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. 1996 Jun 2;133(6):1237–1250. doi: 10.1083/jcb.133.6.1237

The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin

PMCID: PMC2120898  PMID: 8682861

Abstract

Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.

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Selected References

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  1. Allen R. D., Schroeder C. C., Fok A. K. Endosomal system of Paramecium: coated pits to early endosomes. J Cell Sci. 1992 Feb;101(Pt 2):449–461. doi: 10.1242/jcs.101.2.449. [DOI] [PubMed] [Google Scholar]
  2. Cameron P. L., Südhof T. C., Jahn R., De Camilli P. Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis. J Cell Biol. 1991 Oct;115(1):151–164. doi: 10.1083/jcb.115.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carter L. L., Redelmeier T. E., Woollenweber L. A., Schmid S. L. Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis. J Cell Biol. 1993 Jan;120(1):37–45. doi: 10.1083/jcb.120.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Cupers P., Veithen A., Kiss A., Baudhuin P., Courtoy P. J. Clathrin polymerization is not required for bulk-phase endocytosis in rat fetal fibroblasts. J Cell Biol. 1994 Nov;127(3):725–735. doi: 10.1083/jcb.127.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Damke H., Baba T., Warnock D. E., Schmid S. L. Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J Cell Biol. 1994 Nov;127(4):915–934. doi: 10.1083/jcb.127.4.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. De Camilli P., Harris S. M., Jr, Huttner W. B., Greengard P. Synapsin I (Protein I), a nerve terminal-specific phosphoprotein. II. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes. J Cell Biol. 1983 May;96(5):1355–1373. doi: 10.1083/jcb.96.5.1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. De Camilli P., Takei K., McPherson P. S. The function of dynamin in endocytosis. Curr Opin Neurobiol. 1995 Oct;5(5):559–565. doi: 10.1016/0959-4388(95)80059-x. [DOI] [PubMed] [Google Scholar]
  9. De Camilli P., Takei K. Molecular mechanisms in synaptic vesicle endocytosis and recycling. Neuron. 1996 Mar;16(3):481–486. doi: 10.1016/s0896-6273(00)80068-9. [DOI] [PubMed] [Google Scholar]
  10. Donaldson J. G., Finazzi D., Klausner R. D. Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature. 1992 Nov 26;360(6402):350–352. doi: 10.1038/360350a0. [DOI] [PubMed] [Google Scholar]
  11. Fesce R., Grohovaz F., Valtorta F., Meldolesi J. Neurotransmitter release: fusion or 'kiss-and-run'? Trends Cell Biol. 1994 Jan;4(1):1–4. doi: 10.1016/0962-8924(94)90025-6. [DOI] [PubMed] [Google Scholar]
  12. Fried R. C., Blaustein M. P. Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes). J Cell Biol. 1978 Sep;78(3):685–700. doi: 10.1083/jcb.78.3.685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Geuze H. J., Slot J. W., Strous G. J., Peppard J., von Figura K., Hasilik A., Schwartz A. L. Intracellular receptor sorting during endocytosis: comparative immunoelectron microscopy of multiple receptors in rat liver. Cell. 1984 May;37(1):195–204. doi: 10.1016/0092-8674(84)90315-5. [DOI] [PubMed] [Google Scholar]
  14. Hansen S. H., Sandvig K., van Deurs B. Molecules internalized by clathrin-independent endocytosis are delivered to endosomes containing transferrin receptors. J Cell Biol. 1993 Oct;123(1):89–97. doi: 10.1083/jcb.123.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Helms J. B., Rothman J. E. Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF. Nature. 1992 Nov 26;360(6402):352–354. doi: 10.1038/360352a0. [DOI] [PubMed] [Google Scholar]
  16. Herskovits J. S., Burgess C. C., Obar R. A., Vallee R. B. Effects of mutant rat dynamin on endocytosis. J Cell Biol. 1993 Aug;122(3):565–578. doi: 10.1083/jcb.122.3.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Heuser J. E., Reese T. S. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J Cell Biol. 1973 May;57(2):315–344. doi: 10.1083/jcb.57.2.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hinshaw J. E., Schmid S. L. Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature. 1995 Mar 9;374(6518):190–192. doi: 10.1038/374190a0. [DOI] [PubMed] [Google Scholar]
  19. Huttner W. B., Schiebler W., Greengard P., De Camilli P. Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation. J Cell Biol. 1983 May;96(5):1374–1388. doi: 10.1083/jcb.96.5.1374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Killisch I., Steinlein P., Römisch K., Hollinshead R., Beug H., Griffiths G. Characterization of early and late endocytic compartments of the transferrin cycle. Transferrin receptor antibody blocks erythroid differentiation by trapping the receptor in the early endosome. J Cell Sci. 1992 Sep;103(Pt 1):211–232. doi: 10.1242/jcs.103.1.211. [DOI] [PubMed] [Google Scholar]
