Mechanoregulation of the Leukocyte Specific Integrin LFA-1 by the Actin Cytoskeleton

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PROJECT SUMMARY Recruitment of circulating leukocytes to sites of infection plays an important role in the body's defense against pathogens. Mobilization of leukocytes from the blood to the surrounding tissue occurs in a multistep adhesion cascade, which is mediated by several adhesion molecule families. The integrin lymphocyte function- associated 1 (LFA1) mediates firm attachment by binding Intracellular Adhesion Molecules (ICAMs) expressed by the vascular endothlilium and ultimately leads to transmigration through the vessel wall. The applicant, Dr. Travis Moore has preformed important studies in this area in the laboratory of Dr. Timothy Springer. The goal of this application is to deepen Dr. Moore's expertise and findings in this important research area so that it can be developed into a significant independent research program. Dr. Moore recently lead research to show that integrin linkage to the actin cytoskeleton and the tensile force generated along the direction of retrograde actin flow resulted in the orientation of LFA1 on the cell surface. This alignment of LFA1 was substrate dependent and consistent with integrins being tilted in the same direction as retrograde actin flow. These findings strongly support a role for the actin cytoskeleton in the regulation of cellular adhesiveness. Dr. Moore will test the hypothesis that the actin cytoskeleton regulates integrin conformational equilibria and affinity through mechanical force applied to the integrin cytoplasmic domain and acts as an allosteric effector to stabilize the high-affinity, extended-open integrin conformation. This work will utilize innovative methodologies to measure the intrinsic affinities and conformational equilibria of LFA1 and use state-of-the-art super-resolution microscopy to quantitate the actin cytoskeleton linkage to the integrin cytoplasmic tail and directly measure integrin conformation on the cell surface of leukocytes. These experiments will advance our understanding of the regulation of cell adhesiveness and migration, produce novel and innovative methodologies for future research, and expand Dr. Moore's diverse background with expertise in protein chemistry and purification, super-resolution microscopy and computational image analysis, genome modification, and quantitating receptor thermodynamics on the cell surface. Dr. Moore will devote 100 % of his time to this Career Development Award with Dr. Timothy Springer as his mentor at Boston Children's Hospital.