Article |
Address correspondence to Masayuki Komada, Dept. of Life Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. Tel.: 81-45-924-5702. Fax: 81-45-924-5771. E-mail: makomada{at}bio.titech.ac.jp
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: ßIV-spectrin; ankyrin-G; voltage-gated sodium channel; axon initial segment; node of Ranvier
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanisms by which these specific membrane proteins are concentrated at AIS and NR are not yet understood. A number of in vivo and in vitro studies have implicated myelinating glial cells in localizing axonal membrane proteins to NR both in the central and peripheral nervous systems (for review see Peles and Salzer, 2000; Rasband and Trimmer, 2001). Several axonal proteins have also been implicated in this process. It has been suggested that neurofascin and NrCAM may have a role in defining the initial sites for clustering in rat sciatic nerves, because clustering of these cell adhesion molecules precedes that of ankyrin-G and VGSC as well as myelination of the axon (Lambert et al., 1997). However, in cerebellar Purkinje cells of ankyrin-G knockout mice, clustering of neurofascin as well as VGSC at AIS is disrupted, leading to a failure in normal action potential firing (Zhou et al., 1998). These results indicate that ankyrin-G is an essential component for localizing neurofascin and VGSC at these sites. Taken together, these observations suggest that the membrane protein clustering is a sequential process that includes glial and axonal proteins, and that the factors defining the site of protein clustering and the factors stabilizing the once-formed cluster are distinct.
Ankyrins are linked to the spectrin-actin membrane cytoskeleton through binding to ß-spectrins (Davis and Bennett, 1990; Kennedy et al., 1991). Therefore, it is possible that the membrane cytoskeleton at AIS and NR binds ankyrin-G and defines the localization of associated membrane proteins. Indeed, it has been shown that the putative ß-spectrinbinding domain of ankyrin-G alone can be targeted to AIS in cultured neurons (Zhang and Bennett, 1998). As ßII-spectrin is localized to axons (whereas ßI2- and ßIII-spectrins are localized to the cell body and dendrites in neurons) (Riederer et al., 1986; Ohara et al., 1998), ßII-spectrin has been believed to bind ankyrin-G at AIS and NR. However, it has not been thought to be involved in defining the localization of ankyrin-G, as it is present throughout the axon (Riederer et al., 1986). Recently, a new member of the ß-spectrin family, ßIV-spectrin, has been identified (Berghs et al., 2000; Tse et al., 2001). This protein was shown to colocalize with ankyrin-G at AIS and NR (Berghs et al., 2000), raising the possibility that this novel ß-spectrin might bind ankyrin-G and thus be involved in clustering ankyrin-G and associated membrane proteins.
Using gene trap mutagenesis to identify and mutate genes regulated by retinoic acid (Komada et al., 2000), we have identified the ROSA62 mouse strain that harbors a null mutation in the ßIV-spectrin gene. ROSA62 mice exhibit tremors and hindlimb contraction, and the phenotype increases in severity with age. We demonstrate that ßIV-spectrin plays an essential role in localizing ankyrin-G and VGSC at AIS and NR in neurons. Conversely, we show that ßIV-spectrin localization to AIS requires ankyrin-G, indicating a mutual role for ßIV-spectrin and ankyrin-G in stabilizing the membrane protein cluster and the linked membrane skeleton at these sites. Very recently, Parkinson et al. (2001) showed that the spontaneous mouse mutant, quivering (qv) (Yoon and Les, 1957), carries a mutation in the ßIV-spectrin gene. We propose that the phenotype of ROSA62 and qv mice is primarily due to mislocalization of VGSC at AIS and NR.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Neuromuscular defects in ROSA62 mutant mice
Germline chimeric mice were generated by injecting ROSA62 mutant ES cells into blastocysts, and were crossed to 129/S4 and C57BL/6J mice to derive mutants. Heterozygotes exhibited no overt phenotype. Viable homozygous mutant mice were recovered from intercrossing of heterozygotes according to Mendelian expectations and were mostly of normal size. However, by 3 wk of age, homozygous mutants were distinguishable from wild-type and heterozygous mutant littermates by fine tremors. Until 23 mo of age, the homozygotes were grossly normal except for the tremor. They also exhibited clasping of the hindlimbs when held by the tail, a hallmark of ataxia. As they grew older, the phenotype became more severe. By 610 mo of age, they exhibited continuous contraction of hindlimb skeletal muscle and were not able to walk. The same phenotype was observed on congenic 129/S4, mixed 129/S4 x C57BL/6J, and congenic C57BL/6J (>10-generation backcross) genetic background. The following analyses were performed on mixed 129/S4x C57BL/6J genetic background.
