Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin
Figure 3
Cell-based high content imaging of Parkin Ser65 mutations.
HeLa cells transiently expressing GFP-Parkin wild type, S65A, S65D, or S65E mutations were left untreated (0 h) or treated with the uncoupler CCCP for 2 h or 4 h. Cells expressing GFP only or the catalytically inactive GFP-Parkin C431S mutations served as controls. A) Images have been acquired using automated microscopy and show mitochondria (TOM20) in red, GFP-Parkin in green and nuclei (Hoechst) in blue. Quantification of Parkin re-localization is assessed by measuring maximal intensity in a cytoplasmic ring around the nucleus divided by the mean intensity of the nuclear GFP signal. Cytoplasmic ring and inner nuclear regions are schematically shown in the merge image. The GFP ratio is given for each GFP-positive cell in white as an example and reflects Parkin translocation to mitochondria. Untransfected cells or cells below threshold expression of Parkin are marked with a white asterisk and have been excluded from the analysis. B) The average GFP ratios of Parkin wild type and Ser65 mutations per well are shown as a heat map. C–E) Bar graphs give the percentages of cells with a defined mitochondrial translocation of Parkin at 0 h (C), 2 h (D), and 4 h (E) of CCCP treatment. A GFP threshold of 1.8 was chosen that corresponds to the average ratio of Parkin wild type translocation after 2 h CCCP treatment (n>4 plates with at least 4 wells per condition, one-way ANOVA, Tukey's post-hoc, p<0.0001, F = 147.3, ns – not significant, *** p<0.0005). F) Given are representative merge images at higher magnification that show co-localization of GFP-Parkin (green) and mitochondria (anti-TOM20, red). Nuclei (Hoechst) are shown in blue. Scale bars correspond to 10 µM. For images of the individual channels at all time points, see Figure S4.