actin-binding proteins in actin-based motility, in vivo, we used a form of molecular "fractionation", a powerful technique only previously available for in vitro studies. RNAi interference in the Drosophila S2 cell line provided us this form of in vivo "fractionation" by allowing selective depletion of any protein whose cellular function we wished to examine. We first examined two proteins, profilin and ADF/cofilin, whose functions have been well documented through biochemical assays and in vitro systems. We also sought to dissect actin-based motility further and determine if other actin-binding proteins are involved in its mechanism. Our hypothesis is that with over 90 actin-binding proteins implicated in actin-based motility there are other key proteins that remain uncharacterized beyond the five in the current model. Our experiments identified that CAP is an essential protein for actin-based motility in vivo. Through our investigation we also discovered that fascin and other crosslinking proteins, though not essential for motility, might serve a new unique role in regulating the speed and dynamics of Listeria and its actin tail. Our observations have allowed us to identify and dissect the protein make-up of the actin-based motility machine