Structural investigation of the Z-disc by cryo-electron tomography

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The Z-disc is a characteristic feature at the fringe of the sarcomeres, the sub-units of the myofibrils, which associate the myofibrils with the signalling machinery of the cell. Over the last years a large number of proteins were localized in the vicinity of the Z-disc. However, despite the wealth of structural and biophysical information of the main players like α-actinin, Factin, titin and filamin C, little is known about the molecular architecture of the Z-disc. The current knowledge is based on electron-microscopic three-dimensional reconstructions of conventionally-embedded samples, which do not discern individual protein complexes. We will use cryo-electron tomography of vitreous sections and plunge-frozen samples in order to investigate the Z-disc at as-close-to-native conditions as possible. We will compare tomograms from different specimen like rabbit skeletal muscles (Stehle & Pfitzer, P9), mouse heart muscles (Frey, P2) and mouse muscle cells (Fürst & Fleischmann, P1) in healthy and damaged tissue. We will extract sub-tomograms from the Z-disc and classify them according to their different conformational states and interaction partners. This in silico purification should allow for such a high resolution that a reliable fitting of the atomic-resolved structures (Wilmans P5 and Djinovic-Carugo P6) is feasible, and should ultimately produce a hybrid atomic map of the Z-disc.