This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Leukocyte rolling on activated endothelial cells and platelets, during inflammation and thrombosis, allows regional sampling of chemokines and other mediators, which leads to integrin-dependent arrest and emigration of leukocytes into the underlying tissue. Rolling requires the rapid formation and rapid dissociation of selectin-ligand bonds that are subjected to tensile forces applied by wall shear stress. Rolling elocities are remarkably stable over a wide range of shear stresses and are thought to require cellular features that complement the molecular properties of selectin-ligand bonds. The nature of these cellular features ispoorly understood. We have visualized rapid formation of long membrane tethers as neutrophils roll on Pselectin, E-selectin, and L-selectin. We hypothesize that these tethers play a key role in stabilizing rolling velocities by facilitating multiple adhesive contacts and by reducing force on individual selectin-ligand bonds.We propose to study the properties and functions of membrane tethers in the following specific aims:
Aim 1. We will determine whether distinct molecular interactions affect the formation or properties of membrane tethers by comparing the numbers, lengths, and lifetimes of membrane tethers as neutrophils roll on immobilized P-, E-, or L-selectin or on molecularly defined L-selectin ligands. We will also determine whether tethers form during a non-selectin-dependent interaction: rolling of mononuclear leukocytesexpressing integrin a4131 on vascular cell adhesion molecule-1 (VCAM-1).
Aim 2. We will determine whether distinct cellular features affect the formation or properties of membrane tethers by comparing tether properties of neutrophils, HL-60 cells, transfected CHO cells, and ligand-coupled K562 cells, each expressinga common selectin ligand, as they roll on an immobilized selectin. The viscoelastic properties of the cells will be modulated by cytoskeletal-disrupting agents, fixation, and cholesterol-chelating agents.
Aim 3. We will determine whether fluid dynamics affect the formation or properties of membrane tethers by examining the effects of increased wall shear stress, erythrocytes, and altering flow patterns. These studies will provide insights into how specific cellular and molecular properties cooperate to optimize leukocyte rolling over a wide range of hemodynamic conditions.
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