Fig 2: Gain of an extended conformation of gAnkG and ß4-spectrin recruitment to the AIS during neuron development. (A) The 4 DIV and 7 DIV hippocampal neurons were stained for AnkG, ß4-spectrin, or MAP2. Black arrowheads point to the AIS (Scale bar, 20 µm.) (B) Average intensities of AnkG or ß4-spectrin at the AIS are plotted for day 4 neurons (red line) and aligned with 7 DIV neurons (black line). The length of AIS is quantified at 3, 7, and 21 DIV. Mean ± SEM, ****P < 0.0001, 1-way ANOVA followed by Dunnett's multiple comparisons test. n = 10 from 3 independent experiments. (C) PLA of 4 or 7 DIV neurons using antibodies against N- and C-terminal of AnkG. Representative images of AnkG and PLA at the AIS are shown (Scale bar, 20 µm.) (D) PLA signal at the AIS is quantified. Mean ± SEM, ***P = 0.0001, t test. n = 10 from 3 independent experiments. (E) Schematic showing a proposed conformation change of gAnkG and the recruitment of ß4-spectrin in proto-AIS and mature AIS.

Fig 3: Proximity ligation assay of N- and C-ends of gAnkG reveals an extended conformation at the AIS that is lost in mutant gAnkG. (A, Left) Recognition sites of antibody against the N-terminal (AG-N) or C-terminal (AG-C) of gAnkG used in PLA. (A, Right) Immunostaining of the AIS by AG-N and AG-C in 7 DIV hippocampal neurons (Scale bar, 10 µm.) (B) PLA in 7 DIV hippocampal neurons. Bright filled, AnkG and PLA images are displayed from top to bottom. The soma and AIS region of the neurons is labeled with white dash lines. The PLA signal of AIS is enlarged (Scale bar, 10 µm.) (C) PLA of 7 DIV AGE22–23fl/fl neurons cotransfected with plasmids encoding Cre-BFP and either 190 kDa-AnkG-GFP (Left) or gAnkG-GFP (Right). Representative images of GFP and PLA signal of transfected neurons are shown. PLA signals in dendrites and proximal axon are enlarged (Scale bar, 10 µm.) (D) PLA signals at the AIS and dendrite are quantified. Mean ± SEM, ****P = 0.0001, t test, n = 10 from 3 independent experiments; ns, not significantly different. (E) PLA of 7 DIV cultured hippocampal AGE22-23fl/fl neurons cotransfected with Cre-BFP and wild type or indicated gAnkG-GFP mutants. Representative images of GFP and PLA signal of transfected neuron AISs are shown (Scale bar, 10 µm.) (F) Schematic showing a potential conformation change in gAnkG mutations. PLA signals at the AIS are quantified. Mean ± SEM, ****P = 0.0001, 1-way ANOVA followed by Dunnett’s multiple comparisons test, n = 10 from 3 independent experiments.

Fig 4: Suppression of AnkG in uninjured hypoglossal motor neurons Schematic diagram for AAV injection to tongue.Hypoglossal motor neurons labeled by mCherry (red) in injection side of AAVs. The decrease of AnkG (green) immunoreactivity is observed after the infection of AAV-AnkG shRNA but not AAV-scramble shRNA. Dashed lines show hypoglossal nucleus (XII). cc, central canal. Closed arrowheads indicate the AIS, while open arrowheads indicate the region where the AIS is disrupted.CFP-labeled mitochondria in uninjured hypoglossal nerve of Thy1-Mito mouse after the infection of AAV-AnkG shRNA to tongue. Dashed lines show axon.Graph shows the number of CFP-labeled mitochondria in uninjured hypoglossal (XII) axon of Thy1-Mito mouse before and after AAV infection. Data are shown as the mean ± s.e.m. (no significant difference (P = 0.519), determined by two-tailed Student's t-test, n = 3 mice). Data information: Scale bars = 100 µm (top), 20 µm (middle), and 5 µm (bottom) in (B) and 5 µm in (C). Source data are available online for this figure.

Fig 5: Human mutations of gAnkG repress ß4-spectrin recruitment to the AIS. (A) The 3 DIV hippocampal neurons cultured from AGE22-23fl/fl mice were transfected with plasmids encoding Cre-BFP and either wild-type gAnkG-GFP or gAnkG-GFP bearing human mutations T1861M, K2864N, and P2490L. Transfected neurons were fixed and stained with antibody against AnkG (AG), ß4-spectrin (ß4), 186 kDa neurofascin (Nf), or VSVGs. Confocal fluorescent images of transfected neurons are displayed for GFP signal of gAnkG and indicated antibody staining. Blue asterisks indicate the cell body of transfected neurons and black arrowheads point to the AIS from the same neuron (Scale bar, 20 µm.) (B) Average intensity of indicated antibody staining at the AIS is plotted for neurons transfected with wild-type gAnkG (black) or gAnkG with individual human mutation (red for T1861M, blue for P2490L, and green for K2864N). n = 10 and results were repeated in 3 independent neuronal cultures.