Szikora Szilárd, Novák Tibor, Gajdos Tamás, Erdélyi Miklós, Mihály József
Institute of Genetics, Biological Research Centre, Szeged, Hungary.
Department of Optics and Quantum Electronics, University of Szeged, Szeged, Hungary.
Bio Protoc. 2020 Jun 20;10(12):e3654. doi: 10.21769/BioProtoc.3654.
Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organization is an absolute prerequisite to the functionality of these contractile units. Despite the wealth of information collected, the exact spatial arrangement of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed a powerful nanoscopic approach to localize the sarcomeric protein components with a resolution well below the diffraction limit. The ease of sample preparation and the near crystalline structure of the flight muscle sarcomeres make them ideally suitable for single molecule localization microscopy and structure averaging. Our approach allowed us to determine the position of dozens of H-zone and Z-disk proteins with a quasi-molecular, ~5-10 nm localization precision. The protocol described below provides an easy and reproducible method to prepare individual myofibrils for dSTORM imaging. In addition, it includes an in-depth description of a custom made and freely available software toolbox to process and quantitatively analyze the raw localization data.
肌节是高度有序的大分子组件，其中适当的结构组织是这些收缩单元发挥功能的绝对前提。尽管已收集了大量信息，但许多H区和Z盘蛋白的确切空间排列仍不清楚。最近，我们开发了一种强大的纳米级方法，以低于衍射极限的分辨率定位肌节蛋白成分。飞行肌肌节易于制备样品且具有近晶体结构，使其非常适合单分子定位显微镜和结构平均。我们的方法使我们能够以准分子的、约5-10纳米的定位精度确定数十种H区和Z盘蛋白的位置。以下所述方案提供了一种简单且可重复的方法来制备用于dSTORM成像的单个肌原纤维。此外，它还深入描述了一个定制的、可免费获取的软件工具箱，用于处理和定量分析原始定位数据。
