Abstract
Coated vesicles are present in the myoplasm of embryonic chick myotubes grown in vitro. They are most numerous beneath regions of the surface membrane that contain a high density of acetylcholine receptors (AChR). Prolonged exposure of myotubes to saline extract of chick brain increases the number of intracellular AChR and the number of coated vesicles. This suggests that coated vesicles contain AChR, and this hypothesis was tested with horseradish peroxidase-alpha-bungarotoxin (HRP-alpha BTX) conjugates. The conjugates enter saponin-permeabilized cells and, as judged by the inhibition of [125I] alpha BTX binding, they label the entire intracellular AChR pool. Approximately 50% of the coated vesicles contained HRP-alpha BTX reaction product. In addition, reaction product was detected in Golgi cisternae and along membranes that bound a subsurface tubulovesicular network. The majority of labeled vesicles are probably involved in exocytosis rather than endocytosis of AChR because very few coated vesicles were labeled when HRP-alpha BTX was added to the medium bathing intact cells. Moreover, inhibition of protein synthesis with puromycin resulted in a large decrease in the number of labeled vesicles. These results suggest that a subpopulation of coated vesicles ferry newly synthesized AChR to the cell surface.
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- Altman J. Coated vesicles and synaptogenesis. A developmental study in the cerebellar cortex of the rat. Brain Res. 1971 Jul 23;30(2):311–322. doi: 10.1016/0006-8993(71)90081-3. [DOI] [PubMed] [Google Scholar]
- 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]
- Anderson M. J., Cohen M. W., Zorychta E. Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. J Physiol. 1977 Jul;268(3):731–756. doi: 10.1113/jphysiol.1977.sp011879. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Buc-Caron M. H., Nystrom P., Fischbach G. D. Induction of acetylcholine receptor synthesis and aggregation: partial purification of low-molecular-weight activity. Dev Biol. 1983 Feb;95(2):378–386. doi: 10.1016/0012-1606(83)90039-8. [DOI] [PubMed] [Google Scholar]
- 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]
- Daniels M. P., Vogel Z. Immunoperoxidase staining of alpha-bungarotoxin binding sites in muscle endplates shows distribution of acetylcholine receptors. Nature. 1975 Mar 27;254(5498):339–341. doi: 10.1038/254339a0. [DOI] [PubMed] [Google Scholar]
- Devreotes P. N., Fambrough D. M. Acetylcholine receptor turnover in membranes of developing muscle fibers. J Cell Biol. 1975 May;65(2):335–358. doi: 10.1083/jcb.65.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Devreotes P. N., Fambrough D. M. Turnover of acetylcholine receptors in skeletal muscle. Cold Spring Harb Symp Quant Biol. 1976;40:237–251. doi: 10.1101/sqb.1976.040.01.025. [DOI] [PubMed] [Google Scholar]
- Devreotes P. N., Gardner J. M., Fambrough D. M. Kinetics of biosynthesis of acetylcholine receptor and subsequent incorporation into plasma membrane of cultured chick skeletal muscle. Cell. 1977 Mar;10(3):365–373. doi: 10.1016/0092-8674(77)90023-x. [DOI] [PubMed] [Google Scholar]
- Fambrough D. M., Devreotes P. N. Newly synthesized acetylcholine receptors are located in the Golgi apparatus. J Cell Biol. 1978 Jan;76(1):237–244. doi: 10.1083/jcb.76.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fertuck H. C., Salpeter M. M. Localization of acetylcholine receptor by 125I-labeled alpha-bungarotoxin binding at mouse motor endplates. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1376–1378. doi: 10.1073/pnas.71.4.1376. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fertuck H. C., Woodward W., Salpeter M. M. In vivo recovery of muscle contraction after alpha-bungarotoxin binding. J Cell Biol. 1975 Jul;66(1):209–213. doi: 10.1083/jcb.66.1.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fischbach G. D. Synapse formation between dissociated nerve and muscle cells in low density cell cultures. Dev Biol. 1972 Jun;28(2):407–429. doi: 10.1016/0012-1606(72)90023-1. [DOI] [PubMed] [Google Scholar]
- Frank E., Fischbach G. D. ACh receptors accumulate at newly formed nerve-muscle synapses in vitro. Soc Gen Physiol Ser. 1977;32:285–291. [PubMed] [Google Scholar]
- Frank E., Fischbach G. D. Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses. J Cell Biol. 1979 Oct;83(1):143–158. doi: 10.1083/jcb.83.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
