Identification and Characterization of Mitochondrial Subtypes in Caenorhabditis elegans via Analysis of Individual Mitochondria by Flow Cytometry.
Identification and Characterization of Mitochondrial Subtypes in Caenorhabditis elegans via Analysis of Individual Mitochondria by Flow Cytometry.
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DOI：
10.1021/acs.analchem.6b00542
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发表时间：
2016-06-21
影响因子：
7.4
通讯作者：
Dillin A
中科院分区：
文献类型：
--
作者：
Daniele JR;Heydari K;Arriaga EA;Dillin A
线粒体生物能量学涉及许多重要的细胞和生理现象，包括衰老、代谢和抗逆性。线粒体膜电位的异质性（Δψ）是生物体生物能量学的核心，已成功地通过流式细胞术在全细胞中测量，但很少在大型动物模型的分离线粒体中测量。在秀丽隐杆线虫（秀丽隐杆线虫）等小动物模型中进行的类似研究对我们了解人类健康和疾病至关重要，但缺乏分析方法。在这里，我们报告了新的方法学的发展，使得有可能在秀丽隐杆线虫的发展和组织特异性研究中调查Δψ的异质性。本文描述的流式细胞术方法需要改进的基于胶原酶-3的线粒体分离程序，并使用比例荧光探针JC-9标记线粒体。为了证明组织特异性研究的可行性，我们使用了表达蓝色荧光肌肉特异性蛋白的秀丽隐杆线虫菌株，这使得从其他组织的线粒体中识别肌肉线粒体成为可能。该方法首次观察到秀丽隐杆线虫幼虫发育过程中Δψ的关键变化，并提供了直接证据，证明肌肉线粒体的生物能量状态相对于生物体其他部位的对应体有所提高。这些方法的进一步应用可以帮助梳理秀丽隐杆线虫和其他用于研究人类疾病和衰老的小动物模型的生物能量学和其他生物复杂性。
Mitochondrial bioenergetics has been implicated in a number of vital cellular and physiological phenomena, including aging, metabolism, and stress resistance. Heterogeneity of the mitochondrial membrane potential (Δψ), which is central to organismal bioenergetics, has been successfully measured via flow cytometry in whole cells but rarely in isolated mitochondria from large animal models. Similar studies in small animal models, such as Caenorhabditis elegans (C. elegans), are critical to our understanding of human health and disease but lack analytical methodologies. Here we report on new methodological developments that make it possible to investigate the heterogeneity of Δψ in C. elegans during development and in tissue-specific studies. The flow cytometry methodology described here required an improved collagenase-3-based mitochondrial isolation procedure and labeling of mitochondria with the ratiometric fluorescent probe JC-9. To demonstrate feasibility of tissue-specific studies, we used C. elegans strains expressing blue-fluorescent muscle-specific proteins, which enabled identification of muscle mitochondria among mitochondria from other tissues. This methodology made it possible to observe, for the first time, critical changes in Δψ during C. elegans larval development and provided direct evidence of the elevated bioenergetic status of muscle mitochondria relative to their counterparts in the rest of the organism. Further application of these methodologies can help tease apart bioenergetics and other biological complexities in C. elegans and other small animal models used to investigate human disease and aging.