The axon initial segment (AIS), located within the first 30 μm of the axon, has two essential roles in generating action potentials and maintaining axonal identity. AIS assembly depends on a ßIV-spectrin/ankyrin G scaffold, but its macromolecular arrangement is not well understood. Here, we quantitatively determined the AIS nanoscale architecture by using stochastic optical reconstruction microscopy (STORM). First, we directly demonstrate that the 190-nm periodicity of the AIS submembrane lattice results from longitudinal, head-to-head ßIV-spectrin molecules connecting actin rings. Using multicolor 3D-STORM, we resolve the nanoscale organization of ankyrin G: its amino terminus associates with the submembrane lattice, whereas the C terminus radially extends (∼ 32 nm on average) toward the cytosol. This AIS nano-architecture is highly resistant to cytoskeletal perturbations, indicating its role in structural stabilization. Our findings provide a comprehensive view of AIS molecular architecture and will help reveal the crucial physiological functions of this compartment.

The axon initial segment (AIS) is the site of action potential generation and a locus of activity-dependent homeostatic plasticity. A multimeric complex of sodium channels, linked via a cytoskeletal scaffold of ankyrin G and beta IV spectrin to submembranous actin rings, mediates these functions. The mechanisms that specify the AIS complex to the proximal axon and underlie its plasticity remain poorly understood. Here we show phosphorylated myosin light chain (pMLC), an activator of contractile myosin II, is highly enriched in the assembling and mature AIS, where it associates with actin rings. MLC phosphorylation and myosin II contractile activity are required for AIS assembly, and they regulate the distribution of AIS components along the axon. pMLC is rapidly lost during depolarization, destabilizing actin and thereby providing a mechanism for activity-dependent structural plasticity of the AIS. Together, these results identify pMLC/myosin II activity as a common link between AIS assembly and plasticity.