The long-range goal of this project is to explore the physicochemical organization of the B lymphocyte plasma membrane and to learn how changes in this organization may reflect and/or mediate primary events in cellular activation. The first part of this project concern the organization and distribution of lipids on membrane domains. Sizes, lipid composition, and fluidities of lipid domains will be examined using fluorescence photobleaching recovery and scanning fluorescence correlation spectroscopy. Studies of large unilamellar vesicles prepared from B cell membrane lipid will help assess whether cellular lipid organization is dependent on the presence of cell surface protein. Membrane lipid alteration via specific lipid enrichment, via cholesterol depletion through glucocorticoid treatment, and through temperature perturbation will be examined. The second area of work concerns the distribution and interaction of membrane proteins. Scanning fluorescence correlation spectroscopy experiments will explore whether proteins are localized into one or more types of protein-rich regions and whether specific lipid classes preferentially associate with particular proteins. Specific associations occurring between known B cell membrane proteins and other known or as-yet unidentified species will be assessed using fluorescence energy transfer and new immunochemically-targeted photoproximity labeling methods. The aggregation state of specific membrane proteins will be evaluated using time-resolved phosphorescence anisotropy. Finally, immediate changes in B lymphocyte membrane structure resulting from activation by three mechanisms, by anti-IgM treatment, by thymus-independent antigen-like ligands interacting with cells artificially decorated with specific receptors, and by lipopolysaccharide will be examined.
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