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.

Keywords: STED nanoscopy; axon–glia interaction; cytoskeleton; nodes of Ranvier; sciatic nerve.

Fig. 1.

Organization of proteins at nodal gaps of sciatic nerve fibers. Representative STED images (Left) are shown for each protein. The yellow dashed box on the STED image indicates the region in which the 2D AC (Middle) was performed. The y axis of the AC is aligned with the axon. AC intensities are depicted using a red hot look-up table. The vertical line profile AC (Right) is the plot line profile of intensities along a vertical 1-pixel wide line passing through the center of the AC and hence highlights the presence of longitudinal patterns. The gray bars are centered at ±0.2 µm y lag, where the peaks of periodicity are expected. (A) Representative image of axonal betaIV spectrin along with AC and line profile of the AC reveals a predominantly longitudinal periodicity. The dashed white lines indicate the outline of the axon, which has the same orientation in all other panels/proteins. (B) Axonal ankyrin G (monoclonal antibody) shows a predominantly longitudinal periodicity. Comparable results were obtained with both anti-ankyrin G antibodies used in this study. (C) Axonal voltage-gated sodium channels (Pan-Na_v) exhibit longitudinal periodic AC peaks at ∼190 nm (Left) or multiple, hexagonal-like axes of periodicity (Right). More examples of Na_v arrangements at the nodes are provided in Fig. S4A. (D) Subunit 7.2 of axonal voltage-gated potassium channel (K_v7.2 or KCNQ2, N terminus antibody) forms a highly periodic one-dimensional lattice. The two images represent opposing sides of the same node. More examples of K_v7.2 arrangements at the nodes are provided in Fig. S4B. Comparable results were obtained with both anti- K_v7.2 antibodies used in this study. (E) Axonal neurofascin has a ∼190-nm periodic organization but lacks a sharp longitudinal pattern. Neurofascin was detected using a pan-neurofascin antibody; therefore, paranodes are also stained. The red dashed line indicates the position of the node (“N”). (F) Axo-glial NrCAM shows either a mainly longitudinal ∼190 nm periodicity (Left) or a hexagonal-like pattern (Right), although with slightly longer spacing. (G) Glial actin exhibits a hexagonal-like periodic organization. The intensities along the dashed line on the right AC are represented in the vertical line profile AC (gray line). Note, even if actin is present both on the axon and in the microvilli, the signal is mainly produced by glial actin. The red dashed line indicates the position of the node (“N”). (H) Interpeak spacing of line profiles measured from the raw STED images. The box plot includes the 25th, 50th, and 75th percentile, whereas the whiskers indicate the SD. The red dots represent the average values. The periodicity of all proteins is within the same range of 180–200 nm. All image data were smoothed with a 1-pixel low pass Gaussian filter and represents the overlay of several optical sections. (All scale bars, 1 µm.)

Fig. S1.

Confocal images of the proteins analyzed in this study show uniform antibody labeling throughout the samples. (A) Schematic visualization of the structure of a node of Ranvier. The proteins analyzed in this study are listed in the region in which they are present and color-coded according to their function (black, cytoskeletal proteins; blue, channels; red, adhesion molecules). (B) Representative confocal images of the analyzed proteins. Red arrowheads point at nodal gaps. (Scale bars, 5 µm.)

Fig. S2.

