Ultrastructural anatomy of nodes of Ranvier in the peripheral nervous system as revealed by STED microscopy

Significance In vertebrates, the action potential travels along myelin-coated electrically isolated axons and is regenerated at the nodes of Ranvier, which lack myelination and are characterized by a tight interaction between the axon and glial cells. Specific sets of proteins are enriched in each region of the nodes. Thanks to its subdiffraction resolution, stimulated emission depletion (STED) microscopy here uncovers the organization of 12 of these proteins at the nanoscale. The superresolved imaging reveals an extremely fine interplay and alignment of the axonal and glial cytoskeleton, with a defined ∼190-nm periodicity. Furthermore, the results point to the importance of the lateral organization of proteins at nodal gaps, an aspect that is yet unexplored. We used stimulated emission depletion (STED) superresolution microscopy to analyze the nanoscale organization of 12 glial and axonal proteins at the nodes of Ranvier of teased sciatic nerve fibers. Cytoskeletal proteins of the axon (betaIV spectrin, ankyrin G) exhibit a high degree of one-dimensional longitudinal order at nodal gaps. In contrast, axonal and glial nodal adhesion molecules [neurofascin-186, neuron glial-related cell adhesion molecule (NrCAM)] can arrange in a more complex, 2D hexagonal-like lattice but still feature a ∼190-nm periodicity. Such a lattice-like organization is also found for glial actin. Sodium and potassium channels exhibit a one-dimensional periodicity, with the Nav channels appearing to have a lower degree of organization. At paranodes, both axonal proteins (betaII spectrin, Caspr) and glial proteins (neurofascin-155, ankyrin B) form periodic quasi–one-dimensional arrangements, with a high degree of interdependence between the position of the axonal and the glial proteins. The results indicate the presence of mechanisms that finely align the cytoskeleton of the axon with the one of the Schwann cells, both at paranodal junctions (with myelin loops) and at nodal gaps (with microvilli). Taken together, our observations reveal the importance of the lateral organization of proteins at the nodes of Ranvier and pave the way for deeper investigations of the molecular ultrastructural mechanisms involved in action potential propagation, the formation of the nodes, axon–glia interactions, and demyelination diseases.