The Program project is an integrated approach, using state-of-the-art imaging approaches, for advancing our understanding of how antigen receptor inputs are recognized and processed so that a T lymphocyte can interpret a wide range of antigenic stimuli and respond appropriately. The.overall goal of the Imaging Core is to enable the use of very advanced imaging techniques in all five projects of the Program, to study the localization and function of specific proteins during T cell receptor (TCR) engagement and subsequent signaling. Recent advances in digital imaging create new ways to achieve the scientific goals of the project. These advances include new hardware (e.g. on-stage intensified CCD detectors, ultra-fast switching solid state lasers, simultaneous recording from dual cameras, temperature and CO2 environmental enclosures) and software that controls complex data acquisition, analysis and display, enable a more precise spatial temporal analysis of molecular localization and interactions at the single cell level in either live or fixed cells. Transient molecular interactions can be detected by FRET and single proteins can be imaged and studied by Quantum Dots blinking and by TIRF microscopy. Together, these advances enable the study of spatially localized and temporally transient molecular events that may escape conventional biochemical detection. The Imaging Core will be used by all five Projects. It will provide the instrumentation, software and expertise and training to all members of this Program Project. The Imaging Core will assist investigators in the Program in the design, sample preparartion, imaging and analysis of multi-dimensional immuno-fluorescent microscopy and live cell imaging. Project 1 will use the core to analyze TCR organization and clustering, using FRET between fluorophores and quantum dots. Project 2 will use the Core to visualize the Immunological Synapse in activated and anergized T cells. Projects 3, 4 and 5 will use the Core to visualize the localization of Sprouty 1, TFII-I and CARD11, respectively, in the Immunological Synapse (IS). Mutated forms of these proteins will be localized and changes in localizations will be correlated with altered functional responses. The effects of knockdown of these proteins on the IS will be studied too. Since inappropriate TCR signaling can result in ineffective immune surveillance, autoimmunity, or cancer, the results of these imaging studies may provide molecular targets for new therapies designed to treat diseases of the immune system.
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