  21. Koenig J. H., Ikeda K. Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval. J Neurosci. 1989 Nov;9(11):3844–3860. doi: 10.1523/JNEUROSCI.09-11-03844.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kraszewski K., Mundigl O., Daniell L., Verderio C., Matteoli M., De Camilli P. Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin. J Neurosci. 1995 Jun;15(6):4328–4342. doi: 10.1523/JNEUROSCI.15-06-04328.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lamaze C., Schmid S. L. The emergence of clathrin-independent pinocytic pathways. Curr Opin Cell Biol. 1995 Aug;7(4):573–580. doi: 10.1016/0955-0674(95)80015-8. [DOI] [PubMed] [Google Scholar]
  24. Linstedt A. D., Kelly R. B. Synaptophysin is sorted from endocytotic markers in neuroendocrine PC12 cells but not transfected fibroblasts. Neuron. 1991 Aug;7(2):309–317. doi: 10.1016/0896-6273(91)90269-6. [DOI] [PubMed] [Google Scholar]
  25. Lippincott-Schwartz J., Yuan L., Tipper C., Amherdt M., Orci L., Klausner R. D. Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell. 1991 Nov 1;67(3):601–616. doi: 10.1016/0092-8674(91)90534-6. [DOI] [PubMed] [Google Scholar]
  26. Mackay D. J., Nobes C. D., Hall A. The Rho's progress: a potential role during neuritogenesis for the Rho family of GTPases. Trends Neurosci. 1995 Nov;18(11):496–501. doi: 10.1016/0166-2236(95)92773-j. [DOI] [PubMed] [Google Scholar]
  27. Matlin K. S., Reggio H., Helenius A., Simons K. Pathway of vesicular stomatitis virus entry leading to infection. J Mol Biol. 1982 Apr 15;156(3):609–631. doi: 10.1016/0022-2836(82)90269-8. [DOI] [PubMed] [Google Scholar]
  28. Matteoli M., Takei K., Perin M. S., Südhof T. C., De Camilli P. Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons. J Cell Biol. 1992 May;117(4):849–861. doi: 10.1083/jcb.117.4.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Maycox P. R., Link E., Reetz A., Morris S. A., Jahn R. Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling. J Cell Biol. 1992 Sep;118(6):1379–1388. doi: 10.1083/jcb.118.6.1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McPherson P. S., Czernik A. J., Chilcote T. J., Onofri F., Benfenati F., Greengard P., Schlessinger J., De Camilli P. Interaction of Grb2 via its Src homology 3 domains with synaptic proteins including synapsin I. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6486–6490. doi: 10.1073/pnas.91.14.6486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mellman I. Enigma variations: protein mediators of membrane fusion. Cell. 1995 Sep 22;82(6):869–872. doi: 10.1016/0092-8674(95)90018-7. [DOI] [PubMed] [Google Scholar]
  32. Miller T. M., Heuser J. E. Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction. J Cell Biol. 1984 Feb;98(2):685–698. doi: 10.1083/jcb.98.2.685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mundigl O., Matteoli M., Daniell L., Thomas-Reetz A., Metcalf A., Jahn R., De Camilli P. Synaptic vesicle proteins and early endosomes in cultured hippocampal neurons: differential effects of Brefeldin A in axon and dendrites. J Cell Biol. 1993 Sep;122(6):1207–1221. doi: 10.1083/jcb.122.6.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mundigl O., Verderio C., Krazewski K., De Camilli P., Matteoli M. A radioimmunoassay to monitor synaptic activity in hippocampal neurons in vitro. Eur J Cell Biol. 1995 Mar;66(3):246–256. [PubMed] [Google Scholar]
  35. Orci L., Glick B. S., Rothman J. E. A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack. Cell. 1986 Jul 18;46(2):171–184. doi: 10.1016/0092-8674(86)90734-8. [DOI] [PubMed] [Google Scholar]
  36. Orci L., Malhotra V., Amherdt M., Serafini T., Rothman J. E. Dissection of a single round of vesicular transport: sequential intermediates for intercisternal movement in the Golgi stack. Cell. 1989 Feb 10;56(3):357–368. doi: 10.1016/0092-8674(89)90239-0. [DOI] [PubMed] [Google Scholar]
  37. Orci L., Tagaya M., Amherdt M., Perrelet A., Donaldson J. G., Lippincott-Schwartz J., Klausner R. D., Rothman J. E. Brefeldin A, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on Golgi cisternae. Cell. 1991 Mar 22;64(6):1183–1195. doi: 10.1016/0092-8674(91)90273-2. [DOI] [PubMed] [Google Scholar]
  38. Pearse B. M., Robinson M. S. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 1990;6:151–171. doi: 10.1146/annurev.cb.06.110190.001055. [DOI] [PubMed] [Google Scholar]
  39. Pfeffer S. R., Kelly R. B. The subpopulation of brain coated vesicles that carries synaptic vesicle proteins contains two unique polypeptides. Cell. 1985 Apr;40(4):949–957. doi: 10.1016/0092-8674(85)90355-1. [DOI] [PubMed] [Google Scholar]
