Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Frankfurt, Germany.
Optical Imaging Competence Centre, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Methods Mol Biol. 2024;2845:127-140. doi: 10.1007/978-1-0716-4067-8_10.
Selective autophagy of the endoplasmic reticulum (ER-phagy) is a mechanism that is necessary for degrading damaged ER components and preventing cells from experiencing ER stress. Various ER-phagy receptors orchestrate this process by building protein assemblies with dedicated functions. In order to understand the molecular building principles of ER-phagy, it is important to reveal the assembly of ER-phagy receptors in a temporal and functional context. However, direct visualization is hampered by the diffraction limit in light microscopy. Super-resolution microscopy (SRM) can bypass this limitation and resolve single proteins and nanoscale protein clusters in cells. In particular, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful technology that can resolve individual protein clusters in cells and provide information on their molecular composition. Here, we report a step-by-step protocol on how to utilize DNA-PAINT to perform super-resolution imaging of ER-phagy receptors in fixed cells. In addition, we provide a detailed explanation of image generation, cluster analysis, and molecular quantification.
选择性自噬内质网 (ER 自噬) 是一种降解受损内质网成分并防止细胞经历内质网应激的机制。各种 ER 自噬受体通过构建具有特定功能的蛋白质组装来协调这个过程。为了了解 ER 自噬的分子构建原则，揭示 ER 自噬受体在时间和功能方面的组装情况非常重要。然而，由于光显微镜的衍射极限，直接可视化受到阻碍。超分辨率显微镜 (SRM) 可以克服这一限制，在细胞中解析单个蛋白质和纳米级别的蛋白质簇。特别是，DNA 点积累用于成像纳米形貌 (DNA-PAINT) 是一种强大的技术，可以在细胞中解析单个蛋白质簇，并提供关于它们分子组成的信息。在这里，我们报告了一个逐步的方案，介绍如何利用 DNA-PAINT 在固定细胞中进行 ER 自噬受体的超分辨率成像。此外，我们还详细解释了图像生成、聚类分析和分子定量。
