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

Abstract

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 Na v 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.nodes of Ranvier | STED nanoscopy | cytoskeleton | axon-glia interaction | sciatic nerve I n recent years, knowledge of the ultrastructural organization of the axon initial segment (AIS) has been greatly extended by using optical nanoscopy to visualize its molecular composition. Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) microscopy were indeed crucial for the identification of a ∼190-nm periodic organization of the key components of the AIS. In particular, a periodic spatial arrangement was discovered for cytoskeletal proteins (actin, ankyrin G, betaIV spectrin), adhesion molecules (neurofascin), and channels (voltage-gated sodium Na v channels) (1-4). In the distal part of the axon, this periodicity has been found for actin, adducin, ankyrin B, and betaII spectrin in virtually every neuron type from the central and peripheral nervous systems (CNS and PNS) (1, 2, 5, 6). Although myelination of axons does not alter the patterning of the subcortical cytoskeleton (5, 6), it changes its molecular composition, promoting the clustering of specific markers at the nodes of Ranvier, where the action potential is regenerated (reviewed in refs. 7, 8). At nodes of Ranvier, the myelin sheath formed by oligodendrocytes (in the CNS) or Schwann cells (in the PNS) is interrupted. Still, the nodal gap is surrounded by perinodal astrocytes and microvilli stemming from the Schwann cells in the CNS and PNS, respectively (9). Interestingly, the AIS an...

Structural organization of the actin-spectrin–based membrane skeleton in dendrites and soma of neurons

“…It is worth noting that some ion channels and adhesion molecules at the nodes of Ranvier have recently been shown to adopt 2D periodic distributions (14); however, these distributions do not seem to arise from an underlying 2D periodic membrane skeleton but, rather, appear to be imposed by the hexagonal organization of the microvilli of glial cells, as the axonal membrane skeleton at the nodes of Ranvier still adopts a 1D periodic structure (14).…”

“…The MPS also plays a role in shaping the axon morphology, and adducin depletion causes axon enlargement (20). Notably, the MPS is able to organize other associated molecules, such as ankyrin, ion channels, and adhesion molecules, into a periodic distribution along axons (1,(9)(10)(11)14), potentially affect a variety of signaling pathways in axons (1). The MPS can also act as a diffusion barrier and contribute to filtering membrane proteins at the axon initial segment (21).…”

“…In this structure, short actin filaments capped by adducin are organized into repetitive, ring-like structures that wrap around the circumference of the axon underneath the axonal membrane, and adjacent actin rings are connected by spectrin tetramers, forming a long-range quasi-1D periodic structure with a periodicity of ∼190 nm (1). This periodic structure is formed extensively throughout the axonal shaft, including the axon initial segment and the unmyelinated distal axons, as well as the nodes of Ranvier and internodal segments in myelinated axons (1,(9)(10)(11)(12)(13)(14), but appears to be perturbed at synaptic sites (13,15,16). It is a highly prevalent structure observed in many different types of neurons, including both excitatory and inhibitory neurons, as well as neurons in both central and peripheral nervous systems (12,13), and is evolutionarily conserved across diverse animal species, ranging from Caenorhabditis elegans and Drosophila to rodents and humans (13).…”

Wrapped to Adapt: Experience-Dependent Myelination

“…High-throughput electron microscopy techniques have revolutionized our understanding of cortical myelination patterns and promise to offer unprecedented examination of myelination patterns throughout the brain and spinal cord. Increasing access to super-resolution microscopy is beginning to improve our understanding of the molecular ultrastructure of the internode and node (D’Este et al, 2016), while new spectroscopic imaging techniques offer the potential for label-free in vivo myelin imaging (Schain et al, 2014). Techniques for selective manipulation of neural activity, from optogenetics to chemogenetics (DREADDs), represent powerful tools to investigate the role of activity-dependent myelination under physiomimetic conditions in vivo , and targeting circuit-specific manipulations utilizing modern databases of neural connectivity will enable far greater precision in our evaluation of activity-dependent myelination and its potential impact on behavior.…”

The Spectrin-Actin-Based Periodic Cytoskeleton as a Conserved Nanoscale Scaffold and Ruler of the Neural Stem Cell Lineage

“…Although initially noted in neuronal axons as adducin-capped actin rings connected by spectrin tetramers to form a periodic, one-dimensional (1D) lattice of well-defined, ~180- to 190-nm periodicity (Xu et al, 2013), related periodic or quasi-periodic cytoskeletal structures have also been observed in dendrites (D’Este et al, 2015; Han et al, 2017) and certain glial cell types (D’Este et al, 2016, 2017; He et al, 2016). Such periodic nanostructures are markedly different from the traditional view of the actin-based cytoskeleton in common mammalian cell types (e.g., dense filament networks and bundles in fibroblasts and epithelial cells) (Chhabra and Higgs, 2007; Pollard and Cooper, 2009; Xu et al, 2012) as well as the spectrin-actin-based cytoskeleton in erythrocytes (2D triangular lattices of short actin filaments connected by spectrin tetramers) (Baines, 2010; Bennett and Baines, 2001; Bennett and Gilligan, 1993; Fowler, 2013; Pan et al, 2018).…”