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beta-Spectrin is colocalized with both voltage-gated sodium channels and ankyrinG at the adult rat neuromuscular junction.

Wood SJ, Slater CR - J. Cell Biol. (1998)

Bottom Line: At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, beta-spectrin and ankyrin, which may help to maintain the high local density of VGSCs.Double antibody labeling shows that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG at the NMJ.These observations suggest that interactions with beta-spectrin and ankyrinG help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density.

View Article: PubMed Central - PubMed

Affiliation: School of Neurosciences, The Medical School, University of Newcastle upon Tyne NE2 4HH, United Kingdom. s.j.wood@bristol.ac.uk

ABSTRACT
Voltage-gated sodium channels (VGSCs) are concentrated in the depths of the postsynaptic folds at mammalian neuromuscular junctions (NMJs) where they facilitate action potential generation during neuromuscular transmission. At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, beta-spectrin and ankyrin, which may help to maintain the high local density of VGSCs. Here we show in skeletal muscle, using immunofluorescence, that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG, the nodal isoform of ankyrin. In en face views of rat NMJs, acetylcholine receptors (AChRs), and utrophin immunolabeling are organized in distinctive linear arrays corresponding to the crests of the postsynaptic folds. In contrast, beta-spectrin, VGSCs, and ankyrinG have a punctate distribution that extends laterally beyond the AChRs, consistent with a localization in the depths of the folds. Double antibody labeling shows that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG at the NMJ. Furthermore, quantification of immunofluorescence in labeled transverse sections reveals that beta-spectrin is also concentrated in perijunctional regions, in parallel with an increase in labeling of VGSCs and ankyrinG, but not of dystrophin. These observations suggest that interactions with beta-spectrin and ankyrinG help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density.

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Comparison of the distribution of VGSCs and ankyrinG with that of AChRs in en face views of the NMJ. Digitized images are  shown of teased fiber preparations from rat soleus muscles dual labeled with an antibody to either VGSCs (AP1380) or ankyrinG  (AnkG@SpBd) and FITC α-BgTx to identify AChRs. Low magnification images (×50 objective; Bar, 20 μm) show the overall distribution of immunolabeling at the NMJ. At higher magnification (×100 objective; Bar, 10 μm), arrows highlight areas where AChRs are organized in a linear fashion, whereas VGSCs and ankyrinG have a more spotty distribution that clearly extends beyond the AChR region.
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Figure 5: Comparison of the distribution of VGSCs and ankyrinG with that of AChRs in en face views of the NMJ. Digitized images are shown of teased fiber preparations from rat soleus muscles dual labeled with an antibody to either VGSCs (AP1380) or ankyrinG (AnkG@SpBd) and FITC α-BgTx to identify AChRs. Low magnification images (×50 objective; Bar, 20 μm) show the overall distribution of immunolabeling at the NMJ. At higher magnification (×100 objective; Bar, 10 μm), arrows highlight areas where AChRs are organized in a linear fashion, whereas VGSCs and ankyrinG have a more spotty distribution that clearly extends beyond the AChR region.

Mentions: If ankyrinG is an intermediary in the binding of VGSCs and β-spectrin at the NMJ then its distribution would be expected to be similar to that of VGSCs. In low magnification en face views of NMJs, immunolabeling for both VGSCs and ankyrinG is concentrated at the adult NMJ and is approximately codistributed with the AChRs (Fig. 5). On closer examination however, it can be seen that labeling for ankyrinG extends laterally beyond that for AChRs, as does immunolabeling for VGSCs and β-spectrin (Fig. 4). At higher magnification, differences in the detail of the labeling patterns can be seen (Fig. 5, arrows). Whereas AChRs seem to be organized in a characteristic linear pattern corresponding to the crests of the postsynaptic folds, VGSCs and ankyrinG have a more punctate distribution with some features of the labeling pattern being quite different from that of the AChR labeling. Fig. 5 thus confirms and extends the observations of Flucher and Daniels (1989), which found that VGSCs and ankyrin are localized in the depths of the postsynaptic folds. However, we have used different antibodies, in particular the antibody used here to demonstrate ankyrin labeling is specific for the isoform of ankyrin found at the nodes of Ranvier—ankyrinG (Kordeli et al., 1995).


beta-Spectrin is colocalized with both voltage-gated sodium channels and ankyrinG at the adult rat neuromuscular junction.

