Our goal in this project is to understand the role that specific physical characteristics of the adhesive interface have on adhesion and subsequent cell behavior. In particular, our investigation focuses on the microtopography of the cell membrane, the distribution and mobility of receptors, and changes in adhesion molecule affinity, as well as how these attributes change as a result of chemokine stimulus and bond formation between neutrophils and the endothelium. Micromechanical manipulation of single cells into contact with artificial substrates with well-defined adhesion molecule presentation provides unparalleled ability to control both the chemistry and the mechanical forces in relation to adhesive interactions. This approach, combined with newly implemented fluorescence imaging methods, enables us to determine the specific role that cellular mechanics, surface chemistry, and membrane topography play in the formation of adhesive contacts. Building on knowledge of the fundamental contributions of these factors obtained In the previous period, we will extend our investigations to determine how the physical and chemical characteristics of the cell surface and the underiying substrate work to effect changes in adhesive behavior and cell migration. Specifically, we will determine how contact with surfaces presenting immobilized IL8 and adhesion receptors (principally ICAM-1) induces changes in surface topography, and leads via key signaling intermediates (e.g., calcium, RAP-1 and the calcium-dependent guanine nucleotide exchange factor CalDAG GEFl) to integrin activation and adhesion. We will also measure the effects of chemokine stimulus on the distribution, mobility and activation state of adhesive ligands and the stability of the membrane cytoskeletal interface. Finally, we will determine how changes in cell surface microtopgraphy at the interface with its substrate enhance haptotactic signals, and how the distribution and concentration of those haptotactic signals lead to cell spreading and directed cell crawling. These studies will result in a clearer understanding of the mechanisms of neutrophil adhesion and migration on endothelium and its regulation, and thus result in a clearer and more detailed understanding of the inflammatory response in health and disease.
Our ability to control both the chemical and mechanical environment of cells will enable us to understand how mechanics and chemistry work synergistically to determine cell behavior when leukocytes, especially neutrophils, interact with the endothelium. Such interactions are central to the body's response to infection and in inflammatory diseases and similar mechanisms are integral to a wide array of diseases including heart disease and cancer.
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