The objective of the proposed research is to experimentally and theoretically define the motions and organizations of antibodies at substrate-supported planar model membranes, both in the absence and presence of specifically bound cells, using dynamic, laser-based fluorescence microscopy. Emphasis will be placed on understanding the onset of receptor-mediated phagocytosis, in which macrophages bind, ingest and destroy pathogenic organisms. During the initial phase of phagocytosis, antibodies bind both to antigenic sites on the target cell surface and to Fc receptors on the macrophage cell surface. In one set of measurements, the surfaces of pathogenic organisms will be modeled by substrate-supported planar membranes containing hapten- conjugated phospholipids, and the characteristics of hapten-specific antibodies on the membranes (surface concentrations, equilibrium binding curves, surface association and dissociation kinetics, translational and rotational mobilities, and oligomerization states) will be characterized for different antibody, membrane and solution properties. Studies will be conducted both for antibodies that are irreversibly bound to the membranes and for antibodies that are in equilibrium between solution and the membranes. In the latter case, the dynamic conversion between antibodies in solution, antibodies bound monovalently to membranes, and antibodies bound bivalently to membranes will be of particular interest. The requirements (e.g., antibody density, mobility, and oligomerization) for which macrophage-related cells bind to, and are metabolically activated by, planar membranes will be characterized. The effects of macrophage binding and activation on the organization and dynamics of antibodies will also be examined. In a complementary set of measurements, the surfaces of macrophages will be modeled by substrate-supported planar membranes containing purified and reconstituted mouse Fc-gamma-RII. These studies will require the development of methods for forming planar membranes with reconstituted moFc-gamma-RII that is properly oriented and undergoes physiologically relevant degrees of translationa1 mobility. To do this, truncated versions of moFc-gamma-RII that consist of the transmembrane region conjugated to the extracellular region, to the beta1 form of the intracellular region, and to the beta2 form of the intracellular region will be generated. The dynamics of the reconstituted receptors and of anti-hapten antibodies at the planar membranes will be characterized as a function of membrane and solution properties. The requirements for and effects of bound, haptenated liposomes or cells, in the presence of anti- hapten antibodies, will also be characterized. The proposed research will involve the continued development of new techniques in dynamic fluorescence microscopy, including versions of total internal reflection fluorescence microscopy and fluorescence correlation spectroscopy. In addition, the development of a new method for observing highly fluorescent single molecules or particles as they bind and dissociate from surfaces is proposed.
