Semi-automated quantitation of mitophagy in cells and tissues
Semi-automated quantitation of mitophagy in cells and tissues
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DOI：
10.1016/j.mad.2019.111196
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发表时间：
2020-01-01
影响因子：
5.3
通讯作者：
Ganley, Ian G.
中科院分区：
文献类型：
--
作者：
Montava-Garriga, Lambert;Singh, Francois;Ganley, Ian G.
线粒体自噬是一种自然现象，其通过自噬途径引起线粒体的溶酶体降解。近年来，荧光pH敏感的线粒体报告的发展，极大地促进了线粒体自噬的监测，通过区分胞质线粒体或酸性溶酶体。我们最近发表了mito-QC报告基因，它由线粒体外膜定位的串联mCherry-GFP标签组成。这允许通过当GFP信号在线粒体递送至溶酶体时被淬灭时产生的redonly mCherry信号的增加来定量线粒体自噬。在这里，我们为FIJI开发了一个宏，mito-QC计数器，并描述了它的使用，以允许可靠和一致的半自动定量线粒体自噬。在这篇方法文章中，我们一步一步地描述了如何检测和量化线粒体自噬，并表明线粒体自噬水平可以在不同的细胞系和不同的刺激下可靠地计算。最后，我们表明mito-QC计数器可用于定量mitoQC转基因小鼠组织中的线粒体自噬。我们证明骨骼肌线粒体自噬水平与糖酵解活性相关。我们目前的数据表明，FIJI的mito-QC计数器宏能够在体外和体内对线粒体自噬进行稳健的定量。
Mitophagy is a natural phenomenon and entails the lysosomal degradation of mitochondria by the autophagy pathway. In recent years, the development of fluorescent pH-sensitive mitochondrial reporters has greatly facilitated the monitoring of mitophagy by distinguishing between cytosolic mitochondria or those delivered to acidic lysosomes. We recently published the mito-QC reporter, which consists of a mitochondrial outer membrane-localised tandem mCherry-GFP tag. This allows the quantification of mitophagy via the increase in redonly mCherry signal that arises when the GFP signal is quenched upon mitochondrial delivery to lysosomes. Here we develop a macro for FIJI, the mito-QC Counter, and describe its use to allow reliable and consistent semiautomated quantification of mitophagy. In this methods article we describe step-by-step how to detect and quantify mitophagy and show that mitophagy levels can be reliably calculated in different cell lines and under distinct stimuli. Finally, we show that the mito-QC Counter can be used to quantify mitophagy in tissues of mitoQC transgenic mice. We demonstrate that mitophagy levels in skeletal muscle correlates with glycolytic activity. Our present data show that the mito-QC Counter macro for FIJI enables the robust quantification of mitophagy both in vitro and in vivo.