Figure 2.
The stem dendrite of AcD neuron exhibits axon-like MT organization.(A) Left panel: Maximum intensity projection of DIV14 nonAcD (top) and AcD neurons (bottom) transfected with EB3-tdTomato for visualization of MT plus ends. The AIS is live-labeled with anti-NF-CF640R antibody. White arrowhead indicates the AIS and white arrow indicates the axon. Cyan arrowhead indicates regular somatic dendrite (Regular D); yellow and pink arrowhead indicates the AcD and stem dendrite (Stem D) of AcD neuron, respectively. Scale bar is 10 µm. Right panel: 5 min time projection and kymograph of EB3-tdTomato in the AIS, axon, and regular somatic dendrite (Regular D) of nonAcD (top) and AcD neuron (bottom) shown in the left panel. Scale bar is 10 µm. Kymograph scale is 10 s (vertical) and 2 µm (horizontal). Related to Video 4, A and B; and Video 5. (B) 5 min time projection and kymograph of EB3-tdTomato in the AcD and the stem dendrite (Stem D) of AcD neuron shown in A. Scale bar is 5 µm. Kymograph scale is 10 s (vertical) and 2 µm (horizontal). Related to Video 4 A. (C) Percentage of MT plus end orientations in different regions of nonAcD and AcD neurons. Mean ± SEM, three independent cultures, AIS (nonAcD) n = 23, AIS (AcD) n = 17, axon (nonAcD) n = 21, axon (AcD) n = 15, stem dendrite (Stem D) n = 19, AcD n = 17, regular somatic dendrite (Regular D) n = 67. (D) Density of EB3-tdTomato comets in different regions of AcD neurons. Mean ± SEM, three independent cultures, axon n = 16, stem dendrite (Stem D) n = 21, AcD n = 18, regular dendrite (Regular D) n = 32. Grey dot indicates value of individual cell. Orange triangle indicates mean of each independent culture. One-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001. (E) Growth rate of EB3-tdTomato comets in different regions of AcD neurons. Mean ± SEM, 3 independent cultures, axon n = 16, stem dendrite (Stem D) n = 21, AcD n = 18, regular dendrite (Regular D) n = 32. Grey dot indicates the value of individual cell. Orange triangle indicates mean of each independent culture. One-way ANOVA with Tukey’s multiple comparisons test, no significance (n.s.) P > 0.05. (F) Top row: Representative confocal images of DIV10 nonAcD and AcD neurons stained for tyrosinated (tyr) and acetylated (ac) tubulin, and the AIS marker TRIM46. Scale bar is 10 µm. Middle row: Single plane 2D gSTED image of tyrosinated and acetylated MTs corresponding to ROIs in top row. Scale bar is 2.5 µm. ROI1 is the axon (AIS region) of the displayed nonAcD neuron. ROI2 and ROI3 are the axon (AIS region) and stem dendrite of the displayed AcD neuron, respectively. Black arrowhead in ROI3 indicates the start of the axon. Bottom row: Intensity profile of tyrosinated and acetylated MTs indicated by white dashed lines in ROIs shown in middle row. (G) Representative confocal images of nonAcD and AcD neurons stained for tyrosinated (tyr) and acetylated (ac) tubulin, and the AIS marker TRIM46. Scale bar is 10 µm. White arrow in overlay image and black arrow in single-channel image indicates the proximal axon (AIS region). Cyan arrowhead in overlay image indicates the proximal part of the regular dendrite (Regular D); yellow and pink arrowhead in overlay image indicates the proximal part of the AcD and stem dendrite (Stem D) of AcD neuron, respectively. (H) Quantification of acetylated α-tubulin (ac-α-tub) fluorescent intensity in different regions of nonAcD (left) and AcD neurons (right). Mean ± SEM, three independent cultures, nonAcD neuron: proximal axon: n = 29 cells, proximal somatic dendrite (Regular D): n = 52 cells, AcD neuron: proximal axon: n = 28 cells, stem dendrite (Stem D): n = 30 cells, proximal AcD: n = 30 cells, proximal somatic dendrite (Regular D): n = 43 cells. Grey dot indicates value of individual cell. Orange and cyan triangle indicates mean of each independent culture. Mann–Whitney test (two-sided) for nonAcD neurons: not significant (n.s.) P > 0.05. One-way ANOVA with Tukey’s multiple comparisons test for AcD neurons, no significance (n.s.) P > 0.05. (I) Quantification of tyrosinated α-tubulin (tyr-α-tub) fluorescent intensity in different regions of nonAcD (left) and (right) neurons. Mean ± SEM, three independent cultures, nonAcD neuron: proximal axon: n = 29 cells, proximal somatic dendrite (Regular D): n = 53 cells, AcD neuron: proximal axon: n = 30 cells, stem dendrite (Stem D): n = 28 cells, proximal AcD: n = 29 cells, proximal dendrite (Regular D): n = 43 cells. Grey dot indicates value of individual cell. Orange and cyan triangle indicate mean of each independent culture. Mann–Whitney test (two-sided) for nonAcD neurons: ***P < 0.001. One-way ANOVA with Tukey’s multiple comparisons test for AcD neurons, *P < 0.05, **P < 0.01, ***P < 0.001. More detailed statistical information see Data S1.

