German Center for Neurodegenerative Diseases (DZNE), Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Venusberg-Campus 1, Gebäude 99, Bonn, North Rhine-Westphalia 53127, Germany.
Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Bonn, North Rhine-Westphalia 53127, Germany.
Cell Rep Methods. 2021 Apr 2;1(1):100002. doi: 10.1016/j.crmeth.2021.100002. eCollection 2021 May 24.
Mitochondria sustain the energy demand of the cell. The composition and functional state of the mitochondrial oxidative phosphorylation system are informative indicators of organelle bioenergetic capacity. Here, we describe a highly sensitive and reproducible method for a single-cell quantification of mitochondrial CI- and CIV-containing respiratory supercomplexes (CI∗CIV-SCs) as an alternative means of assessing mitochondrial respiratory chain integrity. We apply a proximity ligation assay (PLA) and stain CI∗CIV-SCs in fixed human and mouse brains, tumorigenic cells, induced pluripotent stem cells (iPSCs) and iPSC-derived neural precursor cells (NPCs), and neurons. Spatial visualization of CI∗CIV-SCs enables the detection of mitochondrial lesions in various experimental models, including complex tissues undergoing degenerative processes. We report that comparative assessments of CI∗CIV-SCs facilitate the quantitative profiling of even subtle mitochondrial variations by overcoming the confounding effects that mixed cell populations have on other measurements. Together, our PLA-based analysis of CI∗CIV-SCs is a sensitive and complementary technique for detecting cell-type-specific mitochondrial perturbations in fixed materials.
线粒体维持着细胞的能量需求。线粒体氧化磷酸化系统的组成和功能状态是细胞器生物能量能力的信息指标。在这里，我们描述了一种高度敏感和可重复的单细胞定量检测线粒体 CI 和 CIV 含有呼吸超级复合物 (CI∗CIV-SCs) 的方法，作为评估线粒体呼吸链完整性的替代方法。我们应用邻近连接分析 (PLA) 并在固定的人和鼠脑、致瘤细胞、诱导多能干细胞 (iPSCs) 和 iPSC 衍生的神经前体细胞 (NPCs) 以及神经元中染色 CI∗CIV-SCs。CI∗CIV-SCs 的空间可视化能够检测到各种实验模型中的线粒体损伤，包括经历退行性过程的复杂组织。我们报告说，通过克服混合细胞群对其他测量的混杂影响，对 CI∗CIV-SCs 的比较评估有助于对即使是细微的线粒体变化进行定量分析。总之，我们基于 PLA 的 CI∗CIV-SCs 分析是一种在固定材料中检测细胞特异性线粒体扰动的敏感和互补技术。
