Project Summary/Abstract Actin assembly underlies and drives many biological phenomena. Barbed ends of actin filaments, the major sites of polymerization, are controlled by the heterodimeric actin capping protein (CP). CP is regulated by the direct binding of CPI-motif proteins and the protein V-1. These two classes of regulators, CPI-motif proteins and V-1, bind to opposite sides of CP, and they induce conformational changes in CP that allosterically antagonize the binding of the other class. We are studying the molecular biophysical mechanism of these allosteric regulators. We are also studying the physiological function of CP and its regulators, using biochemical reconstitution with purified components, along with molecular genetic perturbations of living cells. Our biochemical studies will test a novel hypothesis for how CP regulators function in cells. Cells contain stoichiometric amounts of V-1 in micromolar concentrations, sufficient to inhibit nearly all of the cellular CP. V-1 is highly diffusible, and V-1 sterically blocks the ability of CP to cap actin filaments. CPI-motif proteins are targeted to membranes, and their CPI motifs allosterically induce the dissociation of V- 1, thus activating CP locally at the membrane. We are testing this hypothesis by determining the molecular biophysical mechanism of the allostery, and by testing the functions of the CPI-motif proteins with respect to cell motility and migration. We have discovered key differences in the biochemical activities of different families of CPI-motif proteins, and we are now using that information to investigate the allosteric mechanism, by combining single-molecule FRET measurements with molecular dynamics simulations. In addition, we are using purified proteins and lipids in a biochemical reconstitution system that induces actin assembly at a surface, thereby mimicking actin polymerization at cellular membranes. We are also using our discoveries about biochemical activities of CPI motifs to test cellular functions of CPI-motif proteins, using chimeras constructed from different CPI- motif proteins with molecular genetic perturbations and real-time movies of the motility phenomena of living cells. Our cell motility assays employ a system of endothelial cell monolayers with transmigrating immune and cancer cells, mimicking the physiological process of transendothelial migration.