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. 1983 Oct 1;97(4):1043–1054. doi: 10.1083/jcb.97.4.1043

Lipid domains of acetylcholine receptor clusters detected with saponin and filipin

PMCID: PMC2112610  PMID: 6619185

Abstract

The acetylcholine receptor (AChR) clusters of cultured rat myotubes contain two distinct, interdigitating, membrane domains, one enriched in AChR, the other poor in AChR but associated with sites of myotube- substrate contact (Bloch, R.J., and B. Geiger, 1980, Cell, 21:25-35). We have used two cholesterol-specific cytochemical probes, saponin and filipin, to investigate the lipid nature of these membrane domains. When studied with freeze-fracture electron microscopy or fluorescence microscopy, these reagents reacted moderately and preferentially with the AChR-rich domains of AChR clusters. Little or no reaction with the membrane in "contact" domains was seen. In contrast, membrane regions surrounding the AChR clusters reacted extensively with filipin. These results suggest that, in rat myotubes, the composition or the state of the lipids differs between the two membrane domains of the AChR clusters, and between clusters and surrounding membrane. In chick myotubes, AChR clusters do not appear to react with filipin or saponin, although surrounding membrane reacts extensively with these reagents.

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Selected References

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  1. Anderson M. J., Cohen M. W. Nerve-induced and spontaneous redistribution of acetylcholine receptors on cultured muscle cells. J Physiol. 1977 Jul;268(3):757–773. doi: 10.1113/jphysiol.1977.sp011880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Axelrod D. Crosslinkage and visualization of acetylcholine receptors on myotubes with biotinylated alpha-bungarotoxin and fluorescent avidin. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4823–4827. doi: 10.1073/pnas.77.8.4823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Axelrod D., Ravdin P., Koppel D. E., Schlessinger J., Webb W. W., Elson E. L., Podleski T. R. Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4594–4598. doi: 10.1073/pnas.73.12.4594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bittman R., Chen W. C., Anderson O. R. Interaction of filipin 3 and amphotericin B with lecithin-sterol vesicles and cellular membranes. Spectral and electron microscope studies. Biochemistry. 1974 Mar 26;13(7):1364–1373. doi: 10.1021/bi00704a009. [DOI] [PubMed] [Google Scholar]
  5. Bloch R. J. Dispersal and reformation of acetylcholine receptor clusters of cultured rat myotubes treated with inhibitors of energy metabolism. J Cell Biol. 1979 Sep;82(3):626–643. doi: 10.1083/jcb.82.3.626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bloch R. J., Geiger B. The localization of acetylcholine receptor clusters in areas of cell-substrate contact in cultures of rat myotubes. Cell. 1980 Aug;21(1):25–35. doi: 10.1016/0092-8674(80)90111-7. [DOI] [PubMed] [Google Scholar]
  7. Bridgman P. C., Nakajima Y. Distribution of filipin-sterol complexes on cultured muscle cells: cell-substratum contact areas associated with acetylcholine receptor clusters. J Cell Biol. 1983 Feb;96(2):363–372. doi: 10.1083/jcb.96.2.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bridgman P. C., Nakajima Y. Membrane lipid heterogeneity associated with acetylcholine receptor particle aggregates in Xenopus embryonic muscle cells. Proc Natl Acad Sci U S A. 1981 Feb;78(2):1278–1282. doi: 10.1073/pnas.78.2.1278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burden S. J., Sargent P. B., McMahan U. J. Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve. J Cell Biol. 1979 Aug;82(2):412–425. doi: 10.1083/jcb.82.2.412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chang H. W., Bock E. Molecular forms of acetylcholine receptor. Effects of calcium ions and a sulfhydryl reagent on the occurrence of oligomers. Biochemistry. 1977 Oct 4;16(20):4513–4520. doi: 10.1021/bi00639a028. [DOI] [PubMed] [Google Scholar]
