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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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Example of the  effect of antibody dilution  on immunolabeling fluorescence intensity. Transverse  cryostat sections of rat soleus  muscle were dual labeled  with an antibody against  β-spectrin (NCLSPEC2), visualized with a TRITC secondary antibody, and with  FITC α-BgTx to identify the  NMJ. Each point is the mean  (± SEM) fluorescence intensity in junctional (○) and extrajunctional (•) regions of  six muscle fibers. For the  β-spectrin antibody, the dilution at which most of the antigen  binding sites are saturated is 1:30.
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Figure 3: Example of the effect of antibody dilution on immunolabeling fluorescence intensity. Transverse cryostat sections of rat soleus muscle were dual labeled with an antibody against β-spectrin (NCLSPEC2), visualized with a TRITC secondary antibody, and with FITC α-BgTx to identify the NMJ. Each point is the mean (± SEM) fluorescence intensity in junctional (○) and extrajunctional (•) regions of six muscle fibers. For the β-spectrin antibody, the dilution at which most of the antigen binding sites are saturated is 1:30.

Mentions: An important preliminary step in this quantification procedure was to establish an appropriate primary antibody dilution. If the fluorescence intensity is to be proportional to the number of protein molecules, then the primary antibodies should be used at or near saturating concentration. Using serial dilution experiments, an approximately saturating antibody concentration was determined for the primary antibodies to VGSC, ankyrinG, β-spectrin, and dystrophin. Fig. 3 shows an example of a dilution series for the NCLSPEC2 antibody to β-spectrin. Muscle sections were exposed to serial dilutions of antibody and images containing NMJs were analyzed for J and XJ labeling as outlined above (without normalizing the labeling intensity to extrajunctional levels). The mean fluorescence labeling intensity per μm2 in both J and XJ regions was plotted against antibody concentration (Fig. 3). Each point is the mean ± SEM of observations from six muscle fibers. The concentration at which the binding sites appeared saturated was used to label sections throughout the experiments. At this dilution, the greatest difference between labeling intensity in J and XJ regions of the muscle fiber can be seen.


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)

Example of the  effect of antibody dilution  on immunolabeling fluorescence intensity. Transverse  cryostat sections of rat soleus  muscle were dual labeled  with an antibody against  β-spectrin (NCLSPEC2), visualized with a TRITC secondary antibody, and with  FITC α-BgTx to identify the  NMJ. Each point is the mean  (± SEM) fluorescence intensity in junctional (○) and extrajunctional (•) regions of  six muscle fibers. For the  β-spectrin antibody, the dilution at which most of the antigen  binding sites are saturated is 1:30.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Example of the effect of antibody dilution on immunolabeling fluorescence intensity. Transverse cryostat sections of rat soleus muscle were dual labeled with an antibody against β-spectrin (NCLSPEC2), visualized with a TRITC secondary antibody, and with FITC α-BgTx to identify the NMJ. Each point is the mean (± SEM) fluorescence intensity in junctional (○) and extrajunctional (•) regions of six muscle fibers. For the β-spectrin antibody, the dilution at which most of the antigen binding sites are saturated is 1:30.
Mentions: An important preliminary step in this quantification procedure was to establish an appropriate primary antibody dilution. If the fluorescence intensity is to be proportional to the number of protein molecules, then the primary antibodies should be used at or near saturating concentration. Using serial dilution experiments, an approximately saturating antibody concentration was determined for the primary antibodies to VGSC, ankyrinG, β-spectrin, and dystrophin. Fig. 3 shows an example of a dilution series for the NCLSPEC2 antibody to β-spectrin. Muscle sections were exposed to serial dilutions of antibody and images containing NMJs were analyzed for J and XJ labeling as outlined above (without normalizing the labeling intensity to extrajunctional levels). The mean fluorescence labeling intensity per μm2 in both J and XJ regions was plotted against antibody concentration (Fig. 3). Each point is the mean ± SEM of observations from six muscle fibers. The concentration at which the binding sites appeared saturated was used to label sections throughout the experiments. At this dilution, the greatest difference between labeling intensity in J and XJ regions of the muscle fiber can be seen.

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