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Direct, time-resolved observation of protein-scale chemical reactions in living cells

Abstract

This thesis studies protein-scale molecular interactions in the plasma membrane of living mammalian cells. Particular emphasis is paid to fluorescence imaging, as these methods provide high spatial density, high temporal and spatial resolution, chemically-specific information over length scales larger than most cells. In Chapter 1 reactions between the T cell receptor (TCR) and its ligand, peptide-Major Histocompatibility Complex II (pMHC), are studied in hybrid live T cell-supported lipid bilayer (SLB) junctions. The following observations are made: (1) single molecules of pMHC can trigger signaling reactions in T cells, without association of other MHC or receptor clustering, (2) live cell pMHC-TCR kinetics are similar to solution phase measurements and are consistent with in vivo estimates of kinetic thresholds for thymic selection, (3) membrane recruitment of Zeta-chain-associated protein kinase 70 (ZAP70) occurs within seconds of pMHC:TCR engagement, (4) the pMHC:TCR:ZAP70 complex is most likely stoichiometric with a 1:1:10 ratio, is spatially distinct, and is transported as a unit towards the geometric cell center in an actin-dependent process, and (5) a stochastic reaction-diffusion simulation confirms that the interactions observed are the result of pMHC:TCR molecular binding. Together, these observations indicate that signal amplification in T cells occurs downstream of the TCR. In Chapter 2 a hybrid optical/nanofabrication technique is presented for the study of actin-TCR interactions in live T cells. Live cell photoactivated light microscopy (PALM) is used to image single molecules of actin interacting simultaneously with both pMHC-TCR in freely-diffusing membrane and pMHC-TCR in distinct square corrals of laterally fluid membrane segregated by Cr diffusion barriers. Single actin molecule behavior is similar to bulk actin behavior observed using other techniques, but the single molecule approach allows access to previously unseen protein-scale information about the actin network, such as molecular tortuosity. In Chapter 3 a fixed array of gold nanospheres are embedded in a laterally fluid supported membrane, providing a novel tool for studying processes in the plasma membrane of living cells via attachment of immobile moieties to the gold lattice and mobile moieties to the fluid supported membrane. In Chapter 4 synthetic glycoprotein mimics are inserted into the plasma membranes of living cells and their clustering behavior is monitored in the presence of the putative cross-linking galectin proteins using advanced fluorescence fluctuation spectroscopies. This approach provides a novel platform for testing the "galectin lattice" hypothesis, and for studying galectin ligand binding in a physiologically-relevant context.

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