EELS--X-Ray Mapping and Protein Imaging

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The major aim of this Project is to determine the signaling role of local changes in elemental, particularly calcium, concentrations in intercellular signaling, by compositional mapping cryosections at 5 to 10nm resolution with simultaneous scanning transmission electron energy loss spectroscopic imaging and X-ray mapping, the method developed during previous Project Periods. The two hypotheses to the tested are that: 1) the intercalated disc (ICD) space is a regulated, potentially arrhythmogenic compartment and that 2) alterations in the composition of atrial myocytes, particularly increases in cytoplasmic CI, are involved in chronic atrial fibrillation. We will also determine, as our second overall aim, the intracellular associations and translocations of the proteins signaling and regulating smooth muscle functions, using scanning confocal immunofluorescence microscopy and immunoelectronmicroscopy. We will resolve in time and space stimulus dependent translocations of signal proteins to evaluate their physiological significance. The main signaling proteins (and their structural domains) to be localized, and their partner proteins to be co-localized, include mammalian isoform of the regulatory subunit (MYPT1) of smooth muscle myosin phosphatase, RhoA, PDZ RhoGEF, LARG, p120 catenin, telokin, smoothelin and CP1-17. We will also localize membrane associated, cytoplasmic and nuclear LPP in vascular smooth muscle and determine the functional significance and regulation of its Rhokinase-modulated translocations. The functional role of these proteins is determined in Project 1, the molecular structures of RhoGEFs in Project 2 and proteins, including the caged GTP-gamma S.RhoAGDI complex used for photolysis, are provided by Core B.