Figure 4. Drp1 and mitochondrial... | Rockefeller University Press

Figure 4.

$Drp1 and mitochondrial translation are required for the OMM severing and mitochondrial ubiquitination in Acn-treated cells. (A–F) ATG5−/− (A–D), Drp1−/− (E and F), and WT (G) HCT116 cells treated with Acn alone (B and G), CP (C), Acn plus CP (D), Acn plus BafA1 (F), and DMSO (vehicle, A and E) for 6 h were immunolabeled to detect conjugated Ub (FK2 antibody; green), SDHA (red), and Tom20 (blue). In A–F, cells are traced with green lines and nuclei with yellow lines. Red arrowheads indicate examples of mitochondria-associated circular Ub. (G) High magnifications of the mitochondria-associated Ub (green), OMM (Tom20; blue), and IMM/matrix (SDHA; red) from Acn-treated WT HCT116 cells are shown. (H) Fluorescence intensity linescans along mitochondria shown in G. Scale bars represent 20 µm in A–F and 1 µm in G. (I) Quantification of circular mitochondrial ubiquitination in WT, Drp1−/−, and ATG5−/− HCT116 cells treated as indicated in the figure. Data are represented as mean ± SD; n = 3–6 (n = 70 cells per experiment). **, P < 0.01 versus control in WT HCT116 cells; ^, P < 0.01 versus Acn in WT HCT116 cells; ns indicates nonsignificant; one-way ANOVA with Bonferroni correction (α = 0.05). (J) Typical examples of mitochondria with discontinuous OMM from WT HCT116 cells treated with Acn for 6 h and labeled to detect cytochrome c (cyt.c; green), SDHA (red), and Tom20 (blue) are shown. Yellow arrowheads indicate SDHA-positive, cytochrome c–negative mitochondria. Bars represent 1 µm. (K) Control and Acn-treated ATG5−/− HCT116 cells were subjected to cell fractionation and Western blot as indicated in the figure. Actin was used as a loading control. C, cytosol; LM, postmitochondrial LM fractions; M, mitochondria-enriched HM fractions; T, total cell lysate.$

Drp1 and mitochondrial translation are required for the OMM severing and mitochondrial ubiquitination in Acn-treated cells. (A–F) ATG5^−/− (A–D), Drp1^−/− (E and F), and WT (G) HCT116 cells treated with Acn alone (B and G), CP (C), Acn plus CP (D), Acn plus BafA1 (F), and DMSO (vehicle, A and E) for 6 h were immunolabeled to detect conjugated Ub (FK2 antibody; green), SDHA (red), and Tom20 (blue). In A–F, cells are traced with green lines and nuclei with yellow lines. Red arrowheads indicate examples of mitochondria-associated circular Ub. (G) High magnifications of the mitochondria-associated Ub (green), OMM (Tom20; blue), and IMM/matrix (SDHA; red) from Acn-treated WT HCT116 cells are shown. (H) Fluorescence intensity linescans along mitochondria shown in G. Scale bars represent 20 µm in A–F and 1 µm in G. (I) Quantification of circular mitochondrial ubiquitination in WT, Drp1^−/−, and ATG5^−/− HCT116 cells treated as indicated in the figure. Data are represented as mean ± SD; n = 3–6 (n = 70 cells per experiment). **, P < 0.01 versus control in WT HCT116 cells; ^, P < 0.01 versus Acn in WT HCT116 cells; ns indicates nonsignificant; one-way ANOVA with Bonferroni correction (α = 0.05). (J) Typical examples of mitochondria with discontinuous OMM from WT HCT116 cells treated with Acn for 6 h and labeled to detect cytochrome c (cyt.c; green), SDHA (red), and Tom20 (blue) are shown. Yellow arrowheads indicate SDHA-positive, cytochrome c–negative mitochondria. Bars represent 1 µm. (K) Control and Acn-treated ATG5^−/− HCT116 cells were subjected to cell fractionation and Western blot as indicated in the figure. Actin was used as a loading control. C, cytosol; LM, postmitochondrial LM fractions; M, mitochondria-enriched HM fractions; T, total cell lysate.