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[Beta]IV-spectrin regulates sodium channel clustering through ankyrin-G at axon initial segments and nodes of Ranvier.

Komada M, Soriano P - J. Cell Biol. (2002)

Bottom Line: In betaIV-spectrin- neurons, neither ankyrin-G nor voltage-gated sodium channels (VGSC) are correctly clustered at these sites, suggesting that impaired action potential caused by mislocalization of VGSC leads to the phenotype.Conversely, in ankyrin-G- neurons, betaIV-spectrin is not localized to these sites.These results indicate that betaIV-spectrin and ankyrin-G mutually stabilize the membrane protein cluster and the linked membrane cytoskeleton at AIS and NR.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental Biology and Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. makomada@bio.titech.ac.jp

ABSTRACT
beta-Spectrin and ankyrin are major components of the membrane cytoskeleton. We have generated mice carrying a mutation in the betaIV-spectrin gene using gene trapping in embryonic stem cells. Mice homozygous for the mutation exhibit tremors and contraction of hindlimbs. betaIV-spectrin expression is mostly restricted to neurons, where it colocalizes with and binds to ankyrin-G at axon initial segments (AISs) and nodes of Ranvier (NR). In betaIV-spectrin- neurons, neither ankyrin-G nor voltage-gated sodium channels (VGSC) are correctly clustered at these sites, suggesting that impaired action potential caused by mislocalization of VGSC leads to the phenotype. Conversely, in ankyrin-G- neurons, betaIV-spectrin is not localized to these sites. These results indicate that betaIV-spectrin and ankyrin-G mutually stabilize the membrane protein cluster and the linked membrane cytoskeleton at AIS and NR.

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Subcellular localization of βIV-spectrin. Cultured ES cells (A–A′′ and B–B′′), dentate gyrus (C–C′′) and cerebellum (D–D′′) of the brain, and sciatic nerves (E–E′′) were stained with anti–βIV-spectrin antibody (A, B, C, D, and E) together with phalloidin (A′), anti-vinculin (B'), anti–ankyrin-G (C′ and D′), and anti-Nav1.6 (E′). (A′′, B′′, C′′, D′′, and E′′) Merged images. In D′′, the granular layer (G), Purkinje cell layer (P), and molecular layer (M) of the cerebellum are separated by dotted lines. Bars: (A–A,′′ B–B,′′ and E–E′′) 30 μm; (C–C′′ and D–D′′) 100 μm.
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fig3: Subcellular localization of βIV-spectrin. Cultured ES cells (A–A′′ and B–B′′), dentate gyrus (C–C′′) and cerebellum (D–D′′) of the brain, and sciatic nerves (E–E′′) were stained with anti–βIV-spectrin antibody (A, B, C, D, and E) together with phalloidin (A′), anti-vinculin (B'), anti–ankyrin-G (C′ and D′), and anti-Nav1.6 (E′). (A′′, B′′, C′′, D′′, and E′′) Merged images. In D′′, the granular layer (G), Purkinje cell layer (P), and molecular layer (M) of the cerebellum are separated by dotted lines. Bars: (A–A,′′ B–B,′′ and E–E′′) 30 μm; (C–C′′ and D–D′′) 100 μm.

Mentions: Although spectrin was originally identified as the major component of the plasma membrane skeleton, it is also associated with various intracellular organelles and is implicated in maintaining organelle structures and membrane trafficking (for review see De Matteis and Morrow, 2000). Therefore, subcellular localization of endogenous βIV-spectrin was examined by immunofluorescence staining first in ES cells (the only cell line among those tested expressing βIV-spectrin) using the anti–βIV-spectrin antibody. When plated on gelatinized cover glass, ES cells often formed an epithelia-like monolayer of cells that adhered to each other. In these cells, the anti–βIV-spectrin antibody stained subdomains of the adherens junctions which were also stained for F-actin and β-catenin (Fig. 3 , A–A′′; unpublished data). βIV-spectrin completely colocalized with another adherens junction protein, vinculin (Fig. 3, B-B′′). These results suggest that βIV-spectrin functions as a component of the plasma membrane skeleton.


