Depletion of mitochondria in mammalian cells through enforced mitophagy

Clara Correia-Melo, Gabriel Ichim, Stephen W.G. Tait, João F. Passos

Research output: Contribution to journalArticle

11 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)183-194
Number of pages12
JournalNature Protocols
Volume12
Issue number1
DOIs
StatePublished - Jan 1 2017

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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