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Membrane domain organization of myelinated axons requires βII spectrin.

Zhang C, Susuki K, Zollinger DR, Dupree JL, Rasband MN - J. Cell Biol. (2013)

Bottom Line: Surprisingly, the K(+) channels and their associated proteins redistributed into paranodes where they colocalized with intact Caspr-labeled axoglial junctions.Furthermore, electron microscopic analysis of the junctions showed intact paranodal septate-like junctions.Thus, the paranodal spectrin-based submembranous cytoskeleton comprises the paranodal barriers required for myelinated axon domain organization.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030.

ABSTRACT
The precise and remarkable subdivision of myelinated axons into molecularly and functionally distinct membrane domains depends on axoglial junctions that function as barriers. However, the molecular basis of these barriers remains poorly understood. Here, we report that genetic ablation and loss of axonal βII spectrin eradicated the paranodal barrier that normally separates juxtaparanodal K(+) channel protein complexes located beneath the myelin sheath from Na(+) channels located at nodes of Ranvier. Surprisingly, the K(+) channels and their associated proteins redistributed into paranodes where they colocalized with intact Caspr-labeled axoglial junctions. Furthermore, electron microscopic analysis of the junctions showed intact paranodal septate-like junctions. Thus, the paranodal spectrin-based submembranous cytoskeleton comprises the paranodal barriers required for myelinated axon domain organization.

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Paranodal Kv1 K+ channel staining increases with age in βII spectrin–deficient axons. (A–D) Nodes of Ranvier, paranodes, and juxtaparanodes from control (ctrl) and Avil-Cre;SPNB2f/f (cKO) dorsal roots immunostained using antibodies against βIV spectrin (blue), Caspr (green), and Kv1.2 (red), respectively. Immunostaining was performed at postnatal day P7 (A), P14 (B), P28 (C), and at 5 mo of age (D). Arrows indicate aberrant localization of Kv1.2 in nodes (A and D) and paranodes (B and C). Bars, 5 µm. (E) Quantification of the percentage of paranodes with Kv1.2 immunoreactivity as a function of age. Error bars indicate ± SEM. *, P < 0.01.
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fig3: Paranodal Kv1 K+ channel staining increases with age in βII spectrin–deficient axons. (A–D) Nodes of Ranvier, paranodes, and juxtaparanodes from control (ctrl) and Avil-Cre;SPNB2f/f (cKO) dorsal roots immunostained using antibodies against βIV spectrin (blue), Caspr (green), and Kv1.2 (red), respectively. Immunostaining was performed at postnatal day P7 (A), P14 (B), P28 (C), and at 5 mo of age (D). Arrows indicate aberrant localization of Kv1.2 in nodes (A and D) and paranodes (B and C). Bars, 5 µm. (E) Quantification of the percentage of paranodes with Kv1.2 immunoreactivity as a function of age. Error bars indicate ± SEM. *, P < 0.01.

Mentions: The initial assembly of juxtaparanodal, paranodal, and nodal domains in the PNS is thought to depend on interactions between membrane-associated cell adhesion molecules (CAMs; Schafer and Rasband, 2006). For example, juxtaparanodal clustering of Kv1 channels requires trans-interactions between axonal Caspr2 and glial TAG-1, and genetic deletion of either CAM blocks channel clustering (Poliak et al., 2003; Traka et al., 2003). At paranodes, clustering of βII spectrin may depend on Caspr, because Caspr has a cytoplasmic protein 4.1 binding domain, protein 4.1B binds to βII spectrin, and all three proteins are found at paranodes (Ogawa et al., 2006). To determine if paranodal βII spectrin is required for the maintenance of axonal membrane domains, we analyzed Kv1.2 localization in myelinated axons throughout development and into adulthood. As previously reported (Vabnick et al., 1999), we found Kv1.2 immunoreactivity colocalized with paranodal Caspr during the first postnatal week (unpublished data). However, as early as P7 we found instances of aberrant localization of Kv1.2 in cKO myelinated dorsal root axons, including at nodes of Ranvier (Fig. 3 A, arrow). By two weeks of age in control mice, fewer than 20% of paranodes had detectable Kv1.2 immunoreactivity, and this continued to decrease as mice matured (Fig. 3, B–E). In contrast, at two weeks nearly 40% of paranodes in cKO dorsal root axons had detectable Kv1.2 (Fig. 3, B and E), and the frequency of paranodal Kv1.2 continued to increase as cKO mice aged; in 5 mo-old cKO mice nearly 80% of paranodes had detectable Kv1.2 (Fig. 3, D and E). Furthermore, nodal Kv1.2 was often detected in adult cKO dorsal root axons, but was never seen in control mice (Fig. 3 D, arrow). The consistent increase in the number of paranodes with Kv1.2 immunoreactivity with increasing age suggests that βII spectrin plays important rolefor the maintenance of myelinated axon membrane domain organization.


