Semi-automated quantitation of mitophagy in cells and tissues. (January 2020)

• Record Type: Journal Article
• Authors: Montava-Garriga, Lambert; Singh, François; Ball, Graeme; Ganley, Ian G.
• Is Part Of: Mechanisms of ageing and development. Volume 185(2020)
• Publication Date: 2020-01
• Subjects: DFP deferiprone -- ROI region of interest -- stdDev standard deviation -- TEM transition electron microscopy; Mitophagy -- Mitochondria -- Autophagy -- FIJI -- mito-QC -- Mitolysosome; Aging -- Periodicals; Developmental biology -- Periodicals; Aging -- Periodicals; Developmental Biology -- Periodicals; Vieillissement -- Périodiques; Biologie du développement -- Périodiques; Aging; Developmental biology; Periodicals; 612.67
• DOI: 10.1016/j.mad.2019.111196
• Languages: English
• ISSNs: 0047-6374

Abstract

Highlights: 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. Abstract: 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 .