Enhancement of basal mitophagy by USP30 depletion is dependent on PINK1 Enhancement of basal mitophagy by USP30 depletion is dependent on PINK1 Representative.

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Enhancement of basal mitophagy by USP30 depletion is dependent on PINK1 Enhancement of basal mitophagy by USP30 depletion is dependent on PINK1 Representative images of U2OS cells stably expressing mCherry‐GFP‐Fis1(101–152) (U2OS‐MGFIS), transfected with non‐targeting (NT1) or USP30 targeting siRNA (D1, D3) for 72 h prior to imaging. Scale bar 10 μm.Quantification of mitolysosomes (red puncta, “dots”) in U2OS‐MGFIS cells, treated as in (A). Average ± SD; n = 6 independent experiments, 40 cells/experiment; left: one‐way ANOVA and Dunnett's multiple comparison's test, ***P < 0.001; right: two‐way ANOVA and Bonferroni's multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001.Quantification of mitolysosomes in U2OS‐MGFIS cells transfected with siRNAs targeting either USP30 (D1) or ATG7 or both. Average ± SD, n = 3 independent experiments, 40 cells per experiment; one‐way ANOVA and Dunnett's multiple comparison's test, *P < 0.05. Also shown is a representative Western blot.U2OS‐MGFIS cells were treated with siRNA and analysed as in (B). Average ± SD; n = 3 independent experiments; 40 cells per experiment; one‐way ANOVA and Dunnett's multiple comparison's test, *P < 0.05. Also shown is a representative Western blot.Quantification of mitolysosomes in hTERT‐RPE1 cells transfected with non‐targeting (NT1) or USP30 (D1, D3) or PINK1 targeting siRNA and mt‐Keima. Average ± SD; n = 3 independent experiments; 40 cells per experiment; one‐way ANOVA and Dunnett's multiple comparison's test, *P < 0.05, **P < 0.01. Also shown is a representative Western blot.Proposed model: USP30 acts upstream of PINK1 and limits the basal ubiquitylation (U) of outer mitochondrial membrane proteins, which serve as a substrate for PINK1. Phospho (P)‐ubiquitin binds to specialized autophagy adapters (optineurin and NDP52) leading to activation and recruitment of the autophagy machinery including LC3‐decorated autophagic membranes. Source data are available online for this figure. Elena Marcassa et al. EMBO Rep. 2018;embr.201745595 © as stated in the article, figure or figure legend