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机制联系在一起。在过去的几年里，大量的蛋白质被定位在Z圆盘附近。然而，尽管α-Actinin、Factin、Titin和细丝素C等主要成员的结构和生物物理信息丰富，但人们对Z-Disc的分子结构知之甚少。目前的知识是基于常规包埋样品的电子显微镜三维重建，而不是区分单个蛋白质复合体。我们将使用冷冻电子断层扫描技术对玻璃体切片和冰冻样品进行扫描，以便在尽可能接近自然的条件下研究Z-Disc。我们将比较不同标本的断层图像，如兔骨骼肌(Stehle&Pfitzer，P9)、小鼠心肌(Frey，P2)和小鼠肌细胞(Fürst&Fleischmann，P1)在健康和受损组织中的断层图像。我们将从Z-Disc中提取亚断层图像，并根据它们不同的构象状态和相互作用伙伴进行分类。这在电子提纯中应该允许如此高的分辨率，以至于对原子分辨结构(WilmansP5和Djinovic-Carugo P6)的可靠拟合是可行的，并最终应该产生Z-盘的混合原子图。