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Fig. 4 | BMC Biology

Fig. 4

From: mtIF3 is locally translated in axons and regulates mitochondrial translation for axonal growth

Fig. 4

mtIF3-dependent mitochondrial translation elevates ATP generation in axonal growth cone. a, b ATP levels in mtIF3-depleted axonal mitochondria were measured using genetically encoded ATP indicator mito-ATeam1.03. After cell seeding, shRNA and mito-ATeam1.03 vectors were transfected at DIV1. At DIV3, images were sequentially taken before and after drug treatment. BDNF and CHX were treated simultaneously. Drugs in the axonal chamber were treated for 90 min. FRET signals were shown in the pseudo-color image. Expression of shRNA was confirmed by TagRFP657 expression (scale bar, 10 μm). FRET signals were measured by comparing the ratio before and after chemical treatments. Five mitochondria per axon were analyzed. Data represent mean ± SEM (N = 4 replicates and n = 9–12 axons). n.s., not significant; *P < 0.05, **P < 0.01 as determined by two-way ANOVA with Holm-Sidak’s multiple comparisons test. c, d Relative ATP levels in axonal mitochondria of neurons expressing shRNA and mtIF3 (SR). After cell seeding, shRNA, overexpression, and mito-ATeam1.03 vectors were transfected at DIV1. At DIV3, images were sequentially taken before and after BDNF treatment. BDNF in the axonal chamber was treated for 90 min. Pseudo-color shows ATP levels in mitochondria. mCherry and TagRFP657 expression confirms expression of mtIF3 (SR) and shRNA, respectively (scale bar, 10 μm). The increase of relative FRET signals was calculated by comparing before and after chemical treatments. Five mitochondria per axon were analyzed. Data represent mean ± SEM (N = 5 replicates and n = 17–34 axons). Aligned ranks transformation ANOVA detected significant interactions of mtIF3 (SR) overexpression and BDNF on the FRET signals in mtIF3 shRNA groups (but not in control shRNA groups) (P < 0.0001). n.s., not significant; ****P < 0.0001 as determined by Aligned ranks transformation ANOVA with Wilcoxon rank-sum test

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