Abstract 2205 Antibody-directed proximity biotinylation reveals the axon initial segment proteome

The axon initial segment (AIS) is an essential neuronal compartment regulating both neuronal excitability and neuronal polarity. However, the molecular mechanisms regulating these functions remain incompletely understood. For example, few AIS cell surface proteins have been identified. To overcome these challenges, we used antibody-directed proximity biotinylation and mass spectrometry to define the AIS proteome – with special emphasis on AIS cell surface proteins. To determine the distributions of the identified proteins, we used CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We identified Contactin-1 (Cntn1) and SCRIB as previously undescribed AIS cell surface and scaffolding proteins, respectively. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM, 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. The scaffolding protein SCRIB is recruited to the AIS by directly binding to the master organizer of the AIS, AnkyrinG. Our studies demonstrate the power of antibody directed proximity biotinylation, mass-spectrometry, and CRISPR-mediated genome editing to elucidate subcellular proteomes. Supported by grants from the NIH and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.