Neural circuit research group, Korea Brain Research Institute;
J Vis Exp. 2024 Sep 27(211). doi: 10.3791/67020.
Cellular organelles, such as mitochondria and lysosomes, display dynamic structures. Despite the higher resolution of transmission electron microscopy for structural analysis, light microscopy is essential for the visualization of dynamic organelles by target-specific labeling. The following protocol describes a method that combines dual-color correlative light and electron microscopy (CLEM) to observe the interactions between mitochondria and lysosomes. In this study, mitochondria were labeled with mEosEM (Mito-mEosEM) and lysosomes with TMEM192-V5-APEX2. The results obtained from CLEM images enable us to observe the changes in the interactions between mitochondria and lysosomes under external stress conditions. Treatment with bafilomycin (BFA), which inhibits lysosomal function, resulted in an increase in contact between mitochondria and lysosomes, leading to the formation of fragmented mitochondria trapped inside lysosomes. Conversely, treatment with U18666A, which inhibits cholesterol export from lysosomes, caused lysosomes to be surrounded by mitochondria, indicating a distinct form of interaction. This study presents an effective method for observing the interactions between mitochondria and lysosomes in fixed cells. Furthermore, CLEM imaging with dual-color probes offers a powerful tool for future investigations of organelle dynamics and their implications for cell function and pathology.
细胞器官，如线粒体和溶酶体，呈现动态结构。尽管透射电子显微镜在结构分析方面具有更高的分辨率，但光镜对于通过目标特异性标记来可视化动态细胞器是必不可少的。以下方案描述了一种结合双色相关光和电子显微镜（CLEM）的方法，用于观察线粒体和溶酶体之间的相互作用。在这项研究中，线粒体用 mEosEM（Mito-mEosEM）标记，溶酶体用 TMEM192-V5-APEX2 标记。从 CLEM 图像中获得的结果使我们能够观察到在外部应激条件下线粒体和溶酶体之间相互作用的变化。用抑制溶酶体功能的巴弗洛霉素（BFA）处理会导致线粒体和溶酶体之间的接触增加，从而形成被困在溶酶体内部的碎片化线粒体。相反，用抑制胆固醇从溶酶体输出的 U18666A 处理会导致线粒体包围溶酶体，表明存在一种不同的相互作用形式。本研究提供了一种观察固定细胞中线粒体和溶酶体相互作用的有效方法。此外，双色探针的 CLEM 成像为进一步研究细胞器动力学及其对细胞功能和病理学的影响提供了有力工具。
