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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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Pseudo-colored  confocal micrographs of rat  soleus NMJs. Teased fiber  preparations were dual labeled  with FITC α-BgTx (green)  to identify AChRs and antibodies to VGSCs (AP 1380)  or β-spectrin (RBC2/5C4) visualized with TRITC secondary antibodies (red). Both en  face and reconstructed transverse views are shown. The  superimposed en face images  reveal a fringe of VGSC or  β-spectrin labeling that extends laterally beyond the  AChR labeling (Superimposed and Detail). Transverse views (at the position  of the white lines) show that  this fringe is also present on  the cytoplasmic surface of the  AChR domain. This pattern  of labeling is consistent with  the localization of AChRs at  the tops of the postsynaptic  folds and of VGSC and  β-spectrin in the depths of  the folds. Bar, 10 μm.
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Figure 4: Pseudo-colored confocal micrographs of rat soleus NMJs. Teased fiber preparations were dual labeled with FITC α-BgTx (green) to identify AChRs and antibodies to VGSCs (AP 1380) or β-spectrin (RBC2/5C4) visualized with TRITC secondary antibodies (red). Both en face and reconstructed transverse views are shown. The superimposed en face images reveal a fringe of VGSC or β-spectrin labeling that extends laterally beyond the AChR labeling (Superimposed and Detail). Transverse views (at the position of the white lines) show that this fringe is also present on the cytoplasmic surface of the AChR domain. This pattern of labeling is consistent with the localization of AChRs at the tops of the postsynaptic folds and of VGSC and β-spectrin in the depths of the folds. Bar, 10 μm.

Mentions: β-Spectrin, a cytoskeletal protein implicated in the maintenance of VGSC clusters throughout the nervous system, is concentrated at the NMJ (Bloch and Morrow, 1989; Bewick et al., 1992). To see whether it is well placed within the NMJ to interact with VGSCs, the distribution of both VGSCs and β-spectrin was examined using confocal microscopy (Fig. 4). Dual-labeled teased muscle fibers viewed en face show that both VGSCs and β-spectrin are approximately codistributed with AChRs at the NMJ (Fig. 4). The superimposed images show clearly that labeling for both VGSCs and β-spectrin extends laterally ∼0.5 μm beyond that for AChRs. Bewick et al. (1993) reported that in en face views of NMJs a fringe of dystrophin or β-dystroglycan labeling could be seen to extend beyond AChR labeling. These authors interpreted this observed fringe of labeling as representing immunolabeling in the bottom of the postsynaptic folds.


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)

Pseudo-colored  confocal micrographs of rat  soleus NMJs. Teased fiber  preparations were dual labeled  with FITC α-BgTx (green)  to identify AChRs and antibodies to VGSCs (AP 1380)  or β-spectrin (RBC2/5C4) visualized with TRITC secondary antibodies (red). Both en  face and reconstructed transverse views are shown. The  superimposed en face images  reveal a fringe of VGSC or  β-spectrin labeling that extends laterally beyond the  AChR labeling (Superimposed and Detail). Transverse views (at the position  of the white lines) show that  this fringe is also present on  the cytoplasmic surface of the  AChR domain. This pattern  of labeling is consistent with  the localization of AChRs at  the tops of the postsynaptic  folds and of VGSC and  β-spectrin in the depths of  the folds. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2140176&req=5

Figure 4: Pseudo-colored confocal micrographs of rat soleus NMJs. Teased fiber preparations were dual labeled with FITC α-BgTx (green) to identify AChRs and antibodies to VGSCs (AP 1380) or β-spectrin (RBC2/5C4) visualized with TRITC secondary antibodies (red). Both en face and reconstructed transverse views are shown. The superimposed en face images reveal a fringe of VGSC or β-spectrin labeling that extends laterally beyond the AChR labeling (Superimposed and Detail). Transverse views (at the position of the white lines) show that this fringe is also present on the cytoplasmic surface of the AChR domain. This pattern of labeling is consistent with the localization of AChRs at the tops of the postsynaptic folds and of VGSC and β-spectrin in the depths of the folds. Bar, 10 μm.
Mentions: β-Spectrin, a cytoskeletal protein implicated in the maintenance of VGSC clusters throughout the nervous system, is concentrated at the NMJ (Bloch and Morrow, 1989; Bewick et al., 1992). To see whether it is well placed within the NMJ to interact with VGSCs, the distribution of both VGSCs and β-spectrin was examined using confocal microscopy (Fig. 4). Dual-labeled teased muscle fibers viewed en face show that both VGSCs and β-spectrin are approximately codistributed with AChRs at the NMJ (Fig. 4). The superimposed images show clearly that labeling for both VGSCs and β-spectrin extends laterally ∼0.5 μm beyond that for AChRs. Bewick et al. (1993) reported that in en face views of NMJs a fringe of dystrophin or β-dystroglycan labeling could be seen to extend beyond AChR labeling. These authors interpreted this observed fringe of labeling as representing immunolabeling in the bottom of the postsynaptic folds.

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