Membrane domain organization of myelinated axons requires βII spectrin.

Zhang C, Susuki K, Zollinger DR, Dupree JL, Rasband MN - J. Cell Biol. (2013)

Paranodal Kv1 K+ channel staining increases with age in βII spectrin–deficient axons. (A–D) Nodes of Ranvier, paranodes, and juxtaparanodes from control (ctrl) and Avil-Cre;SPNB2f/f (cKO) dorsal roots immunostained using antibodies against βIV spectrin (blue), Caspr (green), and Kv1.2 (red), respectively. Immunostaining was performed at postnatal day P7 (A), P14 (B), P28 (C), and at 5 mo of age (D). Arrows indicate aberrant localization of Kv1.2 in nodes (A and D) and paranodes (B and C). Bars, 5 µm. (E) Quantification of the percentage of paranodes with Kv1.2 immunoreactivity as a function of age. Error bars indicate ± SEM. *, P < 0.01.
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fig3: Paranodal Kv1 K+ channel staining increases with age in βII spectrin–deficient axons. (A–D) Nodes of Ranvier, paranodes, and juxtaparanodes from control (ctrl) and Avil-Cre;SPNB2f/f (cKO) dorsal roots immunostained using antibodies against βIV spectrin (blue), Caspr (green), and Kv1.2 (red), respectively. Immunostaining was performed at postnatal day P7 (A), P14 (B), P28 (C), and at 5 mo of age (D). Arrows indicate aberrant localization of Kv1.2 in nodes (A and D) and paranodes (B and C). Bars, 5 µm. (E) Quantification of the percentage of paranodes with Kv1.2 immunoreactivity as a function of age. Error bars indicate ± SEM. *, P < 0.01.
Mentions: The initial assembly of juxtaparanodal, paranodal, and nodal domains in the PNS is thought to depend on interactions between membrane-associated cell adhesion molecules (CAMs; Schafer and Rasband, 2006). For example, juxtaparanodal clustering of Kv1 channels requires trans-interactions between axonal Caspr2 and glial TAG-1, and genetic deletion of either CAM blocks channel clustering (Poliak et al., 2003; Traka et al., 2003). At paranodes, clustering of βII spectrin may depend on Caspr, because Caspr has a cytoplasmic protein 4.1 binding domain, protein 4.1B binds to βII spectrin, and all three proteins are found at paranodes (Ogawa et al., 2006). To determine if paranodal βII spectrin is required for the maintenance of axonal membrane domains, we analyzed Kv1.2 localization in myelinated axons throughout development and into adulthood. As previously reported (Vabnick et al., 1999), we found Kv1.2 immunoreactivity colocalized with paranodal Caspr during the first postnatal week (unpublished data). However, as early as P7 we found instances of aberrant localization of Kv1.2 in cKO myelinated dorsal root axons, including at nodes of Ranvier (Fig. 3 A, arrow). By two weeks of age in control mice, fewer than 20% of paranodes had detectable Kv1.2 immunoreactivity, and this continued to decrease as mice matured (Fig. 3, B–E). In contrast, at two weeks nearly 40% of paranodes in cKO dorsal root axons had detectable Kv1.2 (Fig. 3, B and E), and the frequency of paranodal Kv1.2 continued to increase as cKO mice aged; in 5 mo-old cKO mice nearly 80% of paranodes had detectable Kv1.2 (Fig. 3, D and E). Furthermore, nodal Kv1.2 was often detected in adult cKO dorsal root axons, but was never seen in control mice (Fig. 3 D, arrow). The consistent increase in the number of paranodes with Kv1.2 immunoreactivity with increasing age suggests that βII spectrin plays important rolefor the maintenance of myelinated axon membrane domain organization.

Bottom Line: Surprisingly, the K(+) channels and their associated proteins redistributed into paranodes where they colocalized with intact Caspr-labeled axoglial junctions.Furthermore, electron microscopic analysis of the junctions showed intact paranodal septate-like junctions.Thus, the paranodal spectrin-based submembranous cytoskeleton comprises the paranodal barriers required for myelinated axon domain organization.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030.

ABSTRACT
The precise and remarkable subdivision of myelinated axons into molecularly and functionally distinct membrane domains depends on axoglial junctions that function as barriers. However, the molecular basis of these barriers remains poorly understood. Here, we report that genetic ablation and loss of axonal βII spectrin eradicated the paranodal barrier that normally separates juxtaparanodal K(+) channel protein complexes located beneath the myelin sheath from Na(+) channels located at nodes of Ranvier. Surprisingly, the K(+) channels and their associated proteins redistributed into paranodes where they colocalized with intact Caspr-labeled axoglial junctions. Furthermore, electron microscopic analysis of the junctions showed intact paranodal septate-like junctions. Thus, the paranodal spectrin-based submembranous cytoskeleton comprises the paranodal barriers required for myelinated axon domain organization.

Show MeSH
Related in: MedlinePlus