Semi-automated quantitation of mitophagy in cells and tissues

• Published in: Mechanisms of ageing and development Vol. 185; p. 111196
• Main Authors: Montava-Garriga, Lambert, Singh, François, Ball, Graeme, Ganley, Ian G.
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier B.V 01.01.2020; Elsevier Science Ireland
• Subjects: Animals; Autophagy; Autophagy - physiology; Biological Transport - physiology; Cell Line; FIJI; Hydrogen-Ion Concentration; Luminescent Proteins; Lysosomes - metabolism; Lysosomes - ultrastructure; Mice; Mice, Transgenic; Microscopy, Fluorescence - methods; mito-QC; Mitochondria; Mitochondria - metabolism; Mitochondria - ultrastructure; Mitochondrial Membranes - metabolism; Mitochondrial Turnover; Mitolysosome; Mitophagy; Mitophagy - physiology; mito-QC; Mitochondria; FIJI; Mitolysosome; stdDev; Mitophagy; ROI; TEM; Autophagy; DFP
• ISSN: 0047-6374; 1872-6216; 1872-6216
• DOI: 10.1016/j.mad.2019.111196

•The mito-QC reporter is a powerful model to monitor mitophagy in vitro and in vivo.•The mito-QC Counter is a new semi-automated tool to quantify mitophagy.•Mitophagy is induced in ARPE19 cells and varies in distinct skeletal muscle regions. 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 red-only 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 semi-automated 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 mito-QC 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.