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

Z盘是肌节边缘的特征，肌节是肌原纤维的亚单位，其将肌原纤维与细胞的信号传导机制相关联。在过去的几年里，大量的蛋白质被定位在Z盘附近。然而，尽管α-辅肌动蛋白、Factin、肌联蛋白和细丝蛋白C等主要参与者的结构和生物物理信息丰富，但对Z盘的分子结构知之甚少。目前的知识是基于电子显微镜三维重建的常规包埋样品，不辨别个别蛋白质复合物。我们将使用冷冻电子断层扫描的玻璃体切片和暴跌冻结的样品，以研究Z盘在尽可能接近自然的条件。我们将比较不同标本的断层图像，如健康和受损组织中的兔骨骼肌（Stehle & Pfitzer，P9），小鼠心肌（Frey，P2）和小鼠肌细胞（Fürst & Fleischmann，P1）。我们将从Z盘中提取子断层图像，并根据它们的不同构象状态和相互作用伙伴对其进行分类。这种计算机纯化应该允许如此高的分辨率，使得原子分辨结构（Wilmans P5和Djinovic-Carugo P6）的可靠拟合是可行的，并且应该最终产生Z盘的混合原子图。

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.