Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1998 Feb;74(2 Pt 1):1061–1073. doi: 10.1016/S0006-3495(98)74030-5

Regulation of exocytotic fusion by cell inflation.

C Solsona 1, B Innocenti 1, J M Fernández 1
PMCID: PMC1302586  PMID: 9533718

Abstract

We have inflated patch-clamped mast cells by 3.8 +/- 1.6 times their volume by applying a hydrostatic pressure of 5-15 cm H2O to the interior of the patch pipette. Inflation did not cause changes in the cell membrane conductance and caused only a small reversible change in the cell membrane capacitance (36 +/- 5 fF/cm H2O). The specific cell membrane capacitance of inflated cells was found to be 0.5 microF/cm2. High-resolution capacitance recordings showed that inflation reduced the frequency of exocytotic fusion events by approximately 70-fold, with the remaining fusion events showing an unusual time course. Shortly after the pressure was returned to 0 cm H2O, mast cells regained their normal size and appearance and degranulated completely, even after remaining inflated for up to 60 min. We interpret these observations as an indication that inflated mast cells reversibly disassemble the structures that regulate exocytotic fusion. Upon returning to its normal size, the cell cytosol reassembles the fusion pore scaffolds and allows exocytosis to proceed, suggesting that exocytotic fusion does not require soluble proteins. Reassembly of the fusion pore can be prevented by inflating the cells with solutions containing the protease pronase, which completely blocked exocytosis. We also interpret these results as evidence that the activity of the fusion pore is sensitive to the tension of the plasma membrane.

Full Text

The Full Text of this article is available as a PDF (182.0 KB).

