Fig. 5: Axonal motility of mitochondria is altered in fl/fl E18 DRG sensory neurons of the CMT2B mutant mice.

E18 DRG sensory neurons from + /+, fl/+, fl/fl embryos were dissected, cultured and labeled with Mito Tracker at DIV4-7 as in Fig. 4. Time-lapsed images of axonal mitochondria were captured by live imaging microscopy. The image series were used to generate kymographs in Image J to quantitate axonal mitochondrial motility. a: Representative kymographs of + /+, fl/+ and fl/fl. White arrows indicate trafficking in anterograde direction, yellow arrows indicate trafficking in retrograde direction. Average velocities of axonal mitochondria moving in the anterograde (The numbers of mitochondria were analyzed are: n = 88 for + /+, n = 103 for fl/+, n = 96 for fl/fl) (b) and retrograde (The numbers of mitochondria were analyzed are: n = 59 for + /+, n = 76 for fl/+, n = 109 for fl/fl) (c) direction. d: % of retrogradely moving mitochondria (The numbers of axons were analyzed are: n = 12 for + /+, n = 10 for fl/+, n = 10 for fl/fl). e: % of total moving mitochondria (The numbers of axons were analyzed are: n = 31 for + /+, n = 26 for fl/+, n = 34 for fl/fl). We also performed JC9 staining to measure MMP by ratiometric analysis. f: representative images of JC9 staining of mitochondria of DRG axons of + /+, fl/+, fl/fl. The green channel and the red channel images as well as the merged images are shown. g: Ratiometric measurements of MMP in + /+, fl/+, fl/fl. Results are shown as mean ± SEM. The numbers of axons analyzed are: n = 12 for + /+, n = 10 for fl/+, n = 10 for fl/fl. The numbers of mitochondria are shown in the graphs. Significance analysis was carried out using Prism. Statistical significances were calculated by One-Way ANOVA. n.s = non significance. All p values are shown in the graphs.