Journal
NATURE PROTOCOLS
Volume 12, Issue 1, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2016.159
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Funding
- Newcastle University
- Foundation for Science and Technology (FCT) through the GABBA Programme, University of Porto, Porto, Portugal
- EMBO advanced long-term postdoctoral fellowship [aALTF 772-2015]
- Biotechnology and Biological Sciences Research Council (BBSRC) [BB/K008374/1]
- Royal Society
- David Phillips Fellowship [BB/H022384/1]
- BBSRC [BB/K017314/1]
- BBSRC [BB/K017314/1, BB/K008374/1, BB/H022384/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/K008374/1, BB/H022384/1, BB/K017314/1] Funding Source: researchfish
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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.
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