The objective of this proposal is to develop a detailed biophysical understanding of the interactions of leukocytes with endothelial cells by quantitating the nanoscale strengths of single molecular bonds involved in the adhesive interactions between these cells. As the interactions have to occur in vivo in an environment of large hydrodynamic stresses, a repertoire of adhesive receptors @ selectins, integrins and immunoglobulin (lg) super family receptors @ are involved in initiation and subsequent strengthening of the adhesive interaction. While the roles of these different receptors in the adhesive function have been well identified, the interplay between mechanical strength, time, and chemistry of the individual adhesive interactions at the single molecular level is largely unknown. The present proposal attempts to bridge this gap in our understanding by using an experimental strategy that the investigators have recently developed to sense molecular attachments and measure single bond forces.
Three specific aims are proposed to test various hypotheses regarding the dynamics of leukocyte adhesion to vascular endothelium. 1) To explore the hypothesis that each receptor-ligand interaction is designed to meet different dynamic loading requirements found in vivo, the investigators will measure the intrinsic relations between mechanical strength and rupture time for single bonds involving selectin and integrin receptors over many orders of magnitude in time scale. 2) To test the hypothesis that the prominent factors governing bond strength arise from unique arrangements of small molecular components, the investigators will compare dynamic strength properties for selectin and integrin interactions with site-specific ligands and ligands that have been engineered through post-translational modification and mutation. 3) To investigate the hypothesis that molecular strength in adhesion depends on chemistry of receptor linkages to cytoskeletal structure as well as ligand-receptor chemistry, the investigators will measure mechanical strengths as a function of rupture times for biofunctional ligand bonds to receptors in situ on cell membranes. The successful accomplishment of these aims is critically dependent on the use of an ultrasensitive force probe with exceptional dynamic range to sense molecular attachments and measure single bond forces over an enormous span of time frame for detachment. The biomembrane force probe BFP, which the investigators have recently developed, meets these requirements. This innovative sensor can be positioned with nanoscale precision and can quantitate forces from the weakest strength of noncovalent bonds ( about0.1 pN) up to the strength of covalent bonds (> 1000pN). Equally essential, the BFP can stress and rupture single molecular attachments over a span of six orders of magnitude in detachment time. By decorating the probe tip with synthetic and recombinant ligands, the investigators plan to measure the dynamic strengths of attachments to selectins and integrins resident on neutrophil membranes and reconstituted on glass microspheres. These studies will provide exciting new and novel biophysical insights into blood-vascular cell adhesion at the molecular level, which will contibute significantly to our understanding of receptor-mediated adhesion in normal immune function and in pathophysiology of inflammation, tissue injury and tumor cell metastasis.
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