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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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Identification of  junctional (J), perijunctional  (PJ), and extrajunctional  (XJ) regions in immunolabeled transverse sections of  rat soleus muscles. For analysis of immunofluorescence  labeling intensity, sections  were dual labeled with FITC  α-BgTx to identify the  AChRs at the NMJ and an  antibody to the protein of interest, in this example polyclonal antibody to VGSCs  (AP1380). Once J, PJ, and  XJ regions were defined as  described in the Materials  and Methods, the mean fluorescence labeling intensity  within each of them was determined. Bar, 20 μm.
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Figure 2: Identification of junctional (J), perijunctional (PJ), and extrajunctional (XJ) regions in immunolabeled transverse sections of rat soleus muscles. For analysis of immunofluorescence labeling intensity, sections were dual labeled with FITC α-BgTx to identify the AChRs at the NMJ and an antibody to the protein of interest, in this example polyclonal antibody to VGSCs (AP1380). Once J, PJ, and XJ regions were defined as described in the Materials and Methods, the mean fluorescence labeling intensity within each of them was determined. Bar, 20 μm.

Mentions: Images of both fluorescein and rhodamine labeling were recorded from transverse sections of muscle fibers that were dual labeled with a primary antibody to the protein of interest and with BgTx. The presence of FITC-α-BgTx binding to AChRs was used to identify the position of the NMJ (Fig. 2), and the corresponding rhodamine image demonstrates the presence of VGSC immunolabeling around the entire muscle fiber membrane. The intensity of fluorescence was measured in each of three regions of the muscle fiber surface (Fig. 2): the NMJ (J); the perijunctional region (PJ) immediately adjacent (0–5 μm) to the NMJ; and an extrajunctional region (XJ) diametrically opposed to the NMJ. For all the images obtained the camera was operating within a range where pixel intensity and exposure time were linearly related. All intensity values were expressed as gray levels per second of exposure time. The total fluorescence intensity within each region was calculated and the value obtained was divided by the area of the region to give the mean intensity of labeling per unit area. A background value determined by applying the same procedure to sections in which the primary antibody had been omitted was subtracted to give the net labeling intensity. For each muscle fiber, the mean fluorescence intensity in J and PJ regions was normalized to that in the XJ region (assigned a value of 1). For each antibody, 10 muscle fibers in each of two soleus muscles were analyzed, and the results expressed as mean normalized fluorescence intensity ± SEM.


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)

Identification of  junctional (J), perijunctional  (PJ), and extrajunctional  (XJ) regions in immunolabeled transverse sections of  rat soleus muscles. For analysis of immunofluorescence  labeling intensity, sections  were dual labeled with FITC  α-BgTx to identify the  AChRs at the NMJ and an  antibody to the protein of interest, in this example polyclonal antibody to VGSCs  (AP1380). Once J, PJ, and  XJ regions were defined as  described in the Materials  and Methods, the mean fluorescence labeling intensity  within each of them was determined. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Identification of junctional (J), perijunctional (PJ), and extrajunctional (XJ) regions in immunolabeled transverse sections of rat soleus muscles. For analysis of immunofluorescence labeling intensity, sections were dual labeled with FITC α-BgTx to identify the AChRs at the NMJ and an antibody to the protein of interest, in this example polyclonal antibody to VGSCs (AP1380). Once J, PJ, and XJ regions were defined as described in the Materials and Methods, the mean fluorescence labeling intensity within each of them was determined. Bar, 20 μm.
Mentions: Images of both fluorescein and rhodamine labeling were recorded from transverse sections of muscle fibers that were dual labeled with a primary antibody to the protein of interest and with BgTx. The presence of FITC-α-BgTx binding to AChRs was used to identify the position of the NMJ (Fig. 2), and the corresponding rhodamine image demonstrates the presence of VGSC immunolabeling around the entire muscle fiber membrane. The intensity of fluorescence was measured in each of three regions of the muscle fiber surface (Fig. 2): the NMJ (J); the perijunctional region (PJ) immediately adjacent (0–5 μm) to the NMJ; and an extrajunctional region (XJ) diametrically opposed to the NMJ. For all the images obtained the camera was operating within a range where pixel intensity and exposure time were linearly related. All intensity values were expressed as gray levels per second of exposure time. The total fluorescence intensity within each region was calculated and the value obtained was divided by the area of the region to give the mean intensity of labeling per unit area. A background value determined by applying the same procedure to sections in which the primary antibody had been omitted was subtracted to give the net labeling intensity. For each muscle fiber, the mean fluorescence intensity in J and PJ regions was normalized to that in the XJ region (assigned a value of 1). For each antibody, 10 muscle fibers in each of two soleus muscles were analyzed, and the results expressed as mean normalized fluorescence intensity ± SEM.

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