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Depletion of mitochondria in mammalian cells through enforced mitophagy

Nature Protocols volume 12, pages 183–194 (2017)Cite this article

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Abstract

Mitochondria are not only the 'powerhouse' of the cell; they are also involved in a multitude of processes that include calcium storage, the cell cycle and cell death. Traditional means of investigating mitochondrial importance in a given cellular process have centered upon depletion of mtDNA through chemical or genetic means. Although these methods severely disrupt the mitochondrial electron transport chain, mtDNA-depleted cells still maintain mitochondria and many mitochondrial functions. Here we describe a straightforward protocol to generate mammalian cell populations with low to nondetectable levels of mitochondria. Ectopic expression of the ubiquitin E3 ligase Parkin, combined with short-term mitochondrial uncoupler treatment, stimulates widespread mitophagy and effectively eliminates mitochondria. In this protocol, we explain how to generate Parkin-expressing, mitochondria-depleted cells from scratch in 23 d, as well as offer a variety of methods for confirming mitochondrial clearance. Furthermore, we describe culture conditions to maintain mitochondrial-depleted cells for up to 30 d with minimal loss of viability, for longitudinal studies. This method should prove useful for investigating the importance of mitochondria in a variety of biological processes.

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Figure 1: Overview of enforced mitophagy protocol.
Figure 2: Validating widespread Parkin-mediated mitophagy.

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Acknowledgements

We thank J. Long and K. Ryan (Cancer Research UK Beatson Institute, Glasgow, United Kingdom) for providing KP-4 cells expressing Parkin. We thank G.P. Nolan (Stanford University, Stanford, California, USA) for providing the pLZRS-empty vector plasmid. C.C.-M. was funded by Newcastle University and the Foundation for Science and Technology (FCT) through the GABBA Programme, University of Porto, Porto, Portugal. G.I. was supported by an EMBO advanced long-term postdoctoral fellowship (aALTF 772–2015). This research was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (grant BB/K008374/1) and the Royal Society (both to S.W.G.T.). S.W.G.T. is a Royal Society University Research Fellow. Work in the J.F.P. laboratory was funded by a David Phillips Fellowship (BB/H022384/1) and a (BBSRC grant BB/K017314/1). We thank C. Winchester (Beaston Institute, Glasgow, Scotland) for reviewing the manuscript.

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Author notes

  1. Clara Correia-Melo and Gabriel Ichim: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Cell and Molecular Biosciences, Newcastle University Institute for Ageing, Campus for Ageing and Vitality, Newcastle University, Newcastle upon Tyne, UK

    Clara Correia-Melo & João F Passos

  2. Cancer Research UK Beatson Institute, Glasgow, UK

    Gabriel Ichim & Stephen W G Tait

  3. Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK

    Gabriel Ichim & Stephen W G Tait

Authors
  1. Clara Correia-Melo
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  2. Gabriel Ichim
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  3. Stephen W G Tait
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  4. João F Passos
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Contributions

C.C.-M., G.I., S.W.G.T. and J.F.P. developed the protocol. C.C.-M., G.I., S.W.G.T. and J.F.P. designed the experiments described herein. G.I. and C.C.-M. performed the experiments, interpreted the results and prepared figures. C.C.-M., G.I., S.W.G.T. and J.F.P. wrote the manuscript.

Corresponding authors

Correspondence to Stephen W G Tait or João F Passos.

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The authors declare no competing financial interests.

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Correia-Melo, C., Ichim, G., Tait, S. et al. Depletion of mitochondria in mammalian cells through enforced mitophagy. Nat Protoc 12, 183–194 (2017). https://doi.org/10.1038/nprot.2016.159

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