Abstract
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.
Highlights
Axon initial segments (AISs) generate action potentials and regulate the polarized distribution of proteins, lipids, and organelles in neurons
Since NF186ΔFIGQY-BirA* does not localize to the AIS, biotinylated proteins can be compared to those identified using NF186-BirA*; proteins that are more abundant in the NF186-BirA* purification are candidate AIS proteins (Fig. 1a)
We found AnkB (Ank2) and β2 spectrin (Sptbn1) were enriched in the NF186-BirA* samples (Fig. 1c, red), since NF186 is found at low densities in distal axons where it interacts with a cytoskeletal complex consisting of AnkB and β2 spectrin[26,27], and β2 spectrin was reported at the AIS28
Summary
Axon initial segments (AISs) generate action potentials and regulate the polarized distribution of proteins, lipids, and organelles in neurons. Axon initial segments (AISs) are located at the interface between somatodendritic and axonal domains of neurons They have two main functions: to cluster and maintain ion channels in high densities for efficient action potential initiation[1], and to control neuronal polarity by regulating the differential distribution and trafficking of proteins, vesicles, organelles, and even lipids between axonal and somatodendritic compartments[2,3,4]. These properties depend on the cytoskeletal and scaffolding proteins AnkyrinG (AnkG) and β4 spectrin. We propose this spatially segregated AIS proteome will be a valuable resource for further study of AIS components, and that this proteome will help overcome the current bottleneck to understanding AIS structure and function
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