Wood SJ, Slater CR - J. Cell Biol. (1998)

Comparison of the distribution of VGSCs and ankyrinG with that of AChRs in en face views of the NMJ. Digitized images are  shown of teased fiber preparations from rat soleus muscles dual labeled with an antibody to either VGSCs (AP1380) or ankyrinG  (AnkG@SpBd) and FITC α-BgTx to identify AChRs. Low magnification images (×50 objective; Bar, 20 μm) show the overall distribution of immunolabeling at the NMJ. At higher magnification (×100 objective; Bar, 10 μm), arrows highlight areas where AChRs are organized in a linear fashion, whereas VGSCs and ankyrinG have a more spotty distribution that clearly extends beyond the AChR region.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2140176&req=5

Figure 5: Comparison of the distribution of VGSCs and ankyrinG with that of AChRs in en face views of the NMJ. Digitized images are shown of teased fiber preparations from rat soleus muscles dual labeled with an antibody to either VGSCs (AP1380) or ankyrinG (AnkG@SpBd) and FITC α-BgTx to identify AChRs. Low magnification images (×50 objective; Bar, 20 μm) show the overall distribution of immunolabeling at the NMJ. At higher magnification (×100 objective; Bar, 10 μm), arrows highlight areas where AChRs are organized in a linear fashion, whereas VGSCs and ankyrinG have a more spotty distribution that clearly extends beyond the AChR region.
Mentions: If ankyrinG is an intermediary in the binding of VGSCs and β-spectrin at the NMJ then its distribution would be expected to be similar to that of VGSCs. In low magnification en face views of NMJs, immunolabeling for both VGSCs and ankyrinG is concentrated at the adult NMJ and is approximately codistributed with the AChRs (Fig. 5). On closer examination however, it can be seen that labeling for ankyrinG extends laterally beyond that for AChRs, as does immunolabeling for VGSCs and β-spectrin (Fig. 4). At higher magnification, differences in the detail of the labeling patterns can be seen (Fig. 5, arrows). Whereas AChRs seem to be organized in a characteristic linear pattern corresponding to the crests of the postsynaptic folds, VGSCs and ankyrinG have a more punctate distribution with some features of the labeling pattern being quite different from that of the AChR labeling. Fig. 5 thus confirms and extends the observations of Flucher and Daniels (1989), which found that VGSCs and ankyrin are localized in the depths of the postsynaptic folds. However, we have used different antibodies, in particular the antibody used here to demonstrate ankyrin labeling is specific for the isoform of ankyrin found at the nodes of Ranvier—ankyrinG (Kordeli et al., 1995).

Bottom Line: At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, beta-spectrin and ankyrin, which may help to maintain the high local density of VGSCs.Double antibody labeling shows that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG at the NMJ.These observations suggest that interactions with beta-spectrin and ankyrinG help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density.

View Article: PubMed Central - PubMed

Affiliation: School of Neurosciences, The Medical School, University of Newcastle upon Tyne NE2 4HH, United Kingdom. s.j.wood@bristol.ac.uk

ABSTRACT
Voltage-gated sodium channels (VGSCs) are concentrated in the depths of the postsynaptic folds at mammalian neuromuscular junctions (NMJs) where they facilitate action potential generation during neuromuscular transmission. At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, beta-spectrin and ankyrin, which may help to maintain the high local density of VGSCs. Here we show in skeletal muscle, using immunofluorescence, that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG, the nodal isoform of ankyrin. In en face views of rat NMJs, acetylcholine receptors (AChRs), and utrophin immunolabeling are organized in distinctive linear arrays corresponding to the crests of the postsynaptic folds. In contrast, beta-spectrin, VGSCs, and ankyrinG have a punctate distribution that extends laterally beyond the AChRs, consistent with a localization in the depths of the folds. Double antibody labeling shows that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG at the NMJ. Furthermore, quantification of immunofluorescence in labeled transverse sections reveals that beta-spectrin is also concentrated in perijunctional regions, in parallel with an increase in labeling of VGSCs and ankyrinG, but not of dystrophin. These observations suggest that interactions with beta-spectrin and ankyrinG help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density.

Show MeSH
Related in: MedlinePlus