Our goal is to understand the molecular mechanisms that drive cell motility and shape change. In particular we are studying the engine that powers these processes, the actin cytoskeleton, using a combination of cell biological, biochemical, and genetic techniques. We are working to determine how actin filament assembly is initiated, how it is regulated by signal transduction pathways, and how it contributes to the forces that drive cell shape change and movement. In addition we are working to understand how the normal functions of the actin cytoskeleton are subverted by bacterial and viral pathogens during infection.
Cellular regulation of actin assembly. Coordinating cell shape change and motility in response to extracellular and intracellular cues requires precise spatial and temporal regulation of actin filament assembly. One key control point in actin assembly is the initiation of new filament formation - a processes called nucleation. Work from our lab and others has established that the Arp2/3 complex, an evolutionarily conserved protein complex, is a critical actin nucleating factor in cells that functions in the vicinity of the plasma membrane to generate new actin filaments and organize them into Y-branched networks. The Arp2/3 complex is a fascinating macromolecular machine that consists of seven subunits including the actin-related proteins Arp2 and Arp3 and five other proteins (ARPC1-5). To determine how these seven polypeptides work in concert to nucleate and organize actin filaments, we have reconstituted recombinant human Arp2/3 complex using the baculovirus expression system. We are using this powerful tool to study the biochemical mechanisms of Arp2/3 complex assembly and function.
The activity of the Arp2/3 complex is regulated in cells by proteins called nucleation promoting factors (NPFs) which bind to the complex and stimulate its nucleating and organizing activities. Cells contain multiple families of NPFs, and we are interested in understanding how different NPFs may regulate the formation of diverse actin-containing structures. One NPF of particular interest is the Wiscott-Aldrich syndrome protein (WASP), encoded by the gene mutated in the inherited immune deficiency disorder Wiscott-Aldrich syndrome. WASP and its related proteins bind to lipids and signal transduction proteins, and we are working to understand how these factors regulate WASP activity at the plasma membrane to modulate cell motility and membrane remodeling. We recently developed a powerful model system for studying the cellular function of WASP family proteins. In this system, WASP-coated beads are introduced into cell cytoplasmic extracts, and actin polymerization at the bead surface generates a force that powers bead motility. We are using this and similar systems to dissect the function and regulation of WASP family proteins during cell shape change and motility.
Interaction between pathogens and the host actin cytoskeleton. The actin cytoskeleton of human cells is targeted during infection by an amazing variety of viral and bacterial pathogens. Studying the interaction between pathogens and the host cell's actin cytoskeleton has provided important insights into the mechanisms that control actin assembly and function. One pathogen of particular interest is Listeria monocytogenes, a bacterium that is internalized by host cells, induces the polymerization of the actin at its surface, and uses the energy derived from polymerization to power intracellular motility and cell to cell spread. Actin polymerization at the L. monocytogenes surface is initiated by the Arp2/3 complex and a bacterial NPF called ActA, and we are investigating the mechanism by which these factors function to nucleate actin assembly. In addition we are working to understand how other bacterial and viral pathogens manipulate the host cytoskeleton during infection.