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. 2002 Jan 21;156(2):337-48.
doi: 10.1083/jcb.200110003. Epub 2002 Jan 21.

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

Masayuki Komada  1 , Philippe Soriano
Affiliations

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

Masayuki Komada et al. J Cell Biol. .

Abstract

beta-Spectrin and ankyrin are major components of the membrane cytoskeleton. We have generated mice carrying a null 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-null 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-null 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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Figures

Figure 1.
Figure 1.
Expression pattern of the ROSA62 gene in mice. Expression pattern of the ROSA62 gene was assessed by X-gal staining of heterozygous mutant mice. (A) Embryonic day 10.5 embryo. Expression is detected in the dorsal root ganglia (DRG) and trigeminal ganglia (TG). (B) Adult brain. High level of expression is detected in the hippocampus (HC) and cerebellum (CL). (C) Adult spinal cord. GM, gray matter; WM, white matter. (D) Adult pancreas. Expression is restricted to the islets of Langerhans (L). (E) Adult testis. Expression is restricted to the round spermatids (RS) and elongated spermatids (ES) facing the lumen (lu) of the seminiferous tubule. Bars: (A and C) 500 μm; (B) 1 mm ; (D) 100 μm; (E) 30 μm .
Figure 2.
Figure 2.
Characterization of βIV-spectrin and its gene products. (A) Schematic diagram of the βIVΣ1- and Σ6-spectrin isoforms. The gene trap vector was inserted between exons encoding the spectrin repeat 12 of the βIV-spectrin gene. (B) Sequence of the 5′ region of βIVΣ6-spectrin cDNA and its comparison with the corresponding region of βIVΣ1-spectrin cDNA. The sequence of Σ6 diverges upstream of the nucleotide 4079 of Σ1 (arrow). (C) Northern blot analysis of βIV-spectrin expression in brains from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice. The regions of the 5′- and 3′-probes are indicated in (A). Positions of the 28S and 18S rRNAs are indicated. (D) Western blot analysis of βIV-spectrin in brain lysates from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice. Positions of size markers are indicated in kD on the left. The sequence data are available from Genbank/EMBL/DDBJ accession no. AB055618 for βIVΣ1-spectrin and AB055619, AB055620, AB055621 for βIVΣ6-spectrin.
Figure 3.
Figure 3.
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.
Figure 4.
Figure 4.
Binding of βIV-spectrin to ankyrin. COS-7 cells were transfected with Myc-tagged βIVΣ6-spectrin (Myc-βIVΣ6) together with GFP (lanes 1), GFP-tagged 270 kD ankyrin-G (AnkG-GFP; lanes 2), or GFP-tagged 220 kD ankyrin-B (AnkB-GFP; lanes 3). Cell lysates were immunoprecipitated with anti-GFP (A and D) or anti-Myc (B and C). Immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-GFP (A and B) or anti-Myc (C and D). Positions of size markers are indicated in kD on the left.
Figure 5.
Figure 5.
Clustering of ankyrin-G at AIS of βIV-spectrin –null 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–null (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–null (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.
Figure 6.
Figure 6.
Clustering of VGSC at AIS of βIV-spectrin –null cerebral, hippocampal, and cerebellar neurons. Cerebral cortex (A, A′, B, and B′), hippocampus (C, C′, D, and D′), and cerebellum (E, E′, F, and F′) of wild-type (A, A′, C, C′, E, and E′) and βIV-spectrin–null (B, B′, D, D′, F, and F′) mice were stained with anti–βIV-spectrin (A, B, C, D, E, and F) together with anti–pan-sodium channel (pan-SC; A′, B′, C′, and D′) or anti-Nav1.6 (E′ and F′). Arrowheads indicate AIS of cerebral (A, A′, and B′), hippocampal pyramidal (C and C′), and Purkinje (E and E') neurons. The granular layer (G), Purkinje cell layer (P), and molecular layer (M) of the cerebellum are separated by dotted lines (E, E′, F, and F′). Mice were killed at 3 mo of age. Bars, 20 μm.
Figure 7.
Figure 7.
Clustering of Nav1.6 at NR of βIV-spectrin –null sciatic nerves. Double staining of wild-type (A, A′, and A′′) and βIV-spectrin–null (B, B′, and B′′) sciatic nerves with anti–βIV-spectrin (A and B) and anti-Nav1.6 (A′ and B′) antibodies. (A′′) and (B′′) are merged images. Arrowheads in A′, B′, A′′, and B′′ indicate some of the Nav1.6-positive NR which are also positive for βIV-spectrin in the wild-type (A). Insets in A′ and B′ show enlarged images of typical NR. In C, the number of Nav1.6-positive NR in wild-type (+/+) and βIV-spectrin–null (−/−) sciatic nerve sections were quantified. Mean numbers per 0.1 mm2 field are shown with SD (n = 9 for each genotype). Mice were killed at 3 mo of age. Bars, 100 μm.
Figure 8.
Figure 8.
Localization of βIV-spectrin at AIS of ankyrin-G–null neurons. Cerebellum (A, A′, B, and B′) and hippocampus (C and C′) of wild-type (A and A′) and cerebellum-specific ankyrin-G– null (B, B′, C, and C′) mice were stained with anti–ankyrin-G (A, B, and C) and anti–βIV-spectrin (A′, B′, and C′) antibodies. In D and E, Purkinje cells from wild-type (D) and ankyrin-G–null (E) mice were double stained for bIV-spectrin (red) and calbindin D-28K (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′). Purkinje cell bodies (Pu) are indicated in (D) and (E). Arrowheads indicate AIS of Purkinje cells (A, A′, and D). In B and B′, the contours of Purkinje cell bodies were observed in the absence of specific staining in the mutant due to background staining. Mice were killed at 5 wk of age. Bars, 50 μm.
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