Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy - PubMed (original) (raw)

Activation of the ubiquitin–proteasome pathway is essential for mitophagy. (A) Co-localization of dispersed mitochondria and LC3B in CCCP-treated cells. HeLa cells expressing Parkin and EGFP-LC3B were treated with 100 n

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bafilomycin A1 and CCCP for 4 h, and stained for Hsp60 (red), EGFP-LC3B (green) and nuclei (DAPI, blue). Enlarged views of the boxed area are shown in the right column. Filled arrowheads mark examples of co-localization between dispersed mitochondria and EGFP-LC3B. The unfilled arrowhead marks an example of a dispersed mitochondrion that does not co-localize with EGFP-LC3B. Quantitation of this experiment is shown in the graph below. Error bars represent standard deviations from three independent experiments. Twenty cells were analyzed for each replicate, and ∼2400 dispersed mitochondria were manually assessed in total. In (**B)**–(D), Parkin-expressing HeLa cells were treated with CCCP in the presence or absence the proteasome inhibitor MG132 (10 μ

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). Cells were immunostained for Hsp60 (green), Tom20 (red) and nuclei (DAPI, blue). (B) MG132 inhibits Tom20 loss after 4 h of persistent CCCP treatment. Inset shows an enlarged view of the boxed area, highlighting the presence of dispersed, Tom20-negative mitochondria when MG132 is not present. The graph on the right shows quantification of this experiment. Each cell was scored into one of the five indicated bins, depending on the number of dispersed mitochondria that are Tom20 negative, but Hsp60 positive. Error bars indicate standard deviations from three independent experiments; 100 cells were scored per experiment. (C) MG132 preserves mitochondrial morphology at 12 h after a 100 min pulse treatment with CCCP. The graph on the right shows quantification of mitochondrial morphology. Cells were scored as having tubular mitochondria, fragmented mitochondria or no mitochondria. Error bars indicate standard deviations from three independent experiments; 500 cells were scored per experiment. (D) MG132 or epoxomicin abrogates CCCP-induced mitophagy. The images show cells 24 h after a 100 min pulse treatment with CCCP in the presence or absence of MG132 (10 μ

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). The graph on the right shows quantification of this experiment and a related one with epoxomicin (2 μ

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). Cells without mitochondria were defined by the complete lack of both Tom20 and Hsp60 signal. Error bars indicate standard deviations from three independent experiments. 1000 cells were scored for each MG132 experiment, and 200 cells were scored for each epoxomicin experiments. Scale bars equal 10 μm for (A)–(D). (E) Degradation of Tom20 in human neuroblastoma SH-SY5Y cells expressing exogenous Parkin. Cells were treated with DMSO (vehicle) or CCCP (20 μ

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) for 4 h. Cells were scored into one of the indicated five bins, depending on the number of dispersed mitochondria lacking Tom20, as described for (B). Error bars indicate standard deviations in three independent experiments; 100 cells were scored per experiment. (F) Epoxomicin abrogates CCCP-induced mitophagy in SH-SY5Y cells expressing Parkin. Cells were treated as in (D) in the presence or absence of epoxomicin (2 μ

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) and stained for Hsp60 and nuclei (DAPI). Cells without mitochondria were identified by complete loss of Hsp60 signal around the DAPI-stained nucleus. Error bars represent standard deviations from three independent experiments; 200 cells were analyzed per experiment.