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

Abstract 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. Competing Interest Statement The authors have declared no competing interest. Data Availability The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.