[Beta]IV-spectrin regulates sodium channel clustering through ankyrin-G at axon initial segments and nodes of Ranvier.

Komada M, Soriano P - J. Cell Biol. (2002)

Subcellular localization of βIV-spectrin. Cultured ES cells (A–A′′ and B–B′′), dentate gyrus (C–C′′) and cerebellum (D–D′′) of the brain, and sciatic nerves (E–E′′) were stained with anti–βIV-spectrin antibody (A, B, C, D, and E) together with phalloidin (A′), anti-vinculin (B'), anti–ankyrin-G (C′ and D′), and anti-Nav1.6 (E′). (A′′, B′′, C′′, D′′, and E′′) Merged images. In D′′, the granular layer (G), Purkinje cell layer (P), and molecular layer (M) of the cerebellum are separated by dotted lines. Bars: (A–A,′′ B–B,′′ and E–E′′) 30 μm; (C–C′′ and D–D′′) 100 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199236&req=5

fig3: Subcellular localization of βIV-spectrin. Cultured ES cells (A–A′′ and B–B′′), dentate gyrus (C–C′′) and cerebellum (D–D′′) of the brain, and sciatic nerves (E–E′′) were stained with anti–βIV-spectrin antibody (A, B, C, D, and E) together with phalloidin (A′), anti-vinculin (B'), anti–ankyrin-G (C′ and D′), and anti-Nav1.6 (E′). (A′′, B′′, C′′, D′′, and E′′) Merged images. In D′′, the granular layer (G), Purkinje cell layer (P), and molecular layer (M) of the cerebellum are separated by dotted lines. Bars: (A–A,′′ B–B,′′ and E–E′′) 30 μm; (C–C′′ and D–D′′) 100 μm.
Mentions: Although spectrin was originally identified as the major component of the plasma membrane skeleton, it is also associated with various intracellular organelles and is implicated in maintaining organelle structures and membrane trafficking (for review see De Matteis and Morrow, 2000). Therefore, subcellular localization of endogenous βIV-spectrin was examined by immunofluorescence staining first in ES cells (the only cell line among those tested expressing βIV-spectrin) using the anti–βIV-spectrin antibody. When plated on gelatinized cover glass, ES cells often formed an epithelia-like monolayer of cells that adhered to each other. In these cells, the anti–βIV-spectrin antibody stained subdomains of the adherens junctions which were also stained for F-actin and β-catenin (Fig. 3 , A–A′′; unpublished data). βIV-spectrin completely colocalized with another adherens junction protein, vinculin (Fig. 3, B-B′′). These results suggest that βIV-spectrin functions as a component of the plasma membrane skeleton.

Bottom Line: In betaIV-spectrin- neurons, neither ankyrin-G nor voltage-gated sodium channels (VGSC) are correctly clustered at these sites, suggesting that impaired action potential caused by mislocalization of VGSC leads to the phenotype.Conversely, in ankyrin-G- neurons, betaIV-spectrin is not localized to these sites.These results indicate that betaIV-spectrin and ankyrin-G mutually stabilize the membrane protein cluster and the linked membrane cytoskeleton at AIS and NR.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental Biology and Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. makomada@bio.titech.ac.jp

ABSTRACT
beta-Spectrin and ankyrin are major components of the membrane cytoskeleton. We have generated mice carrying a mutation in the betaIV-spectrin gene using gene trapping in embryonic stem cells. Mice homozygous for the mutation exhibit tremors and contraction of hindlimbs. betaIV-spectrin expression is mostly restricted to neurons, where it colocalizes with and binds to ankyrin-G at axon initial segments (AISs) and nodes of Ranvier (NR). In betaIV-spectrin- neurons, neither ankyrin-G nor voltage-gated sodium channels (VGSC) are correctly clustered at these sites, suggesting that impaired action potential caused by mislocalization of VGSC leads to the phenotype. Conversely, in ankyrin-G- neurons, betaIV-spectrin is not localized to these sites. These results indicate that betaIV-spectrin and ankyrin-G mutually stabilize the membrane protein cluster and the linked membrane cytoskeleton at AIS and NR.

Show MeSH
Related in: MedlinePlus