The stem dendrite of AcD neuron exhibits axon-like MT organization . (A) Left panel: Maximum intensity projection of DIV14 nonAcD (top) and AcD neurons (bottom) transfected with EB3-tdTomato for visualization of MT plus ends. The AIS is live-labeled with anti-NF-CF640R antibody. White arrowhead indicates the AIS and white arrow indicates the axon. Cyan arrowhead indicates regular somatic dendrite (Regular D); yellow and pink arrowhead indicates the AcD and stem dendrite (Stem D) of AcD neuron, respectively. Scale bar is 10 µm. Right panel: 5 min time projection and kymograph of EB3-tdTomato in the AIS, axon, and regular somatic dendrite (Regular D) of nonAcD (top) and AcD neuron (bottom) shown in the left panel. Scale bar is 10 µm. Kymograph scale is 10 s (vertical) and 2 µm (horizontal). Related to Video 4, A and B; and Video 5. (B) 5 min time projection and kymograph of EB3-tdTomato in the AcD and the stem dendrite (Stem D) of AcD neuron shown in A. Scale bar is 5 µm. Kymograph scale is 10 s (vertical) and 2 µm (horizontal). Related to Video 4 A. (C) Percentage of MT plus end orientations in different regions of nonAcD and AcD neurons. Mean ± SEM, three independent cultures, AIS (nonAcD) n = 23, AIS (AcD) n = 17, axon (nonAcD) n = 21, axon (AcD) n = 15, stem dendrite (Stem D) n = 19, AcD n = 17, regular somatic dendrite (Regular D) n = 67. (D) Density of EB3-tdTomato comets in different regions of AcD neurons. Mean ± SEM, three independent cultures, axon n = 16, stem dendrite (Stem D) n = 21, AcD n = 18, regular dendrite (Regular D) n = 32. Grey dot indicates value of individual cell. Orange triangle indicates mean of each independent culture. One-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001. (E) Growth rate of EB3-tdTomato comets in different regions of AcD neurons. Mean ± SEM, 3 independent cultures, axon n = 16, stem dendrite (Stem D) n = 21, AcD n = 18, regular dendrite (Regular D) n = 32. Grey dot indicates the value of individual cell. Orange triangle indicates mean of each independent culture. One-way ANOVA with Tukey’s multiple comparisons test, no significance (n.s.) P > 0.05. (F) Top row: Representative confocal images of DIV10 nonAcD and AcD neurons stained for tyrosinated (tyr) and acetylated (ac) tubulin, and the AIS marker TRIM46. Scale bar is 10 µm. Middle row: Single plane 2D gSTED image of tyrosinated and acetylated MTs corresponding to ROIs in top row. Scale bar is 2.5 µm. ROI1 is the axon (AIS region) of the displayed nonAcD neuron. ROI2 and ROI3 are the axon (AIS region) and stem dendrite of the displayed AcD neuron, respectively. Black arrowhead in ROI3 indicates the start of the axon. Bottom row: Intensity profile of tyrosinated and acetylated MTs indicated by white dashed lines in ROIs shown in middle row. (G) Representative confocal images of nonAcD and AcD neurons stained for tyrosinated (tyr) and acetylated (ac) tubulin, and the AIS marker TRIM46. Scale bar is 10 µm. White arrow in overlay image and black arrow in single-channel image indicates the proximal axon (AIS region). Cyan arrowhead in overlay image indicates the proximal part of the regular dendrite (Regular D); yellow and pink arrowhead in overlay image indicates the proximal part of the AcD and stem dendrite (Stem D) of AcD neuron, respectively. (H) Quantification of acetylated α-tubulin (ac-α-tub) fluorescent intensity in different regions of nonAcD (left) and AcD neurons (right). Mean ± SEM, three independent cultures, nonAcD neuron: proximal axon: n = 29 cells, proximal somatic dendrite (Regular D): n = 52 cells, AcD neuron: proximal axon: n = 28 cells, stem dendrite (Stem D): n = 30 cells, proximal AcD: n = 30 cells, proximal somatic dendrite (Regular D): n = 43 cells. Grey dot indicates value of individual cell. Orange and cyan triangle indicates mean of each independent culture. Mann–Whitney test (two-sided) for nonAcD neurons: not significant (n.s.) P > 0.05. One-way ANOVA with Tukey’s multiple comparisons test for AcD neurons, no significance (n.s.) P > 0.05. (I) Quantification of tyrosinated α-tubulin (tyr-α-tub) fluorescent intensity in different regions of nonAcD (left) and (right) neurons. Mean ± SEM, three independent cultures, nonAcD neuron: proximal axon: n = 29 cells, proximal somatic dendrite (Regular D): n = 53 cells, AcD neuron: proximal axon: n = 30 cells, stem dendrite (Stem D): n = 28 cells, proximal AcD: n = 29 cells, proximal dendrite (Regular D): n = 43 cells. Grey dot indicates value of individual cell. Orange and cyan triangle indicate mean of each independent culture. Mann–Whitney test (two-sided) for nonAcD neurons: ***P < 0.001. One-way ANOVA with Tukey’s multiple comparisons test for AcD neurons, *P < 0.05, **P < 0.01, ***P < 0.001. More detailed statistical information see Data S1.

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