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    Mitochondrial fusion is essential to maintain genomic stability and physiological functions of mitochondria. Since mitochondrial fusion and fission work in concert to regulate mitochondrial morphology and functions, it has been challenging to quantitatively measure the direct roles of mitochondrial fusion in apoptosis and cancer progression. Here, we report the development of a high-throughput in vitro method to quantify mitochondrial fusion through single mitochondria analysis by a laboratory-built nano-flow cytometer (nFCM). Isolated mitochondria expressing green fluorescent protein (GFP-mito) or discosoma red fluorescent protein (DsRed-mito) were mixed together, induced to fuse, and analyzed by nFCM. A particle exhibiting both green and red fluorescence was identified as an event of heterotypic fusion, and the efficiency of heterotypic fusion was used as a surrogate of overall fusion efficiency. The as-developed method was applied to reveal the interplay between mitochondrial fusion and apoptosis without the interference of fission. We show that cytosolic components promoted mitochondrial fusion, and this upregulation was diminished during apoptosis. Combined with the translocation of Bid and Bax from cytosol to mitochondria, these findings suggest that cytosolic pro-apoptotic Bcl-2 family proteins could be the positive mediators of mitochondrial fusion. On the other hand, fusion also renders mitochondria more resistant to membrane potential collapse upon apoptosis induction. Our data suggest that disruption of mitochondrial fusion could be a potent strategy for cancer therapy. Furthermore, the as-developed method offers an effective approach to identify fusion inhibitors, including betulinic acid and antimycin A, giving reasons for their powerful utility in cancer treatment. Copyright © 2020 Elsevier B.V. All rights reserved.

    Citation

    Jingyi Xu, Liyun Su, Jinyan Han, Kaimin Gao, Miaomiao Zhang, Shuo Wang, Chaoxiang Chen, Xiaomei Yan. Rapid and quantitative in vitro analysis of mitochondrial fusion and its interplay with apoptosis. Talanta. 2021 Jan 15;222:121523

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    PMID: 33167233

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