PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
 
J Cell Biol. 1995 December 2; 131(6): 1747–1758.
PMCID: PMC2120667

Calcium-regulated exocytosis is required for cell membrane resealing

Abstract

Using confocal microscopy, we visualized exocytosis during membrane resealing in sea urchin eggs and embryos. Upon wounding by a laser beam, both eggs and embryos showed a rapid burst of localized Ca(2+)- regulated exocytosis. The rate of exocytosis was correlated quantitatively with successfully resealing. In embryos, whose activated surfaces must first dock vesicles before fusion, exocytosis and membrane resealing were inhibited by neurotoxins that selectively cleave the SNARE complex proteins, synaptobrevin, SNAP-25, and syntaxin. In eggs, whose cortical vesicles are already docked, vesicles could be reversibly undocked with externally applied stachyose. If cortical vesicles were undocked both exocytosis and plasma membrane resealing were completely inhibited. When cortical vesicles were transiently undocked, exposure to tetanus toxin and botulinum neurotoxin type C1 rendered them no longer competent for resealing, although botulinum neurotoxin type A was still ineffective. Cortical vesicles transiently undocked in the presence of tetanus toxin were subsequently fusion incompetent although to a large extent they retained their ability to redock when stachyose was diluted. We conclude that addition of internal membranes by exocytosis is required and that a SNARE-like complex plays differential roles in vesicle docking and fusion for the repair of disrupted plasma membrane.

