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Fig. 1 | BMC Biology

Fig. 1

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

Fig. 1

BDNF induces local protein synthesis of mtIF3 in axon growth cone. a mtIF3 mRNAs were detected in both cell bodies and axons of primary hippocampal neurons at DIV4. RNA samples were purified from the isolated lysates of cell bodies and axons. RT-PCR was performed using each pair of gene-specific primers. β-actin existing in both lysates and γ-actin detected in cell bodies indicate that both cell bodies and axons were fractionated without contamination [19]. b Primary hippocampal neurons were seeded into the cell body chamber. Axons reached the other side of the device through the microgroove. To locally stimulate axons, the only axonal chamber was treated with drugs. c Kymographs of newly synthesized mtIF3-Dendra2 fusion proteins in axons. Neurons were cultured on microfluidic devices and transfected with an expression vector for the mtIF3-Dendra2 protein at DIV3. Fluorescent intensity of mtIF3-Dendra2 fusion was measured in mitochondrial areas at DIV4 (horizontal scale bar, 5 μm; vertical scale bar, 5 min). The red arrow indicates images before photoconversion. The existing mtIF3-Dendra2 was photo-converted from green to red over the 20 μm path from the axon tip. Green signal is non-photoconverted mtIF3-Dendra2 fluorescence and red signal is photo-converted fluorescence. Recovered green signal from existing red mitochondria was analyzed. Images were then taken at 5-min intervals for 90 min. BDNF (30 ng/ml), and anisomycin (20 μM) were added to the axonal chamber at a 0-min timepoint. d Quantification of newly synthesized mtIF3-Dendra2 proteins. The green fluorescence was measured from mitochondria at the very end of the axonal tip. The relative intensity was calculated by normalizing the values at each time point to the value at a 0-min time point. Data represent mean ± SEM (N = 3 replicates and n = 6–9 axons). *P < 0.05, **P < 0.01 as determined by two-way repeated-measures ANOVA with Holm-Sidak’s multiple comparisons test. Asterisk indicates statistical significance from 5′UTRmtIF3-mtIF3-Dendra2-3′UTRmtIF3 with BDNF treatment for 5′UTRmtIF3-mtIF3-Dendra2-3′UTRmtIF3 without treatment or with BDNF and anisomycin treatments. No statistical significance was observed among mtIF3-Dendra2 groups without UTRs. e Pseudo-color images of locally synthesized Dendra2 reporter in axonal tip (scale bar, 10 μm). The experiment was performed similarly as in panel c. f Quantification of newly synthesized Dendra2 reporters in axonal tip after drug treatment. Data represent mean ± SEM (N = 3 replicates and n = 6–10 axons). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as determined by two-way repeated-measures ANOVA with Holm-Sidak’s multiple comparisons test. Asterisk indicates statistical significance from 5′UTRmtIF3-Dendra2-3′UTRmtIF3 with BDNF treatment for 5′UTRmtIF3-Dendra2-3′UTRmtIF3 without treatment or BDNF and anisomycin treatments. No statistical significance was observed between 5′UTRGAPDH-Dendra2-3′UTRGAPDH groups

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