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-DISC是肉瘤的边缘的特征，即肌原纤维的子单位，将肌原纤维与细胞的信号机械相关联。在过去的几年中，大量蛋白质被定位在Z-DISC附近。但是，尽管主要参与者（如α-肌动蛋白，FACTIN，TITIN和FILAMIN C）拥有大量的结构和生物物理信息，但对Z-DISC的分子结构知之甚少。当前的知识基于传统样品的电子微观三维重建，这些重建不辨别单个蛋白质复合物。我们将使用玻璃体切片和暴发样品的冷冻电子断层扫描，以便在固定条件下调查Z盘。我们将在健康和受损的组织中比较不同标本（例如兔骨骼肌（Stehle＆Pfitzer，p9），小鼠心脏肌肉（Frey，P2）和小鼠肌肉细胞（Fürst＆Fleischmann，p1）等不同标本的断层图。我们将从Z-DISC中提取子图像图，并根据其不同的宪法状态和互动伙伴对其进行分类。在硅纯化中，这应该允许如此高的分辨率，以至于可靠地拟合原子分辨结构（Wilmans P5和Djinovic-Carugo P6）是可行的，并且最终应产生Z-DISC的混合原子图。