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

Abstract

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we used antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we used CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We found Contactin-1 (Cntn1) among the previously unknown AIS proteins we identified. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS-extracellular matrix, and is required for AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

Figure 1.. Proximity-dependent biotinylation using Nfasc antibodies.

a, Illustration of the antibody-directed proximity biotinylation strategy. Anti-Nfasc antibodies bind to Nfasc, while HRP-conjugated secondary antibodies bind to the Nfasc antibodies. Addition of biotin phenol (biotin tyramide) in an H₂O₂ containing diluent results HRP-mediated conversion of the biotin phenol to an active radical biotin phenoxyl that covalently adds the tyramide biotin to extracellular tyrosine residues. Omission of the primary anti-Nfasc serves as a control. After stringent solubilization and affinity capture by streptavidin-conjugated magnetic beads. Biotinylated proteins are then identified by mass spectrometry. b, Fluorescence imaging of DIV14 rat hippocampal neurons labeled by Nfasc-BAR or a control condition (no primary Ab). Nfasc fluorescence (green) enrichment defines the AIS. Biotinylated proteins were detected using Alexa594-conjugated streptavidin. Scale bars, 20 μm.

Figure 2.. NF186 proximity proteomes across neuronal development.

a, Volcano plots showing the log₂-fold changes of proteins versus the statistical significance −log₁₀(pvalue) identified using Nfasc-directed proximity biotinylation (N=3). p<0.05 was used as a cutoff for significance (horizontal dashed line). Some identified proteins are indicated (corresponding gene names listed), with those previously reported as AIS cell surface proteins in red, respectively. Inset images show immunofluorescence labeling of NF186 at different timepoints of hippocampal neuron development in vitro. Lower panels: magnified images show the Nfasc-labeled AIS at each time point. Scale bars, 20 μm. b, Heatmaps showing log₂-fold changes at each timepoint for all 285 proteins that satisfied two filtering criteria [(1) normalized PSMs > 10; (2) log₂FC (Nfasc/Ctrl) > 2] for at least one of five timepoints, rank-ordered by the slope of the linear regression of their log₂ fold enrichment over time. c, Expanded heat map showing gene names for the proteins (1–50 and 51–100) with the largest rate of increase in PSM count (B). Data shown are from N = 3 replicates for each timepoint (see Figure S2).

Figure 3.. Nfasc-BAR identifies known AIS membrane and membrane-associated proteins.

a, Known AIS membrane and membrane associated proteins and their log₂FC (Nfasc/Ctrl). b, Illustration of membrane topology, average PSMs (at DIV14), and the number of extracellular tyrosine residues for three different AIS and membrane proteins. Figure generated using Biorender. c, d, Scatter plot of the number of peptide spectral matches (PSMs) for each biotinylated protein identified by mass spectrometry as compared to the number of tyrosine residues present in each protein’s extracellular domain, shown at different scales. Proteins in red were previously reported at the AIS. e, Proximity plot showing biotinylated proteins (at DIV14) ordered by extracellular (EC) tyrosine/PSM ratio. The plot is an estimate of abundance and proximity to the HRP secondary antibody bound to the Nfasc primary antibody. Each protein is represented by a circle with size proportional to the number of PSMs identified for that protein. Proteins analzyed in subsequent experiments are indicated by their gene names. EC Tyrosine/PSM ratio: 0–0.5 green, 0.5–1.0 yellow, 1.0–1.5 blue, 1.5–2.0 red.

Figure 4.. Tagging of endogenous membrane proteins.

a, Proteins whose distribution was tested using endogenous protein tagging. The heatmap shows the increase in expression level is shown as a function of days in vitro (see also Figure 2c). The presence of the tagged protein in AIS, axon, and dendrite is indicated. b, Schematic of the knock-in vector for in vitro CRISPR-mediated endogenous gene tagging. DRS, donor recognition sites. c-k, Examples of smFP-V5 tagged proteins (red) enriched at the AIS (c, d, f, k), the axon (e, g), dendrites (j), or in multiple domains (e, g, h, i). AIS are labeled for β4 spectrin (green) and are indicated by an arrowhead. Scale bar, 20 μm.

Figure 5.. Cntn1 is a bona fide AIS protein.

