title: The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells.
abstract: Mitophagy is a selective pathway, which targets and delivers mitochondria to the lysosomes for degradation. Depolarization of mitochondria by the protonophore CCCP is a strategy increasingly used to experimentally trigger not only mitophagy, but also bulk autophagy. Using live-cell fluorescence microscopy we found that treatment of HeLa cells with CCCP caused redistribution of mitochondrially targeted dyes, including DiOC6, TMRM, MTR, and MTG, from mitochondria to the cytosol, and subsequently to lysosomal compartments. Localization of mitochondrial dyes to lysosomal compartments was caused by retargeting of the dye, rather than delivery of mitochondrial components to the lysosome. We showed that CCCP interfered with lysosomal function and autophagosomal degradation in both yeast and mammalian cells, inhibited starvation-induced mitophagy in mammalian cells, and blocked the induction of mitophagy in yeast cells. PARK2/Parkin-expressing mammalian cells treated with CCCP have been reported to undergo high levels of mitophagy and clearance of all mitochondria during extensive treatment with CCCP. Using correlative light and electron microscopy in PARK2-expressing HeLa cells, we showed that mitochondrial remnants remained present in the cell after 24 h of CCCP treatment, although they were no longer easily identifiable as such due to morphological alterations. Our results showed that CCCP inhibits autophagy at both the initiation and lysosomal degradation stages. In addition, our data demonstrated that caution should be taken when using organelle-specific dyes in conjunction with strategies affecting membrane potential.
authors.affiliation: Department of Biochemistry and Molecular Biology; Monash University; Clayton campus; Victoria, Australia; Monash Micro Imaging; Monash University; Clayton campus; Victoria, Australia.
authors.forename: Benjamin S
authors.initials: BS
authors.lastname: Padman
authors.forename: Markus
authors.initials: M
authors.lastname: Bach
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Lucarelli
authors.forename: Mark
authors.initials: M
authors.lastname: Prescott
authors.forename: Georg
authors.initials: G
authors.lastname: Ramm
mesh_terms: D001343:Autophagy / Q000187:drug effects*; D001692:Biological Transport / Q000187:drug effects; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology*; D004396:Coloring Agents / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008564:Membrane Potentials / Q000187:drug effects; D026901:Membrane Transport Proteins / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D063306:Mitophagy / Q000187:drug effects; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D011956:Receptors, Cell Surface / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D004396:Coloring Agents; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011956:Receptors, Cell Surface; C084008:TOMM20 protein, human; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein
keywords: CCCP; DiOC6; MitoTracker; PARK2; Parkin; TMRM; lysosomal chloride; mitophagy