Fig 2: NF single guide RNA (sgRNA) successfully depletes NF enrichment in the AIS. (A,B) Representative images of DIV14 sgRNA control (A) or NF sgRNA (B) neurons stained for mCherry, AnkG, and NF using an antibody targeting an extracellular domain of NF. Red arrows indicate the AIS of transfected neurons. Scale bar: 10 µm. (C) Ratio of mean fluorescence intensity of NF at the AIS using an externally targeting antibody in sgRNA control or NF sgRNA expressing neurons, normalized to untransfected neurons in the same image (n = 15 for both conditions; ***p < 0.001, Student’s t-test). (D,E) Representative images of DIV14 sgRNA control (D) or NF sgRNA (E) neurons stained for mCherry, AnkG, and NF using an antibody targeting an intracellular domain of NF. Red arrows indicate the AIS of transfected neurons. Scale bar: 10 µm. (F) Ratio of mean fluorescence intensity for external NF at the AIS using an internally targeting antibody in sgRNA control or NF sgRNA expressing neurons, normalized to untransfected neurons in the same image (n = 15 for both conditions; ***p < 0.001, Student’s t-test). Error bars indicate mean ± SEM.

Fig 3: Nav1.6 overexpression rescues AnkG enrichment, but not translocation. (A,D) Representative images for NF sgRNA + Nav1.6-GFP co-transfected neurons stained for mCherry, GFP, and AnkG (A) or panNav (D). Red arrows indicate the AIS of transfected neurons. Scale bars: 10 µm. (B,E) Ratio of mean fluorescence intensity for AnkG (B) and Nav (E) at the AIS in NF sgRNA (originally from Figure 3) or NF sgRNA + Nav1.6-GFP expressing neurons normalized to untransfected neurons in the same image (B: NF sgRNA n = 63; NF sgRNA + Nav1.6-GFP n = 18; ***p < 0.001, Student’s t-test. E: NF sgRNA n = 61; NF sgRNA + Nav1.6-GFP n = 28; **p = 0.0099, Student’s t-test). (C,F) Distance from the soma to start of AIS for AnkG (C) and Nav (F) in NF sgRNA (data originally from Figure 4) or NF sgRNA + Nav1.6-GFP neurons (C: NF sgRNA n = 44; NF sgRNA + Nav1.6-GFP n = 44; asterisks indicate statistical significance from sgRNA Ctl.; NF sgRNA *p = 0.012, NF sgRNA + Nav1.6-GFP **p = 0.0073, Kruskal-Wallis ANOVA followed by Dunn’s post-test with Bonferroni correction. F: NF sgRNA n = 43; NF sgRNA + Nav1.6-GFP n = 26). sgRNA Ctl. is displayed as a dashed line for comparison. Error bars indicate mean ± SEM. (G) To-scale distribution of AnkG (black) and Nav (red) in NF sgRNA (left) and NF sgRNA + Nav1.6-GFP (right) neurons. Color saturation of bars are indicative of average labeling intensity of the population. Dashed black line indicates start of AnkG enrichment in sgRNA control (data originally from Figure 4). Left error bar indicates SEM for distance from the soma, right error bar indicates SEM for length; data taken from (B,C) and (E,F).

Fig 4: Pk2 binds to AnkG, leading to MT bundling.(A) Schematic of the AnkG270, the AnkG190 isoform domains of AnkG, and the short AnkG-MBD. N, N terminus; C, C terminus; MBD, membrane-binding domain; SBD, spectrin-binding domain. (B and C) coIP of AnkG with Flag-Pk2. Both AnkG isoforms interact with Pk2 but not with immunoglobulin G (IgG)–R serum. (D) coIP of the short AnkG-MBD with Flag-Pk2. (E) Schematic of the full-length Pk2 protein with the different domains and the corresponding amino acid numbers and the GST constructs used. (F) GST pulldown experiments showing that Pk2 interacts with AnkG via the LIM and the C-terminal domains, the latter including the C2 region. (G) Pk2 coimmunoprecipitates with AnkG in vivo but not with IgG control serum (Ig). Asterisks indicate the two bands immunoprecipitated. (H and I) COS7 cells transfected with Flag-Pk2 (H) or AnkG480-GFP (I). (J to J?) Flag-Pk2 (magenta) and AnkG480-GFP (green) promote the bundling of MTs, strongly immunoreactive to tyrosinated tubulin [cyan in (J?)]. Note the relocalization of Pk2 and AnkG to the MT bundles. (K to L?) MT bundling is lost upon coexpression of Flag-Pk2 with AnkG480NN, an AnkG480 construct that can no longer bind to MTs. Scale bars, 10 µm. Statistics, Table 1.

Fig 5: Pk2 and AnkG colocalize during and after AIS formation.(A to C) Illustration of Pk2 (magenta) and AnkG (green) labeling at DIV4 (A), DIV7 (B), and DIV10 (C), respectively. Dotted circles outline the cell body, and brackets indicate the AIS. (D to F) Linescans of normalized fluorescence intensity at the AIS of Pk2 and AnkG at DIV4 (D), DIV7 (E), and DIV10 (F). The two proteins colocalize at the AIS at all analyzed stages. Values are normalized to the maximum intensity of each marker. Scale bars, 10 µm. Statistics, Table 1.