Spectrins and ankyrinB constitute a specialized paranodal cytoskeleton

Abstract

Paranodal junctions of myelinated nerve fibers are important for saltatory conduction and function as paracellular and membrane protein diffusion barriers flanking nodes of Ranvier. The formation of these specialized axoglial contacts depends on the presence of three cell adhesion molecules: neurofascin 155 on the glial membrane and a complex of Caspr and contactin on the axon. We isolated axonal and glial membranes highly enriched in these paranodal proteins and then used mass spectrometry to identify additional proteins associated with the paranodal axoglial junction. This strategy led to the identification of three novel components of the paranodal cytoskeleton: ankyrinB, alphaII spectrin, and betaII spectrin. Biochemical and immunohistochemical analyses revealed that these proteins associate with protein 4.1B in a macromolecular complex that is concentrated at central and peripheral paranodal junctions in the adult and during early myelination. Furthermore, we show that the paranodal localization of ankyrinB is disrupted in Caspr-null mice with aberrant paranodal junctions, demonstrating that paranodal neuron-glia interactions regulate the organization of the underlying cytoskeleton. In contrast, genetic disruption of the juxtaparanodal protein Caspr2 or the nodal cytoskeletal protein betaIV spectrin did not alter the paranodal cytoskeleton. Our results demonstrate that the paranodal junction contains specialized cytoskeletal components that may be important to stabilize axon-glia interactions and contribute to the membrane protein diffusion barrier found at paranodes.

Figure 1.

Isolation of detergent-resistant, low-density paranodal protein–lipid complexes. A, Sucrose density gradient analysis of the detergent-insoluble fraction from rat optic nerve membranes immunoblotted using pan NF (the band corresponding to NF-155 is indicated), Caspr, and contactin antibodies. The higher-molecular-weight band shown in the pan NF blot corresponds to NF-186, demonstrating the enrichment for paranodal proteins over nodal proteins. B, Silver staining analysis of sucrose density gradient fractions from detergent-resistant optic nerve membranes. The silver-stained gel shows a complex mixture of proteins, most of which float in fractions 3–5 on the gradient. For mass spectrometry, fractions 4 and 5 (asterisk) were pooled. Ten protein bands were cut and then analyzed by mass spectrometry (see brackets at the right of the gel).

Figure 2.

Cofractionation of paranodal and cytoskeletal proteins in detergent-resistant, low-density protein–lipid complexes isolated from rat optic nerves. A, Five proteins identified by mass spectrometry of proteins isolated from optic nerve lipid rafts. Percentage coverage indicates the percentage of the protein sequence covered by the identified tryptic peptides. B, Immunoblots showing that cytoskeletal proteins are detected in low-density fractions from sucrose gradient centrifugation of detergent-insoluble optic nerve membranes. Numbers on the bottom represent the fraction number collected, and fraction 14 corresponds to the pellet at the bottom of the gradient. C, Association of αII spectrin, βII spectrin, protein 4.1B, and ankyrinB in mouse brain. Solubilized mouse brain membranes were subjected to immunoprecipitation to the various cytoskeletal proteins as indicated across the top. Immunoblots were performed with antibodies to βII spectrin, protein 4.1B, and ankyrinB as indicated on the right of each panel.

Figure 3.

Cytoskeletal proteins are localized to paranodal junctions in the optic and sciatic nerves. Longitudinal sections of optic (A–E) and sciatic (F–K) nerves or cross sections of sciatic nerve (L–N) were labeled with the antibodies indicated in each panel. The merged images are shown on the right. A, Pan Nav (green) and Caspr (red). B, Pan Nav (green) and protein 4.1B

Figure 4.

Retraction of myelin membranes does not alter the paranodal junction localization of ankyrinB or βII spectrin. A, B, Sciatic nerve fibers that were treated with collagenase to cause myelin retraction. The nerve fiber is shown using differential interference contrast (DIC). Immunolabeling for βIV spectrin (red), ankyrinB (green), and MBP (blue; only in A) is shown as indicated. C, Collagenase-treated nerve fiber immunostained using antibodies against βIV spectrin (red) and βII spectrin (green). D, Paranodal ankyrinB (green) and paranodal pan NF (red) immunoreactivity is retained after myelin retraction. E, Acid stripping of nerve fibers after myelin retraction eliminates paranodal pan NF (red) staining but does not affect paranodal ankyrinB (green) immunoreactivity. Arrows indicate the edge of the retracted myelin. Arrowheads indicate paranodal junctions. Asterisks indicate nodes of Ranvier. Scale bars: A–D, 10 μm; E, 5 μm.

Figure 5.

Cytoskeletal proteins localize to paranodal junctions during paranode formation. A, Double immunostaining of rat optic nerve at P5, P11, and P13 using antibodies against pan Nav (green) and βII spectrin (red) or protein 4.1B (red). Inset shows detail of individual nodes and paranodes labeled for pan Nav and βII spectrin. B, Double immunostaining of rat sciatic nerve at P2, P5, and P13 using antibodies against βIV spectrin (green) and αII spectrin, pan Nav (green) and βII spectrin (red), or pan Nav (green) and protein 4.1B (red). C, Caspr (green) and ankyrinB (red) immunostaining of sciatic nerve paranodes during developmental myelination at P1, P2, P4, P8, and P13. Scale bars: A, B, 10 μm; C, 3 μm.

Figure 6.

AnkyrinB localization is disrupted in Caspr^−/− mice. A, Caspr (green) and ankyrinB (red) immunostaining of sciatic nerve. Aa, WT mouse sciatic nerve. Ab, Caspr^−/− mouse sciatic nerve. Ac, Caspr2^−/− mouse sciatic nerve. B, Caspr2 (green) and ankyrinB (red) immunostaining of mouse sciatic nerve. Ba, WT mouse sciatic nerve. Bb, Caspr^−/− mouse sciatic nerve. Bc, Caspr2^−/− mouse sciatic nerve. Fluorescence intensity profiles were generated by measuring fluorescence intensity along a 35-μm-long line drawn through the juxtaparanodes, paranodes, and node. Scale bars, 5 μm.

Figure 7.

Disruption of the nodal cytoskeleton does not alter localization of βII spectrin or ankyrinB. A, Cultured hippocampal neurons immunostained for βIV spectrin (red) and βII spectrin (green). βIV spectrin labels the axon initial segment, whereas βII spectrin labels the axon (arrowheads). B, Cultured hippocampal neurons immunostained for pan Nav (red) and βII spectrin (green). Fluorescence intensity profiles in A and B show a reduction in βII immunoreactivity at the axon initial segment. C, D, WT and qv^3J optic nerves (C) immunostained for Nav1.6 (green) and βII spectrin (red). Arrowheads show the nodal gap in βII spectrin immunostaining. Scale bars: A, B, 10 μm; C, 3 μm.