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

通过冷冻电子断层扫描对 Z 盘进行结构研究

• 批准号: 175307392
• 负责人: Professor Dr. Achilleas Frangakis
• 金额: --
• 依托单位: Institut für Biophysik
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/175307392?language=en
• 关键词: Structural; investigation; disc; cryo; electron

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.

Z 盘是肌节边缘的一个特征，肌节是肌原纤维的亚单位，它将肌原纤维与细胞的信号传导机制联系起来。在过去的几年里，大量蛋白质定位在肌原纤维附近。然而，尽管α-肌动蛋白、肌动蛋白、肌动蛋白和丝蛋白 C 等主要参与者的结构和生物物理信息丰富，但人们对 Z 盘的分子结构知之甚少。目前的知识基于传统嵌入样品的电子显微镜三维重建，它不能辨别单个蛋白质复合物，我们将使用玻璃体切片和冷冻样品的冷冻电子断层扫描来进行研究。我们将在尽可能接近原始条件下比较不同样本的断层图，例如兔子骨骼肌 (Stehle & Pfitzer, P9)、小鼠心肌。健康和受损组织中的 (Frey, P2) 和小鼠肌肉细胞 (Fürst & Fleischmann, P1) 我们将从 Z 盘中提取子断层图，并根据其不同的构象状态和相互作用伙伴对其进行分类。应该允许如此高分辨率，以至于原子分辨结构（Wilmans P5 和 Djinovic-Carugo P6）的可靠拟合是可行的，并且最终应该产生一个混合原子图Z 盘。