Double periodicity of the AnkyrinG-associated complex in the axon initial segment

Abstract

The axon initial segment (AIS), situated within the first 20 to 60 µm of the axon, is essential for action potential generation and maintenance of axonal identity [M. N. Rasband, Nat. Rev. Neurosci. 11 , 552–562 (2010)]. Its structure relies on the beta (β)-IV-spectrin/AnkyrinG (AnkG) scaffold arranged periodically underneath the plasma membrane, harboring diverse membrane proteins [K. Xu, G. Zhong, X. Zhuang, Science 339 , 452–456 (2013); K. L. Hedstrom, Y. Ogawa, M. N. Rasband, J. Cell Biol. 183 , 635–640 (2008)]. 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 labeling efficiency. Here, using expansion microscopy and cryoelectron 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 are situated within each period and with their C termini separated by ~80 nm. By contrast, the AnkG-associated cell-adhesion protein neurofascin-186 appears in clusters of varying sizes that are consistent with the periodic organization of AnkG pairs, yet suggesting a more complex molecular arrangement between the two molecules. Altogether, our approach provides insights into AIS molecular organization and protein stoichiometry.