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.

Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD

“…These results suggest that the mitophagy protein PINK1 potentially condamage by decreasing ΔΨ m , induce accumulation of PINK1 and recruitment of Parkin to mitochondria, mitochondrial dysfunction is considered an initiating event for mitophagy (11,46,47); however, this remains controversial (48). A recent study investigated the cellular fate of mitochondria damaged by the action of oxidative phosphorylation inhibitors, including CCCP, and suggested that mitophagy is not directly induced by mitochondrial damage (48,49). It has also been reported that expression of unfolded proteins in the matrix causes accumulation of PINK1, resulting in activation of mitophagy independently of the loss of ΔΨ m (50).…”

The pharmacological regulation of cellular mitophagy

“…20 Moreover, traditional protonophores are not specific for mitochondria and have protonophoric activity on other membranes, particularly the plasma membrane. 23 As a result, they mediate a variety of off-target effects, including disruption of the microtubule cytoskeleton 24 , inhibition of lysosomal activity 25 and activation of ion channels, 26 which leads to relatively high levels of toxicity and a narrow therapeutic window. Notably, FCCP is cytotoxic, even at concentrations that are not high enough to dissipate ΔΨm and thus induce mitophagy.…”

Peroxisomal protein PEX 13 functions in selective autophagy

“…At an early time point during mitophagy, we observed Parkin colocalization with TOMM20 even after PEX13 knockdown, suggesting that Parkin recruitment to the mitochondria is similar in PEX13‐deficient cells and control cells (Fig EV2B). We confirmed the role of PEX13 in mitophagy using a combination of more selective inhibitors of mitochondrial respiration, oligomycin, and antimycin A (OA) (as CCCP may have direct effects on lysosomal function 19) and by measuring the clearance of mitochondrial double‐stranded DNA (mtDNA) (as the proteasomal system can contribute to the degradation of mitochondrial outer membrane proteins such as TOMM20 but not to mtDNA 20). Our results indicate that four different siRNAs targeting PEX13 block OA‐induced mtDNA clearance as effectively as ATG7 siRNA (Fig 2C and D).…”