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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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Loss of βII spectrin disrupts paranodal membrane barriers. (A) Immunoblot analysis of dorsal roots using antibodies against nodal, paranodal, and juxtaparanodal proteins. (B) Immunostaining of 5-mo-old dorsal and ventral roots from control and Avil-Cre;SPNB2f/f mice using antibodies against Kv1.2 and βIV spectrin. Control axons have a pronounced paranodal gap in immunoreactivity between Kv1.2 and βIV spectrin (arrows), but βII spectrin–deficient axons have paranodal Kv1.2 (arrowheads) that is directly adjacent to nodal βIV spectrin staining. Bar, 10 µm. (C) Immunostaining of 5-mo-old dorsal and ventral roots shows overlap between caspr and Kv1.2 in Avil-Cre;SPNB2f/f dorsal roots. Line scans of immunofluorescence intensity for the depicted nodes are shown at the right. Kv1.2 is shown in red, caspr in green, and βIV spectrin is shown in blue. Bar, 10 µm. (D) Immunostaining of dorsal roots shows juxtaparanodal Caspr2 (red) is excluded from Caspr-labeled paranodes (green) in control but not Avil-Cre;SPNB2f/f (cKO) mice. Bar, 5 µm. (E) Immunostaining of dorsal roots shows juxtaparanodal TAG-1 (red) is excluded from Caspr-labeled paranodes (green) in control but not cKO mice. Instead, TAG-1 can be found at both paranodes and nodes. Bar, 5 µm. (F and G) Toluidine blue–stained dorsal root ganglia and dorsal roots from control (SPNB2f/f) and Avil-Cre;SPNB2f/f mice show no signs of neurodegeneration. Bars, 20 µm.
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fig2: Loss of βII spectrin disrupts paranodal membrane barriers. (A) Immunoblot analysis of dorsal roots using antibodies against nodal, paranodal, and juxtaparanodal proteins. (B) Immunostaining of 5-mo-old dorsal and ventral roots from control and Avil-Cre;SPNB2f/f mice using antibodies against Kv1.2 and βIV spectrin. Control axons have a pronounced paranodal gap in immunoreactivity between Kv1.2 and βIV spectrin (arrows), but βII spectrin–deficient axons have paranodal Kv1.2 (arrowheads) that is directly adjacent to nodal βIV spectrin staining. Bar, 10 µm. (C) Immunostaining of 5-mo-old dorsal and ventral roots shows overlap between caspr and Kv1.2 in Avil-Cre;SPNB2f/f dorsal roots. Line scans of immunofluorescence intensity for the depicted nodes are shown at the right. Kv1.2 is shown in red, caspr in green, and βIV spectrin is shown in blue. Bar, 10 µm. (D) Immunostaining of dorsal roots shows juxtaparanodal Caspr2 (red) is excluded from Caspr-labeled paranodes (green) in control but not Avil-Cre;SPNB2f/f (cKO) mice. Bar, 5 µm. (E) Immunostaining of dorsal roots shows juxtaparanodal TAG-1 (red) is excluded from Caspr-labeled paranodes (green) in control but not cKO mice. Instead, TAG-1 can be found at both paranodes and nodes. Bar, 5 µm. (F and G) Toluidine blue–stained dorsal root ganglia and dorsal roots from control (SPNB2f/f) and Avil-Cre;SPNB2f/f mice show no signs of neurodegeneration. Bars, 20 µm.

Mentions: We next determined if loss of axonal βII spectrin affects the amount or localization of nodal, paranodal, or juxtaparanodal proteins. Immunoblots showed no difference in the amounts of proteins between dorsal roots from control and cKO mice when probed using antibodies against nodal (PanNav), paranodal (Caspr), or juxtaparanodal (Kv1.2, Caspr2, and Tag-1) proteins (Fig. 2 A). In ventral and control dorsal roots we observed a prominent gap in immunoreactivity between Kv1 channels and nodal βIV spectrin that corresponds to the paranode (Fig. 2 B, arrows). In contrast, immunostaining of βII spectrin–deficient axons showed that Kv1.2 channels, normally restricted to juxtaparanodes, were located in paranodal regions adjacent to the nodes of Ranvier (Fig. 2 B, arrowheads). Immunofluorescence and line scans through ventral and control dorsal root nodes of Ranvier showed sharp transitions in the distributions of juxtaparanodal and paranodal proteins (Fig. 2 C, arrows). Remarkably, dorsal root axons lacking βII spectrin had paranodal Kv1.2 immunoreactivity despite intact Caspr staining (Fig. 2 C, arrowheads). Similar to Kv1.2, other components of the juxtaparanodal K+ channel complex including Caspr2, TAG-1, and protein 4.1B also redistributed into paranodal (Fig. 2, D and E; protein 4.1B not depicted) and even nodal regions in cKO axons (Fig. 2 E). Interestingly, although ankB is found at paranodes and can bind to βII spectrin (Ogawa et al., 2006), its localization was not affected by loss of axonal βII spectrin (not depicted).


Membrane domain organization of myelinated axons requires βII spectrin.

