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