Rolling of leukocytes in the vasculature is a facile adhesive interaction that enables efficient surveillance of endothelium for inflammatory signals, and is a requisite step for diapedesis. We will investigate the molecular and biophysical basis of leukocyte rolling of vascular endothelium through selectins and alpha4 integrins. Leukocyte rolling velocity is regulated within a marrow range, despite wide variation in ligand density, wall shear stress, and the exponential increase in receptor off-rates. with force on the receptor: ligand bond or tether. This enables uniform surveillance of endothelium despite wide variation in wall shear stress in vivo. We will extend studies on shear-enhanced bond formation to P-selectin and mild periodate-treated peripheral node addressin (PNAd) substrates, and generalize the concept that the number of bonds between the leukocyte and substrate increase with increasing wall shear stress. The kinetic and mechanical properties of the MadCAM-1 and alpha4beta7 interaction will be measured, and compared to those of selectins and the VCAM-1:alpha4beta1 interaction. We will test the hypothesis that mucin- like domains n selectin ligands and MadCAM-1 are important in the shear threshold phenomenon and shear-induced bond formation. A novel Ca2+- dependent interaction site responsible for rolling through alpha4 integrins will be defined structurally on alpha4beta7 and MadCAM-1. We will examine the mechanical properties of the microvillous tethers that connect the body of a leukocyte to a ligand-bearing substrate. The function of transmembrane and cytoplasmic domains that resist force and hence prevent extraction or """"""""up-rooting"""""""" of receptors from the cell membrane will be examined. The long-term goal is to use rolling adhesive interactions, in which the behavior of a small number of receptor-ligand bonds can be measured, as a model to understand the specialized kinetic and mechanical properties of adhesive molecules, and the molecules underlying cell adhesion in general.
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