Ein kyro-elektronenmikroskopischer Ansatz zur Identifizierung der molekularen Struktur der nativen Schlitzmembran
Final Report Abstract
In recent decades the SD has emerged as a key target of glomerular disease and proteinuria. Although the composition and function of the SD has been described before, the native molecular architecture and function of its constituent parts have remained elusive. Here we used mammalian and avian animal models to reconcile the different SD models. The crux of our findings is the characterization of the molecular landscape of the mammalian kidney slit diaphragm (SD) which allowed to propose a mechanism for the function of the filtration process. For this purpose, we analyzed dedicated transgenic mouse models on different resolution levels by electron microscopy (ranging from molecular resolution obtained by cryo-electron tomography to accessing larger regions of interest by dual beam scanning electron microscopy) and correlated the results with light microscopy superresolution data. Electron tomography revealed two populations of strands within the SD: one with an average length of ~23 (variance ~10nm) and another with an average length of ~45 nm (variance ~10nm). The extreme variability seen in individual strand lengths is in striking contrast to the minimal flexibility usually observed in cadherin-based junctions such as desmosomes or adherens junctions. The measurements of the inter-foot process distances in capillaries without blood flow correspond well to the extracellular lengths of both NEPH1 and NEPHRIN. Substantiated by data from the group of Tobias Huber (Freiburg University) and by superresolution light microscopy data, we propose that NEPH1 and NEPHRIN form a spring-like, multi-layered interpodocyte arrangement that accounts for the dynamic adjustments of the SD required to respond to different blood pressures and variable glomerular capillary wall extensions. We have already published the correlative protocols used in our work and the image processing algorithms developed to gain high resolution threedimensional reconstructions. The concept of a spring-like, multi-layered inter-podocyte slit that could be directly drawn from our structural data, immediately pointed to the function of the SD. The molecular machine of a strong, yet flexible, cell-cell contact that forms an adjustable barrier to proteins, thereby ensuring that the renal filter does not clog at its outermost part enlightens once more nature as an ingenious engineer.
Publications
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(2014): Correlative light- and electron microscopy with chemical tags. In: J. Struct. Biol. 186, 205–213
Perkovic M, Kunz M, Endesfelder U, Bunse S, Wigge C, Yu Z, Hodirnau VV, Scheffer MP, Seybert A, Malkusch S, Schuman EM, Heilemann M, Frangakis AS
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A flexible, multilayered protein scaffold maintains the slit in between glomerular podocytes. JCI Insight. 2016 Jun 16; 1(9): e86177
Grahammer F, Wigge C, Schell C, Kretz O, Patrakka J, Schneider S, Klose M, Kind J, Arnold SJ, Habermann A, Bräuniger R, Rinschen MM, Völker L, Bregenzer A, Rubbenstroth D, Boerries M, Kerjaschki D, Miner JH, Walz G; Benzing T, Fornoni A, Frangakis A, Hube