Simulations of defined patterns and sensitivity of AC in the identification of periodic structures. (A) Background-free simulated images of a random pattern (27 molecules per square micrometer), of a 2D hexagonal lattice, and of a one-dimensional longitudinal lattice with 190-nm periodicity (simulated image, Left). Corresponding ACs (AutoCorr, Middle) and profile of intensities along a 1-pixel vertical line passing through the center of the AC (vertical line profile AC, Right) are shown. The gray bars are centered at ±0.2 µm y lag, where the peaks of periodicity are expected. No periodic peaks are visible in the random pattern, whereas both the 2D and one-dimensional lattices reveal a high periodicity and peak amplitude. In the case of the 2D hexagonal lattice, the vertical line profile has a ∼330-nm periodicity (black line), however the profile along a 30° tilted line (dashed line on the AutoCorr image) generates ∼190-nm peaks (gray line). Simulated images are 1 × 2 µm, and therefore, their size is comparable to the size of nodal gaps. (Scale bars, 500 nm.) (B) Simulation of the vertical line profile of the AC for different densities of molecules for the random pattern and different lattice filling factors for the other patterns. The lattice filling factor is the probability of having a lattice position occupied by a molecule. Because the AC is symmetrical, only half of the graph is depicted. For the one-dimensional lattice, molecules have been placed along a line every ∼40 nm. Indeed, in red blood cells, subcortical actin filaments are ∼40 nm long (28). For each parameter, the average line profile AC (thicker colored lines), the variance (thinner colored lines), and the profiles of each of the 50 simulations (gray lines) are shown. The AC analysis does not show any peak regardless of the density of molecules in the case of a random pattern. On the contrary, one-dimensional and 2D lattices exhibit periodic peaks already at low filling values (0.1 and 0.2, respectively), therefore proving the high sensitivity of AC. (C) Simulation of a mixed population of molecules. The 2D hexagonal lattice (filling factor = 1) was overlapped with different densities of randomly positioned molecules. For each parameter, the average line profile AC (thicker blue lines) with respect to the 2D hexagonal lattice (green lines) and the profiles of each of the 50 simulations (gray lines) are shown. The amplitude of peaks in the AC analysis is attenuated by the simultaneous presence of randomly positioned molecules and the effect increases with their concentration. A high concentration of random molecules overlaid to a perfect lattice completely hides the lattice, making it not detectable by the AC. Therefore, the presence of peaks in the AC is a robust measurement, as it can detect the presence of periodic structures almost regardless of the density of molecules and even in the presence of molecules that are not incorporated in the lattice. Simulated images on the right are 1 × 2 µm in size.

Fig. S3.

Auto- and cross-correlation of linear structures do not exhibit periodic peaks. (A) Two-color STED images of a node and paranodes labeled with acetylated tubulin (Left) and neurofascin (Middle) and the overlay of the channels (Right). Microtubules form linear structures running along the axon. On the right, the regions of the node are indicated. (B) AC (for single-channel images) and cross-correlation (for the merged image) of the region indicated by the yellow dashed box in A (y axis along the axon). AC of microtubules does not show any periodic feature, whereas a one-dimensional order is present for neurofascin. (C) Vertical line profiles of intensities along the y axis of the panels shown in B show the absence of periodic features for microtubules, whereas neurofascin exhibits a periodic arrangement. Cross-correlation does not indicate the presence of any pattern in the relative positioning of the two proteins, therefore proving the specificity of the signal observed in the following experiments. Gray bars highlight the ±0.2 µm y lag. All image data were smoothed with a 1-pixel low-pass Gaussian filter. (All scale bars, 1 µm.)

Fig. S4.

Nanoscale organization of proteins at the nodal gap is often inhomogeneous and asymmetrical. (A) Examples of nodes stained with a pan-Na_v channel antibody. When possible, the opposite sides of the same node (side 1 and side 2) are shown. The staining often appears inhomogeneous and asymmetrical on the opposite side. (B) Examples of nodes stained with a K_v7.2 antibody directed against the N terminus. The opposite sides of the same node (side 1 and side 2) are shown. Different geometries in the organization of the nodes are visible on the opposite side of the nodes. The third node – side 1, as presented in Fig. S5A. (C) Costainings of Na_v channels (magenta) and K_v7.2 (N terminus, green) indicate that the grooves (arrows) are depleted from both proteins. (D) Costaining of Na_v channels (magenta) and neurofascin (green) shows a similar behavior of the two proteins and the lack of both species from the groove (arrow). On the right, the regions of the node are indicated. All image data were smoothed with a 1-pixel low-pass Gaussian filter and represent the overlay of several optical sections. (All scale bars, 500 nm.)

Fig. S5.

K_v7.2 channels intercalate with both ankyrin G and Na_v channels. Two-color STED images of nodes labeled for (A) K_v7.2 (N terminus, Left) and ankyrin G (monoclonal antibody, Middle) or (B) K_v7.2 (C terminus, Left) and pan-Na_v (Middle). Single-color images and overlays are shown. For both combinations of proteins, an alternating pattern is observed. (C and D) AC (for single-channel images) and cross-correlation (CC, for the merged image) of the regions indicated by the yellow dashed boxes in A and B. The y axis is aligned with the axon. A longitudinal periodicity can be observed for all AC and CC. (E and F) Vertical line profile of intensities along the panels shown in C and D, respectively. The gray bars highlight the ±0.2 µm y lag. All proteins show peaks at the same position, and cross-correlation indicates a phase shift of the proteins. The node in A is also presented in Fig. S4B (third image). All image data were smoothed with a 1-pixel low-pass Gaussian filter and represent the overlay of several optical sections. (All scale bars, 1 µm.)