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

Loss of βII spectrin disrupts paranodal membrane barriers. (A) Immunoblot analysis of dorsal roots using antibodies against nodal, paranodal, and juxtaparanodal proteins. (B) Immunostaining of 5-mo-old dorsal and ventral roots from control and Avil-Cre;SPNB2f/f mice using antibodies against Kv1.2 and βIV spectrin. Control axons have a pronounced paranodal gap in immunoreactivity between Kv1.2 and βIV spectrin (arrows), but βII spectrin–deficient axons have paranodal Kv1.2 (arrowheads) that is directly adjacent to nodal βIV spectrin staining. Bar, 10 µm. (C) Immunostaining of 5-mo-old dorsal and ventral roots shows overlap between caspr and Kv1.2 in Avil-Cre;SPNB2f/f dorsal roots. Line scans of immunofluorescence intensity for the depicted nodes are shown at the right. Kv1.2 is shown in red, caspr in green, and βIV spectrin is shown in blue. Bar, 10 µm. (D) Immunostaining of dorsal roots shows juxtaparanodal Caspr2 (red) is excluded from Caspr-labeled paranodes (green) in control but not Avil-Cre;SPNB2f/f (cKO) mice. Bar, 5 µm. (E) Immunostaining of dorsal roots shows juxtaparanodal TAG-1 (red) is excluded from Caspr-labeled paranodes (green) in control but not cKO mice. Instead, TAG-1 can be found at both paranodes and nodes. Bar, 5 µm. (F and G) Toluidine blue–stained dorsal root ganglia and dorsal roots from control (SPNB2f/f) and Avil-Cre;SPNB2f/f mice show no signs of neurodegeneration. Bars, 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3824014&req=5

fig2: Loss of βII spectrin disrupts paranodal membrane barriers. (A) Immunoblot analysis of dorsal roots using antibodies against nodal, paranodal, and juxtaparanodal proteins. (B) Immunostaining of 5-mo-old dorsal and ventral roots from control and Avil-Cre;SPNB2f/f mice using antibodies against Kv1.2 and βIV spectrin. Control axons have a pronounced paranodal gap in immunoreactivity between Kv1.2 and βIV spectrin (arrows), but βII spectrin–deficient axons have paranodal Kv1.2 (arrowheads) that is directly adjacent to nodal βIV spectrin staining. Bar, 10 µm. (C) Immunostaining of 5-mo-old dorsal and ventral roots shows overlap between caspr and Kv1.2 in Avil-Cre;SPNB2f/f dorsal roots. Line scans of immunofluorescence intensity for the depicted nodes are shown at the right. Kv1.2 is shown in red, caspr in green, and βIV spectrin is shown in blue. Bar, 10 µm. (D) Immunostaining of dorsal roots shows juxtaparanodal Caspr2 (red) is excluded from Caspr-labeled paranodes (green) in control but not Avil-Cre;SPNB2f/f (cKO) mice. Bar, 5 µm. (E) Immunostaining of dorsal roots shows juxtaparanodal TAG-1 (red) is excluded from Caspr-labeled paranodes (green) in control but not cKO mice. Instead, TAG-1 can be found at both paranodes and nodes. Bar, 5 µm. (F and G) Toluidine blue–stained dorsal root ganglia and dorsal roots from control (SPNB2f/f) and Avil-Cre;SPNB2f/f mice show no signs of neurodegeneration. Bars, 20 µm.
Mentions: We next determined if loss of axonal βII spectrin affects the amount or localization of nodal, paranodal, or juxtaparanodal proteins. Immunoblots showed no difference in the amounts of proteins between dorsal roots from control and cKO mice when probed using antibodies against nodal (PanNav), paranodal (Caspr), or juxtaparanodal (Kv1.2, Caspr2, and Tag-1) proteins (Fig. 2 A). In ventral and control dorsal roots we observed a prominent gap in immunoreactivity between Kv1 channels and nodal βIV spectrin that corresponds to the paranode (Fig. 2 B, arrows). In contrast, immunostaining of βII spectrin–deficient axons showed that Kv1.2 channels, normally restricted to juxtaparanodes, were located in paranodal regions adjacent to the nodes of Ranvier (Fig. 2 B, arrowheads). Immunofluorescence and line scans through ventral and control dorsal root nodes of Ranvier showed sharp transitions in the distributions of juxtaparanodal and paranodal proteins (Fig. 2 C, arrows). Remarkably, dorsal root axons lacking βII spectrin had paranodal Kv1.2 immunoreactivity despite intact Caspr staining (Fig. 2 C, arrowheads). Similar to Kv1.2, other components of the juxtaparanodal K+ channel complex including Caspr2, TAG-1, and protein 4.1B also redistributed into paranodal (Fig. 2, D and E; protein 4.1B not depicted) and even nodal regions in cKO axons (Fig. 2 E). Interestingly, although ankB is found at paranodes and can bind to βII spectrin (Ogawa et al., 2006), its localization was not affected by loss of axonal βII spectrin (not depicted).

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