This project examines the specialized molecular and cellular mechanisms that enable tethering, rolling, and firm adhesion of leukocytes on vascular endothelium in shear flow. The long term goal is to acquire a deep understanding of adhesion in vascular flow, and enhance treatment of vascular diseases and development of therapeutics directed to vascular adhesion molecules. The structuralbasis for the ability of integrin a4(37 to mediate both rolling and firm adhesion will be studied with crystal structures designed to capture a4(37 in both its low affinity (rolling) and high affinity (firm adhesion) conformational states bound to its ligand MAdCAM-1 and small molecule antagonists currently in clinical development. Measurements of solution binding kinetics will complement structural studies and previous transient tether kinetic measurements. In selectins, the hypothesis that force-induced pivoting at the interface between the lectin and EGF domains stabilizes a high affinity state will be examined with mutations,structures, shear flow, and solution kinetics studies. The relation of selectin conformational change to catch-bonds, mechanical strength, and shear- enhanced bond formation will be examined. Comparisons between rolling of yeast and leukocytes will provide insights into the importance of mucin-like domains and cell deformability and microvilli. Regulation by tethering to ligand in shear flow of the conformation of the I domain of integrin ccL|32 will be examined in systems in which force application is either coupled or not to allosteric pathways that induce the high affinity state. A novel single-molecule system will be developed for studying the biophysics of receptor-ligand bonds in which the receptor and ligand are present within a single polyprotein. Unique force-extension curves of other modules in the same polyprotein will provide internal signatures that verify identification of receptor- ligand unbinding events. Both off-rate and on-rate under force will be examined, illuminating the role of conformational change, catch-bonds, and mechanical specializations of receptor-ligand bonds that enable adhesion despite the large forces exerted on tethered leukocytes in the vasculature.
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