Expression pattern of the ROSA62 gene in mice
Expression of ß-galactosidase activity in gene trap mutant mice is driven by the promoter of the trapped gene (Friedrich and Soriano, 1991). Therefore, ROSA62 heterozygous mutant embryos and adult tissues were isolated and stained with X-gal to determine the expression pattern of the trapped gene.
In embryonic day 10.5 embryos, expression was restricted to cranial and dorsal root ganglia (Fig. 1 A). In adult mice, expression was detected in many regions of the brain, with the highest level in the hippocampus and cerebellum (Fig. 1 B). Expression was also detected in the spinal cord (Fig. 1 C). In contrast, no ß-galactosidase activity was detected in skeletal muscle (unpublished data), suggesting that the phenotype of ROSA62 mutant mice is primarily due to a defect in neurons but not skeletal muscle.
|
Mutation in ßIV-spectrin in ROSA62 mutant mice
To identify the gene trapped in this mutant, the ROSA62ßgeo* fusion cDNA was cloned from ROSA62 mutant ES cells by 5'-RACE, and full-length cDNAs were subsequently cloned by screening cDNA libraries. Sequencing of the cDNAs revealed that the trapped gene encodes ßIV-spectrin, for which the human homologue has been recently identified (Berghs et al., 2000; Tse et al., 2001). Two ßIV-spectrin splice isoforms, 1 and
6, were cloned (Fig. 2 A). ßIV
1-spectrin was 2561 amino acids long, consisted of an NH2-terminal actin-binding domain, 17 spectrin repeats, a variable region, and a COOH-terminal plecstrin homology (PH) domain, and had 95% overall sequence identity to its human homologue. The actin-binding domain, the entire spectrin repeats, and the PH domain of ßIV
1-spectrin were
70, 40, and 55% identical in amino acid sequences, respectively, to those of ßI-, ßII-, and ßIII-spectrins, with the highest sequence identity to ßII-spectrin.
|
Expression of ßIV-spectrin in mice and its absence in ROSA62 mutant
To determine if the gene trap mutation leads to a null allele, Northern and Western blot analyses were performed. By Northern blot analysis with a ßIV-spectrin cDNA fragment encoding the 3' region (3'-probe in Fig. 2 A), four ßIV-spectrin transcripts (9, 7, 6.5, and 5 kb in size) were detected in the brain of wild-type and heterozygous mutant mice (Fig. 2 C). Using a 5'-probe specific to ßIV
1-spectrin (Fig. 2 A), only the 9-kb transcript was detected (Fig. 2 C). As the
1 and
6 cDNAs we cloned are 8.7 and 4.7 kb long, respectively, mRNAs encoding these isoforms likely correspond to the 9- and 5-kb transcripts, respectively. In the homozygous mutant, all of the transcripts detected with the 3'-probe were absent (Fig. 2 C). Using the 5'-probe, a transcript which is
9 kb in size was detected (unpublished data). As this transcript did not hybridize with the 3' probe, it likely represents the fusion transcript between the 5' end of ßIV-spectrin and the reporter. Western blot analysis was performed using a polyclonal antibody raised against the variable region of ßIV-spectrin which has no homology to other proteins. In the wild-type brain, the antibody detected a minor
300-kD band and a major
160-kD band (Fig. 2 D). The faint band of
140 kD may be another splice isoform or a degradation product. From the calculated molecular mass (289 kD for
1 and 141 kD for
6), the
300- and
160-kD bands most likely correspond to ßIV
1- and
6-spectrins, respectively. Interestingly, the truncated
6 isoform was much highly expressed than the full-length
1 isoform both at the level of mRNA as well as protein. The same bands were detected in the heterozygotes, but were less abundant, and they were absent from the homozygous mutant extract (Fig. 2 D). A truncated ßIV-spectrin containing the NH2-terminal portion might conceivably be produced in the homozygotes. However, the absence of any mRNAs and proteins detected by the 3' probe and the antibody, respectively, as well as the gene trap insertion site indicates that it lacks the spectrin repeats 1217, the variable region, and the PH domain. Therefore, the gene trap insertion has most likely created a functionally null allele of the ßIV-spectrin gene.
