Visualization of spatial organization of membrane rafts onthe plasma membrane of CD4+ T lymphocytes in response to a foreign antigen

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Description: The plasma membranes of eukaryotic cells show phase separation with liquid-order phases enriched in cholesterol and sphingolipids, also known as membrane rafts, constituting about 50% of the plasma membrane. These membrane domains participate in a number of cellular processes including signal transduction. Current models of membrane rafts predict that these cell surface domains are 10-200 nm in diameter, short lived and often beyond the resolution of conventional light microscopy. Imaging techniques with high resolution allowing analysis of membrane rafts on a single raft as well as sub-population basis are required to assess the role of these nanodomains in cell signaling. A major goal of my thesis was to develop scanning electron microscopic techniques to analyze membrane rafts on the surface of CD4 + T cells. Two raft markers, Thy-1, Ly-6A.2 on the plasma membrane of un-stimulated and stimulated CD4 + T lymphocytes were targeted with specific monoclonal antibodies and 10 nm colloidal gold followed by their detection and visualization using the scanning electron microscope (SEM). I studied the distribution of the rafts on the plasma membrane of CD4 + T cell line in response to a specific antigen. While I successfully labeled and visualized membrane rafts by SEM, I did not observe mobilization of Thy-1 and Ly-6A.2 - enriched membrane rafts to the immunological synapse. In addition, I have developed a novel flow cytometry-based method to quantify membrane rafts and measure their size using fluorescently conjugated antibodies against the raft markers after isolating them from the cells in the absence of a detergent. Based on these studies the size range of membrane rafts is 146 to 160 nm in diameter and about 57 to 65% of raft fractions have either Ly-6A.2 or Thy-1 present in them.
Language: English
Format: Degree Work