Figure 3.
AcD neurons have similar MPS organization of the AIS as in nonAcD neurons. (A) Top row: Representative confocal images of DIV14 nonAcD (left) and AcD (right) neurons stained with the AIS marker βIV-spectrin and F-actin probe Phalloidin-Atto647N. Orange arrowhead indicates the stem dendrite of the displayed AcD neuron. Yellow dashed rectangle indicates the AIS. Scale bar is 10 µm. Bottom row: Single plane 2D gSTED image of F-actin in the AIS of nonAcD and AcD neurons (corresponding zoom-ins of yellow dashed rectangle in top row). Scale bar is 1 µm. Red arrowhead indicates F-actin patch. Red dashed line indicates the profile of F-actin rings shown in B. (B) Intensity profile of periodic F-actin structures along the longitudinal axis of the AIS in nonAcD and AcD neurons; corresponding to black dashed lines in A bottom row. (C) Quantification of distance between F-actin rings at the AIS of nonAcD and AcD neurons. Mean ± SEM, three independent cultures, nonAcD: n = 45 profiles from 12 cells, AcD: n = 67 profiles from 18 cells. Grey dot indicates value of individual cell. Orange and cyan triangle indicates mean of each independent culture. Mann–Whitney test (two-sided): not significant (n.s.) P > 0.05. More detailed statistical information see Data S1. (D) Representative widefield (left) and dSTORM (right) images of DIV14 AcD neurons stained with the AIS marker βIV-spectrin and AnkG, F-actin probe Phalloidin-Alexa647+ and MAP2. Scale bar is 20 µm on widefield image and 5 µm on dSTORM image. Orange arrowhead indicates the stem dendrite. Yellow dashed square indicates the AIS. dSTORM image corresponds to the yellow dashed rectangle in widefield image. (E) Dual-color 3D dSTORM image of F-actin and βIV-spectrin at the AIS of the displayed AcD neuron. Left panel: corresponding zoom-in of the yellow dashed square on dSTORM image shown in D right panel. Yellow dashed square indicates the zoom-in region shown in right panel. Right panel: corresponding zoom-in of the yellow dashed square in left panel. Scale bar is 5 µm. Red arrowhead indicates F-actin patch. Red dashed line indicates the intensity profile of F-actin rings and βIV-spectrin shown in F. (F) Intensity profile of periodical F-actin (cyan) and βIV-spectrin (orange) structures along the longitudinal axis of the AIS in AcD neurons; corresponding to red dashed lines in E right panel. (G) Auto-correlation curve of F-actin (cyan) and βIV-spectrin (orange) at the AIS of AcD neurons. Three independent cultures, AcD: n = 39 profiles from eight cells. Space between βIV-spectrin bands is ∼196 nm. Space between F-actin bands is ∼200 nm. (H) Cross-correlation curve of F-actin and βIV-spectrin at the AIS of AcD neurons. Three independent cultures, AcD: n = 39 profiles from eight cells.

AcD neurons have similar MPS organization of the AIS as in nonAcD neurons. (A) Top row: Representative confocal images of DIV14 nonAcD (left) and AcD (right) neurons stained with the AIS marker βIV-spectrin and F-actin probe Phalloidin-Atto647N. Orange arrowhead indicates the stem dendrite of the displayed AcD neuron. Yellow dashed rectangle indicates the AIS. Scale bar is 10 µm. Bottom row: Single plane 2D gSTED image of F-actin in the AIS of nonAcD and AcD neurons (corresponding zoom-ins of yellow dashed rectangle in top row). Scale bar is 1 µm. Red arrowhead indicates F-actin patch. Red dashed line indicates the profile of F-actin rings shown in B. (B) Intensity profile of periodic F-actin structures along the longitudinal axis of the AIS in nonAcD and AcD neurons; corresponding to black dashed lines in A bottom row. (C) Quantification of distance between F-actin rings at the AIS of nonAcD and AcD neurons. Mean ± SEM, three independent cultures, nonAcD: n = 45 profiles from 12 cells, AcD: n = 67 profiles from 18 cells. Grey dot indicates value of individual cell. Orange and cyan triangle indicates mean of each independent culture. Mann–Whitney test (two-sided): not significant (n.s.) P > 0.05. More detailed statistical information see Data S1. (D) Representative widefield (left) and dSTORM (right) images of DIV14 AcD neurons stained with the AIS marker βIV-spectrin and AnkG, F-actin probe Phalloidin-Alexa647+ and MAP2. Scale bar is 20 µm on widefield image and 5 µm on dSTORM image. Orange arrowhead indicates the stem dendrite. Yellow dashed square indicates the AIS. dSTORM image corresponds to the yellow dashed rectangle in widefield image. (E) Dual-color 3D dSTORM image of F-actin and βIV-spectrin at the AIS of the displayed AcD neuron. Left panel: corresponding zoom-in of the yellow dashed square on dSTORM image shown in D right panel. Yellow dashed square indicates the zoom-in region shown in right panel. Right panel: corresponding zoom-in of the yellow dashed square in left panel. Scale bar is 5 µm. Red arrowhead indicates F-actin patch. Red dashed line indicates the intensity profile of F-actin rings and βIV-spectrin shown in F. (F) Intensity profile of periodical F-actin (cyan) and βIV-spectrin (orange) structures along the longitudinal axis of the AIS in AcD neurons; corresponding to red dashed lines in E right panel. (G) Auto-correlation curve of F-actin (cyan) and βIV-spectrin (orange) at the AIS of AcD neurons. Three independent cultures, AcD: n = 39 profiles from eight cells. Space between βIV-spectrin bands is ∼196 nm. Space between F-actin bands is ∼200 nm. (H) Cross-correlation curve of F-actin and βIV-spectrin at the AIS of AcD neurons. Three independent cultures, AcD: n = 39 profiles from eight cells.

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