Subcellular localization of ßIV-spectrin
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 ßIV-spectrin was examined by immunofluorescence staining first in ES cells (the only cell line among those tested expressing ßIV-spectrin) using the antißIV-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 antißIV-spectrin antibody stained subdomains of the adherens junctions which were also stained for F-actin and ß-catenin (Fig. 3, AA''; unpublished data). ßIV-spectrin completely colocalized with another adherens junction protein, vinculin (Fig. 3, B-B''). These results suggest that ßIV-spectrin functions as a component of the plasma membrane skeleton.
|
Binding of ßIV-spectrin to ankyrin-G
ßI- and ßII-spectrins have been shown to bind ankyrin-R and ankyrin-B (Davis and Bennett, 1990; Kennedy et al., 1991). This fact, together with colocalization of ßIV-spectrin with ankyrin-G at AIS and NR, suggested that ßIV-spectrin might bind ankyrin-G. To test this possibility, Myc-tagged ßIV6-spectrin (Myc-ßIV
6) was cotransfected with green fluorescent protein (GFP), GFP-tagged 270-kD ankyrin-G (AnkG-GFP), or GFP-tagged 220-kD ankyrin-B (AnkB-GFP) into COS-7 cells. Transfected cells were lysed and immunoprecipitated, and then immunoblotted with anti-Myc or anti-GFP antibody. Whereas Myc-ßIV
6 was expressed at a similar level in the three transfectants (Fig. 4 C), GFP and AnkB-GFP were much more highly expressed than AnkG-GFP (Fig. 4 A). Consistent with the presence in ßIV
6-spectrin of the spectrin repeat 15, which has been mapped as an ankyrin-binding domain in ßI- and ßII-spectrins (Kennedy et al., 1991), the anti-Myc antibody coprecipitated both AnkG-GFP and AnkB-GFP but not GFP alone (Fig. 4 B). However, significantly more AnkG-GFP was brought down than AnkB-GFP (Fig. 4 B). Similarly, AnkG-GFP coprecipitated Myc-ßIV
6 more efficiently than AnkB-GFP (Fig. 4 D). These results indicate that ßIV-spectrin binds to ankyrin-G with high affinity, and to ankyrin-B to a lesser extent.
|
As expected from the Northern and Western blot analyses (Fig. 2, C and D), no antißIV-spectrin staining was detected above background level in the ßIV-spectrin mutant cerebellar Purkinje and hippocampal pyramidal neurons (Fig. 5, B and D). Compared with wild-type neurons where ankyrin-G colocalized with ßIV-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 ßIV-spectrinnull neurons (Fig. 5, B', D', and F). In mutant Purkinje cells with faint antiankyrin-G staining, staining was not restricted to the AIS but was spread over the rest of the axon (unpublished data). Although antiankyrin-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).
