Double Periodicity of the AnkyrinG-Associated Complex in the Axon Initial Segment

The axon initial segment (AIS), situated within the first 20-60 µm of the axon, is essential for action potential generation and maintenance of axonal identity. Its structure relies on the beta (β)-IV-spectrin/AnkyrinG (AnkG) scaffold arranged periodically underneath the plasma membrane, harbouring diverse membrane proteins. Although a ∼190-nm cytoskeletal periodic organization is well established, the precise stoichiometry and spatial arrangement of AIS proteins within the ∼190-nm spatial period remain rudimentary, mostly for lack of sufficient spatial resolution and labelling efficiency. Here, using expansion microscopy and cryo-electron tomography, which overcome these technical limitations, we present data on the organization of the AnkG-associated complex within the ∼190-nm spatial period. We demonstrate that exactly two AnkG molecules with their C-termini separated by ∼80 nm are situated within each period. By contrast, the AnkG-associated cell-adhesion protein neurofascin-186 appears in clusters of varying sizes that are consistent with the periodic organisation of AnkG pairs, yet suggest a more complex molecular arrangement between the two molecules. Altogether, our novel approach provides new insights into AIS molecular organisation and protein stoichiometry.