  40. Rabinowitz S., Horstmann H., Gordon S., Griffiths G. Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages. J Cell Biol. 1992 Jan;116(1):95–112. doi: 10.1083/jcb.116.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Robinson M. S., Kreis T. E. Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of brefeldin A and G protein activators. Cell. 1992 Apr 3;69(1):129–138. doi: 10.1016/0092-8674(92)90124-u. [DOI] [PubMed] [Google Scholar]
  42. Robinson M. S., Watts C., Zerial M. Membrane dynamics in endocytosis. Cell. 1996 Jan 12;84(1):13–21. doi: 10.1016/s0092-8674(00)80988-5. [DOI] [PubMed] [Google Scholar]
  43. Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
  44. Schekman R., Orci L. Coat proteins and vesicle budding. Science. 1996 Mar 15;271(5255):1526–1533. doi: 10.1126/science.271.5255.1526. [DOI] [PubMed] [Google Scholar]
  45. Schmalzing G., Richter H. P., Hansen A., Schwarz W., Just I., Aktories K. Involvement of the GTP binding protein Rho in constitutive endocytosis in Xenopus laevis oocytes. J Cell Biol. 1995 Sep;130(6):1319–1332. doi: 10.1083/jcb.130.6.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schweizer F. E., Betz H., Augustine G. J. From vesicle docking to endocytosis: intermediate reactions of exocytosis. Neuron. 1995 Apr;14(4):689–696. doi: 10.1016/0896-6273(95)90213-9. [DOI] [PubMed] [Google Scholar]
  47. Seaman M. N., Ball C. L., Robinson M. S. Targeting and mistargeting of plasma membrane adaptors in vitro. J Cell Biol. 1993 Dec;123(5):1093–1105. doi: 10.1083/jcb.123.5.1093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shpetner H. S., Vallee R. B. Identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules. Cell. 1989 Nov 3;59(3):421–432. doi: 10.1016/0092-8674(89)90027-5. [DOI] [PubMed] [Google Scholar]
  49. Stoorvogel W., Oorschot V., Geuze H. J. A novel class of clathrin-coated vesicles budding from endosomes. J Cell Biol. 1996 Jan;132(1-2):21–33. doi: 10.1083/jcb.132.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Südhof T. C., De Camilli P., Niemann H., Jahn R. Membrane fusion machinery: insights from synaptic proteins. Cell. 1993 Oct 8;75(1):1–4. [PubMed] [Google Scholar]
  51. Takei K., McPherson P. S., Schmid S. L., De Camilli P. Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma S in nerve terminals. Nature. 1995 Mar 9;374(6518):186–190. doi: 10.1038/374186a0. [DOI] [PubMed] [Google Scholar]
  52. Takei K., Stukenbrok H., Metcalf A., Mignery G. A., Südhof T. C., Volpe P., De Camilli P. Ca2+ stores in Purkinje neurons: endoplasmic reticulum subcompartments demonstrated by the heterogeneous distribution of the InsP3 receptor, Ca(2+)-ATPase, and calsequestrin. J Neurosci. 1992 Feb;12(2):489–505. doi: 10.1523/JNEUROSCI.12-02-00489.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Thomas-Reetz A. C., De Camilli P. A role for synaptic vesicles in non-neuronal cells: clues from pancreatic beta cells and from chromaffin cells. FASEB J. 1994 Feb;8(2):209–216. doi: 10.1096/fasebj.8.2.7907072. [DOI] [PubMed] [Google Scholar]
  54. Tijssen P., Kurstak E. Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays. Anal Biochem. 1984 Feb;136(2):451–457. doi: 10.1016/0003-2697(84)90243-4. [DOI] [PubMed] [Google Scholar]
  55. Tooze S. A., Weiss U., Huttner W. B. Requirement for GTP hydrolysis in the formation of secretory vesicles. Nature. 1990 Sep 13;347(6289):207–208. doi: 10.1038/347207a0. [DOI] [PubMed] [Google Scholar]
  56. Whitney J. A., Gomez M., Sheff D., Kreis T. E., Mellman I. Cytoplasmic coat proteins involved in endosome function. Cell. 1995 Dec 1;83(5):703–713. doi: 10.1016/0092-8674(95)90183-3. [DOI] [PubMed] [Google Scholar]
  57. Wong D. H., Brodsky F. M. 100-kD proteins of Golgi- and trans-Golgi network-associated coated vesicles have related but distinct membrane binding properties. J Cell Biol. 1992 Jun;117(6):1171–1179. doi: 10.1083/jcb.117.6.1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wood S. A., Park J. E., Brown W. J. Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell. 1991 Nov 1;67(3):591–600. doi: 10.1016/0092-8674(91)90533-5. [DOI] [PubMed] [Google Scholar]
  59. Ye W., Lafer E. M. Bacterially expressed F1-20/AP-3 assembles clathrin into cages with a narrow size distribution: implications for the regulation of quantal size during neurotransmission. J Neurosci Res. 1995 May 1;41(1):15–26. doi: 10.1002/jnr.490410104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. van der Bliek A. M., Redelmeier T. E., Damke H., Tisdale E. J., Meyerowitz E. M., Schmid S. L. Mutations in human dynamin block an intermediate stage in coated vesicle formation. J Cell Biol. 1993 Aug;122(3):553–563. doi: 10.1083/jcb.122.3.553. [DOI] [PMC free article] [PubMed] [Google Scholar]

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