This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. T lymphocytes undergo a change from the resting state to the activated state when specific receptors and co-receptors on their surface are stimulated. These activation signals induce a polarization of the T-cell involving redistribution of the actin-cytoskeleton and plasma membrane domains commonly known as lipid rafts. One method that will be used to look for induction of T-cell polarization as well as localization of Nef-GFPis lipid rafts enriched in cholesterol, sphingolipids, and glycosylphosphatidylinositol s with HIV-1 Nef-GFP. Another method will be HIV-1 infection of Jurkat cells in order to observe the induction of T-cell polarization. HIV-1 infected cells will be fixed with 3% paraformaldehyde (rendering them uninfectious), stained, and mounted under glass coverslips. Confocal (GPI)-linked proteins. The first goal of the project is to develop a system in which Jurkat T-cells can artificially be activated and lipid raft polarization visualized. Lipid raft markers such as GM1 glycosphingolipid, CD59 (a GPI-linked protein), and two different fluorescent lipid analogs will be used to stain lipid rafts. Induction of T-cell activation will be accomplished using antibodies to the T-cell receptor complex and subsequent cross-linking with a secondary antibody. The second goal of the project is to investigate the role that HIV-1 Nef protein might play in causing T-cell activation. Transient transfection of Jurkat cell microscopy will be useful in these studies to observe the redistribution of lipid raft markers and localization of Nef-GFP. Ultimately, if HIV-1 Nef is found to induce polarization, the structural determinants of Nef that are required for this activity will be investigated.
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