Fig. S6.

Periodic organization of NrCAM and K_v1.2 along the AIS of hippocampal neurons. STED image of AIS labeled with phalloidin to stain F-actin (Left), (A) NrCAM (31 d in vitro) or (B) K_v1.2 (29 d in vitro) (Middle), and the overlay of the two channels (Right). (C and D) AC (for single channel images) and cross-correlation (for the merged image) of the regions indicated by the yellow dashed boxes in A and B (y axis along the axon) show a strong longitudinal pattern. (E and F) Vertical line profiles of intensities along the y axis of the panels shown in C and D show a weak NrCAM periodicity that is out-of-phase with actin, whereas K_v1.2 has a pronounced, in-phase organization. Gray bars highlight the ±0.2 µm y lag. All image data were smoothed with a 1-pixel low-pass Gaussian filter. (All scale bars, 1 µm.)

Fig. S7.

Correlation between actin and neurofascin at the nodes of Ranvier. (A) STED image of a node stained with phalloidin to mark F-actin (Left), neurofascin (Middle), and the overlay of the two channels (Right). On the right, the regions of the node are indicated. PN, paranode. Same node as shown in Fig. 3. (B) AC (for single-channel images) and cross-correlation (for the merged image) of the region indicated by the yellow dashed box in A do not show a predominantly longitudinal organization. (C) Vertical line profile of intensities along the panels shown in B reveals periodic peaks at ∼190 nm (gray bars centered at ±200 µm y lag) and an out-of-phase cross-correlation. All image data were smoothed with a 1-pixel low-pass Gaussian filter and represent the overlay of several optical sections. (Scale bar, 1 µm.)

Fig. 2.

Organization of proteins at paranodes of sciatic nerve fibers. Representative STED images of proteins at the paranodes (Upper). The red dashed line indicates the position of the node (“N”). The yellow dashed box on the STED image marks the region in which the AC was performed (Lower Left). The y axis of the AC is always aligned with the axon. (Lower Right) The plot line profiles of intensities along a vertical 1-pixel wide line passing through the center of the AC, highlighting the presence of longitudinal patterns. The gray bars are positioned at ±0.2 µm y lag, where the peaks of periodicity are expected. (A) Axonal betaII spectrin, (B) axonal Caspr, (C) glial neurofascin-155, and (D) glial ankyrin B (monoclonal antibody) all show a long-range ∼190-nm longitudinal periodic arrangement. The high background makes the ankyrin B pattern less pronounced but still discernable (see also Fig. 3 B, D, and F). Comparable results were obtained with both anti-ankyrin B antibodies used in this study. In C neurofascin was detected using a pan-neurofascin antibody; therefore, the node is also stained. (E) Interpeak spacing of line profiles measured on raw STED images. The box plot includes the 25th, 50th, and 75th percentile, whereas the whiskers indicate the SD. The red dots represent the average values. The periodicity of all proteins is within the same range of 180–206 nm. All image data were smoothed with a 1-pixel low-pass Gaussian filter and represent the overlay of several optical sections. (All scale bars, 1 µm.)

Fig. S8.

The spectrin scaffold is uninterrupted at the transition between node and paranodes. (A) STED image of a sciatic nerve in which both betaII spectrin (paranodes) and betaIV spectrin (node) have been labeled. (B) Profile of intensities for the two channels along the dashed line (5-pixel width) in A shows the continuity of the spectrin lattice at the transition between paranodes and node. Image data were smoothed with a 1-pixel low-pass Gaussian filter and represent the overlay of several optical sections. (Scale bar, 1 µm.)

Fig. 3.

