Institute of Neuronal Cell Biology, Technical University of Munich, Munich, Germany.
Institute for Diabetes and Obesity, Helmholtz Zentrum München, Munich, Germany.
Nat Protoc. 2023 Jul;18(7):2181-2220. doi: 10.1038/s41596-023-00831-w. Epub 2023 Jun 16.
Mitochondria are key bioenergetic organelles involved in many biosynthetic and signaling pathways. However, their differential contribution to specific functions of cells within complex tissues is difficult to dissect with current methods. The present protocol addresses this need by enabling the ex vivo immunocapture of cell-type-specific mitochondria directly from their tissue context through a MitoTag reporter mouse. While other available methods were developed for bulk mitochondria isolation or more abundant cell-type-specific mitochondria, this protocol was optimized for the selective isolation of functional mitochondria from medium-to-low-abundant cell types in a heterogeneous tissue, such as the central nervous system. The protocol has three major parts: First, mitochondria of a cell type of interest are tagged via an outer mitochondrial membrane eGFP by crossing MitoTag mice to a cell-type-specific Cre-driver line or by delivery of viral vectors for Cre expression. Second, homogenates are prepared from relevant tissues by nitrogen cavitation, from which tagged organelles are immunocaptured using magnetic microbeads. Third, immunocaptured mitochondria are used for downstream assays, e.g., to probe respiratory capacity or calcium handling, revealing cell-type-specific mitochondrial diversity in molecular composition and function. The MitoTag approach enables the identification of marker proteins to label cell-type-specific organelle populations in situ, elucidates cell-type-enriched mitochondrial metabolic and signaling pathways, and reveals functional mitochondrial diversity between adjacent cell types in complex tissues, such as the brain. Apart from establishing the mouse colony (6-8 weeks without import), the immunocapture protocol takes 2 h and functional assays require 1-2 h.
线粒体是参与许多生物合成和信号通路的关键生物能细胞器。然而，用当前的方法很难剖析它们在复杂组织中的特定细胞功能中的差异贡献。本方案通过使用 MitoTag 报告小鼠，从组织背景中直接对细胞类型特异性线粒体进行体外免疫捕获，解决了这一需求。虽然其他现有的方法是为批量线粒体分离或更丰富的细胞类型特异性线粒体开发的，但该方案是针对从中枢神经系统等异质组织中从中等到低丰度细胞类型中选择性分离功能线粒体进行了优化。该方案有三个主要部分：第一，通过将 MitoTag 小鼠与细胞类型特异性 Cre 驱动系或 Cre 表达的病毒载体交叉，使感兴趣的细胞类型的线粒体通过外膜 eGFP 进行标记。第二，通过氮空化从相关组织中制备匀浆，使用磁性微珠从匀浆中免疫捕获标记的细胞器。第三，免疫捕获的线粒体用于下游分析，例如，探测呼吸能力或钙处理，揭示分子组成和功能上具有细胞类型特异性的线粒体多样性。MitoTag 方法可用于鉴定标记细胞类型特异性细胞器群体的标记蛋白，阐明细胞类型丰富的线粒体代谢和信号通路，并揭示复杂组织（如大脑）中相邻细胞类型之间的功能线粒体多样性。除了建立小鼠品系（无需进口的 6-8 周）外，免疫捕获方案耗时 2 小时，功能测定需要 1-2 小时。
