Figure S7.
Short-term inactivation of NM-II causes the axon diameter expansion without affecting the microtubule structure or docking mitochondria. (a) Schematic cartoon showing the structure of a 6-well microfluidic device. (b) Time-lapse images showing the distribution of Af488 dye in the middle chamber of a 6-well microfluidic device, indicating its restriction capacity. Scale bar = 50 µm. DIC, differential interference contrast microscopy. (c) Axons of DIV14 hippocampal neurons were treated with blebbistatin (10 µM) in the middle chamber for 90 min. Representative images of the 3D SIM time-lapse images of axons before (Pre) and after blebbistatin treatment (+BLB). Value of z axis is color-coded. Scale bar = 5 µm. Magnified images from the boxed regions and the corresponding Imaris surface rendered images are shown in panels on the right. Scale bars = 1 µm. (d and e) Quantification of changes in the axon diameter (d) and volume (e) before and after blebbistatin treatment. Data represent mean ± SEM. For diameter quantification, n = 10 (Pre) and 10 (+BLB) axons were analyzed; and for volume quantification, n = 8 axons (Pre), n = 8 (+BLB) axons were analyzed from three independent cultures (***, P < 0.001, two-tailed paired t test). (f) DIV14 hippocampal neurons were treated with blebbistatin (10 µM, 60 min) and then fixed and stained for endogenous F-actin (phalloidin) and β-III-tubulin. Two-color SIM was used to resolve the actomyosin and microtubule structures, respectively. Scale bars = 1 µm. Quantification of changes in the microtubule intensity (g) and bundle width (h) in control and blebbistatin-treated axons. Data represent mean ± SEM. For intensity quantification, n = 33 (Pre) and 34 (+BLB) axons; for bundle width quantification, n = 33 axons (Pre) and n = 37 axons (+BLB) from three independent cultures (two-tailed paired t test). (i) Hippocampal neurons transfected with mito-TagRFP (red) and Lifeact-GFP (green) were imaged at the level of their axons before and after blebbistatin treatment. Kymographs of mitochondria movements from boxed regions are shown in the lower panels. Asterisks indicate moving mitochondria. x bar = 10 µm; y bar = 10 s. (j) Quantification of average speed of mitochondria transport in i. The frequency distributions of the trajectories with different speeds are shown to note the effect on docking (0 and 0.1 µm/s) and moving mitochondria. Data represent mean ± SEM; n = 7 (Pre) and 8 (+BLB) axons from three independent cultures (**, P < 0.01, Student’s t test). (k) Quantification of average speed of CTB-positive carriers with and without blebbistatin treatment, as shown in transport in Fig. 7 a. The frequency distribution of the trajectories with different speed are shown to note the effect on slow and fast CTB carriers, respectively. Data represent mean ± SEM; n = 3 (Pre) and 3 (+BLB) experiments from three independent preparations (*, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student’s t test). n.s., not significant.

Short-term inactivation of NM-II causes the axon diameter expansion without affecting the microtubule structure or docking mitochondria. (a) Schematic cartoon showing the structure of a 6-well microfluidic device. (b) Time-lapse images showing the distribution of Af488 dye in the middle chamber of a 6-well microfluidic device, indicating its restriction capacity. Scale bar = 50 µm. DIC, differential interference contrast microscopy. (c) Axons of DIV14 hippocampal neurons were treated with blebbistatin (10 µM) in the middle chamber for 90 min. Representative images of the 3D SIM time-lapse images of axons before (Pre) and after blebbistatin treatment (+BLB). Value of z axis is color-coded. Scale bar = 5 µm. Magnified images from the boxed regions and the corresponding Imaris surface rendered images are shown in panels on the right. Scale bars = 1 µm. (d and e) Quantification of changes in the axon diameter (d) and volume (e) before and after blebbistatin treatment. Data represent mean ± SEM. For diameter quantification, n = 10 (Pre) and 10 (+BLB) axons were analyzed; and for volume quantification, n = 8 axons (Pre), n = 8 (+BLB) axons were analyzed from three independent cultures (***, P < 0.001, two-tailed paired t test). (f) DIV14 hippocampal neurons were treated with blebbistatin (10 µM, 60 min) and then fixed and stained for endogenous F-actin (phalloidin) and β-III-tubulin. Two-color SIM was used to resolve the actomyosin and microtubule structures, respectively. Scale bars = 1 µm. Quantification of changes in the microtubule intensity (g) and bundle width (h) in control and blebbistatin-treated axons. Data represent mean ± SEM. For intensity quantification, n = 33 (Pre) and 34 (+BLB) axons; for bundle width quantification, n = 33 axons (Pre) and n = 37 axons (+BLB) from three independent cultures (two-tailed paired t test). (i) Hippocampal neurons transfected with mito-TagRFP (red) and Lifeact-GFP (green) were imaged at the level of their axons before and after blebbistatin treatment. Kymographs of mitochondria movements from boxed regions are shown in the lower panels. Asterisks indicate moving mitochondria. x bar = 10 µm; y bar = 10 s. (j) Quantification of average speed of mitochondria transport in i. The frequency distributions of the trajectories with different speeds are shown to note the effect on docking (0 and 0.1 µm/s) and moving mitochondria. Data represent mean ± SEM; n = 7 (Pre) and 8 (+BLB) axons from three independent cultures (**, P < 0.01, Student’s t test). (k) Quantification of average speed of CTB-positive carriers with and without blebbistatin treatment, as shown in transport in Fig. 7 a. The frequency distribution of the trajectories with different speed are shown to note the effect on slow and fast CTB carriers, respectively. Data represent mean ± SEM; n = 3 (Pre) and 3 (+BLB) experiments from three independent preparations (*, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student’s t test). n.s., not significant.

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