Full Text

The Full Text of this article is available as a PDF (3.4M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Baitinger C, Alderton J, Poenie M, Schulman H, Steinhardt RA. Multifunctional Ca2+/calmodulin-dependent protein kinase is necessary for nuclear envelope breakdown. J Cell Biol. 1990 Nov;111(5 Pt 1):1763–1773. [PMC free article] [PubMed]
  • Bennett MK, Calakos N, Scheller RH. Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science. 1992 Jul 10;257(5067):255–259. [PubMed]
  • Bennett MK, Scheller RH. Molecular correlates of synaptic vesicle docking and fusion. Curr Opin Neurobiol. 1994 Jun;4(3):324–329. [PubMed]
  • Betz WJ, Bewick GS. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science. 1992 Jan 10;255(5041):200–203. [PubMed]
  • Betz WJ, Mao F, Bewick GS. Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J Neurosci. 1992 Feb;12(2):363–375. [PubMed]
  • Billah MM, Finean JB, Coleman R, Michell RH. Permeability characteristics of erythrocyte ghosts prepared under isoionic conditions by a glycol-induced osmotic lysis. Biochim Biophys Acta. 1977 Mar 17;465(3):515–526. [PubMed]
  • Binz T, Blasi J, Yamasaki S, Baumeister A, Link E, Südhof TC, Jahn R, Niemann H. Proteolysis of SNAP-25 by types E and A botulinal neurotoxins. J Biol Chem. 1994 Jan 21;269(3):1617–1620. [PubMed]
  • Bodemann H, Passow H. Factors controlling the resealing of the membrane of human erythrocyte ghosts after hypotonic hemolysis. J Membr Biol. 1972;8(1):1–26. [PubMed]
  • Celis JE. Microinjection of somatic cells with micropipettes: comparison with other transfer techniques. Biochem J. 1984 Oct 15;223(2):281–291. [PubMed]
  • Chandler DE, Heuser J. Membrane fusion during secretion: cortical granule exocytosis in sex urchin eggs as studied by quick-freezing and freeze-fracture. J Cell Biol. 1979 Oct;83(1):91–108. [PMC free article] [PubMed]
  • Chandler DE, Whitaker M, Zimmerberg J. High molecular weight polymers block cortical granule exocytosis in sea urchin eggs at the level of granule matrix disassembly. J Cell Biol. 1989 Sep;109(3):1269–1278. [PMC free article] [PubMed]
  • Chang DC, Reese TS. Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. Biophys J. 1990 Jul;58(1):1–12. [PubMed]
  • Chilcote TJ, Galli T, Mundigl O, Edelmann L, McPherson PS, Takei K, De Camilli P. Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells. J Cell Biol. 1995 Apr;129(1):219–231. [PMC free article] [PubMed]
  • Clarke MS, Khakee R, McNeil PL. Loss of cytoplasmic basic fibroblast growth factor from physiologically wounded myofibers of normal and dystrophic muscle. J Cell Sci. 1993 Sep;106(Pt 1):121–133. [PubMed]
  • Dan Y, Poo MM. Quantal transmitter secretion from myocytes loaded with acetylcholine. Nature. 1992 Oct 22;359(6397):733–736. [PubMed]
  • Dayanithi G, Ahnert-Hilger G, Weller U, Nordmann JJ, Gratzl M. Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin. Neuroscience. 1990;39(3):711–715. [PubMed]
  • de Paiva A, Dolly JO. Light chain of botulinum neurotoxin is active in mammalian motor nerve terminals when delivered via liposomes. FEBS Lett. 1990 Dec 17;277(1-2):171–174. [PubMed]
  • DeBello WM, Betz H, Augustine GJ. Synaptotagmin and neurotransmitter release. Cell. 1993 Sep 24;74(6):947–950. [PubMed]
  • Ferro-Novick S, Jahn R. Vesicle fusion from yeast to man. Nature. 1994 Jul 21;370(6486):191–193. [PubMed]
  • Geppert M, Goda Y, Hammer RE, Li C, Rosahl TW, Stevens CF, Südhof TC. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell. 1994 Nov 18;79(4):717–727. [PubMed]
  • Girod R, Popov S, Alder J, Zheng JQ, Lohof A, Poo MM. Spontaneous quantal transmitter secretion from myocytes and fibroblasts: comparison with neuronal secretion. J Neurosci. 1995 Apr;15(4):2826–2838. [PubMed]
  • Hayashi T, McMahon H, Yamasaki S, Binz T, Hata Y, Südhof TC, Niemann H. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 1994 Nov 1;13(21):5051–5061. [PubMed]
  • Heidelberger R, Heinemann C, Neher E, Matthews G. Calcium dependence of the rate of exocytosis in a synaptic terminal. Nature. 1994 Oct 6;371(6497):513–515. [PubMed]
  • Hoffman JF. On red blood cells, hemolysis and resealed ghosts. Adv Exp Med Biol. 1992;326:1–15. [PubMed]
  • HOFFMAN JF, TOSTESON DC, WHITTAM R. Retention of potassium by human erythrocyte ghosts. Nature. 1960 Jan 16;185:186–187. [PubMed]
  • Hubbard JI, Jones SF, Landau EM. On the mechanism by which calcium and magnesium affect the release of transmitter by nerve impulses. J Physiol. 1968 May;196(1):75–86. [PubMed]
  • Hunt JM, Bommert K, Charlton MP, Kistner A, Habermann E, Augustine GJ, Betz H. A post-docking role for synaptobrevin in synaptic vesicle fusion. Neuron. 1994 Jun;12(6):1269–1279. [PubMed]
  • Jahn R, Niemann H. Molecular mechanisms of clostridial neurotoxins. Ann N Y Acad Sci. 1994 Sep 15;733:245–255. [PubMed]
