Fig. 2.

YFP-Parkin accumulates on a portion of mitochondria in COXICA65 cybrid cells. The parental 143B cell with 100% wild-type mtDNA and COXICA65 cybrid cell with ~75% mutant mtDNA (G->A transition at 6,930 nt in cytochrome c oxidase subunit I) transfected with YFP-Parkin (green) (A) or YFP-Parkin plus vMIA-myc (B) were incubated without (A) or with 100 μM sodium azide (B) for 4 h. Cells were fixed and immunostained for Tom20 (mitochondria, red). Arrows in B represent YFP-Parkin localized to a subset of mitochondria in zoom image of white box of COXICA65 cells. (C) 143B and COXICA65 coexpressing YFP-Parkin (green), vMIA-myc and mito-CFP (white; blue in the merged image) were stained with 2.5 nM of the potentiometric mitochondrial dye TMRE (red) for 1 h. (D) Cells in each condition were scored for the presence of YFP-tagged wild-type (WT) or mutant Parkin on mitochondria. Greater than 70 cells were counted in each sample. The mean and SD were calculated from three replicates. (Scale bars, 10 μm in the full-size images and 2 μm in the magnified images.)