Correlation between the glial proteins at paranodes. Two-color STED images of a node and paranodes in which (A) actin and neurofascin or (B) ankyrin B (monoclonal antibody) and neurofascin have been labeled. Single color images and overlay are shown. In A, neurofascin labeling is incomplete and only the part close to the node was stained. The nodal gap from the same image is depicted in Fig. S7. The red dashed line indicates the position of the node (“N”). (C and D) AC (for single-channel images) and cross-correlation (CC, for the merged image) of the regions indicated by the yellow dashed boxes in A and B reveal periodic longitudinal patterns for both AC and CC. The y axis is aligned with the axon. (E and F) Vertical line profiles of intensities along the panels shown in C and D confirm the ∼200-nm periodic organization of the glial proteins (gray bars positioned at ±0.2 µm y lag) and reveal an out-of-phase (for actin and neurofascin-155) or in-phase (for ankyrin B and neurofascin-155) cross-correlation. All image data were smoothed with a 1-pixel low-pass Gaussian filter and represent the overlay of several optical sections. (All scale bars, 1 µm.)

Fig. 4.

Nanoscale alignment of the axonal and glial cytoskeleton at paranodes. Two-color STED images of paranodes labeled for (A) axonal Caspr and glial neurofascin, (B) axonal betaII spectrin and glial neurofascin, and (C) axonal betaII spectrin and glial ankyrin B (polyclonal antibody). Single-color images and overlays are shown. The red dashed line indicates the position of the node (“N”). (D–F) AC (for single-channel images) and cross-correlation (CC, for the merged image) of the regions indicated by the yellow dashed boxes in A–C. The y axis is aligned with the axon. A longitudinal periodicity can be observed for all AC and CC. (G–I) Vertical line profile of intensities along the panels shown in D, E, and F, respectively. The gray bars highlight the ±0.2 µm y lag. All proteins show peaks at the expected position, and CC indicates in-phase colocalization (G and I) or a slight phase shift (H). All image data were smoothed with a 1-pixel low-pass Gaussian filter and represent the overlay of several optical sections. (All scale bars, 1 µm.)

Fig. S9.

K_v1.2 channels do not show a periodic organization at juxtaparanodes and are complementary to neurofascin. STED image of a nerve stained for K_v1.2 (Left), neurofascin (Middle), and the overlay of the two channels (Right). On the right, the regions of the node are indicated. JuxtaPN, juxtaparanodes; N, node; PN, paranode. (Bottom) Close-up of the region indicated by the yellow dashed box reveals almost no overlay of the two proteins. All image data were smoothed with a 1-pixel low-pass Gaussian filter and represent the overlay of several optical sections. (Scale bar, 1 µm.)

Fig. 5.

Proposed model for the molecular organization of nodes of Ranvier in the PNS. The molecular organization of each different compartment (node, paranode, and juxtaparanode) is displayed to highlight both the vertical (Upper, side view) and the lateral (Lower, top view) positioning of the glial and axonal cytoskeleton. The upper part of the top view is an overlay of the axonal and glial proteins and highlights the influence of the glia on the organization of the nodal gaps. The lower part shows the organization of the axonal components only, without the influence of the glia. Actin and spectrin (and ankyrin G at the nodal gap) constitute a continuous ∼190-nm periodic axonal subcortical scaffold onto which the other components (channels and adhesion molecules) assemble at discrete positions possibly because of the interplay with the glia. At nodes, these components can feature a 2D hexagonal-like periodic organization, also characteristic of proteins present in the microvilli (top view, Upper), or a one-dimensional longitudinal periodicity (top view, Lower). Positioning of nodal K_v7.2 follows almost exclusively a one-dimensional order and does not seem to be influenced by the microvilli. At paranodes, glial and axonal proteins show a highly regular one-dimensional periodic organization that probably reflects the position of myelin loops. The glial and axonal cytoskeleton are aligned and the actin/spectrin scaffold might be densely occupied. It appears that actin bundles are positioned at the side of myelin loops as they alternate neurofascin bands. At juxtaparanodes, actin and spectrin are periodically arranged. However, K_v1.2 channels do not exhibit a specific pattern but are complementary to neurofascin. AnkB, ankyrin B; AnkG, ankyrin G; βIIspec, betaII spectrin; βIVspec, betaIV spectrin; K_v1.2, K_v1.2 channels; K_v7.2, K_v7.2 (KCNQ2); Na_v, Na_v channels; NF155, neurofascin-155; NF186, neurofascin-186.