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Fig. 5 | BMC Molecular and Cell Biology

Fig. 5

From: Temporal integration of mitochondrial stress signals by the PINK1:Parkin pathway

Fig. 5

Dissociation of Parkin from the mitochondria occurs at a slower rate than PINK1. a-c HeLa cells expressing EYFP-Parkin and mito-mCherry were imaged by live cell fluorescence microscopy in the presence of 10 μM CCCP, which was washed out after either 20 or 60 min. Timecourse images of cells exposed to 10 μM CCCP for 60 min are shown in (a). In these cells, EYFP-Parkin (green) colocalizes with the mitochondrial marker (mito-mCherry) within 60 min after initial CCCP exposure and remains colocalized for at least 60 min after CCCP removal (120 min timepoint). Thin white lines in the zoomed images mark the trajectory of the line scan measurement and the intensity values for EYFP-Parkin and mito-mCherry fluorescence are shown in (b). Quatification of the change in EYFP-Parkin levels at the mitochondria are shown as (c). Trajectories represent average fold change in mitochondrial EYFP-Parkin from 3 biological repeats with a minimum of 44 cells per condition. d-g HeLa cells expressing EYFP-Parkin and stained with 10 nM TMRM were imaged by live cell microscopy in the presence of 10 μM CCCP. After 60 min, the concentration of CCCP was reduced to (d) 5 μM or (e-g) 0 μM to either partially or fully restore mitochondrial membrane potential for the final 60 min of the experiment. For (d + e) a representation of the CCCP treatment regime (left), quantification of average TMRM fluorescence (center), and average fold change of EYFP-Parkin localized to mitochondria (right) is shown. Error is represented as the S.E. Representative fluorescence microscopy images and single cell trajectories for mitochondrial EYFP-Parkin and TMRM levels for cells exposed to 10 μM CCCP for 60 min followed by 0 μM CCCP for the following 60 min is shown as (f) and (g), respectively

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