The broad, long term goal of this project is to determine the submicroscopic physical factors that govern adhesive interactions between leukocytes and vascular endothelial cells and that couple adhesion to cell function. These adhesive interactions are consequences of binding specific ligands to integrin, immunoglobulin (Ig) super family, and selectin receptors expressed by leukocytes and endothelial cells, and are central to normal and pathophysiological processes in blood circulation. The five Specific Aims are motivated by contemporary questions and major uncertainties in neutrophil-vascular endothelial cell interactions as well as interactions between model membrane structures and will yield fundamental insights into mechanisms relevant to all cell adhesion processes. SA l. To characterize intrinsic kinetics of receptor-ligand bond dissociation; SA 2. To correlate ligand receptor binding and inflammatory stimuli to transformations in mechanical properties of cell cortical structures; SA 3. To examine the strength of attachments in relation to receptor-ligand avidity; SA 4. To evaluate the threshold strength for receptor anchoring in model membranes; SA 5. To examine the influence of membrane steric and electrostatic topography on receptor extraction. The approach will be to physically probe the structural properties involved in adhesion at the level of the single receptor-ligand complexes and to test functional changes in cell material properties subsequent to molecular adhesion events and stimulation by chemotactic agents. A novel ultrasensitive force sensor will be used that can be positioned with nanoscale resolution and can measure forces over a range from below the strength of the weakest chemical bonds (0.01 pN) up to the strength of covalent bonds (greater than or equal to 1000 pN). The force sensor is a capsular assembly in which a microbead probe is chemically glued to a red blood cell or a synthetic lipid bilayer vesicle held by micropipet suction, driven by microprocessor-controlled piezo translation, and detected optically by digitized video images (diffraction and reflection interference) processed by microcomputer. Using this sensor, individual receptors in neutrophil,endothelial and transfected cell surfaces will be probed by their endogenous adhesive ligands, specific monoclonal antibodies and other ligands attached to the microbead. This research will establish a new realm of biophysical insight into blood- vascular cell adhesion at the molecular level and will contribute significantly to scientific understanding of the roles of receptor- mediated adhesion and cell surface structure in immune function, the development of drugs that target receptor molecules, and in the restoration of sufficient immune responses in tumor tissues that will aid immunotherapy in cancer treatment.