|
|
|
Localization of ßIV-spectrin at AIS of ankyrin-Gnull neurons
Next, we examined whether localization of ßIV-spectrin to AIS requires ankyrin-G. To test this, we examined the localization of ßIV-spectrin in cerebellar neurons of mutant mice generated by Zhou et al. (1998) in which the cerebellum-specific form of ankyrin-G is knocked out. Compared with wild-type cerebellum where AIS of both Purkinje and granular neurons were positive for ankyrin-G and ßIV-spectrin (Fig. 8, A, A', and D), ßIV-spectrin staining as well as ankyrin-G staining was mostly lost in the knockout mice (Fig. 8, B, B', and E). ßIV-spectrin localization was normal in the mutant in other regions than the cerebellum, such as the hippocampus, where ankyrin-G was expressed (Fig. 8, C and C'; unpublished data), indicating that ankyrin-G is a prerequisite for the correct ßIV-spectrin localization in AIS.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular mechanisms underlying the ßIV-spectrinnull phenotype
Mislocalization of VGSC to AIS and NR in the ßIV-spectrin mutant suggested that the neurological phenotypes of the mutant are, at least in part, due to impaired firing and propagation of the action potential in the central and peripheral nervous systems. This is supported by mouse mutants in which expression or localization of VGSC is affected. Nav1.6, also called Scn8a, is the major isoform of VGSC -subunit localized at AIS and NR of cerebellar neurons as well as the sciatic nerve (S. Jenkins and V. Bennett, personal communication; Caldwell et al., 2000). It has been shown that in mice with mutations in the Nav1.6 gene, action potential firing is impaired in Purkinje cells (Raman et al., 1997). Similarly in cerebellum-specific ankyrin-G knockout mice, mislocalization of VGSC to AIS is associated with the impaired action potential firing in Purkinje cells (Zhou et al., 1998). As ankyrin-G is not correctly localized to AIS in the ßIV-spectrin mutant, localization of other ankyrin-G-binding membrane proteins such as neurofascin and NrCAM is also likely to be affected. Therefore, the possibility that mislocalization of these proteins also contributes to the phenotype cannot be excluded. In this context, it will be interesting to compare the ßIV-spectrin mutant phenotype with that of the NrCAM knockout mice, in which a possible defect in AIS and NR has not been examined yet (Moré et al., 2001).
Although disruption of the ankyrin-G gene is restricted to the cerebellum in the mutant mice generated by Zhou et al. (1998), the resulting phenotype is more severe than that observed in our ßIV-spectrin mutant. The ankyrin-G mutant mice frequently fall down when prodded to walk, and in some cases exhibit premature death following uncontrollable jumping and convulsions (Zhou et al., 1998). These phenotypes were not observed in the ßIV-spectrin mutant. Faint ankyrin-G and Nav1.6 staining at AIS in some Purkinje cells of the ßIV-spectrin mutant might indicate that the milder phenotype is due to the trace amount of VGSC remaining in cerebellar neurons. Taking into account the fact that the Nav1.6-null mice die before 4 wk of age due to muscular atrophy (Duchen and Stefani, 1971; Burgess et al., 1995), incomplete mislocalization of VGSC associated with relatively mild phenotype in the ßIV-spectrin mutant is likely.
Role for ßIV-spectrin in membrane protein clustering at AIS and NR
Mislocalization of ankyrin-G and VGSC in the ßIV-spectrin mutant raises the question of whether ßIV-spectrin is required for targeting these membrane proteins to AIS and NR or for stabilizing their localization at these sites. The observation that some ankyrin-G and VGSC labeling still occurs in some AIS and NR in the mutant tends to support the latter, although it is also possible that trace amounts of other ß-spectrins at AIS and NR partially compensate for ßIV-spectrin function. The increased severity of the ßIV-spectrin mutant phenotype with age is also consistent with the role for ßIV-spectrin in stabilizing the membrane protein cluster. Conversely, ßIV-spectrin was also excluded from AIS of Purkinje cells in region-specific ankyrin-G knockout mice, suggesting that ankyrin-G in turn stabilizes the ßIV-spectrin localization at these sites. High binding affinity between ßIV-spectrin and ankyrin-G, as shown by coimmunoprecipitation experiments, must underlie this mutual stabilization. As ßIV-spectrin localization to AIS was almost completely disrupted in the ankyrin-G mutant, ankyrin-G may also play a role in targeting ßIV-spectrin to AIS and NR during development of neurons. Initial clustering of ankyrin-G at these sites is thought to be regulated by other axonal proteins such as VGSC, neurofascin, or NrCAM, that are already clustered at these sites by a glial signal (for review see Peles and Salzer, 2000; Rasband and Trimmer, 2001). Therefore, it is possible that ankyrin-G, which is recruited to these sites by other membrane proteins, recruits ßIV-spectrin, and once ßIV-spectrin forms a complex with the membrane proteins, it in turn serves to stabilize the protein clustering at these sites.
