Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1986 Aug 1;103(2):535–544. doi: 10.1083/jcb.103.2.535

Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. II. Autoradiographic evidence for its uptake into motor nerves by acceptor-mediated endocytosis

PMCID: PMC2113823  PMID: 3015983

Abstract

Using pharmacological (Simpson, L.L., 1980, J. Pharmacol. Exp. Ther. 212:16-21) and autoradiographic techniques (Black, J.D., and J.O. Dolly, 1986, J. Cell Biol., 103:521-534), it has been shown that botulinum neurotoxin (BoNT) is translocated across the motor nerve terminal membrane to reach a postulated intraterminal target. In the present study, the nature of this uptake process was investigated using electron microscopic autoradiography. It was found that internalization is acceptor-mediated and that binding to specific cell surface acceptors involves the heavier chain of the toxin. In addition, uptake was shown to be energy and temperature-dependent and to be accelerated by nerve stimulation, a treatment which also shortens the time course of the toxin-induced neuroparalysis. These results, together with the observation that silver grains were often associated with endocytic structures within the nerve terminal, suggested that acceptor-mediated endocytosis is responsible for toxin uptake. This proposal is supported further by the fact that lysosomotropic agents, which are known to interfere with the endocytic pathway, retard the onset of BoNT-induced neuroparalysis and also affect the distribution of silver grains at nerve terminals treated with 125I-BoNT. Possible recycling of BoNT acceptors (an important aspect of acceptor-mediated endocytosis of toxins) at motor nerve terminals was indicated by comparing the extent of labeling in the presence and absence of metabolic inhibitors. On the basis of these collective results, it is concluded that BoNT is internalized by acceptor-mediated endocytosis and, hence, the data support the proposal that this toxin inhibits release of acetylcholine by interaction with an intracellular target.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BURGEN A. S. V., DICKENS F., ZATMAN L. J. The action of botulinum toxin on the neuro-muscular junction. J Physiol. 1949 Aug;109(1-2):10–24. doi: 10.1113/jphysiol.1949.sp004364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Black J. D., Dolly J. O. Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. I. Ultrastructural autoradiographic localization and quantitation of distinct membrane acceptors for types A and B on motor nerves. J Cell Biol. 1986 Aug;103(2):521–534. doi: 10.1083/jcb.103.2.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boquet P., Duflot E. Tetanus toxin fragment forms channels in lipid vesicles at low pH. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7614–7618. doi: 10.1073/pnas.79.24.7614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boquet P., Pappenheimer A. M., Jr Interaction of diphtheria toxin with mammalian cell membranes. J Biol Chem. 1976 Sep 25;251(18):5770–5778. [PubMed] [Google Scholar]
  5. Ceccarelli B., Hurlbut W. P., Mauro A. Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J Cell Biol. 1973 May;57(2):499–524. doi: 10.1083/jcb.57.2.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Collier R. J., Kandel J. Structure and activity of diphtheria toxin. I. Thiol-dependent dissociation of a fraction of toxin into enzymically active and inactive fragments. J Biol Chem. 1971 Mar 10;246(5):1496–1503. [PubMed] [Google Scholar]
  7. Dasgupta B. R., Sugiyama H. Molecular forms of neurotoxins in proteolytic Clostridium botulinum type B cultures. Infect Immun. 1976 Sep;14(3):680–686. doi: 10.1128/iai.14.3.680-686.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dolly J. O., Black J., Williams R. S., Melling J. Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization. Nature. 1984 Feb 2;307(5950):457–460. doi: 10.1038/307457a0. [DOI] [PubMed] [Google Scholar]
  9. Dolly J. O., Halliwell J. V., Black J. D., Williams R. S., Pelchen-Matthews A., Breeze A. L., Mehraban F., Othman I. B., Black A. R. Botulinum neurotoxin and dendrotoxin as probes for studies on transmitter release. J Physiol (Paris) 1984;79(4):280–303. [PubMed] [Google Scholar]
