EELS--X-Ray Mapping and Protein Imaging

该项目的主要目的是确定元素局部变化的信号作用， 特别是钙，细胞间信号的浓度，通过成分映射冷冻切片在5至10纳米的分辨率，同时扫描透射电子能量损失光谱成像和X射线映射，在以前的项目期间开发的方法。测试的两个假设是：1）闰盘（ICD）空间是一个受调节的、潜在的致心律失常隔室，2）心房肌细胞组成的改变，特别是细胞质CI的增加，与慢性房颤有关。我们还将确定，作为我们的第二个总体目标，细胞内协会和易位的蛋白质信号和调节平滑肌功能，使用扫描共聚焦免疫荧光显微镜和免疫电子显微镜。我们将解决在时间和空间刺激依赖的信号蛋白易位，以评估其生理意义。待定位的主要信号蛋白（及其结构域）及其待共定位的配偶体蛋白包括平滑肌肌球蛋白磷酸酶的调节亚基（MYPT 1）的哺乳动物同种型、RhoA、PDZ RhoGEF、LARG、p120连环蛋白、telokin、smoothelin和CP 1 -17。我们还将定位血管平滑肌中的膜相关、细胞质和细胞核LPP并确定其功能 Rhokinase调控易位意义和调控。这些蛋白质的功能作用在项目1中确定，项目2中RhoGEFs的分子结构和蛋白质，包括用于光解的笼状GTP-γ S.RhoAGDI复合物，由核心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.