Using Expansion Microscopy to Visualize and Characterize the Morphology of Mitochondrial Cristae
Using Expansion Microscopy to Visualize and Characterize the Morphology of Mitochondrial Cristae
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DOI：
10.3389/fcell.2020.00617
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发表时间：
2020-07-15
影响因子：
5.5
通讯作者：
Kozjak-Pavlovic, Vera
中科院分区：
文献类型：
--
作者：
Kunz, Tobias C.;Goetz, Ralph;Kozjak-Pavlovic, Vera
线粒体是生物学过程不可或缺的双膜结合细胞器，如细胞凋亡，细胞信号传导和许多重要代谢产物的产生，其中包括在称为氧化磷酸化（OXPHOS）的过程中产生的ATP。内膜包含称为嵴的褶皱，其增加膜表面，从而增加OXPHOS所需的膜结合蛋白的量。这些褶皱引起了极大的兴趣，不仅因为它们对能量转换的重要性，而且因为形态学的变化与癌症，糖尿病，神经退行性疾病，衰老和感染等广泛的疾病有关。由于相对的嵴膜之间的距离通常低于100 nm，常规荧光成像不能提供足以分辨这些结构的分辨率。出于这个原因，各种高度专业化的超分辨率方法，包括dSTORM，PALM，STED和SIM已被应用于嵴可视化。扩展显微镜（ExM）提供了在常规共聚焦显微镜上进行超分辨率显微镜检查的可能性，其通过将样品嵌入到各向同性扩展4-4.5倍的可溶胀水凝胶中，将常规共聚焦显微镜的分辨率提高到60-70 nm，其可以使用SIM进一步横向增加到类似于30 nm。在这里，我们证明，线粒体肌酸激酶MtCK的表达连接到标记蛋白GFP（MtCK-GFP），它定位于外部和内部线粒体膜之间的空间，可以用作嵴标记。将ExM应用于用该构建体标记的线粒体上，使得能够可视化嵴的形态变化和线粒体蛋白相对于嵴的定位研究，而不需要专门的设置。我们首次提出了结合特定的线粒体膜间隙标记和ExM作为研究线粒体内部结构的工具。
Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods includingdSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4-4.5, improving the resolution to 60-70 nm on conventional confocal microscopes, which can be further increased to similar to 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria.