The leukocyte adhesion molecule LFA-1 plays a crucial role in all forms and levels of immunity. Leukocytes are able to turn LFA-1 function on or off on a minute-by-minute basis, giving them precise control over their adhesions with each other and other cells. The rapid regulation of LFA-1 function is mediated by protein kinase C, and does not require changes in cell surface density of LFA-1 or its ligand, ICAM-1. Rather, the regulation of the avidity of LFA-1 for ICAM-1 involves poorly defined configurational changes. Elucidation of these configurational changes is the goal of this research. Highly plausible changes would be homotypic clustering of LFA-1 to increase avidity for ICAM-1 and anchoring to the cytoskeleton. The approach proposed is to quantitate clustering by flow cytometric analysis of fluorescence resonance energy transfer, a method proven capable of estimating mean intermolecular distances on cell surfaces. The ability to quantitate clustering will make it possible to explore its mechanisms, including the roles of divalent cations and cytoskeleton. Anchoring to the cytoskeleton will be evaluated by solubility in Triton X-100. Cellular adhesion plays a critical role in many developmental and physiological processes, including immune function. The molecular mechanisms by which cells recognize and adhere to appropriate partners is a subject of great current interest. The proposed research will explore an innovative approach to the study of mechanisms by which the strength of adhesion between cells is regulated.
