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Depletion of mitochondria in mammalian cells through enforced mitophagy

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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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.

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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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