Fig. 4

Partial recovery of mitochondrial membrane potential is sufficient to induce PINK1 dissociation from the mitochondria. a fluorescence microscopy images of PINK1-EGFP (green) and TMRM (red) fluorescence in HeLa cells maintained in a constant concentration of TMRM (10 nM) and treated with 10 μM CCCP for 60 min to completely depolarize the mitochondria. The mitochondria were then partially repolarized by decreasing the CCCP concentration to 5 μM for the following 60 min, as depicted in the schematic (b). The scale bar in (a) represents 50 μm. c-e quantification of (c) average TMRM fluorescence in single cells (d) PINK1-EGFP fluorescence in 10 representative single cells and (e) the population average for PINK1-EGFP fluorescence. f For the experiment depicted in (a-e), the PINK1-EGFP responses of individual cells were separated into categories based on the magnitude of ΔΨM recovery (complete, moderate, or small) after CCCP concentration was decreased from 10 μM to 5 μM. The experiment depicted in (a-f) was performed as 3 discrete biological repeats with data gathered from a total of 24 individual cells. Error is represented as the standard deviation. g HeLa cells were treated with 10 μM CCCP for 60 min and then the mitochondria were partially or fully repolarized by decreasing the concentration of CCCP to 5, 2.5, or 0 μM or they were left in 10 μM CCCP as a control as depicted in the schematic (left). Endogenous PINK1 and actin levels were determined at the indicated timepoints during the treatment regime by western blotting (right)