Our long-term objective is to develop a detailed biophysical understanding of leukocyte adhesion at the molecular level, relating adhesive bond strength to chemical interactions and establishing how adhesive bond stress is transmitted through the cell membrane to the interior connections between receptor tails and the cell cytostructure where it can impact specific biochemical pathways. Our current research has provided significant insight into the mechanical strengths of leukocyte adhesion bonds and their kinetics under force, (i) first for interactions between the sialo mucin ligand PSGL-1 and selectins that initiate leukocyte attachments to vessel walls, (ii) second for interactions between super immunoglobulin (Ig)family ligands and integrins that signal and stabilize leukocyte adhesion enabling emigration into tissues. We have developed novel methods to immobilize ligands on the tip of ultrasensitive force probes and test the mechanical strengths of individual bonds to receptors either immobilized on microspheres or expressed on cells. Exploiting an exciting new approach and established cell lines with strategic alterations in receptor- cytoskeletal interactions, the principal objective of this application is to """"""""move inside the cell"""""""", first establishing the extent to which cytostructural proteins regulate the mechanical strength of an adhesion complex, and then determining the mechanical role that these linkages play in the leukocyte signalling processes important for recruitment to sites of inflammation and injury. The studies are designed to test three hypotheses. Hypothesis: leukocyte selectin and integrin interactions have function-specific mechanical designs that govern bond formation and release under conditions of low stress interior to a cell- surface contact, thereby impacting bond proliferation and determining the fate of the initial adhesion event. Hypothesis: mechanical strengths of molecular adhesion complexes are governed by the weakest link in entire sequence of protein interactions from the outside adhesive bond to the inside bonds that connect receptor-tail domains to the cell cytostructure. Hypothesis: the linkages that anchor an integrin adhesion complex to the cell structure are key mechanical effectors of """"""""outside-in"""""""" and """"""""inside-out"""""""" signaling in leukocytes and represent an important feed-back process to regulate adhesion strength.
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