ßIV-spectrin has a variable region between the spectrin repeat 17 and the PH domain. This domain was termed ERQES domain because it contains four repeats of those five amino acid residues in human (Berghs et al., 2000). This domain (300 amino acids) is much larger than the corresponding regions of other ß-spectrins (
100 amino acids) and it is rich in proline (15%) and positively (20%), and negatively charged (16%) residues. This domain has no sequence homology to any other proteins including other ß-spectrins in databases, suggesting that it has a function specific to ßIV-spectrin. One possibility is that it serves as an interaction domain with an unknown protein to regulate protein clustering at AIS and NR.
The major ßIV-spectrin isoform, 6
In this study, we cloned cDNAs encoding two splice isoforms for ßIV-spectrin, 1 and
6, from three independent libraries. The
1 isoform is a full-length type consisting of an actin-binding domain, 17 spectrin repeats, a variable region, and a PH domain, and has been reported previously in human (Berghs et al., 2000; Tse et al., 2001). The other isoform,
6, is an NH2-terminal truncation lacking the actin-binding domain and the spectrin repeats 19 and a part of 10. Although various splice isoforms have been reported for other ß-spectrins, this type has not been identified, suggesting that this is a unique isoform for ßIV-spectrin. Other groups have identified four additional ßIV-spectrin isoforms (
2
5) in human (Berghs et al., 2000; Tse et al., 2001). It is noteworthy that among these isoforms,
6 appears to be the major isoform at least in the brain, as the size of the
6 cDNA (4.7 kb) and its protein product (141 kD) roughly correspond to that of the major bands in Northern blotting (
5 kb) and in Western blotting (
160 kD), respectively. On the contrary, the size of mRNAs encoding
2,
3, and
4 are much larger than 5 kb (
9 kb) (Berghs et al., 2000), whereas the size of mRNA encoding
5 is expected to be much smaller than 5 kb (
2.5 kb) (Tse et al., 2001). By Western blotting with antibodies raised against the variable region and the COOH-terminal region of ßIV-spectrin, Berghs et al. (2000) also detected two major truncated isoforms with similar molecular mass (ßIV-spectrin 160 and 140) which were not characterized further. One of these proteins is likely to be the
6 isoform.
In a classical model of the spectrin-actin membrane skeleton, a tetramer consisting of two - and two ß-spectrin molecules is linked to the actin filament through the actin-binding domain of ß-spectrin (for review see Bennett and Gilligan, 1993; Winkelmann and Forget, 1993). As
6 lacks the actin-binding domain as well as part of the spectrin repeats, it is unclear how this isoform fits into the model. Although it is impossible to distinguish
1 and
6 in tissues by immunostaining, as our antißIV-spectrin antibody recognizes the variable region, Western blotting suggested that immunostaining in neurons with the antibody mostly reflects the expression of
