This project supports studies on signal transduction through the high affinity IgE receptor, FcepsilonRI, of mast cells. Early work (1993-2000) focused on biochemical and pharmacological analyses to identify protein components of the signaling pathways activated by FceRI crosslinking and to define phosphorylations and other modifications that control their interactions. In the current award period (2000-2004), high resolution transmission electron microscopy (TEM) mapping using cytoplasmic face-up membrane sheets and antibody-coated nanoprobes showed for the first time that crosslinked FcepsilonRI, the receptor-associated tyrosine kinases, Lyn and Syk, and downstream signaling proteins segregate during signaling to distinct membrane domains. Statistical analyses revealed patterns of protein topographical segregation during signaling that are consistent with, although more complex than, the patterns of protein functional segregation already inferred from biochemical studies. The main hypothesis for the new award period is that FceRI crosslinkinq causes receptors and signaling proteins to segregate within the plasma membrane into topographically distinct modules that may launch separate response pathways. Spatially-resolved TEM, image analysis and computational technologies will be used to investigate the role of IgE binding in stabilizing the multichain (alpha beta gamma2) FcepsilonRI against disassembly and, in a major effort, to map and model the time evolution of topographical relationships between receptor and multiple signaling proteins in antigen-activated cells. siRNA technology will be introduced to determine if FceRI signaling is confined to the plasma membrane or, alternatively, if signaling extends to the endosomal compartment. Genetic and pharmacological interventions, testing predictions from network modeling, will link topography to signaling activities. Time-resolved fluorescence methods (FRET, FLIM) will quantify dynamic interactions between key proteins during signaling. The integrated data will help to define the rules of membrane reorganization induced by ligand-receptor interaction and to reveal how this reorganization affects the activity of signaling networks.
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