Understanding how the dynamics and distributions of membrane molecules relate to the regulation of cellular function is the long- term goal of this project. Several instrumental techniques will be used to study the lateral and rotational motions of molecules involved in lymphocyte stimulation by antigens and mitogens. Fluorescence photobleaching recovery examines molecular lateral motions, while time-resolved phosphorescence anisotropy and polarized fluorescence depletion measure protein rotational relaxation. Both fluorescence energy transfer and scanning fluorescence correlation spectroscopy provide information on the distributions of specific molecules on cell surfaces. One research goal concerns the mechanisms by which B cells present antigens to T helper cells. Lateral and rotational motions of Ia on B cells capable of presenting antigenic peptides to antigen-specific T cells will be studied to identify possible changes in Ia aggregation or conformation caused by peptide binding. Polyclonal and monoclonal hapten-specific B cells will be used to present antigen to T cells. The relation between antigen structure and concentration and subsequent presentation efficiency will be examined to learn how antigen encounter with the B cell membrane initiates antigen processing and expression of antigenic peptides. Antigen-Ia-T cell receptor complexes will be examined using environmentally-sensitive fluorescent peptides and fluorescence energy transfer methods to probe how T cell encounter modifies antigen-Ia interactions. The second area of work deals with T-independent antigen and mitogen interaction with B cells. Lateral diffusion of sIg-antigen aggregates on cloned DNP-specific B cells as functions of antigen concentration and epitope density will be measured by fluorescence photobleaching recovery and compared with accompanying B cell differentiative responses. The size and composition of antigen-immunoglobulin aggregates formed under conditions leading to stimulation and tolerance of the same DNP- specific cell lines will be measured for comparison with recently- completed models. Flow cytometric techniques will be used together with scanning fluorescence correlation spectroscopy. The state of B cell lipids during high zone tolerance induced by T- independent antigens and during activation by lipopolysaccharide will be explored by fluorescence photobleaching recovery.
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