The organization of the T-cell in its resting and activated state, specifically of plasma-membrane molecules involved in the triggering of the T-cell, is a long-standing and contentious research topic in molecular immunology. Understanding the molecular mechanisms involved at the nanoscale, i.e. at spatial scales below the resolution limit of conventional optical microscopy (< 200 nm) will find answers to still open questions, and offer new routes to immunotherapy. The proposed multidisciplinary project aims to bring close together super-resolution STED microscopy and membrane biophysics with molecular immunology. The proposed studies will follow a holistic approach and include the investigation of the functional associations of a multitude of molecules in T-cell triggering, ranging from the T-cell receptor and co-receptors, over other proteins such as kinases and phosphatases, to lipids and the cortical cytoskeleton. Using super-resolution STED and single-molecule microscopy (specifically STED and fluorescence correlation spectroscopy, STED-FCS) I will directly observe, determine, follow and evaluate nanoscale molecular interactions of these molecules from the resting state and early activation of the T-cell until the constitution of the immunological synapse. Besides a sophisticated design of the experimental conditions such as appropriate live-cell fluorescence labeling, biochemical treatments and choice of activating the T-cell, I will improve the STED-FCS technology towards multi-color observations allowing the determination of nanoscale dynamics and interactions of different specific molecules at the same time. With new molecular interactions highlighted, I will be able to better understand how T-cells sense antigen presenting cells and what molecular ramifications are involved during the constitution of the immunological synapse.
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