Semi-automated quantitation of mitophagy in cells and tissues

Lambert Montava-Garriga, Francois Singh, Graeme Ball, Ian Ganley (Lead / Corresponding author)

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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.
Original languageEnglish
Article number111196
Pages (from-to)1-12
Number of pages12
JournalMechanisms of Ageing and Development
Volume185
Early online date13 Dec 2019
DOIs
Publication statusPublished - Jan 2020

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Keywords

  • mitophagy
  • mitochondria
  • autophagy
  • FIJI
  • mito-QC
  • mitolysosome
  • Mitochondria
  • Mitolysosome
  • Mitophagy
  • Autophagy

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