  11. Christian C. N., Daniels M. P., Sugiyama H., Vogel Z., Jacques L., Nelson P. G. A factor from neurons increases the number of acetylcholine receptor aggregates on cultured muscle cells. Proc Natl Acad Sci U S A. 1978 Aug;75(8):4011–4015. doi: 10.1073/pnas.75.8.4011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cohen S. A., Pumplin D. W. Clusters of intramembrane particles associated with binding sites for alpha-bungarotoxin in cultured chick myotubes. J Cell Biol. 1979 Aug;82(2):494–516. doi: 10.1083/jcb.82.2.494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fischbach G. D., Cohen S. A. The distribution of acetylcholine sensitivity over uninnervated and innervated muscle fibers grown in cell culture. Dev Biol. 1973 Mar;31(1):147–162. doi: 10.1016/0012-1606(73)90326-6. [DOI] [PubMed] [Google Scholar]
  14. Friend D. S. Plasma-membrane diversity in a highly polarized cell. J Cell Biol. 1982 May;93(2):243–249. doi: 10.1083/jcb.93.2.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Froehner S. C., Gulbrandsen V., Hyman C., Jeng A. Y., Neubig R. R., Cohen J. B. Immunofluorescence localization at the mammalian neuromuscular junction of the Mr 43,000 protein of Torpedo postsynaptic membranes. Proc Natl Acad Sci U S A. 1981 Aug;78(8):5230–5234. doi: 10.1073/pnas.78.8.5230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gonzalez-Ros J. M., Llanillo M., Paraschos A., Martinez-Carrion M. Lipid environment of acetylcholine receptor from Torpedo californica. Biochemistry. 1982 Jul 6;21(14):3467–3474. doi: 10.1021/bi00257a033. [DOI] [PubMed] [Google Scholar]
  17. Hall Z. W., Lubit B. W., Schwartz J. H. Cytoplasmic actin in postsynaptic structures at the neuromuscular junction. J Cell Biol. 1981 Sep;90(3):789–792. doi: 10.1083/jcb.90.3.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Heuser J. E., Salpeter S. R. Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane. J Cell Biol. 1979 Jul;82(1):150–173. doi: 10.1083/jcb.82.1.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hirokawa N., Heuser J. E. Internal and external differentiations of the postsynaptic membrane at the neuromuscular junction. J Neurocytol. 1982 Jun;11(3):487–510. doi: 10.1007/BF01257990. [DOI] [PubMed] [Google Scholar]
  20. Kallai-Sanfacon M. A., Reed J. K. Characterization of the lipid and polypeptide components of a tetrodotoxin binding membrane fraction from Electrophorus electricus. J Membr Biol. 1980 Jun 15;54(3):173–181. doi: 10.1007/BF01870233. [DOI] [PubMed] [Google Scholar]
  21. Land B. R., Podleski T. R., Salpeter E. E., Salpeter M. M. Acetylcholine receptor distribution on myotubes in culture correlated to acetylcholine sensitivity. J Physiol. 1977 Jul;269(1):155–176. doi: 10.1113/jphysiol.1977.sp011897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Markelonis G. J., Oh T. H. A protein fraction from peripheral nerve having neurotrophic effects on skeletal muscle cells in culture. Exp Neurol. 1978 Jan 15;58(2):285–295. doi: 10.1016/0014-4886(78)90141-3. [DOI] [PubMed] [Google Scholar]
  23. Montesano R. Inhomogeneous distribution of filipin-sterol complexes in smooth muscle cell plasma membrane. Nature. 1979 Jul 26;280(5720):328–329. doi: 10.1038/280328a0. [DOI] [PubMed] [Google Scholar]
  24. Nakajima Y., Bridgman P. C. Absence of filipin-sterol complexes from the membranes of active zones and acetylcholine receptor aggregates at frog neuromuscular junctions. J Cell Biol. 1981 Feb;88(2):453–458. doi: 10.1083/jcb.88.2.453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Norman A. W., Spielvogel A. M., Wong R. G. Polyene antibiotic - sterol interaction. Adv Lipid Res. 1976;14:127–170. [PubMed] [Google Scholar]
  26. Orci L., Miller R. G., Montesano R., Perrelet A., Amherdt M., Vassalli P. Opposite polarity of filipin-induced deformations in the membrane of condensing vacuoles and zymogen granules. Science. 1980 Nov 28;210(4473):1019–1021. doi: 10.1126/science.7434010. [DOI] [PubMed] [Google Scholar]
