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], located within the first 30 µm of the axon, has two essential roles: generating the action potential, and maintaining axonal identity. AIS assembly depends on an ankyrin G / ßIV-spectrin scaffold, but its macromolecular arrangement is unknown, precluding the mechanistic understanding of its functions. We quantitatively determined the AIS nanoscale architecture using STochastic Optical Reconstruction Microscopy [STORM]. First, we directly demonstrated the existence of an AIS 190-nm periodic submembrane lattice composed of alternated actin rings and ßIV-spectrin dimers. Next, we used antibodies against different domains of ankyrin G to map its 3D nanoscale positioning: multicolor STORM demonstrated that the two large isoforms of ankyrin G are radially oriented across the AIS, with a 30-nm radial extent. The robustness of the AIS nano-architecture features against cytoskeletal or pharmacological perturbations suggest their structural role in the overall stability of the compartment. This organized and robust nanoscale architecture likely underpins the AIS functions in physiological and pathological contexts.axon initial segment | cytoskeleton | ankyrin G | super-resolution microscopy