  • Krause TL, Fishman HM, Ballinger ML, Bittner GD. Extent and mechanism of sealing in transected giant axons of squid and earthworms. J Neurosci. 1994 Nov;14(11 Pt 1):6638–6651. [PubMed]
  • Li C, Ullrich B, Zhang JZ, Anderson RG, Brose N, Südhof TC. Ca(2+)-dependent and -independent activities of neural and non-neural synaptotagmins. Nature. 1995 Jun 15;375(6532):594–599. [PubMed]
  • McMahon HT, Ushkaryov YA, Edelmann L, Link E, Binz T, Niemann H, Jahn R, Südhof TC. Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein. Nature. 1993 Jul 22;364(6435):346–349. [PubMed]
  • Mcneil PL. Incorporation of macromolecules into living cells. Methods Cell Biol. 1989;29:153–173. [PubMed]
  • McNeil PL, Ito S. Gastrointestinal cell plasma membrane wounding and resealing in vivo. Gastroenterology. 1989 May;96(5 Pt 1):1238–1248. [PubMed]
  • McNeil PL, Khakee R. Disruptions of muscle fiber plasma membranes. Role in exercise-induced damage. Am J Pathol. 1992 May;140(5):1097–1109. [PubMed]
  • Miyake K, McNeil PL. Vesicle accumulation and exocytosis at sites of plasma membrane disruption. J Cell Biol. 1995 Dec;131(6 Pt 2):1737–1745. [PMC free article] [PubMed]
  • Mochida S, Poulain B, Weller U, Habermann E, Tauc L. Light chain of tetanus toxin intracellularly inhibits acetylcholine release at neuro-neuronal synapses, and its internalization is mediated by heavy chain. FEBS Lett. 1989 Aug 14;253(1-2):47–51. [PubMed]
  • Neher E, Zucker RS. Multiple calcium-dependent processes related to secretion in bovine chromaffin cells. Neuron. 1993 Jan;10(1):21–30. [PubMed]
  • Pellegrini LL, O'Connor V, Betz H. Fusion complex formation protects synaptobrevin against proteolysis by tetanus toxin light chain. FEBS Lett. 1994 Oct 24;353(3):319–323. [PubMed]
  • Ribchester RR, Mao F, Betz WJ. Optical measurements of activity-dependent membrane recycling in motor nerve terminals of mammalian skeletal muscle. Proc Biol Sci. 1994 Jan 22;255(1342):61–66. [PubMed]
  • Ryan TA, Reuter H, Wendland B, Schweizer FE, Tsien RW, Smith SJ. The kinetics of synaptic vesicle recycling measured at single presynaptic boutons. Neuron. 1993 Oct;11(4):713–724. [PubMed]
  • Schiavo G, Benfenati F, Poulain B, Rossetto O, Polverino de Laureto P, DasGupta BR, Montecucco C. Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature. 1992 Oct 29;359(6398):832–835. [PubMed]
  • Schiavo G, Santucci A, Dasgupta BR, Mehta PP, Jontes J, Benfenati F, Wilson MC, Montecucco C. Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds. FEBS Lett. 1993 Nov 29;335(1):99–103. [PubMed]
  • Schiavo G, Rossetto O, Montecucco C. Clostridial neurotoxins as tools to investigate the molecular events of neurotransmitter release. Semin Cell Biol. 1994 Aug;5(4):221–229. [PubMed]
  • Schiavo G, Shone CC, Bennett MK, Scheller RH, Montecucco C. Botulinum neurotoxin type C cleaves a single Lys-Ala bond within the carboxyl-terminal region of syntaxins. J Biol Chem. 1995 May 5;270(18):10566–10570. [PubMed]
  • Schwarz TL. Genetic analysis of neurotransmitter release at the synapse. Curr Opin Neurobiol. 1994 Oct;4(5):633–639. [PubMed]
  • Spira ME, Benbassat D, Dormann A. Resealing of the proximal and distal cut ends of transected axons: electrophysiological and ultrastructural analysis. J Neurobiol. 1993 Mar;24(3):300–316. [PubMed]
  • Steinhardt RA, Bi G, Alderton JM. Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. Science. 1994 Jan 21;263(5145):390–393. [PubMed]
  • Terasaki M. Visualization of exocytosis during sea urchin egg fertilization using confocal microscopy. J Cell Sci. 1995 Jun;108(Pt 6):2293–2300. [PubMed]
  • Tsong TY. Electroporation of cell membranes. Biophys J. 1991 Aug;60(2):297–306. [PubMed]
  • Vogel SS, Delaney K, Zimmerberg J. The sea urchin cortical reaction. A model system for studying the final steps of calcium-triggered vesicle fusion. Ann N Y Acad Sci. 1991;635:35–44. [PubMed]
  • Weaver JC. Electroporation: a general phenomenon for manipulating cells and tissues. J Cell Biochem. 1993 Apr;51(4):426–435. [PubMed]
  • Whitaker M. Exocytosis in sea urchin eggs. Ann N Y Acad Sci. 1994 Mar 9;710:248–253. [PubMed]
  • Xie XY, Barrett JN. Membrane resealing in cultured rat septal neurons after neurite transection: evidence for enhancement by Ca(2+)-triggered protease activity and cytoskeletal disassembly. J Neurosci. 1991 Oct;11(10):3257–3267. [PubMed]
  • Yawo H, Kuno M. Calcium dependence of membrane sealing at the cut end of the cockroach giant axon. J Neurosci. 1985 Jun;5(6):1626–1632. [PubMed]
  • Yu QC, McNeil PL. Transient disruptions of aortic endothelial cell plasma membranes. Am J Pathol. 1992 Dec;141(6):1349–1360. [PubMed]
  • Zimmerberg J, Whitaker M. Irreversible swelling of secretory granules during exocytosis caused by calcium. Nature. 1985 Jun 13;315(6020):581–584. [PubMed]

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