Phosphorylation of Mitochondrial Polyubiquitin by PINK1 Promotes Parkin Mitochondrial Tethering (original) (raw)
Figure 6
Mitochondrially targeted phospho-mimetic 4× Ub anchors Parkin.
(A) FLIP analysis. Mitochondria of HeLa cells transfected with GFP-Parkin S65E (green) along with Tom70MTS, Tom70MTS-4Ub SA or SE were visualized with MitoTracker Red (red). Representative live cell images obtained before and after pulse photobleaching in the circled region was shown. Higher magnification images of the dashed square area were also shown in separated color channels at the bottom. (B) The ratios of averaged fluorescence intensity in mitochondrial and cytosolic regions (Mito/Cyto ratio, 4 µm2 each) were graphed (mean ±SEM, n = 4–6). ** p<0.01 by two-tailed unpaired Student's _t_-test. (C) FRAP analysis. HeLa cells transfected with GFP-Parkin WT along with Tom70MTS-4Ub SA or SE were treated with 2 µM a proteasome inhibitor MG132 for 3 hrs to avoid degradation of activated Parkin and mitochondria were visualized with MitoTracker Red. MG132 treatment had little effect on the molecular behavior of GFP-Parkin (see S6 Figure). Representative images of cells expressing GFP-Parkin were obtained before photobleaching (Pre) of a ROI (circle) and at the indicated times after photobleaching. (D) The relative fluorescence intensity (RFI) of GFP-Parkin at the mitochondria in a ROI (circle) was measured in FRAP analysis performed as in (C). RFI is represented as the mean ±SEM (n≥3). Scale bars = 5 µm in (A, C).