6. These results indicate an unknown structure of the membrane skeleton specific for AIS and NR.
Quivering, a spontaneous mutation in ßIV-spectrin in mice
The ßIV-spectrin gene has been mapped to the human chromosome 19q13.13 and mouse chromosome 7 near the centromere (Berghs et al., 2000; Tse et al., 2001; unpublished data). Very recently, Parkinson et al. (2001) reported that the spontaneous mouse mutation "quivering" (qv), which has been mapped to chromosome 7 at 14.5 cM from the centromere (Yoon and Les, 1957), carries a mutation in the ßIV-spectrin gene. Qv is an autosomal recessive mutation that causes various phenotypes including tremors, progressive ataxia with hindlimb paralysis, and deafness (Yoon and Les, 1957) (MGD; http://www.informatics.jax.org). Although we have not examined deafness in our ßIV-spectrin mutant, other neurological phenotypes in the qv/qv mice are consistent with our findings. The allele of ßIV-spectrin that we have generated is a null allele, based on Northern and Western blot analyses, whereas at least five of seven known spontaneous qv mutations are the results of single nucleotide changes or a small deletion/insertion (Parkinson et al., 2001). Parkinson et al. (2001) compared three alleles in a test for auditory brainstem function and suggest that various mutations may result in different degrees of severity of the phenotype. Therefore, it will be interesting to perform a molecular analysis of the different qv alleles (for example, the 6 isoform can be normally expressed from the qvlnd allele) and to compare the phenotypic severity with the allele presented in this work.
The results presented in our study strongly suggest that the ROSA62 and the qv/qv phenotype, and the impaired action potential firing and propagation in the auditory neurons in the qv/qv mice, are primarily due to mislocalization of VGSC to AIS and NR. Parkinson et al. (2001) also showed mislocalization of the potassium channel KCNA1 in axons of qv/qv mice. However, this is likely to be a secondary defect, as KCNA1 is not localized to AIS and NR but to the juxtaparanodal regions flanking NR by a distinct mechanism involving binding to a cell recognition molecule Caspr2 via an unidentified PDZ domain protein (Wang et al., 1993; Poliak et al., 1999). The demonstration of an essential role for a region-specific spectrin membrane skeleton in neurons might provide new insights into the molecular mechanisms underlying certain neuropathological conditions in human.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
X-gal staining and histology
X-gal staining was performed as described (Komada et al., 2000). For staining adult tissues, mice were perfused transcardially with 4% formaldehyde. Brains and spinal cords were sectioned at 100 µm using a vibratome before staining. After staining, pancreas and testis were embedded in paraffin, sectioned at 5 µm, and counterstained with Nuclear Fast Red.
cDNA cloning and Northern blot analysis
Total RNA was prepared as described (Komada et al., 2000), and poly(A)+ RNA was purified using oligo(dT)-cellulose. ROSA62ßgeo* fusion cDNA was amplified from poly(A)+ RNA of ROSA62 mutant ES cells by 5'-RACE as described (Hildebrand and Soriano, 1999). The 5'-RACE product was then used to screen mouse ES cell, embryonic day 15 embryo, and adult brain libraries to obtain full-length cDNAs. Northern blot analysis was performed using standard procedures.
Generation of antibody, Western blotting, and immunoprecipitation
A chicken antibody (Aves Lab) was raised against the variable region of ßIV-spectrin (amino acid 21712345 of ßIV1-spectrin) which was expressed in Escherichia coli as a glutathione S-transferase fusion protein. Purified IgY fraction from immunized chicken was absorbed against the ßIV-spectrinnull brain sliced using a vibratome. For Western blotting, a brain was homogenized in 1 ml lysis buffer (20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 1% Nonidet P-40, 1 mM PMSF, and 2 µg/ml aprotinin). After removing insoluble materials by low-speed centrifugation (1,000 g for 5 min), 20 µl of the lysate was run on SDS-PAGE. After transferring proteins to a Trans-Blot polyvinylidene difluoride membrane (Bio-Rad Laboratories), ßIV-spectrin was detected using the antißIV-spectrin antibody (1:400), peroxidase-conjugated antichicken IgY antibody (1:10,000; Jackson ImmunoResearch Laboratories), and the ECL reagent (Amersham Pharmacia Biotech).