- Ishikawa H. Formation of elaborate networks of T-system tubules in cultured skeletal muscle with special reference to the T-system formation. J Cell Biol. 1968 Jul;38(1):51–66. doi: 10.1083/jcb.38.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jessell T. M., Siegel R. E., Fischbach G. D. Induction of acetylcholine receptors on cultured skeletal muscle by a factor extracted from brain and spinal cord. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5397–5401. doi: 10.1073/pnas.76.10.5397. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuffler S. W., Yoshikami D. The distribution of acetylcholine sensitivity at the post-synaptic membrane of vertebrate skeletal twitch muscles: iontophoretic mapping in the micron range. J Physiol. 1975 Jan;244(3):703–730. doi: 10.1113/jphysiol.1975.sp010821. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lentz T. L., Chester J. Localization of acetylcholine receptors in central synapses. J Cell Biol. 1977 Oct;75(1):258–267. doi: 10.1083/jcb.75.1.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
- MILEDI R. Junctional and extra-junctional acetylcholine receptors in skeletal muscle fibres. J Physiol. 1960 Apr;151:24–30. [PMC free article] [PubMed] [Google Scholar]
- Merlie J. P., Heinemann S., Lindstrom J. M. Acetylcholine receptor degradation in adult rat diaphragms in organ culture and the effect of anti-acetylcholine receptor antibodies. J Biol Chem. 1979 Jul 25;254(14):6320–6327. [PubMed] [Google Scholar]
- Novikoff A. B., Novikoff P. M., Quintana N., Davis C. Diffusion artificats in 3,3'-diaminobenzidine cytochemistry. J Histochem Cytochem. 1972 Sep;20(9):745–749. doi: 10.1177/20.9.745. [DOI] [PubMed] [Google Scholar]
- Orida N., Poo M. M. Electrophoretic movement and localisation of acetylcholine receptors in the embryonic muscle cell membrane. Nature. 1978 Sep 7;275(5675):31–35. doi: 10.1038/275031a0. [DOI] [PubMed] [Google Scholar]
- Peng H. B., Nakajima Y., Bridgman P. C. Development of the postsynaptic membrane in Xenopus neuromuscular cultures observed by freeze-fracture and thin-section electron microscopy. Brain Res. 1980 Aug 25;196(1):11–31. doi: 10.1016/0006-8993(80)90713-1. [DOI] [PubMed] [Google Scholar]
- 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]
- Rees R. P., Bunge M. B., Bunge R. P. Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture. J Cell Biol. 1976 Feb;68(2):240–263. doi: 10.1083/jcb.68.2.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothman J. E., Bursztyn-Pettegrew H., Fine R. E. Transport of the membrane glycoprotein of vesicular stomatitis virus to the cell surface in two stages by clathrin-coated vesicles. J Cell Biol. 1980 Jul;86(1):162–171. doi: 10.1083/jcb.86.1.162. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothman J. E., Fine R. E. Coated vesicles transport newly synthesized membrane glycoproteins from endoplasmic reticulum to plasma membrane in two successive stages. Proc Natl Acad Sci U S A. 1980 Feb;77(2):780–784. doi: 10.1073/pnas.77.2.780. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Salpeter M. M., Plattner H., Rogers A. W. Quantitative assay of esterases in end plates of mouse diaphragm by electron microscope autoradiography. J Histochem Cytochem. 1972 Dec;20(12):1059–1068. doi: 10.1177/20.12.1059. [DOI] [PubMed] [Google Scholar]
- Schuetze S. M., Frank E. F., Fischbach G. D. Channel open time and metabolic stability of synaptic and extrasynaptic acetylcholine receptors on cultured chick myotubes. Proc Natl Acad Sci U S A. 1978 Jan;75(1):520–523. doi: 10.1073/pnas.75.1.520. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stelzner D. J., Martin A. H., Scott G. L. Early stages of synaptogenesis in the cervical spinal cord of the chick embryo. Z Zellforsch Mikrosk Anat. 1973;138(4):475–488. doi: 10.1007/BF00572291. [DOI] [PubMed] [Google Scholar]
- Ternynck T., Avrameas S. Polyacrylamide-protein immunoadsorbents prepared with glutaraldehyde. FEBS Lett. 1972 Jun 1;23(1):24–28. doi: 10.1016/0014-5793(72)80274-6. [DOI] [PubMed] [Google Scholar]
- Vogel Z., Maloney G. J., Ling A., Daniels M. P. Identification of synaptic acetylcholine receptor sites in retina with peroxidase-labeled alpha-bungarotoxin. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3268–3272. doi: 10.1073/pnas.74.8.3268. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weibel E. R. Stereological principles for morphometry in electron microscopic cytology. Int Rev Cytol. 1969;26:235–302. doi: 10.1016/s0074-7696(08)61637-x. [DOI] [PubMed] [Google Scholar]
- 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]