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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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The distribution of ankyrinG and β-spectrin in rat soleus muscle fibers. Transverse serial sections through the same  NMJ were double labeled with a polyclonal antibody to ankyrinG  (AnkG@SpBd; visualized with TRITC-secondary antibody), and  FITC α-BgTx to identify the NMJ, and monoclonal antibody for  β-spectrin (NCLSPEC2) visualized with FITC secondary antibody. β-Spectrin and ankyrinG are concentrated at the NMJ and  in perijunctional regions (arrows). Immunolabeling for ankyrinG  is also seen in the myoplasm. Bar, 50 μm.
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Figure 8: The distribution of ankyrinG and β-spectrin in rat soleus muscle fibers. Transverse serial sections through the same NMJ were double labeled with a polyclonal antibody to ankyrinG (AnkG@SpBd; visualized with TRITC-secondary antibody), and FITC α-BgTx to identify the NMJ, and monoclonal antibody for β-spectrin (NCLSPEC2) visualized with FITC secondary antibody. β-Spectrin and ankyrinG are concentrated at the NMJ and in perijunctional regions (arrows). Immunolabeling for ankyrinG is also seen in the myoplasm. Bar, 50 μm.

Mentions: If VGSC clustering is mediated via an interaction between ankyrinG and β-spectrin, then ankyrinG would also be expected to be concentrated at the NMJ and in the perijunctional region. The distribution of ankyrinG immunolabeling in the muscle fiber membrane was therefore examined and compared with that of the cytoskeletal proteins β-spectrin, utrophin, and dystrophin. Transverse sections were dual labeled with ankyrinG antibody and BgTx to allow the NMJ to be identified (Fig. 8). AnkyrinG appeared concentrated at the NMJ and in adjacent regions, compared with extrajunctional regions. Subsequent serial sections were double antibody labeled and revealed that like VGSCs, the perijunctional concentration of ankyrinG immunolabeling is mirrored by β-spectrin (Fig. 8), but not by dystrophin or utrophin (data not shown). Thus ankyrinG and β-spectrin are colocalized at the NMJ and in the perijunctional region where the concentration of ankyrinG appears to parallel that of β-spectrin.


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)

The distribution of ankyrinG and β-spectrin in rat soleus muscle fibers. Transverse serial sections through the same  NMJ were double labeled with a polyclonal antibody to ankyrinG  (AnkG@SpBd; visualized with TRITC-secondary antibody), and  FITC α-BgTx to identify the NMJ, and monoclonal antibody for  β-spectrin (NCLSPEC2) visualized with FITC secondary antibody. β-Spectrin and ankyrinG are concentrated at the NMJ and  in perijunctional regions (arrows). Immunolabeling for ankyrinG  is also seen in the myoplasm. Bar, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140176&req=5

Figure 8: The distribution of ankyrinG and β-spectrin in rat soleus muscle fibers. Transverse serial sections through the same NMJ were double labeled with a polyclonal antibody to ankyrinG (AnkG@SpBd; visualized with TRITC-secondary antibody), and FITC α-BgTx to identify the NMJ, and monoclonal antibody for β-spectrin (NCLSPEC2) visualized with FITC secondary antibody. β-Spectrin and ankyrinG are concentrated at the NMJ and in perijunctional regions (arrows). Immunolabeling for ankyrinG is also seen in the myoplasm. Bar, 50 μm.
Mentions: If VGSC clustering is mediated via an interaction between ankyrinG and β-spectrin, then ankyrinG would also be expected to be concentrated at the NMJ and in the perijunctional region. The distribution of ankyrinG immunolabeling in the muscle fiber membrane was therefore examined and compared with that of the cytoskeletal proteins β-spectrin, utrophin, and dystrophin. Transverse sections were dual labeled with ankyrinG antibody and BgTx to allow the NMJ to be identified (Fig. 8). AnkyrinG appeared concentrated at the NMJ and in adjacent regions, compared with extrajunctional regions. Subsequent serial sections were double antibody labeled and revealed that like VGSCs, the perijunctional concentration of ankyrinG immunolabeling is mirrored by β-spectrin (Fig. 8), but not by dystrophin or utrophin (data not shown). Thus ankyrinG and β-spectrin are colocalized at the NMJ and in the perijunctional region where the concentration of ankyrinG appears to parallel that of β-spectrin.

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