  10. Donovan J. J., Simon M. I., Montal M. Insertion of diphtheria toxin into and across membranes: role of phosphoinositide asymmetry. Nature. 1982 Aug 12;298(5875):669–672. doi: 10.1038/298669a0. [DOI] [PubMed] [Google Scholar]
  11. Dreyer F., Schmitt A. Transmitter release in tetanus and botulinum A toxin-poisoned mammalian motor endplates and its dependence on nerve stimulation and temperature. Pflugers Arch. 1983 Nov;399(3):228–234. doi: 10.1007/BF00656720. [DOI] [PubMed] [Google Scholar]
  12. Geuze H. J., Slot J. W., Strous G. J., Lodish H. F., Schwartz A. L. Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell. 1983 Jan;32(1):277–287. doi: 10.1016/0092-8674(83)90518-4. [DOI] [PubMed] [Google Scholar]
  13. Goldstein J. L., Anderson R. G., Brown M. S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. doi: 10.1038/279679a0. [DOI] [PubMed] [Google Scholar]
  14. HUGHES R., WHALER B. C. Influence of nerve-ending activity and of drugs on the rate of paralysis of rat diaphragm preparations by Cl. botulinum type A toxin. J Physiol. 1962 Feb;160:221–233. doi: 10.1113/jphysiol.1962.sp006843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Helenius A., Kartenbeck J., Simons K., Fries E. On the entry of Semliki forest virus into BHK-21 cells. J Cell Biol. 1980 Feb;84(2):404–420. doi: 10.1083/jcb.84.2.404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hoch D. H., Romero-Mira M., Ehrlich B. E., Finkelstein A., DasGupta B. R., Simpson L. L. Channels formed by botulinum, tetanus, and diphtheria toxins in planar lipid bilayers: relevance to translocation of proteins across membranes. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1692–1696. doi: 10.1073/pnas.82.6.1692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kozaki S. Interaction of botulinum type A, B and E derivative toxins with synaptosomes of rat brain. Naunyn Schmiedebergs Arch Pharmacol. 1979 Jul;308(1):67–70. doi: 10.1007/BF00499721. [DOI] [PubMed] [Google Scholar]
  18. Kozaki S., Togashi S., Sakaguchi G. Separation of Clostridium botulinum type A derivative toxin into two fragments. Jpn J Med Sci Biol. 1981 Apr;34(2):61–68. doi: 10.7883/yoken1952.34.61. [DOI] [PubMed] [Google Scholar]
  19. Leppla S., Dorland R. B., Middlebrook J. L. Inhibition of diphtheria toxin degradation and cytotoxic action by chloroquine. J Biol Chem. 1980 Mar 25;255(6):2247–2250. [PubMed] [Google Scholar]
  20. Marsh M., Bolzau E., Helenius A. Penetration of Semliki Forest virus from acidic prelysosomal vacuoles. Cell. 1983 Mar;32(3):931–940. doi: 10.1016/0092-8674(83)90078-8. [DOI] [PubMed] [Google Scholar]
  21. Matlin K. S., Reggio H., Helenius A., Simons K. Infectious entry pathway of influenza virus in a canine kidney cell line. J Cell Biol. 1981 Dec;91(3 Pt 1):601–613. doi: 10.1083/jcb.91.3.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Matsuda M., Yoneda M. Isolation and purification of two antigenically active, "complimentary" polypeptide fragments of tetanus neurotoxin. Infect Immun. 1975 Nov;12(5):1147–1153. doi: 10.1128/iai.12.5.1147-1153.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maxfield F. R., Willingham M. C., Davies P. J., Pastan I. Amines inhibit the clustering of alpha2-macroglobulin and EGF on the fibroblast cell surface. Nature. 1979 Feb 22;277(5698):661–663. doi: 10.1038/277661a0. [DOI] [PubMed] [Google Scholar]
  24. Mellman I., Plutner H., Ukkonen P. Internalization and rapid recycling of macrophage Fc receptors tagged with monovalent antireceptor antibody: possible role of a prelysosomal compartment. J Cell Biol. 1984 Apr;98(4):1163–1169. doi: 10.1083/jcb.98.4.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Olsnes S., Sandvig K., Eiklid K., Pihl A. Properties and action mechanism of the toxic lectin modeccin: interaction with cell lines resistant to modeccin, abrin, and ricin. J Supramol Struct. 1978;9(1):15–25. doi: 10.1002/jss.400090103. [DOI] [PubMed] [Google Scholar]
  26. Pastan I. H., Willingham M. C. Journey to the center of the cell: role of the receptosome. Science. 1981 Oct 30;214(4520):504–509. doi: 10.1126/science.6170111. [DOI] [PubMed] [Google Scholar]
  27. Pastan I. H., Willingham M. C. Receptor-mediated endocytosis of hormones in cultured cells. Annu Rev Physiol. 1981;43:239–250. doi: 10.1146/annurev.ph.43.030181.001323. [DOI] [PubMed] [Google Scholar]
