? Endothelial cell (EC) movement is initiated by angiogenic growth factors, which trigger a sequence of spatially polarized intracellular events including the activation of motility-regulating small GTPases and the assembly of actin-dependent, force-generating systems at the cell anterior. Our primary interest is the role of the plasma membrane in cell movement. We have shown that membrane microviscosity is a key determinant of motility, and that basic fibroblast growth factor increases EC plasma membrane microviscosity as measured by fluorescence recovery after photobleaching (FRAP). Spatial analysis shows a highly polarized gradient of microviscosity in plasma membranes of rapidly migrating EC, with a leading edge that is substantially more viscous than the trailing edge. An important role of cholesterol in generation of this membrane microviscosity gradient is suggested by an increase in cholesterol content of the membrane, by gradient reversal upon cholesterol removal, and by relocalization of a fluorescent cholesterol analog, NBD-cholesterol, to the front of moving ECs. In studies of the mechanism that drives membrane polarization we have observed that caveolin-1, an intracellular cholesterol transport protein, is also highly polarized and accumulates in the rear of migrating ECs. In studies of the mechanism by which membrane physical properties regulate motility, we have found that increased membrane microviscosity increases the binding of Racl to plasma membranes in the front of moving ECs. We have also found that the ability of actin to deform large unitamellar vesicles is decreased when microviscosity is high, i.e., at an elevated cholesterol-to-phospholipid ratio. From these data we propose as a hypothesis that angiogenic growth factors alter cholesterol synthesis and trafficking to increase the membrane microviscosity at the leading edge of the moving cell. We further propose that increased microviscosity increases cell movement by increasing Racl-binding to the plasma membrane and by altering the barrier properties to improve the efficiency by which actin filaments propel the cell forward. We will test this hypothesis in three Specific Aims: (1) Determine the molecular mechanism regulating polarization of membrane microviscosity during EC movement, (2) determine the mechanism by which microviscosity regulates Racl binding to membranes and (3) determine the role of membrane microviscosity in regulation of actin filament formation and function. The experiments will make use of cultured cells expressing GFP-tagged These studies will provide basic information on mechanisms regulating cell motility and may lead, in the long-term, to molecular strategies to inhibit or enhance cell migration. Pharmacological agents based on these results may be useful for inhibition of tumor angiogenesis or to enhance collateral blood vessel formation and the healing of synthetic vascular grafts. ? ?
