Figure 1.
Loss of DJ-1 results in multiple mitochondrial abnormalities. (A) M17 human dopaminergic neuroblastoma cells were stably transduced with control shRNA (lane 1) or DJ-1 shRNA (lane 2) and cell lysates blotted for DJ-1 (arrow). Markers on the right of the blot are in kilodaltons. (B) Δψm estimated in living cells using flow cytometry. Control cells (upper panel) or DJ-1 shRNA (lower panel) cell lines were stained with TMRE (x-axis) and DAPI for nuclei (y-axis). Flow cytometry was performed, and intensity of each signal for 10 000 cells was plotted and proportions of cells in each quadrant were calculated. (C) Δψm was estimated using live cell confocal imaging. After staining for TMRE, live cells were imaged and fluorescence pseudocolored to show differences in intensity. (D) Quantification of fluorescence in arbitrary units (a.u.) shows that DJ-1-deficient cells have significantly lower TMRE signal (P < 0.001 by t-test with Welsh's correction for unequal variances, n = 43–50 measurements per line). (E) Mitochondrial morphology was imaged in living cells transfected with mito-YFP plasmid. Mitochondria were elongated in control shRNA cells (upper panels) and at higher power (inset and right) showed a connected morphology. In DJ-1-deficient cells (lower panels), there were many small, disconnected mito-YFP-positive structures (inset and right). Scale bar is 2 μm. (F) Recovery of fluorescence was measured over a 12 s period after photobleaching a small region of mitochondria. Fluorescence recovery over time is plotted after normalization to both background fluorescence and non-photobleached mitochondrial fluorescence for 30 cells, showing decreased recovery of DJ-1-deficient cells (red symbols) compared with control lines (black symbols). Error bars show the SEM. (G) Mobile fraction of mito-YFP was lower in DJ-1-deficient mitochondria compared with controls. DJ-1-deficient cells had significantly lower FRAP for mito-YFP (P < 0.05 by t-test, n = 60 cells from duplicate experiments). (H) Control shRNA cells (upper panels) or DJ-1 shRNA cells (lower panels) either without treatment (left panels) or after treatment with 10 μm CCCP for 3 h (right panels) were transfected with LC3-GFP (green) and stained for the mitochondrial marker TOM20 (red). Arrows show the accumulation of LC3-GFP-positive punctatae near mitochondria in the cells. Scale bar is 10 μm. (I) Counts of LC3-GFP-positive punctatae per cell in n > 50 cells in three independent experiments showing the proportion of cells with less than 6 (open bars), between 6 and 15 (stippled bars), between 16 and 30 (striped bars) or more than 30 (filled bars) punctatae per cell, with error bars showing the SEM between experiments. Proportions of cells in each category were different by χ2-test comparing control and DJ-1 shRNA cells in each condition, untreated or with CCCP; ***P < 0.001; ns, not significant.