We investigate class I MHC molecules both as probes of surface membrane organization and in their own right, functioning in antigen presentation. We propose to integrate these two topics by investigating the way in which changes in membrane organization affect antigen presentation to effector cells by class I MHC molecules. Lateral organization and antigen presentation to cytotoxic lymphocytes, CTL, and to natural killer cells will be examined in cells deprived of cholesterol, in cells incubated with excess b2-microglobulin, which disperses MHC I clusters, in cells with defective membrane skeletons or truncated MHC molecules, and in cells in which the insulin-dependent phosphorylation of MHC-I molecules is compromised. In a second part of the project, we will use fluorescence photobleaching recovery, FPR, to probe the stability of interactions between MHC I molecules and TcR, as a function of peptide bound to MHC I molecules. These measurements will speak to mechanisms for recruitment of MHC-I and TcR to contact zones between effector and target. In a third part of the project, we will investigate interactions of nascent MHC I molecules with the TAP complex. These interactions have been inferred from the biochemistry of detergent-extracted membranes. We will use endogenously fluorescent MHC I molecules tagged with A. victoria green fluorescent protein, GFP, to probe TAP/MHC-I and MHC-I interactions in unperturbed endoplasmic reticulum, e.r. Lateral diffusion of GFP-class I molecules and energy transfer between class I molecules tagged with different GFP's will be compared in the e.r. of wild-type, TAP-deficient and TAP-overexpressing cells. These experiments will indicate the stability of the TAP/MHC-I complex, the extent of clustering of class I molecules by the TAP complex, and the allele-dependence of TAP/MHC-I association. Finally, patterns of lateral organization of MHC I and other cell surface proteins and lateral associations of class I molecules will be studied by fluorescence photobleaching recovery, fluorescence resonance energy transfer and by the newly-developed near-field scanning optical microscopy.
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