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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Mitochondrial Degradation
Lysosomes
Mitochondria
Autophagy
Mitochondrial Membranes
Transgenic Mice
Skeletal Muscle

Keywords

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

Cite this

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title = "Semi-automated quantitation of mitophagy in cells and tissues",
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.",
keywords = "mitophagy, mitochondria, autophagy, FIJI, mito-QC, mitolysosome, Mitochondria, Mitolysosome, Mitophagy, Autophagy",
author = "Lambert Montava-Garriga and Francois Singh and Graeme Ball and Ian Ganley",
note = "The work was funded (and acknowledged) by the MRC (grant MC_UU_00018/2).",
year = "2020",
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language = "English",
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TY - JOUR

T1 - Semi-automated quantitation of mitophagy in cells and tissues

AU - Montava-Garriga, Lambert

AU - Singh, Francois

AU - Ball, Graeme

AU - Ganley, Ian

N1 - The work was funded (and acknowledged) by the MRC (grant MC_UU_00018/2).

PY - 2020/1

Y1 - 2020/1

N2 - 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.

AB - 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.

KW - mitophagy

KW - mitochondria

KW - autophagy

KW - FIJI

KW - mito-QC

KW - mitolysosome

KW - Mitochondria

KW - Mitolysosome

KW - Mitophagy

KW - Autophagy

U2 - 10.1016/j.mad.2019.111196

DO - 10.1016/j.mad.2019.111196

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JO - Mechanisms of Ageing and Development

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