The use and discovery of small molecules that affect the actin cytoskeleton

The long-term goal of this thesis was to explore the use of small molecule inhibitors for actin-related phenomena in cells. This thesis is defined by two bodies of work: the first, the characterization of a compound that affects the actin cytoskeleton and the second, the use of a small molecule screening approach to understand cell migration and discover compounds that affect cell migration and morphology.; I first describe the characterization of the molecular effects of 2,3 butanedione monoxime (BDM) a compound previously thought to inhibit non-muscle myosin II. We found that BDM treatment caused rapid de-localization of proteins, including the Arp2/3 complex, involved in actin polymerization at the leading edge. These results suggest an alternative interpretation for how BDM affects cellular processes and is of particular interest given that BDM has been found to not affect non-muscle myosin II directly. In the second half of the thesis, I developed a high-throughput cell migration screen based on a classic wound-healing assay and performed an image-based screen of multiple small molecule libraries in an effort to chemically dissect the processes underlying cell migration and develop new tools for research. We found many compounds with interesting effects in this screen and after characterizing their effects in several sub-assays we chose one compound, Filopodine (3-pyridyl-indole), for follow-up. I characterized the effect of this small molecule and identified its molecular target as Rho-kinase (ROCK).