a, Schematic of the knockout vector including 3 sgRNAs targeting the gene of interesting. The AAV generated using this vector were used for in vitro transduction of neurons. b, c, Immunostaining for Cntn1 (green), AnkG (red) and Map2 (blue) after transduction with AAV to Cas9 and control (b) or Cntn1 (c) sgRNAs. Neurons transduced with the Cntn1 sgRNAs lacked AIS Cntn1, but retained robust AnkG at the AIS. AIS are indicated by the arrowheads. Scale bar, 10 μm. d, Schematic of the Cntn1-myc overexpression vector used for in vitro and in vivo infection of neurons. e, Transduction of cultured hippocampal neurons using AAV to express Myc-tagged Cntn1. Cntn1-myc (red) is enriched at the AIS (arrowhead) where it colocalizes with β4 spectrin (green). The somatodendritic domain is identified using antibodies against Map2 (blue). Scale bar, 10 μm. f, In vivo transduction of cortical neurons using AAV to express Myc-tagged Cntn1. Cntn1-myc (red) is enriched at the AIS (arrowhead) where it colocalizes with β4 spectrin (green). Nuclei are labeled using Hoechst dye (blue). Scale bar, 10 μm. g, Schematic of the knock-in vector for in vivo CRISPR-mediated endogenous tagging of Cntn1. DRS, donor recognition sites. AAV were delivered by intracerebroventricular (ICV) injection at P0. h, In vivo transduction of cortical neurons for CRISPR-dependent genome editing to tag endogenous Cntn1 using smFP-V5 (red). The smFP-V5 tagged Cntn1 colocalizes with β4 spectrin (green) at the AIS (arrowhead). Nuclei are labeled using Hoechst dye (blue). Scale bar, 10 μm.

Figure 6.. Cntn1 is recruited to the AIS through interactions with AnkG-binding L1-family cell adhesion molecules.

a,Cntn1-myc (red) is targeted to the AIS and 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 fail to localize at the AIS. 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. d, Cultured hippocampal neurons transduced with AAV to express Cas9 and control, Nfasc, NrCAM, or Nfasc+NrCAM gRNAs. Neurons were labeled using antibodies against HA as a transduction marker (to label the spaghetti monster fluorescent protein tagged with HA, smFP-HA; blue), β4 spectrin (green), and Cntn1-myc (red). Scale bar, 10 μm. e, Quantification of the percentage of transduced neurons with AIS Cntn1-myc. 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. f, Immunostaining of cultured hippocampal neurons using antibodies against Tenascin R (Tnr; red), β4 spectrin (green), and β3 Tubulin (blue). Scale bar, 25 μm. g, Cultured hippocampal neurons transduced with AAV to express Cas9 and control, Nfasc, or Cntn1 gRNAs. Neurons were labeled using antibodies against Tnr (red), β4 spectrin (green), and Nfasc (blue). Scale bar, 10 μm. h, 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.

Figure 7.. Cntn1 is required for axo-axonic innervation of Purkinje neuron AIS.

a, Illustration of a Purkinje neuron (black) with a basket cell (red) forming the cerebellar pinceau on the AIS. b, Immunostaining of P17 cerebellar pinceau in control and Cntn1 −/− mouse brain using antibodies against Kv1.2 (red) and PSD95 (green) to label the pinceau, and Nfasc (blue) to label the Purkinje neuron AIS. Scale bar, 10 μm. c, Violin plot of the mean Kv1.2 intensity of the cerebellar pinceau in control and Cntn1 −/− mice. N=3 control and 2 Cntn1 −/− mice. The number of pinceau analyzed is indicated.

Figure 8.. Pyramidal neuron Cntn1 is important for AIS synaptic innervation by ChCs.

a, Illustration of the knockout and labeling strategy for PyN and ChCs. PyNs are electroporated at E15.5 using plasmids to express Cas9 and 3X sgRNA-smFP (HA tag) to delete expression on Cntn1. ChCs are labeled by expression of red fluorescent protein (RFP) using inducible Cre (CreER) in Nkx2.1-CreER mice at E18.5. b, Illustration of ChC (red) innervation of PyN (blue/green) AIS. c, Representative images of PyNs innervated at their AIS by ChC cartridges (red) in layer II of the Ogawa, Lim et al. somatosensory cortex from Nkx2.1-CreER;Ai9 mice co-electroporated at E15.5 with a plasmid expressing Cas9 and a plasmid expressing smFP-HA and a control sgRNA or Cntn1 sgRNA; mice were sacrificed at P17. AISs and PyNs are visualized by immunostaining for β4 spectrin (blue) and HA (green), respectively. Stars in C indicate HA+ PyNs and arrows indicate ChC innervation of PyN AISs. Arrowheads in C indicate AIS of transfected with Cntn1 sgRNA and that lack innervation by ChC cartridges. Scale bar, 10 μm. d, Quantification of the percentage of HA+ PyNs innervated by single RFP+ ChCs at P17. 12 ChCs and 15–66 HA+ PyNs per ChC from 3 animals were analyzed for each condition. Data are mean ± SEM. e, f, Representative images of HA+ PyN AISs from Nkx2.1-CreER;Ai9 mice electroporated at E15.5 with plasmids indicated in (a) and sacrificed at P17. Inhibitory synapses are visualized by immunostaining for the GABAergic postsynaptic marker gephyrin (Gphn; red; e) or the GABAergic presynaptic marker VGAT (red; g). AISs (blue) are visualized by immunostaining for AnkG in e and β4 spectrin in g. Scale bars, 2 μm. f, h, Quantification of the average number of gephyrin (f) or VGAT (h) puncta per μm of HA+ PyN AIS at P17. 23–40 AISs from 4 fields of view from 3 animals were analyzed for each condition. Data are mean ± SEM.