title: 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.
authors.affiliation: Institute for Cell and Molecular Biosciences, Newcastle University Institute for Ageing, Campus for Ageing and Vitality, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: Clara
authors.initials: C
authors.lastname: Correia-Melo
authors.affiliation: Cancer Research UK Beatson Institute, Glasgow, UK.|Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Ichim
authors.affiliation: Cancer Research UK Beatson Institute, Glasgow, UK.|Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK.
authors.forename: Stephen W G
authors.identifier: http://orcid.org/0000-0001-7697-132X
authors.initials: SW
authors.lastname: Tait
authors.affiliation: Institute for Cell and Molecular Biosciences, Newcastle University Institute for Ageing, Campus for Ageing and Vitality, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: João F
authors.initials: JF
authors.lastname: Passos
mesh_terms: D000818:Animals; D001343:Autophagy; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D016922:Cellular Senescence; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics
chemical_list: D044767:Ubiquitin-Protein Ligases