In this proposal, we combine methodologies and expertise from the fields of immunology, biophysics and cell biology to address the question of whether the organizational state of the lymphocyte plasma membrane lipid is related to maturational and/or functional events in the immune system. For this study, we will first characterize the nature of lipid heterogeneity (as reflected by lateral domain formation, transbilayer asymmetry, alteration in fluidity) in several T and B lymphocyte model systems of maturation and function. We will also selectively perturb membrane lipid organization and examine the effect of these alterations on specific endpoints of immune function. A unique focus is brought to this proposal because of recent observations from this laboratory concerning unexpectedly large differences in the amount of the cytoskeletal protein spectrin associated with the inner surface of the plasma membrane among lymphocyte subsets in vivo and in vitro. We have also determined that lymphocyte activation results in alterations in the distribution pattern of spectrin. Because of spectrin's important role in the regulation of membrane fluidity and lipid organization, this observation may provide a valuable indicator of to inherent differences in membrane state among various lymphocyte subsets. In this proposal, we test the hypothesis that lipid heterogeneity is coincidentally linked to patterns of spectrin distribution and therefore also to lymphocyte function and state of maturation. Strong support for this hypothesis is provided by our preliminary studies in which we show that perturbants of lipid organization cause rapid alterations in spectrin association with the plasma membrane. To determine the state of lipid organization, we will utilize several techniques including phospholipid exchange, fluorescent probe partitioning, anisotropy and life-time spectroscopic and microscopic measurements, electron microscopy and deuterium NMR. Lymphocyte function and maturation will be monitored by measurement of antibody secretion, receptor-ligand internalization of B cells, mitogen and antigenspecific activation of T cells, and immunofluorescence of cell surface receptors. Immunolocalization of spectrin at the ultrastructural level has already been accomplished and this technique will be used extensively in this proposal. We expect that these studies will contribute to a better understanding of how lipid asymmetry is associated with lymphocyte function and ultimately provide us with knowledge required to predict the effects of changes in extracellular lipids upon lymphocyte function.
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