Figure 4.
Actomyosin-II, not MT, mediates the reversible axon beading induced by mild mechanical stress. (A) Representative SEM images illustrate axon beading induced by low-speed (50 μl/min) flux. Red and blue arrows indicate the fluctuation of axon diameter. The bracket indicates the 10-μm length of the axon for each sampling. Bar = 1 μm. (B) Quantification of axon diameter fluctuations, calculated as the ratio of the longest to shortest diameters per 10 μm length of the axon in control (Ctrl) and fluxed axons (N = 80, 86). (C) Time-lapse SIM images capture dynamic changes in axons labeled with SiR-Actin (0.2 μM, 2 h) on DIV12 (Lukinavičius et al., 2014; Wang et al., 2020a). The bracketed ROI (1#) is magnified in the right panels, revealing actin ring diameter fluctuations before, during, and after flux. Further magnification highlights contracted (blue spots) and dilated (red spots) rings. Flux duration was indicated with a blue box. Bar = 5 μm (left), 1 μm (middle), and 1 μm (right). (D) Quantification of diameter changes in contracted (blue) and dilated (red) actin rings (N = 317, 139 rings). (E) Quantification of actin ring diameters before, during, and after flux (N = 456 rings). (F) The diagram summarizing the proportion of actin ring diameters undergoing contraction or expansion during and after flux. (G and H) Representative TEM images show the cytoskeletal changes in (G) control and (H) fluxed axons at 50 μl/min. Intact MT tracks are highlighted with yellow arrows, and organelles (multivesicular body, MVB; autophagosome, AP) are marked with blue arrows. Beading (red) and non-beading (blue) regions were indicated with brackets and amplified in the bottom panels. Representative images of non-beading (i and ii), beading with MT (iii), and beading without MT (iv) axons are shown. Bar = 200 nm. (I) Quantification of the percentage of axonal segments with either non-beading, beading with MTs, or beading without MTs in control and fluxed axons. N = 3 preparations. (J) Quantification of the percentage of axon beads containing organelles. N = 3 preparations. Data presented as mean ± SEM. Statistical analyses were performed using unpaired two-tailed Student’s t test for B, I, and J, within groups paired two-tailed t test for D, between groups unpaired two-tailed t test for D, and paired two-tailed unpaired t test for E. *P < 0.05, **P < 0.01, ***P < 0.001.

Actomyosin-II, not MT, mediates the reversible axon beading induced by mild mechanical stress. (A) Representative SEM images illustrate axon beading induced by low-speed (50 μl/min) flux. Red and blue arrows indicate the fluctuation of axon diameter. The bracket indicates the 10-μm length of the axon for each sampling. Bar = 1 μm. (B) Quantification of axon diameter fluctuations, calculated as the ratio of the longest to shortest diameters per 10 μm length of the axon in control (Ctrl) and fluxed axons (N = 80, 86). (C) Time-lapse SIM images capture dynamic changes in axons labeled with SiR-Actin (0.2 μM, 2 h) on DIV12 (Lukinavičius et al., 2014; Wang et al., 2020a). The bracketed ROI (1#) is magnified in the right panels, revealing actin ring diameter fluctuations before, during, and after flux. Further magnification highlights contracted (blue spots) and dilated (red spots) rings. Flux duration was indicated with a blue box. Bar = 5 μm (left), 1 μm (middle), and 1 μm (right). (D) Quantification of diameter changes in contracted (blue) and dilated (red) actin rings (N = 317, 139 rings). (E) Quantification of actin ring diameters before, during, and after flux (N = 456 rings). (F) The diagram summarizing the proportion of actin ring diameters undergoing contraction or expansion during and after flux. (G and H) Representative TEM images show the cytoskeletal changes in (G) control and (H) fluxed axons at 50 μl/min. Intact MT tracks are highlighted with yellow arrows, and organelles (multivesicular body, MVB; autophagosome, AP) are marked with blue arrows. Beading (red) and non-beading (blue) regions were indicated with brackets and amplified in the bottom panels. Representative images of non-beading (i and ii), beading with MT (iii), and beading without MT (iv) axons are shown. Bar = 200 nm. (I) Quantification of the percentage of axonal segments with either non-beading, beading with MTs, or beading without MTs in control and fluxed axons. N = 3 preparations. (J) Quantification of the percentage of axon beads containing organelles. N = 3 preparations. Data presented as mean ± SEM. Statistical analyses were performed using unpaired two-tailed Student’s t test for B, I, and J, within groups paired two-tailed t test for D, between groups unpaired two-tailed t test for D, and paired two-tailed unpaired t test for E. *P < 0.05, **P < 0.01, ***P < 0.001.

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