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Depletion of mitochondria in mammalian cells through enforced mitophagy

Lookup NU author(s): Dr Clara Correia Melo, Dr Joao Passos

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Abstract

© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.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.

Publication metadata

Author(s): Correia-Melo C, Ichim G, Tait SWG, Passos JF

Publication type: Article

Publication status: Published

Journal: Nature Protocols

Year: 2017

Volume: 12

Issue: 1

Pages: 183-194

Print publication date: 01/01/2017

Online publication date: 22/12/2016

Acceptance date: 02/04/2016

ISSN (print): 1754-2189

ISSN (electronic): 1750-2799

Publisher: Nature Publishing Group

URL: https://doi.org/10.1038/nprot.2016.159

DOI: 10.1038/nprot.2016.159

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