Figure 6.
Periodic actin rings correlate more extensively with the head domain than the rod domain of the NM-II filaments along axons. (a) Cartoon showing the binding site of antibodies against the NM-II head domain (αNM-II (nt)) and rod domain (αNM-II (ct)), respectively. (b) Prior to fixation, DIV14 rat hippocampal neurons were labeled with SiR-actin, fixed and stained for endogenous head domain (αNM-II (nt)), and imaged with dual-color SIM. Bracketed regions with persistent actin rings (Ring+) or without actin rings (Ring−) are magnified in the bottom panels, respectively. Scale bars = 1 µm (top) and 500 nm (bottom). (c) Following fixation, DIV14 rat hippocampal neurons were stained for endogenous F-actin (Phalloidin) and head domain (αNM-II (nt)), and imaged with dual-color SIM. Bracketed regions with persistent actin rings (Ring+) or without actin rings (Ring−) are magnified in the bottom panels, respectively. Scale bars = 1 µm (top) and 500 nm (bottom). (d) Triple-color SIM image of endogenous F-actin (SiR-actin), head domain (αNM-II (nt)), and rod domain (αNM-II (ct)) of the NM-II along the axon of hippocampal neuron. Bracketed regions are magnified on the right. αNM-II (nt) puncta overlapping with actin rings are annotated with filled arrowheads. αNM-II (ct) puncta not overlapping with actin rings are annotated with hollow arrowheads. Scale bars = 1 µm (left) and 200 nm (right). (e) Comparison of colocalization between actin rings with either the head domain (αNM-II (nt)) or the rod domain (αNM-II (ct)) of NM-II filaments. (f) Quantification of the spacing between adjacent NM-II puncta in the axonal segments with preserved periodic actin rings. Data represent mean ± SEM; n = 34 (nt) and 33 (ct) axon segments for fraction of the F-actin overlapping with NM-II. (g) The NM-II and actin structures resolved with 3D SIM (top) were rendered into surface (middle) using Imaris software; boxed regions are magnified to show the accuracy of the rendering. Colocalization of the bracketed region is shown in the bottom panels. Comparison of colocalization between NM-II and actin rings before and after the 60 min blebbistatin treatment. Scale bars = 0.5 µm. (h) Quantification of the spacing between adjacent NM-II puncta in the axonal segments with preserved periodic actin rings in control and blebbistatin-treated neurons. Data represent mean ± SEM; n = 211 (nt) and 285 (ct) in f; n = 262 (ctrl) and 261 (blebbistatin) in i. Values represent numbers of axonal actin rings analyzed. (i) The Mander’s coefficient reflecting the colocalization rate. b and d are from different ROIs of the same SIM image. Data represent mean ± SEM; n = 30 (control) and 30 (blebbistatin) axon segments for fraction of the F-actin overlapping with NM-II. Data are from three independent cultures (**, P < 0.01; ***, P < 0.001, two-tailed unpaired t test). n.s., not significant.

Periodic actin rings correlate more extensively with the head domain than the rod domain of the NM-II filaments along axons. (a) Cartoon showing the binding site of antibodies against the NM-II head domain (αNM-II (nt)) and rod domain (αNM-II (ct)), respectively. (b) Prior to fixation, DIV14 rat hippocampal neurons were labeled with SiR-actin, fixed and stained for endogenous head domain (αNM-II (nt)), and imaged with dual-color SIM. Bracketed regions with persistent actin rings (Ring+) or without actin rings (Ring−) are magnified in the bottom panels, respectively. Scale bars = 1 µm (top) and 500 nm (bottom). (c) Following fixation, DIV14 rat hippocampal neurons were stained for endogenous F-actin (Phalloidin) and head domain (αNM-II (nt)), and imaged with dual-color SIM. Bracketed regions with persistent actin rings (Ring+) or without actin rings (Ring−) are magnified in the bottom panels, respectively. Scale bars = 1 µm (top) and 500 nm (bottom). (d) Triple-color SIM image of endogenous F-actin (SiR-actin), head domain (αNM-II (nt)), and rod domain (αNM-II (ct)) of the NM-II along the axon of hippocampal neuron. Bracketed regions are magnified on the right. αNM-II (nt) puncta overlapping with actin rings are annotated with filled arrowheads. αNM-II (ct) puncta not overlapping with actin rings are annotated with hollow arrowheads. Scale bars = 1 µm (left) and 200 nm (right). (e) Comparison of colocalization between actin rings with either the head domain (αNM-II (nt)) or the rod domain (αNM-II (ct)) of NM-II filaments. (f) Quantification of the spacing between adjacent NM-II puncta in the axonal segments with preserved periodic actin rings. Data represent mean ± SEM; n = 34 (nt) and 33 (ct) axon segments for fraction of the F-actin overlapping with NM-II. (g) The NM-II and actin structures resolved with 3D SIM (top) were rendered into surface (middle) using Imaris software; boxed regions are magnified to show the accuracy of the rendering. Colocalization of the bracketed region is shown in the bottom panels. Comparison of colocalization between NM-II and actin rings before and after the 60 min blebbistatin treatment. Scale bars = 0.5 µm. (h) Quantification of the spacing between adjacent NM-II puncta in the axonal segments with preserved periodic actin rings in control and blebbistatin-treated neurons. Data represent mean ± SEM; n = 211 (nt) and 285 (ct) in f; n = 262 (ctrl) and 261 (blebbistatin) in i. Values represent numbers of axonal actin rings analyzed. (i) The Mander’s coefficient reflecting the colocalization rate. b and d are from different ROIs of the same SIM image. Data represent mean ± SEM; n = 30 (control) and 30 (blebbistatin) axon segments for fraction of the F-actin overlapping with NM-II. Data are from three independent cultures (**, P < 0.01; ***, P < 0.001, two-tailed unpaired t test). n.s., not significant.

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