Figure 3. MFN2 R400Q impairs conformational shifting and suppresses mitochondrial Parkin recruitment, but not mitochondrial motility in neuronal axons.

(A) Forster resonance energy transfer (FRET) studies of conformational switching in disease-linked MFN2 mutants expressed in MFN double knockout (DKO) murine embryonic fibroblasts (MEFs). Antagonist peptide normally induces closed conformation (increase in FRET) and agonist peptide open conformation (decrease in FRET). (B) Mitochondrial motility in cultured mouse dorsal root ganglion neuronal processes: representative kymographs are to the right (raw data above; motile mitochondrial emphasized below [red, antegrade; blue, retrograde]).Scale bar 10 μm, Data are n~3 5 DRGs for each independent experiment, statistic is one-way ANOVA. (C) mcParkin (red) recruitment to mitochondria in MFN DKO MEFs expressing MFN2 mutants before (baseline) and after FCCP (left, representative confocal micrographs; right, group quantitative data). p-Values calculated using ANOVA and Tukey’s test.

Figure 3—figure supplement 1. Effects of engineered synthetic MFN2 mutants on mitochondrial Parkin recruitment and mitochondrial motility in neuronal axons.

(A) Mitochondrial motility in cultured dorsal root ganglion (DRG) neuronal processes. Group quantitative data are to the left; representative kymographs are to the right. β-gal and WT MFN2 are from Figure 3. p-Values calculated using ANOVA and Tukey’s test. (B) Representative confocal micrograph showing Parkin localization (red) to mitochondria (green) after FCCP treatment. Arrows point to some co-localization events (yellow). Scale bar 10 μm, Data are n~3 5 DRGs for each independent experiment. (C) Parkin aggregation at mitochondria in MFN2 double knockout (DKO) murine embryonic fibroblasts (MEFs) in the absence (-) and presence (+) of FCCP; representative images to the right. MFN2 K109A is GTPase-defective; MFN2 EE constitutively binds Parkin; MFN2 AA cannot bind Parkin.