Fig. 7
Mitochondrial and cellular stress cause association of Wt a-syn with mitochondria, disrupted by perturbation of helices 1 and 2. RBL cells co-expressing low levels of Wt a-syn a, b or V70P a-syn c, d together with Mito-cameleon (green) were incubated for 30 min at 37o in BSS without a, c or with b, d 10 µM CCCP then fixed, and a-syn was immunostained (red) for visualization with confocal microscopy; scale bar = 10 μm. Alternatively, RBL cells expressing Wt a-syn were labeled with MitoTracker Red, washed with cold PBS and permeabilized with 0.001% digitonin for 3 min e. Cells were then fixed, and a-syn was immunostained (green) and visualized as for other samples. Traces below micrographs in a–e are fluorescence intensities across the arrow-line drawn on the images for both a-syn and mitochondrial marker channels. f Averaged Mander’s overlap coefficients (MOC) for a-syn and mitochondrial marker were calculated for specified samples as represented in a box plot. The box shows 25th–75th percentile of the data; the midline shows the median, and the small square shows the average. ***P-values < 0.001, NS, not statistically significant (P-values > 0.05). g Samples prepared as described in e were visualized at super resolution with structure illumination microscopy, using fiduciary beads in each sample to ensure the channel alignment; scale bar is 5 μm for the image and 2 μm for the inset