Selected References

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

  1. Alvarez de Toledo G., Fernández-Chacón R., Fernández J. M. Release of secretory products during transient vesicle fusion. Nature. 1993 Jun 10;363(6429):554–558. doi: 10.1038/363554a0. [DOI] [PubMed] [Google Scholar]
  2. Breckenridge L. J., Almers W. Currents through the fusion pore that forms during exocytosis of a secretory vesicle. 1987 Aug 27-Sep 2Nature. 328(6133):814–817. doi: 10.1038/328814a0. [DOI] [PubMed] [Google Scholar]
  3. Chandler D. E., Heuser J. E. Arrest of membrane fusion events in mast cells by quick-freezing. J Cell Biol. 1980 Aug;86(2):666–674. doi: 10.1083/jcb.86.2.666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Curran M. J., Brodwick M. S. Ionic control of the size of the vesicle matrix of beige mouse mast cells. J Gen Physiol. 1991 Oct;98(4):771–790. doi: 10.1085/jgp.98.4.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dai J., Sheetz M. P. Mechanical properties of neuronal growth cone membranes studied by tether formation with laser optical tweezers. Biophys J. 1995 Mar;68(3):988–996. doi: 10.1016/S0006-3495(95)80274-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Du X. Y., Sorota S. Cardiac swelling-induced chloride current depolarizes canine atrial myocytes. Am J Physiol. 1997 Apr;272(4 Pt 2):H1904–H1916. doi: 10.1152/ajpheart.1997.272.4.H1904. [DOI] [PubMed] [Google Scholar]
  7. Evans E. A., Waugh R., Melnik L. Elastic area compressibility modulus of red cell membrane. Biophys J. 1976 Jun;16(6):585–595. doi: 10.1016/S0006-3495(76)85713-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fernandez J. M., Neher E., Gomperts B. D. Capacitance measurements reveal stepwise fusion events in degranulating mast cells. 1984 Nov 29-Dec 5Nature. 312(5993):453–455. doi: 10.1038/312453a0. [DOI] [PubMed] [Google Scholar]
  9. Fernandez J. M., Villalón M., Verdugo P. Reversible condensation of mast cell secretory products in vitro. Biophys J. 1991 May;59(5):1022–1027. doi: 10.1016/S0006-3495(91)82317-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fernández-Chacón R., Alvarez de Toledo G. Cytosolic calcium facilitates release of secretory products after exocytotic vesicle fusion. FEBS Lett. 1995 Apr 24;363(3):221–225. doi: 10.1016/0014-5793(95)00319-5. [DOI] [PubMed] [Google Scholar]
  11. Fidler N., Fernandez J. M. Phase tracking: an improved phase detection technique for cell membrane capacitance measurements. Biophys J. 1989 Dec;56(6):1153–1162. doi: 10.1016/S0006-3495(89)82762-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Graf J., Rupnik M., Zupancic G., Zorec R. Osmotic swelling of hepatocytes increases membrane conductance but not membrane capacitance. Biophys J. 1995 Apr;68(4):1359–1363. doi: 10.1016/S0006-3495(95)80308-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gustin M. C., Zhou X. L., Martinac B., Kung C. A mechanosensitive ion channel in the yeast plasma membrane. Science. 1988 Nov 4;242(4879):762–765. doi: 10.1126/science.2460920. [DOI] [PubMed] [Google Scholar]
  14. Hide I., Bennett J. P., Pizzey A., Boonen G., Bar-Sagi D., Gomperts B. D., Tatham P. E. Degranulation of individual mast cells in response to Ca2+ and guanine nucleotides: an all-or-none event. J Cell Biol. 1993 Nov;123(3):585–593. doi: 10.1083/jcb.123.3.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hochmuth F. M., Shao J. Y., Dai J., Sheetz M. P. Deformation and flow of membrane into tethers extracted from neuronal growth cones. Biophys J. 1996 Jan;70(1):358–369. doi: 10.1016/S0006-3495(96)79577-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Joshi C., Fernandez J. M. Capacitance measurements. An analysis of the phase detector technique used to study exocytosis and endocytosis. Biophys J. 1988 Jun;53(6):885–892. doi: 10.1016/S0006-3495(88)83169-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kawagoe K. T., Jankowski J. A., Wightman R. M. Etched carbon-fiber electrodes as amperometric detectors of catecholamine secretion from isolated biological cells. Anal Chem. 1991 Aug 1;63(15):1589–1594. doi: 10.1021/ac00015a017. [DOI] [PubMed] [Google Scholar]
  18. Kemble G. W., Danieli T., White J. M. Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion. Cell. 1994 Jan 28;76(2):383–391. doi: 10.1016/0092-8674(94)90344-1. [DOI] [PubMed] [Google Scholar]
  19. Kwok R., Evans E. Thermoelasticity of large lecithin bilayer vesicles. Biophys J. 1981 Sep;35(3):637–652. doi: 10.1016/S0006-3495(81)84817-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liu Z. Y., Young J. I., Elson E. L. Rat basophilic leukemia cells stiffen when they secrete. J Cell Biol. 1987 Dec;105(6 Pt 2):2933–2943. doi: 10.1083/jcb.105.6.2933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Malhotra V., Orci L., Glick B. S., Block M. R., Rothman J. E. Role of an N-ethylmaleimide-sensitive transport component in promoting fusion of transport vesicles with cisternae of the Golgi stack. Cell. 1988 Jul 15;54(2):221–227. doi: 10.1016/0092-8674(88)90554-5. [DOI] [PubMed] [Google Scholar]
  22. Marszalek P. E., Markin V. S., Tanaka T., Kawaguchi H., Fernandez J. M. The secretory granule matrix-electrolyte interface: a homologue of the p-n rectifying junction. Biophys J. 1995 Oct;69(4):1218–1229. doi: 10.1016/S0006-3495(95)80004-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Marszalek P., Farrell B., Fernandez J. M. Ion-exchange gel regulates neurotransmitter release through the exocytotic fusion pore. Soc Gen Physiol Ser. 1996;51:211–222. [PubMed] [Google Scholar]
  24. Matsuda N., Hagiwara N., Shoda M., Kasanuki H., Hosoda S. Enhancement of the L-type Ca2+ current by mechanical stimulation in single rabbit cardiac myocytes. Circ Res. 1996 Apr;78(4):650–659. doi: 10.1161/01.res.78.4.650. [DOI] [PubMed] [Google Scholar]
  25. Monck J. R., Alvarez de Toledo G., Fernandez J. M. Tension in secretory granule membranes causes extensive membrane transfer through the exocytotic fusion pore. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7804–7808. doi: 10.1073/pnas.87.20.7804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Monck J. R., Fernandez J. M. The exocytotic fusion pore and neurotransmitter release. Neuron. 1994 Apr;12(4):707–716. doi: 10.1016/0896-6273(94)90325-5. [DOI] [PubMed] [Google Scholar]
  27. Monck J. R., Oberhauser A. F., Alvarez de Toledo G., Fernandez J. M. Is swelling of the secretory granule matrix the force that dilates the exocytotic fusion pore? Biophys J. 1991 Jan;59(1):39–47. doi: 10.1016/S0006-3495(91)82196-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Morgan A., Burgoyne R. D. Is NSF a fusion protein? Trends Cell Biol. 1995 Sep;5(9):335–339. doi: 10.1016/s0962-8924(00)89059-5. [DOI] [PubMed] [Google Scholar]
  29. Nanavati C., Fernandez J. M. The secretory granule matrix: a fast-acting smart polymer. Science. 1993 Feb 12;259(5097):963–965. doi: 10.1126/science.8438154. [DOI] [PubMed] [Google Scholar]
  30. Nanavati C., Markin V. S., Oberhauser A. F., Fernandez J. M. The exocytotic fusion pore modeled as a lipidic pore. Biophys J. 1992 Oct;63(4):1118–1132. doi: 10.1016/S0006-3495(92)81679-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Needham D., