Molecular regulation of the actin cytoskeleton

The Nolen lab investigates the regulation of the cytoskeleton, a molecular framework that provides physical support for cells. One of our primary interests is actin, a highly conserved eukaryotic protein that polymerizes into two-stranded helical filaments. Rearrangements of the actin filament network are critical for cellular processes that require a change in cell shape, such as motility, uptake and release of materials and cell division. To regulate these processes, cells utilize a myriad of proteins to control polymerization, depolymerization, severing, capping and crosslinking of actin filaments.Actin filaments are generated de novo by actin nucleator proteins, which bring the first few actin monomers together to form a template for filament elongation. Arp2/3 complex, a 225kD assembly of seven subunits (Arp3, Arp2, ARPC1-5) is one such nucleator. Arp2/3 complex is intrinsically inactive, and activation requires binding to an activator, such as a WASp/Scar family protein, and recruitment of the complex to the side of a pre-existing actin filament. Once activated, the complex nucleates the growth of a new filament which is anchored to the pre-existing filament at a 78˚ angle. This process results in the formation of tightly crosslinked, highly branched filament networks.

Arp2/3 complex is essential in the formation of invadopdia, cellular structures essential for the migration of tumor cells through the basal lamina into the blood stream. Invading bacterial and viral pathogens usurp Arp2/3 complex in host cells to escape detection by the immune system. Despite the biomedical importance of the complex, many fundamental questions about how it functions remain unanswered. For instance, how do activator proteins like WASp/Scar activate Arp2/3 complex? How is the proposed activating conformational change accomplished and how does this change promote formation of the filament nucleus?

We use a combination of biochemistry, biophysics, x-ray crystallography and molecular dynamics simulations to investigate these questions, and our ultimate goal is to understand how phenomena observed at the cellular level are controlled at the molecular level. Therefore, we are especially interested in experimental systems in which we can test our findings in vivo. Schizosaccharomyces pombe provides one such system, since it is genetically tractable, can be used for production of the large quantities of protein required for x-ray crystallography and is suitable for microscopy.