For coimmunoprecipitation experiments, COS-7 cells were transfected with Myc-ßIV6 together with GFP, AnkG-GFP, or AnkB-GFP, gifts of V. Bennett (Duke University Medical Center, Durham, NC) using the FuGENE 6 Transfection Reagent (Roche). 2 d after transfection, cells were lysed and the lysates were immunoprecipitated with anti-Myc antibody 9E10 or anti-GFP (Molecular Probes). Immunoprecipitates were used for Western blotting with the same antibodies.
Immunofluorescence staining
ES cells were plated on gelatin-coated cover glass, fixed with 4% formaldehyde, permeabilized with 0.2% Triton X-100, and stained with the antißIV-spectrin (1:400) together with mouse anti-vinculin antibody (1:400; Sigma-Aldrich) by standard procedures. For staining F-actin, FITC-conjugated phalloidin (Sigma-Aldrich) was included in incubation with secondary antibody. Brains and spinal cords were recovered from mice perfused transcardially with 1% formaldehyde, cryoprotected with 30% sucrose, embedded in the OCT compound (Tissue Tec), and sectioned at 10 µm using a cryostat. The sections were stained with the antißIV-spectrin (1:400), mouse antiankyrin-G (1:200), a gift of V. Bennett), mouse antipan-sodium channel (10 µg/ml; Sigma-Aldrich), rabbit anti-Nav1.6 (Scn8a) (10 µg/ml; Alomone Labs), and rabbit anti-calbindin D-28K (1:1,000; Chemicon) antibodies. Secondary antibodies used were Alexa594-conjugated antichicken IgY, Alexa594-conjugated antimouse IgG, Alexa488-conjugated antimouse IgG, and Alexa488-conjugated antirabbit IgG antibodies (Molecular Probes). Fluorescent images were captured using a confocal microscope.
Cerebellum-specific ankyrin-G knockout mice originally generated by Zhou et al. (1998) were provided by H. Kamiguchi and V. Bennett.
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
This work was supported by National Institute of Child Health and Human Development grant HD24875 to P. Soriano.
Submitted: 1 October 2001
Revised: 21 November 2001
Accepted: 7 December 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bennett, V., and D.M. Gilligan. 1993. The spectrin-based membrane skeleton and micron-scale organization of the plasma membrane. Annu. Rev. Cell Biol. 9:2766.[CrossRef]
Berghs, S., D. Aggujaro, R. Dirkx, Jr., E. Maksimova, P. Stabach, J.-M. Hermel, J.-P. Zhang, W. Philbrick, V. Slepnev, T. Ort, and M. Solimena. 2000. ßIV spectrin, a new spectrin localized at axon initial segments and nodes of Ranvier in the central and peripheral nervous system. J. Cell Biol. 151:9851001.
Caldwell, J.H., K.L. Schaller, R.S. Lasher, E. Peles, and S.R. Levinson. 2000. Sodium channel Nav1.6 is localized at nodes of Ranvier, dendrites, and synapses. Proc. Natl. Acad. Sci. USA. 97:56165620.
Davis, L.H., and V. Bennett. 1990. Mapping the binding sites of human erythrocyte ankyrin for the anion exchanger and spectrin. J. Biol. Chem. 265:1058910596.
Davis, J.Q., S. Lambert, and V. Bennett. 1996. Molecular composition of the node of Ranvier: identification of ankyrin-binding cell adhesion molecules neurofascin (mucin+/third FNIII domain-) and NrCAM at nodal axon segments. J. Cell Biol. 135:13551367.[Abstract]
De Matteis, M.A., and J.S. Morrow. 2000. Spectrin tethers and mesh in the biosynthetic pathway. J. Cell Sci. 113:23312343.
