200 kV Transmission Electron Microscope

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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.