200 kV Transmission Electron Microscope

细胞之间的沟通和信息交换在很大程度上是由质膜中的多蛋白复合体介导的(S)。因此，对这种复合体的全面分析是了解细胞和器官的结构和运作的基础，这是弗莱堡大学生命科学及其几个合作研究中心的中心研究重点。申请人的实验室利用新开发的蛋白质组学技术，成功地破译了一系列膜蛋白复合体和网络的相互作用，即蛋白质构建块的集合，并提供了对它们的时空动力学的见解。全面了解这些复杂的蛋白质-蛋白质相互作用的意义需要广泛的技术，包括电生理学、基于荧光的方法、微型蛋白质组学和电子显微镜(EM)。事实上，EM是这些研究的关键，因为它具有独特的能力，可以定量地解析内源蛋白质复合体及其亚基组成的亚细胞分布，并在空间上具有高分辨率(&lt；20 nm)。这种高空间分辨率特别是在十二烷基硫酸钠消化的冷冻断裂复制品(SDS-FRL)中实现的，其中表面蛋白得到稳定，膜蛋白可以被标记。这项技术是在申请人的实验室建立的，事实证明，这项技术在分辨率和灵敏度方面优于其他技术，此外，还可以同时使用多个标记。这项高端EM技术将进一步完善，并在一系列资助和新项目中使用。这些包括：(1)啮齿动物脑内谷氨酸受体的生物发生和活性依赖的动力学，(2)天然Trp-C通道的结构和功能，(3)肾脏足细胞裂隙-横隔膜的结构、功能和动力学，(4)钙通道相关蛋白网络的组成和动力学及其G-蛋白偶联受体的调节，以及(5)质膜钙通道的调节和亚细胞分布。EM-和SDS-FRL技术的预期发展和改进的重要目标是(1)提高空间分辨率(&lt；通过使用更小的金粒子，(2)建立了多个金粒子与纳米体和新的基于蛋白质的探针的使用，以及(3)改进了膜间隔的表示。为了成功完成这些项目，本申请中要求的变速器-EM是强制性的。

Communication and exchange of information between cells is largely mediated by multi-protein complexes in the plasma membrane(s). Comprehensive analysis of such complexes is, therefore, fundamental for understanding structure and operation of cells and organs, a central research focus in the Life Sciences at the University of Freiburg and several of its collaborative research centers.Using newly developed proteomic technologies, the applicant’s lab has successfully deciphered the interactomes, the ensemble of protein building blocks, for a series of membrane protein complexes and networks and provided insights into their spatiotemporal dynamics. Comprehensive understanding of the significance of these complex protein-protein interactions requires a broad spectrum of techniques including electrophysiology, fluorescence-based approaches, micro-proteomics and electron-microscopy (EM). In fact, EM is key for these investigations due to its unique ability to resolve subcellular distribution of endogenous protein complexes and their subunit composition in a quantitative manner and with high resolution in space (< 20 nm).This high spatial resolution is achieved, in particular, in SDS-digested freeze fracture replicas (SDS-FRL) where surface proteins are stabilized and membrane proteins made accessible for labelling. This technology is established in the applicant’s lab and proved to be superior to other technologies with respect to resolution and sensitivity, and, in addition, enables the use of multiple labels at the same time.This high-end EM technique will be further refined and used in series of funded and new projects. These include: (1) biogenesis and activity-dependent dynamics of glutamate receptors in the rodent brain, (2) structure and function of native TRP-C channels, (3) structure, function and dynamics of the slit-diaphragm of podocytes in the kidney, (4) composition and dynamics of calcium channel-associated protein networks and their regulation by G-protein coupled receptors, as well as (5) regulation and subcellular distribution of plasma membrane Ca2+ ATPases.Important goals of the intended developments and refinements in EM- and SDS-FRL technologies are (1) improvement of spatial resolution (< 10 nm) by using smaller gold particles, (2) establishing the use of multiple gold particles coupled to nano-bodies and novel protein-based probes and (3) improved representation of membrane compartments. For successful completion of these projects the transmission-EM requested in this application is mandatory.