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

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 use 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 use CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We identify Contactin-1 (Cntn1) as an AIS cell surface protein. 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 regulates AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

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

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.

Saltatory Conduction: Jumping to New Conclusions.

Rapid and efficient saltatory action potential conduction depends on the myelin sheath and clustered Na+ channels at nodes of Ranvier. A new study convincingly shows that the periaxonal space is a necessary conductive component to accurately model myelinated axon physiology and saltatory conduction.

Mapping axon initial segment structure and function by multiplexed proximity biotinylation.

Axon initial segments (AISs) generate action potentials and regulate the polarized distribution of proteins, lipids, and organelles in neurons. While the mechanisms of AIS Na+ and K+ channel clustering are understood, the molecular mechanisms that stabilize the AIS and control neuronal polarity remain obscure. Here, we use proximity biotinylation and mass spectrometry to identify the AIS proteome. We target the biotin-ligase BirA* to the AIS by generating fusion proteins of BirA* with NF186, Ndel1, and Trim46; these chimeras map the molecular organization of AIS intracellular membrane, cytosolic, and microtubule compartments. Our experiments reveal a diverse set of biotinylated proteins not previously reported at the AIS. We show many are located at the AIS, interact with known AIS proteins, and their loss disrupts AIS structure and function. Our results provide conceptual insights and a resource for AIS molecular organization, the mechanisms of AIS stability, and polarized trafficking in neurons.