Figure S2.
The reversible axon beading consumes ATP and is not caused by the disruption of MT tracks. (A) Cartoon showing the unipolar or bipolar NM-II filaments represented by double- or triple-dot immunostaining signals revealed by antibodies recognizing the C-terminal (gray) and N-terminal (red) of the NM-IIB heavy chain. (B) STED image showing the distribution of the assembled NM-II filaments beneath the plasm membrane of the axon. The unipolar and bipolar NM-II filaments are indicated with arrowheads and arrows, respectively. Bar = 1 μm (left) and 300 nm (right). (C) Rat hippocampal neurons cultured in an AoC device were transfected with Lifeact-GFP on DIV5–6. On DIV7–8, after being pretreated with either an MT polymerization inhibitor (+Nocodazole; 50 μΜ) or MT stabilizer (+Taxol; 10 μΜ) for 30 min, neurons were subjected to 50 μl/min flux for 180 s. Time-lapse images showing the deformation of the same axons before (B), during (D), and after (A) the flux, with the automatically detected beading segments shown in magenta. Bar = 5 μm. (D and E) Quantification of (D) the dynamic beading process and (E) peak bead number are presented (N = 11, 15, 13) (F) Quantification of the peak number of axon beads formed in response to the 50 μl/min flux for 180 s of Fig. 5 B (N = 41, 30, 49, 29, 23). (G) Neurons expressing Lifeact-GFP were pretreated with oligomycin (1 μM) for 30 min, followed by 50 μl/min flux for 180 s. Time-lapse images illustrate axon deformation before (B), during (D), and after (A) flux, with automatically detected beading shown in magenta. Bar = 10 μm. (H and I) Quantification of (H) normalized bead number and (I) peak bead number (N = 41, 14). (J) Schematic timeline for flux-induced axon stress with acute blebbistatin treatment. Blebbistatin (+BLB, 50 μM) was administered concurrently with 50 μl/min flux for 180 s, followed by an approximate 10-min recovery. Axon dynamics were monitored via live-imaging microscopy throughout. (K) Time-lapse images depict axon deformation before (B), during (D), and after (A) flux, with automatically detected beading in magenta. Bar = 10 μm. (L and M) Quantification of (L) dynamic beading process and (M) reversibility index (N = 16, 17). (N) Western blot showing the level of the p-MRLC, total MRLC, βΙΙ-Spectrin, and GAPDH following Calyculin A (+CA) treatment in cultured cortical neurons. (O) Quantification of the p-MRLC intensity in control (Ctrl) and CA-treated neuron axons (N = 41, 45, 45). Data represent mean ± SEM; in E, F, M, and O, unpaired two-tailed Student’s t test; in I, Mann-Whitney test; *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available for this figure: SourceData FS2.

The reversible axon beading consumes ATP and is not caused by the disruption of MT tracks. (A) Cartoon showing the unipolar or bipolar NM-II filaments represented by double- or triple-dot immunostaining signals revealed by antibodies recognizing the C-terminal (gray) and N-terminal (red) of the NM-IIB heavy chain. (B) STED image showing the distribution of the assembled NM-II filaments beneath the plasm membrane of the axon. The unipolar and bipolar NM-II filaments are indicated with arrowheads and arrows, respectively. Bar = 1 μm (left) and 300 nm (right). (C) Rat hippocampal neurons cultured in an AoC device were transfected with Lifeact-GFP on DIV5–6. On DIV7–8, after being pretreated with either an MT polymerization inhibitor (+Nocodazole; 50 μΜ) or MT stabilizer (+Taxol; 10 μΜ) for 30 min, neurons were subjected to 50 μl/min flux for 180 s. Time-lapse images showing the deformation of the same axons before (B), during (D), and after (A) the flux, with the automatically detected beading segments shown in magenta. Bar = 5 μm. (D and E) Quantification of (D) the dynamic beading process and (E) peak bead number are presented (N = 11, 15, 13) (F) Quantification of the peak number of axon beads formed in response to the 50 μl/min flux for 180 s of Fig. 5 B (N = 41, 30, 49, 29, 23). (G) Neurons expressing Lifeact-GFP were pretreated with oligomycin (1 μM) for 30 min, followed by 50 μl/min flux for 180 s. Time-lapse images illustrate axon deformation before (B), during (D), and after (A) flux, with automatically detected beading shown in magenta. Bar = 10 μm. (H and I) Quantification of (H) normalized bead number and (I) peak bead number (N = 41, 14). (J) Schematic timeline for flux-induced axon stress with acute blebbistatin treatment. Blebbistatin (+BLB, 50 μM) was administered concurrently with 50 μl/min flux for 180 s, followed by an approximate 10-min recovery. Axon dynamics were monitored via live-imaging microscopy throughout. (K) Time-lapse images depict axon deformation before (B), during (D), and after (A) flux, with automatically detected beading in magenta. Bar = 10 μm. (L and M) Quantification of (L) dynamic beading process and (M) reversibility index (N = 16, 17). (N) Western blot showing the level of the p-MRLC, total MRLC, βΙΙ-Spectrin, and GAPDH following Calyculin A (+CA) treatment in cultured cortical neurons. (O) Quantification of the p-MRLC intensity in control (Ctrl) and CA-treated neuron axons (N = 41, 45, 45). Data represent mean ± SEM; in E, F, M, and O, unpaired two-tailed Student’s t test; in I, Mann-Whitney test; *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available for this figure: SourceData FS2.

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