The alpha4beta1 (VLA-4) integrin plays a major role in the recruitment of hematopoietic cells to various VCAM-1 expressing inflamed endothelial sites and the bone marrow. Recent studies suggest that both the nature and strength of preformed associations between the alpha4 and beta1 subunits of VLA-4 with the actin cytoskeleton can determine its ability to support adhesive interactions at subsecond contacts to its endothelial ligand under shear flow. Indeed, preliminary collaborative work with the U.S. PI indicates that a specific alpha subunit association with the cytoskeletal adaptor paxillin provides a crucial cytoskeletal constraint of VLA-4 necessary for its ability to mediate adhesive tethers to VCAM-1 under physiological shear flow. The proposed studies will investigate the regulation of these dynamic adhesive events by the cytoskeletal environment of VLA-4 in T cells and its implications in in situ chemokine stimulation of VLA-4 adhesiveness at endothelial contacts. Novel kinetic approaches will monitor by real-time videomicroscopy millisecond avidity generation events mounted by VLA-4 integrinNCAM-1 bonds under shear flow. Computerized analysis of both the frequency and duration of these tethers under different stimulatory and adhesive contexts can serve as a powerful tool in following the contribution of specific VLA-4 cytoskeletal partners to VLA-4 adhesive properties in shear flow. The first part of this proposal is aimed at elucidating how the constitutive association of VLA-4 with paxillin and its adhesive outcome are regulated by alpha4 tail phosphorylation. In the second part of the work, we will expand this approach to study how key elements on the beta1 subunit of VLA-4 which mediate the integrin's associations with talin and filamin, two major integrin linkers to the actin cytoskeleton, modulate the earliest stages of VLA-4 tether stabilization under shear flow. The final part is aimed at unraveling the specific regulatory roles of the two beta1 subunit NXPY motifs, key elements in integrin tail functions, in subsecond VLA-4 avidity generation events. Elucidation of specialized roles for specific integrin-associated cytoskeletal effectors may identify new regulatory pathways for rapid avidity modulation of alpha4 integrins at both endothelial and extracellular matrix short-lived adhesive contacts. New insights gained into the earliest events of VLA-4 avidity regulation have important implications for hematopoietic and tumor cell migration through primary lymphoid organs and sites of inflammation. This research will be done in Israel at the Weizmann Institute of Science in collaboration with Dr. Mark Ginsberg as an extension of NIH grants AR 27214 and HL 31950.