Duchen, L.W., and E. Stefani. 1971. Electrophysiological studies of neuromuscular transmission in hereditary "motor end-plate disease" of the mouse. J. Physiol. 212:535548.[Medline]
Friedrich, G., and P. Soriano. 1991. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 5:15131523.[Abstract]
Kennedy, S.P., S.L. Warren, B.G. Forget, and J.S. Morrow. 1991. Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid ß-spectrin. J. Cell Biol. 115:267277.[Abstract]
Komada, M., D.J. McLean, M.D. Griswold, L.D. Russell, and P. Soriano. 2000. E-MAP-115, encoding a microtubule-associated protein, is a retinoic acid-inducible gene required for spermatogenesis. Genes Dev. 14:13321342.
Kordeli, E., S. Lambert, and V. Bennett. 1995. AnkyrnG: a new ankyrin gene with neural-specific isoforms localized at the axonal initial segment and node of Ranvier. J. Biol. Chem. 270:23522359.
Lambert, S., J.Q. Davis, and V. Bennett. 1997. Morphogenesis of the node of Ranvier: co-clusters of ankyrin and ankyrin-binding integral proteins define early developmental intermediates. J. Neurosci. 17:70257036.
Moré, M.I., F.-P. Kirsch, and F.G. Rathjen. 2001. Targeted ablation of NrCAM or ankyrin-B results in disorganized lens fibers leading to cataract formation. J. Cell Biol. 154:187196.
Parkinson, N.J., C.L. Olsson, J.L. Hallows, J. McKee-Johnson, B.P. Keogh, K. Noben-Trauth, S.G. Kujawa, and B.L. Tempel. 2001. Mutant ß-spectrin 4 causes auditory and motor neuropathies in quivering mice. Nat. Genet. 29:6165.[CrossRef][Medline]
Poliak, S., L. Gollan, R. Martinez, A. Custer, S. Einheber, J.L. Salzer, J.S. Trimmer, P. Shrager, and E. Peles. 1999. Caspr2, a new member of the neurexin superfamily, is localized at the juxtaparanodes of myelinated axons and associates with K+ channels. Neuron. 24:10371047.[Medline]
Rasband, M.N., and J.S. Trimmer. 2001. Developmental clustering of ion channels at and near the node of Ranvier. Dev. Biol. 236:516.[CrossRef][Medline]
Rasband, M.N., E. Peles, J.S. Trimmer, S.R. Levinson, S.E. Lux, and P. Shrager. 1999. Dependence of nodal sodium channel clustering on paranodal axoglial contact in the developing CNS. J. Neurosci. 19:75167528.
Riederer, B.M., I.S. Zagon, and S.R. Goodman. 1986. Brain spectrin (240/235) and brain spectrin (240/235E): two distinct spectrin subtypes with different locations within mammalian neural cells. J. Cell Biol. 102:20882097.[Abstract]
Tse, W.T., J. Tang, O. Jin, C. Korsgren, K.M. John, A.L. Kung, B. Gwynn, L.L. Peters, and S.E. Lux. 2001. A new spectrin, ßIV, has a major truncated isoform that associates with promyelocytic leukemia protein nuclear bodies and the nuclear matrix. J. Biol. Chem. 276: 2397423985.
Wang, H., D.D. Kunkel, T.M. Martin, P.A. Schwartzkroin, and B.L. Tempel. 1993. Heteromultimeric K+ channels in terminal and juxtaparanodal regions of neurons. Nature. 365:7579.[CrossRef][Medline]
Winkelmann, J.C., and B.G. Forget. 1993. Erythroid and nonerythroid spectrins. Blood. 81:31733185.[Abstract]
Yoon, C.H., and E.P. Les. 1957. Quivering, a new first chromosome mutation in mice. J. Hered. 48:176180.
Zhang, X., and V. Bennett. 1998. Restriction of 480/270-kD ankyrinG to axon proximal segments requires multiple ankyrinG-specific domains. J. Cell Biol. 142:15711581.
Zhou, D., S. Lambert, P.L. Malen, S. Carpenter, L.M. Boland, and V. Bennett. 1998. AnkyrinG is required for clustering of voltage-gated Na channels at axon initial segments and for normal action potential firing. J. Cell Biol. 143:12951304.