Fig. 7: Cntn1 is recruited to the AIS through interactions with AnkG-binding L1-family cell adhesion molecules. | Nature Communications

Fig. 7: Cntn1 is recruited to the AIS through interactions with AnkG-binding L1-family cell adhesion molecules.

From: Antibody-directed extracellular proximity biotinylation reveals that Contactin-1 regulates axo-axonic innervation of axon initial segments

Fig. 7

a Cntn1-myc (red) colocalizes with β4 spectrin (green). Cntn1 consists of 6 N-terminal Immunoglobulin (Ig)-like domains and 4 C-terminal Fibronectin type III (FNIII) domains. Scale bar, 10 µm. b Cntn1-myc with N-terminal and internal deletions of the first 4 Ig-like domains. Scale bar, 10 µm. c Cntn1-myc localization to the AIS does not depend on the last 2 Ig-like domains or any FNIII ___domain. Scale bar, 10 µm. For experiments in (b and c), neurons were transfected at DIV15 and fixed one day later at DIV16. d The percentage of neurons with Cntn1-myc (full) or Cntn1-myc truncation variants (variants 1-10) at the AIS. N = 2, 4, 4, 4, 6, 5, 6, 5, 3, and 3 FOV for constructs 1–10, respectively; individual data points and mean are shown. Data are from two independent experiments. e Hippocampal neurons transduced with AAV to express Cas9 and control, Nfasc, NrCAM, or Nfasc+NrCAM gRNAs at DIV0 (these AAVs also express smFP-HA). At DIV12 neurons were transduced with Cntn1-myc AAV, and fixed at DIV21. Neurons were labeled using antibodies against HA as a transduction marker (blue), β4 spectrin (green), and Cntn1-myc (red). Scale bar, 10 µm. f Quantification of the percentage of transduced neurons with AIS Cntn1-myc. N = 3 independent experiments, 5-13 FOV per experiment. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Error bars, ±SEM. The total number of neurons analyzed is also indicated. g Immunostaining of cultured hippocampal neurons using antibodies against Tenascin R (Tnr; red), β4 spectrin (green), and β3 Tubulin (blue). N = 3 independent experiments. Scale bar, 25 µm. h Cultured hippocampal neurons transduced at DIV0 with AAV to express Cas9 and control, Nfasc, or Cntn1 gRNAs. Neurons were labeled at DIV21 using antibodies against Tnr (red), β4 spectrin (green), and Nfasc (blue). Scale bar, 10 µm. i Quantification of the percentage of transduced neurons with AIS Tnr. N = 3 independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Error bars, ±SEM. The total number of neurons analyzed is also indicated.

Back to article page