Nanoscale Architecture of the Axon Initial Segment Reveals an Organized and Robust Scaffold

116

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...

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confidence: 99%

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

D’Este

Kamin

Balzarotti

et al. 2016

116

“…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).…”

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confidence: 99%

“…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).…”

mentioning

confidence: 99%

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

Han

Zhou

Xia

et al. 2017

124

158

Actin, spectrin, and associated molecules form a membraneassociated periodic skeleton (MPS) in neurons. In the MPS, short actin filaments, capped by actin-capping proteins, form ring-like structures that wrap around the circumference of neurites, and these rings are periodically spaced along the neurite by spectrin tetramers, forming a quasi-1D lattice structure. This 1D MPS structure was initially observed in axons and exists extensively in axons, spanning nearly the entire axonal shaft of mature neurons. Such 1D MPS was also observed in dendrites, but the extent to which it exists and how it develops in dendrites remain unclear. It is also unclear whether other structural forms of the membrane skeleton are present in neurons. Here, we investigated the spatial organizations of spectrin, actin, and adducin, an actin-capping protein, in the dendrites and soma of cultured hippocampal neurons at different developmental stages, and compared results with those obtained in axons, using superresolution imaging. We observed that the 1D MPS exists in a substantial fraction of dendritic regions in relatively mature neurons, but this structure develops slower and forms with a lower propensity in dendrites than in axons. In addition, we observed that spectrin, actin, and adducin also form a 2D polygonal lattice structure, resembling the expanded erythrocyte membrane skeleton structure, in the somatodendritic compartment. This 2D lattice structure also develops substantially more slowly in the soma and dendrites than the development of the 1D MPS in axons. These results suggest membrane skeleton structures are differentially regulated across different subcompartments of neurons.actin | spectrin | adducin | super-resolution imaging | STORM I t was recently discovered that actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) structure in neurons (1). As revealed by superresolution stochastic optical reconstruction microscopy (STORM) (1), this structure contains molecules homologous to those present in the erythrocyte membrane skeleton, including spectrin (2-4), actin, and actincapping proteins such as adducin (5, 6), but adopts a structural organization that is distinct from the polygonal lattice structure of the erythrocyte membrane skeleton (7,8). 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, 9-14), 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 neuro...

“…During neuronal development, MPS originates from the axonal region proximal to the soma and propagates to distal axonal terminals (16). At a relatively late stage during development, specific isoforms of ankyrin and spectrin molecules, ankyrin-G and βIV spectrin, are recruited to the axon initial segment (AIS) (17,18), and these molecules are also assembled into the MPS structure, adopting a similar periodic distribution (16,19). As in the AIS, this periodic structure is also present in the nodes of Ranvier (20).…”

mentioning

confidence: 99%

Prevalent presence of periodic actin–spectrin-based membrane skeleton in a broad range of neuronal cell types and animal species

Zhou

et al. 2016

150

110

Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal-and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens.ctin is critically involved in the regulation of neuronal polarization, differentiation, and growth of neuronal processes, cargo trafficking, and plasticity of synapses (1-3). Spectrin is an actin-binding protein that is important for the development and stabilization of axons and maintenance of neuronal polarization (4-6). In Caenorhabditis elegans, spectrin is important for the stability and integrity of axons under mechanical stress (4, 6) and for mechanosensation (6), and spectrin depletion results in axon breakage during animal locomotion (4). In Drosophila, spectrin has been shown to be involved in axonal path finding (7) and stabilization of presynaptic terminals (8). In mice, spectrin null mutations are embryonically lethal, and neurons with spectrin knockdown display defects in axonal initial segment assembly (5, 9, 10).Actin and spectrin form a 2D polygonal lattice structure underneath the membrane of erythrocytes (11). Recently, a novel form of actin-spectrin-based submembrane skeleton structure was discovered in neuronal axons (12) using superresolution STORM imaging (13,14). This membrane-associated periodic skeleton (MPS) has been observed in both fixed and live cultured neurons (12, 15, 16) and in brain tissue sections (12). In this structure, short actin filaments are organized into repetitive, ring-like structures that wrap around the circumference o...