Chapter 23 - Detection of mitophagy in mammalian cells, mice, and yeast
Section snippets
Rationale
Mitophagy in mammalian cultured cells can be monitored using Mito-Keima to measure the relative abundance of a mitochondrial protein in lysosomes (also called mitolysosomes) by flow cytometry or fluorescence microscopy. In the setting of a constant lysosomal degradation rate, an increase in mitolysosomes reflects an increase in mitophagic flux. The Mito-Keima reporter comprises a mitochondrial matrix targeting sequence fused to the N-terminus of the pH-sensitive fluorescent protein, monomeric
Rationale
There is growing appreciation that defective mitophagy might contribute to normal aging as well as to various neurodegenerative and cardiovascular diseases (Fivenson et al., 2017; Sun, Youle, & Finkel, 2016). Although cell biological observations suggest several potential mechanisms of mitophagy (Lazarou et al., 2015; Youle & Narendra, 2011), it remains largely unknown whether mitophagy is activated in vivo and how it is regulated. A better understanding of the physiological and pathological
Rationale
In the budding yeast Saccharomyces cerevisiae, mitophagy requires Atg32, a mitochondrial outer membrane-anchored protein that recruits the autophagy machinery to mitochondria and promotes the formation of autophagosomes enclosing mitochondria (Kanki, Wang, Cao, Baba, & Klionsky, 2009; Okamoto, Kondo-Okamoto, & Ohsumi, 2009). Yeast cells undergo a dramatic increase in mitophagy during prolonged respiration-driven growth in media containing a non-fermentable carbon source, such as glycerol, which
Summary
Mitophagy can be measured in mammalian cultured cells using the ratiometric Mito-Keima probe by flow cytometry or confocal microscopy (provided that the rate of lysosomal degradation is constant). PINK1/Parkin-dependent mitophagy can be assayed by Parkin translocation to mitochondria and pS65-Ub accumulation. In addition, the use of the mt-Keima mouse to facilitate the analysis of mitophagy can provide mitophagic flux data in both physiological and pathological conditions. By following the
Acknowledgments
We thank Maric Dragan and the NINDS Flow Cytometry Core Facility for technical assistance with FACS experiments, Chunxin Wang and Richard Youle for sharing cell lines stably expressing Mito-Keima and Parkin, and Yi-Ting Liu and Xiaoping Huang in the Narendra lab for helpful discussions and assistance with sample preparation. We are also grateful to Toren Finkel for the mt-Keima mice, Toren Finkel and Daniela Malide for helpful discussions, and Atsushi Miyawaki for the original mt-Keima
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The GET pathway serves to activate Atg32-mediated mitophagy by ER targeting of the Ppg1-Far complex
2023, Life Science AllianceMitochondria autophagy: a potential target for cancer therapy
2021, Journal of Drug Targeting
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These authors contributed equally to this work and are listed in alphabetical order.