  28. Pearse B. M., Bretscher M. S. Membrane recycling by coated vesicles. Annu Rev Biochem. 1981;50:85–101. doi: 10.1146/annurev.bi.50.070181.000505. [DOI] [PubMed] [Google Scholar]
  29. Pearse B. M. Clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1255–1259. doi: 10.1073/pnas.73.4.1255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. ROTH T. F., PORTER K. R. YOLK PROTEIN UPTAKE IN THE OOCYTE OF THE MOSQUITO AEDES AEGYPTI. L. J Cell Biol. 1964 Feb;20:313–332. doi: 10.1083/jcb.20.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sandvig K., Olsnes S. Diphtheria toxin entry into cells is facilitated by low pH. J Cell Biol. 1980 Dec;87(3 Pt 1):828–832. doi: 10.1083/jcb.87.3.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sandvig K., Olsnes S. Entry of the toxic proteins abrin, modeccin, ricin, and diphtheria toxin into cells. I. Requirement for calcium. J Biol Chem. 1982 Jul 10;257(13):7495–7503. [PubMed] [Google Scholar]
  33. Sandvig K., Olsnes S., Pihl A. Inhibitory effect of ammonium chloride and chloroquine on the entry of the toxic lectin modeccin into HeLa cells. Biochem Biophys Res Commun. 1979 Sep 27;90(2):648–655. doi: 10.1016/0006-291x(79)91284-1. [DOI] [PubMed] [Google Scholar]
  34. Sandvig K., Olsnes S. Rapid entry of nicked diphtheria toxin into cells at low pH. Characterization of the entry process and effects of low pH on the toxin molecule. J Biol Chem. 1981 Sep 10;256(17):9068–9076. [PubMed] [Google Scholar]
  35. Schneider Y. J., Trouet A. Effect of chloroquine and methylamine on endocytosis of fluorescein-labelled controlled IgG and of anti-(plasma membrane) IgG by cultured fibroblasts. Eur J Biochem. 1981 Aug;118(1):33–38. doi: 10.1111/j.1432-1033.1981.tb05482.x. [DOI] [PubMed] [Google Scholar]
  36. Simpson L. L. Ammonium chloride and methylamine hydrochloride antagonize clostridial neurotoxins. J Pharmacol Exp Ther. 1983 Jun;225(3):546–552. [PubMed] [Google Scholar]
  37. Simpson L. L. Fragment C of tetanus toxin antagonizes the neuromuscular blocking properties of native tetanus toxin. J Pharmacol Exp Ther. 1984 Mar;228(3):600–604. [PubMed] [Google Scholar]
  38. Simpson L. L. Kinetic studies on the interaction between botulinum toxin type A and the cholinergic neuromuscular junction. J Pharmacol Exp Ther. 1980 Jan;212(1):16–21. [PubMed] [Google Scholar]
  39. Simpson L. L. Studies on the binding of botulinum toxin type A to the rat phrenic nerve-hemidiaphragm preparation. Neuropharmacology. 1974 Aug;13(8):683–691. doi: 10.1016/0028-3908(74)90014-8. [DOI] [PubMed] [Google Scholar]
  40. Simpson L. L. The interaction between aminoquinolines and presynaptically acting neurotoxins. J Pharmacol Exp Ther. 1982 Jul;222(1):43–48. [PubMed] [Google Scholar]
  41. Simpson L. L. The origin, structure, and pharmacological activity of botulinum toxin. Pharmacol Rev. 1981 Sep;33(3):155–188. [PubMed] [Google Scholar]
  42. Steinman R. M., Mellman I. S., Muller W. A., Cohn Z. A. Endocytosis and the recycling of plasma membrane. J Cell Biol. 1983 Jan;96(1):1–27. doi: 10.1083/jcb.96.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tycko B., Maxfield F. R. Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin. Cell. 1982 Mar;28(3):643–651. doi: 10.1016/0092-8674(82)90219-7. [DOI] [PubMed] [Google Scholar]
  44. Vidal G. The oldest eukaryotic cells. Sci Am. 1984 Feb;250(2):48–57. doi: 10.1038/scientificamerican0284-48. [DOI] [PubMed] [Google Scholar]
  45. Williams R. S., Tse C. K., Dolly J. O., Hambleton P., Melling J. Radioiodination of botulinum neurotoxin type A with retention of biological activity and its binding to brain synaptosomes. Eur J Biochem. 1983 Mar 15;131(2):437–445. doi: 10.1111/j.1432-1033.1983.tb07282.x. [DOI] [PubMed] [Google Scholar]
  46. de Duve C. Lysosomes revisited. Eur J Biochem. 1983 Dec 15;137(3):391–397. doi: 10.1111/j.1432-1033.1983.tb07841.x. [DOI] [PubMed] [Google Scholar]
  47. de Duve C., de Barsy T., Poole B., Trouet A., Tulkens P., Van Hoof F. Commentary. Lysosomotropic agents. Biochem Pharmacol. 1974 Sep 15;23(18):2495–2531. doi: 10.1016/0006-2952(74)90174-9. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES