XB-ART-30169J Cell Biol July 1, 1983; 97 (1): 217-23.
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Cytoskeletal components of the vertebrate neuromuscular junction: vinculin, alpha-actinin, and filamin.
We have used immunocytochemical methods to investigate the cytoskeletal constituents of the vertebrate neuromuscular junction. Specific, affinity-purified antibodies to three cytoskeletal proteins, vinculin, alpha-actinin, and filamin, bound to neuromuscular junctions in sections of normal rat, mouse, chick, and Xenopus muscles. All three antibodies bound to the synaptic regions of denervated rat muscle fibers, indicating that the proteins recognized by these antibodies are associated with postsynaptic structures. The three proteins are present at the neuromuscular junction in muscle fibers of embryonic and neonatal animals, and therefore, may play an important role in its differentiation.
PubMed ID: 6408100
PMC ID: PMC2112479
Species referenced: Xenopus laevis
Genes referenced: actn1 avd flnb ttl vcl
Article Images: [+] show captions
|FIGURE 1. Specificity of the antibodies for their respective antigens. Multiwell plates were coated with 50 ng of the protein designated as antigen, in 50 #1 of buffered saline. All remaining, nonspecific binding sites for protein were then saturated with 8SA. Solutions in buffered saline of antivinculin (48 ng/ml), anti-a-actinin (144 ng/ml), antifilamin (208 ng/ml), and antimyosin (160 ng/ml) were introduced in 50-ttl aliquots and incubated for 1.5 h at 37 °. Unbound antibody was washed out, and goat-antirabbit IgG coupled to alkaline phosphatase (270 ng/ml) was introduced and incubated, as described. Bound antibody-linked alkaline phosphatase was assayed using p-nitrophenyl phosphate (0.6 mg/ml in diethanolamine buffer, pH 9.8). More details are given in reference 9. F, filamin; V, vinculin; A, a-actinin; M, myosin; R, tropomyosin; G, gizzard actin; S, skeletal muscle actin; T, tubulin; and O, blank. Similar results were obtained with sixfold higher antibody concentrations. The results show that each antibody is highly specific for its appropriate antigen.|
|FIGURE 2. Antibodies against vinculin, e~-actinin, and filamin stain the neuromuscular junction of rat diaphragm. Frozen transverse sections were cut through junctional regions of normal, adult rat diaphragm. Sections were reacted with (a and b), antivinculin (12/~g/ml); (c and d), anti-a-actinin (36 #g/ml); (e and f) antifilamin (34 ttg/ml). Counterstaining was with R-a-BuTx and either fGAR in a-d, or with biotinylated GAR and fluoresceinated avidin, in e-f. After staining, samples were mounted in Elvanol and observed under epi-illumination to visualize fluorescein (b, d, f) and tetramethylrhodamine (a, c, e). The results show that the three antibodies react with junctional regions, labeled with R-cz- Bul-x. Bar in t', 25/~m.|
|FIGURE 3. Specificity of antivincuJin staining of the neuromuscular junction. The procedure given in the legend to Fig. 1 was followed, but only antivinculin was used. b, d, f, h, and j show fluorescein fluorescence obtained after staining with fGAR. a, c, e, g, and i show staining of the AChR in the same sections with R-a-BuTx. the antibodies used were (a and b) antivinculin (12 pg/ ml); (c and d) nonspecific IgG from antivinculin antiserum (12 pg/ml), which shows essentially no staining; (e and t~ antivinculin (0.38 #g/ml); (g and h) antivinculin (0.38 #g/ml) preadsorbed with purified vinculin (7.6 #g/ml) for 15 rain at 22*, which shows significantly lower staining; (i and j) antivinculin (12 pg/ml) staining of a section through the junctional region of a rat diaphragm that had been denervated 3 wk before freezing and sectioning. The results show that antivinculin staining of the neuromuscular junction is specific and that it persists in long-term denervated muscle. Note the appearance of intense extrajunctional stain in denervated muscle (j). Bars, 20 pro; the bar in d applies to a-d; in h, to eh; and in i, to i and i.|
|FIGURE 4 Antibody stainingofjunctional regions from newborn rats. Frozen sections (6 #m) through diaphragm obtained from newborn (<24-h-old) rats are shown. Conditions are as described in the legend to Fig. 1. The antibodies are (b), antivinculin; (d), anti-cz-actinin; (/), antifilamin, a, c, and e show corresponding R-a-BuTx staining of the same sections. The results indicate that junctional regions of newborn diaphragm show antivinculin, anti-a-actinin, and antifilamin staining. Note, however, that extrajunctional staining is also apparent (e.g., t3. Bar in h, 10 ~.m.|
|FIGURE 5 Staining of neuromuscular junctions of mouse, chicken, and Xenopus. Frozen sections through junctional regions of mouse diaphragm (a, d, and g), chicken posterior latissimus dorsi (b, e, and h), and Xenopus sartorius (c, f, and i) muscles were stained with antibodies. Methods were as described in Fig. 1, except that fluorescein-avidin was used only in antifilamin staining of mouse muscle and staining of chicken muscle used lO-fold lower concentrations of antivinculin and ninefold lower concentrations of fGAR. R-a-BuTx stained regions (not shown) in all cases coincided with the brightly staining, membraneassociated structures pictured here. The antibodies used were antivinculin, (a-c); anti-a-actinin, (d-f); antifilamin, (g-i). The results show that, except for anti-c~-actinin reaction with Xenopus muscle (f), junctional regions of all three species are stained by the antibodies. Some weak extrajunctional staining is also apparent in a and e; staining of the contractile apparatus is evident in f, and, more faintly, in d. Bars, 20 pm. The bar in g also applies to a and d, and the bar in i applies to the rest of the panels.|
References [+] :
Bader, Density and distribution of alpha-bungarotoxin-binding sites in postsynaptic structures of regenerated rat skeletal muscle. 1981, Pubmed