Fig. 3

Local protein synthesis of mtIF3 is necessary for mitochondrial translation in axonal growth cone. a Timeline for mito-riboBiFC experiments. After cell seeding, shRNA and overexpression vectors were transfected at DIV1. At DIV3, images were sequentially taken before and after drug treatment. BDNF, CHX, and CA were treated simultaneously. Drugs in the axonal chamber were treated for 90 min. b, c Visualization of mitochondrial translation in mtIF3-depleted axon growth cones by mito-riboBiFC. Mitochondria were marked by mitochondria-targeted mTFP1. Transfection of shRNA was confirmed by TagRFP657 expression (scale bar, 10 μm). Mito-riboBiFC signals from mitochondrial staining by mito-mTFP1 were quantified, and the relative intensity of BiFC increase upon drug treatment was measured. Five mitochondria per axon were analyzed. Data represent mean ± SEM (N = 3 replicates and n = 11–21 axons). Aligned ranks transformation ANOVA detected significant interaction effects of mtIF3 depletion and BDNF on the increase of relative BiFC intensity (P = 0.0180). n.s., not significant; *P < 0.05, **P < 0.01 as determined by Wilcoxon rank-sum test. d, e Representative images of mito-riboBiFC in axon growth cones expressing shRNA and mtIF3 (SR) (scale bar, 10 μm). Mito-riboBiFC signals from mitochondrial staining mito-mTFP1 were analyzed before and after CA treatment. Five mitochondria per axon were analyzed. Data represent mean ± SEM (N = 6 replicates and n = 24–54 axons). Aligned ranks transformation ANOVA detected significant interactions of mtIF3 (SR) overexpression and BDNF on mito-riboBiFC in mtIF3 shRNA groups (but not in control shRNA groups) (P = 0.0123). n.s., not significant; **P < 0.01, ***P < 0.001, ****P < 0.0001 as determined by Aligned ranks transformation ANOVA with Wilcoxon rank-sum test