  27. Pauli B., Weinstein R. S., Soble L. W., Alroy J. Freeze-fracture of monolayer cultures. J Cell Biol. 1977 Mar;72(3):763–769. doi: 10.1083/jcb.72.3.763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Peng H. B., Nakajima Y. Membrane particle aggregates in innervated and noninnervated cultures of Xenopus embryonic muscle cells. Proc Natl Acad Sci U S A. 1978 Jan;75(1):500–504. doi: 10.1073/pnas.75.1.500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Perrelet A., Garcia-Segura L. M., Singh A., Orgi L. Distribution of cytochemically detectable cholesterol in the electric organ of Torpedo marmorata. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2598–2602. doi: 10.1073/pnas.79.8.2598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Podleski T. R., Axelrod D., Ravdin P., Greenberg I., Johnson M. M., Salpeter M. M. Nerve extract induces increase and redistribution of acetylcholine receptors on cloned muscle cells. Proc Natl Acad Sci U S A. 1978 Apr;75(4):2035–2039. doi: 10.1073/pnas.75.4.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Popot J. L., Demel R. A., Sobel A., Van Deenen L. L., Changeux J. P. Interaction of the acetylcholine (nicotinic) receptor protein from Torpedo marmorata electric organ with monolayers of pure lipids. Eur J Biochem. 1978 Apr;85(1):27–42. doi: 10.1111/j.1432-1033.1978.tb12209.x. [DOI] [PubMed] [Google Scholar]
  32. Prives J., Fulton A. B., Penman S., Daniels M. P., Christian C. N. Interaction of the cytoskeletal framework with acetylcholine receptor on th surface of embryonic muscle cells in culture. J Cell Biol. 1982 Jan;92(1):231–236. doi: 10.1083/jcb.92.1.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pumplin D. W., Drachman D. B. Myasthenic patients' IgG causes redistribution of acetylcholine receptors: freeze-fracture studies. J Neurosci. 1983 Mar;3(3):576–584. doi: 10.1523/JNEUROSCI.03-03-00576.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Richler C., Yaffe D. The in vitro cultivation and differentiation capacities of myogenic cell lines. Dev Biol. 1970 Sep;23(1):1–22. doi: 10.1016/s0012-1606(70)80004-5. [DOI] [PubMed] [Google Scholar]
  35. Robinson J. M., Karnovsky M. J. Evaluation of the polyene antibiotic filipin as a cytochemical probe for membrane cholesterol. J Histochem Cytochem. 1980 Feb;28(2):161–168. doi: 10.1177/28.2.6766487. [DOI] [PubMed] [Google Scholar]
  36. Robinson J. M., Karnovsky M. J. Specializations in filopodial membranes at points of attachment to the substrate. J Cell Biol. 1980 Dec;87(3 Pt 1):562–568. doi: 10.1083/jcb.87.3.562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sealock R. Cytoplasmic surface structure in postsynaptic membranes from electric tissue visualized by tannic-acid-mediated negative contrasting. J Cell Biol. 1982 Feb;92(2):514–522. doi: 10.1083/jcb.92.2.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sealock R. Visualization at the mouse neuromuscular junction of a submembrane structure in common with Torpedo postsynaptic membranes. J Neurosci. 1982 Jul;2(7):918–923. doi: 10.1523/JNEUROSCI.02-07-00918.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sytkowski A. J., Vogel Z., Nirenberg M. W. Development of acetylcholine receptor clusters on cultured muscle cells. Proc Natl Acad Sci U S A. 1973 Jan;70(1):270–274. doi: 10.1073/pnas.70.1.270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Träuble H., Sackmann E. Studies of the crystalline-liquid crystalline phase transition of lipid model membranes. 3. Structure of a steroid-lecithin system below and above the lipid-phase transition. J Am Chem Soc. 1972 Jun 28;94(13):4499–4510. doi: 10.1021/ja00768a015. [DOI] [PubMed] [Google Scholar]
  41. Wolf D. E., Edidin M., Handyside A. H. Changes in the organization of the mouse egg plasma membrane upon fertilization and first cleavage: indications from the lateral diffusion rates of fluorescent lipid analogs. Dev Biol. 1981 Jul 15;85(1):195–198. doi: 10.1016/0012-1606(81)90250-5. [DOI] [PubMed] [Google Scholar]
  42. Yee A. G., Fischbach G. D., Karnovsky M. J. Clusters of intramembranous particles on cultured myotubes at sites that are highly sensitive to acetylcholine. Proc Natl Acad Sci U S A. 1978 Jun;75(6):3004–3008. doi: 10.1073/pnas.75.6.3004. [DOI] [PMC free article] [PubMed] [Google Scholar]

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