EELS--X-Ray Mapping and Protein Imaging

这个项目的主要目的是确定元素局部变化的信号作用， 特别是细胞间信号中的钙浓度，通过同时扫描透射式电子能量损失光谱成像和X射线成像的5至10 nm分辨率的成分图冰冻切片，该方法是在以前的项目期开发的。对受试者的两个假设是：1)间盘(ICD)间隙是一个调节的、潜在的致心律失常的间隔；2)心房肌细胞成分的改变，特别是胞浆CI的增加，参与了慢性房颤的发生。我们还将使用扫描共聚焦免疫荧光显微镜和免疫电子显微镜来确定，作为我们的第二个总体目标，信号和调节平滑肌功能的蛋白质在细胞内的相互作用和移位。我们将在时间和空间上解决依赖刺激的信号蛋白易位，以评估它们的生理意义。待定位的主要信号蛋白(及其结构域)及其共定位的伙伴蛋白包括哺乳动物肌球蛋白磷酸酶调节亚基(MYPT1)、RhoA、PDZ Rhogef、LARG、p120连环蛋白、端粒蛋白、平滑蛋白和CP1-17。我们还将定位膜相关的、胞浆的和核的LPP在血管平滑肌中的位置，并确定其功能 其Rhokinase型易位的意义和调控。这些蛋白质的功能作用在项目1中确定，项目2中的RhoGEF的分子结构和蛋白质，包括用于光解的笼状GTP-伽马S.RhoAGDI复合体，由Core B提供。

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.