FIGURE 5:
Silencing gigaxonin in normal fibroblasts impairs mitochondrial motility. (A) Three different shRNAs caused a reduction in the protein levels of gigaxonin in control fibroblasts, as demonstrated by immunoblotting with anti-gigaxonin at 72 h after silencing (see Materials and Methods). (B) Left, silencing gigaxonin for longer periods with shRNA #3 (up to 18 d) causes vimentin IF aggregation (green). Right, higher-magnification view of the same aggregate showing mitochondria (red; nucleus, blue). Scale bars, 10 μm. Trajectories of individual mitochondria in (C) control (expressing scrambled sequence) and (D) gigaxonin-silenced cells. (E) Box plot of net displacement of mitochondria in control and gigaxonin-silenced (Gigaxonin KD) cells. The box represents 25th and 75th percentiles, the median is denoted by the horizontal line, the mean is indicated by a small dot, and whiskers indicate SD. (F) Bar graph shows that there are significant differences in the total distance traveled by mitochondria in control and gigaxonin-silenced cells (p < 0.002). (G, H) Instantaneous velocities of individual mitochondria in control and gigaxonin-silenced cells (each color represents a different mitochondrion). We analyzed 68 mitochondria (stained with MitoTracker Red) in GAN fibroblasts and 62 in control fibroblasts (see Materials and Methods).