Depletion of mitochondria in mammalian cells through enforced mitophagy

Correia-Melo, C., Ichim, G., Tait, S. and Passos, J. F. (2017) Depletion of mitochondria in mammalian cells through enforced mitophagy. Nature Protocols, 12(1), pp. 183-194. (doi:10.1038/nprot.2016.159) (PMID:28005069)

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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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Ichim, Dr Gabriel and Tait, Professor Stephen
Authors: Correia-Melo, C., Ichim, G., Tait, S., and Passos, J. F.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Journal Name:Nature Protocols
Publisher:Nature Publications
ISSN:1754-2189
ISSN (Online):1750-2799
Published Online:22 December 2016
Copyright Holders:Copyright © 2017 Nature Publications
First Published:First published in Nature Protocols 12(1):183-194
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
607521A new approach to understanding mitochondrial functions in cell death, autophagy and beyondStephen TaitBiotechnology and Biological Sciences Research Council (BBSRC)BB/K008374/1RI CANCER SCIENCES