Nanoscale organization of betaII-spectrin within segments of the membrane-associated periodic skeleton in mouse sciatic nerve axons

Abstract The actin/spectrin membrane-associated periodic skeleton (MPS) is a ubiquitous cytoskeletal structure essential for axonal integrity and function. Primarily studied in cultured neurons, the MPS has been extensively modeled as actin rings spaced by spectrin tetramers, the latter assumed to be regularly and densely distributed across the axonal perimeter. However, its nanoscale organization within native tissue environments remains poorly understood. In this study, we investigated the three-dimensional organization of βII-spectrin in the mouse sciatic nerve using 3D-dSTORM and STED super-resolution microscopy on thin transversal cryosections. By implementing a custom quantitative analysis pipeline, we resolved the sub-diffraction architecture of the MPS across myelinated axons of diverse diameters. We confirm that βII-spectrin is localized to the inner face of the axonal plasma membrane and maintains a longitudinal periodicity of approximately 170 nm, consistent with previous observations. Crucially, 3D-dSTORM revealed that βII-spectrin along the axonal perimeter is organized in discrete nanoscale clusters with a median effective radius of 25 nm, compatible with the size of an individual spectrin tetramer visualized by indirect immunolabeling. The number of these clusters scales linearly with the axonal perimeter, maintaining a constant membrane occupancy of ∼20% across varying axon diameters. Moreover, these clusters exhibit a non-random spatial distribution with a characteristic center-to-center nearest-neighbor distance of ∼200 nm. These findings challenge simplified models of the MPS based on cultured systems and demonstrate that the MPS in peripheral nerves is composed of discrete structural units. This modular, dispersed organization may provide the structural flexibility required to withstand the mechanical demands of the peripheral nervous system while maintaining a stable periodic scaffold.