Fig. 9

Parkin levels at the mitochondria build in response to repetitive cycles of de- and repolarization. a-c HeLa cells expressing EYFP-Parkin and mito-mCherry were exposed to 60 min pulses of 10 μM CCCP every 100 min, as depicted in the schematic (a), and imaged by live cell fluorescence microscopy. b images of representative cells from the experiment. Scale bar represent 50 μm. c population average trajectories of the fold change in mitochondrial EYFP-Parkin fluorescence based on 3 biological repeats and a total of 23 cells. Error is represented as the S.E. d-f the experiment depicted in (a-c) was repeated using 30 min pulses of 10 μM CCCP separated by (d) 30 min or (e) 120 min and the trajectories of the fold change in mitochondrial EYFP-Parkin fluorescence for 4 representative cells is shown. The grey bars represent CCCP treatment times. f the peak amplitude of mitochondrial Parkin after exposure to the second pulse of CCCP is expressed as a percentage of the amplitude of peak mitochondrial Parkin levels after the first pulse of CCCP. Data is from a minimum of 30 cells per condition across at least 4 biological repeats. Statistical differences in peak amplitude was appraised using a Mann-Whitney test and significance is indicated as follows: ***, p < 0.001. g-h, EYFP-Parkin-expressing HeLa cells were exposed to CCCP, as depicted in the schematic (g), fixed and stained with DAPI (blue) and anti-TOM20 antibodies (red), and imaged by confocal microscopy. h The percentage of Parkin-expressing cells showing fused, fragmented, aggregated, or cleared mitochondria was determined based on 3 biological repeats with ≥70 cells/condition. Error is represented as the S.E. Representative images for each of the scored groups are shown above the graph. Scale bar represents 20 μm