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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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Clustering of ankyrin-G at AIS of βIV-spectrin– cerebellar and hippocampal neurons. Cerebellum (A, A′, B, and B′) and hippocampus (C, C′, D, and D′) of wild-type (A, A′, C, and C') and βIV-spectrin– (B, B′, D, and D′) mice were stained for βIV-spectrin and ankyrin-G as indicated. In E and F, Purkinje cells from wild-type (E) and βIV-spectrin– (F) mice were double stained for ankyrin-G (red) and calbindin D-28K to visualize their cell bodies (green), and are shown at higher magnification. The granular layer (G), Purkinje cell layer (P), and molecular layer (M) of the cerebellum are separated by dotted lines (A, A′, B, and B′), and Purkinje cell bodies (Pu) are indicated in E and F. AIS of Purkinje cells are indicated by arrowheads (A, A′, E, and F). Arrows in D and D′ indicate the pyramidal cell layer of the hippocampus, where AIS of pyramidal neurons are detected in wild-type mice (arrowheads in C and C′). Mice were killed at 3 mo of age. Bars, 50 μm.
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fig5: Clustering of ankyrin-G at AIS of βIV-spectrin– cerebellar and hippocampal neurons. Cerebellum (A, A′, B, and B′) and hippocampus (C, C′, D, and D′) of wild-type (A, A′, C, and C') and βIV-spectrin– (B, B′, D, and D′) mice were stained for βIV-spectrin and ankyrin-G as indicated. In E and F, Purkinje cells from wild-type (E) and βIV-spectrin– (F) mice were double stained for ankyrin-G (red) and calbindin D-28K to visualize their cell bodies (green), and are shown at higher magnification. The granular layer (G), Purkinje cell layer (P), and molecular layer (M) of the cerebellum are separated by dotted lines (A, A′, B, and B′), and Purkinje cell bodies (Pu) are indicated in E and F. AIS of Purkinje cells are indicated by arrowheads (A, A′, E, and F). Arrows in D and D′ indicate the pyramidal cell layer of the hippocampus, where AIS of pyramidal neurons are detected in wild-type mice (arrowheads in C and C′). Mice were killed at 3 mo of age. Bars, 50 μm.

Mentions: As expected from the Northern and Western blot analyses (Fig. 2, C and D), no anti–βIV-spectrin staining was detected above background level in the βIV-spectrin mutant cerebellar Purkinje and hippocampal pyramidal neurons (Fig. 5, B and D) . Compared with wild-type neurons where ankyrin-G colocalized with βIV-spectrin at AIS (Fig. 5, A, A′, C, C′, and E), staining for ankyrin-G was undetectable or very faint at these sites in most βIV-spectrin– neurons (Fig. 5, B′, D′, and F). In mutant Purkinje cells with faint anti–ankyrin-G staining, staining was not restricted to the AIS but was spread over the rest of the axon (unpublished data). Although anti–ankyrin-G staining was still restricted to the AIS in some neurons of the mutant, it was much weaker than in wild-type neurons (Fig. 5, E and F).


[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)

Clustering of ankyrin-G at AIS of βIV-spectrin– cerebellar and hippocampal neurons. Cerebellum (A, A′, B, and B′) and hippocampus (C, C′, D, and D′) of wild-type (A, A′, C, and C') and βIV-spectrin– (B, B′, D, and D′) mice were stained for βIV-spectrin and ankyrin-G as indicated. In E and F, Purkinje cells from wild-type (E) and βIV-spectrin– (F) mice were double stained for ankyrin-G (red) and calbindin D-28K to visualize their cell bodies (green), and are shown at higher magnification. The granular layer (G), Purkinje cell layer (P), and molecular layer (M) of the cerebellum are separated by dotted lines (A, A′, B, and B′), and Purkinje cell bodies (Pu) are indicated in E and F. AIS of Purkinje cells are indicated by arrowheads (A, A′, E, and F). Arrows in D and D′ indicate the pyramidal cell layer of the hippocampus, where AIS of pyramidal neurons are detected in wild-type mice (arrowheads in C and C′). Mice were killed at 3 mo of age. Bars, 50 μm.
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Related In: Results  -  Collection

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fig5: Clustering of ankyrin-G at AIS of βIV-spectrin– cerebellar and hippocampal neurons. Cerebellum (A, A′, B, and B′) and hippocampus (C, C′, D, and D′) of wild-type (A, A′, C, and C') and βIV-spectrin– (B, B′, D, and D′) mice were stained for βIV-spectrin and ankyrin-G as indicated. In E and F, Purkinje cells from wild-type (E) and βIV-spectrin– (F) mice were double stained for ankyrin-G (red) and calbindin D-28K to visualize their cell bodies (green), and are shown at higher magnification. The granular layer (G), Purkinje cell layer (P), and molecular layer (M) of the cerebellum are separated by dotted lines (A, A′, B, and B′), and Purkinje cell bodies (Pu) are indicated in E and F. AIS of Purkinje cells are indicated by arrowheads (A, A′, E, and F). Arrows in D and D′ indicate the pyramidal cell layer of the hippocampus, where AIS of pyramidal neurons are detected in wild-type mice (arrowheads in C and C′). Mice were killed at 3 mo of age. Bars, 50 μm.
Mentions: As expected from the Northern and Western blot analyses (Fig. 2, C and D), no anti–βIV-spectrin staining was detected above background level in the βIV-spectrin mutant cerebellar Purkinje and hippocampal pyramidal neurons (Fig. 5, B and D) . Compared with wild-type neurons where ankyrin-G colocalized with βIV-spectrin at AIS (Fig. 5, A, A′, C, C′, and E), staining for ankyrin-G was undetectable or very faint at these sites in most βIV-spectrin– neurons (Fig. 5, B′, D′, and F). In mutant Purkinje cells with faint anti–ankyrin-G staining, staining was not restricted to the AIS but was spread over the rest of the axon (unpublished data). Although anti–ankyrin-G staining was still restricted to the AIS in some neurons of the mutant, it was much weaker than in wild-type neurons (Fig. 5, E and F).

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