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)

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 > School 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

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
60752	1	A new approach to understanding mitochondrial functions in cell death, autophagy and beyond	Stephen Tait	Biotechnology and Biological Sciences Research Council (BBSRC)	BB/K008374/1	RI CANCER SCIENCES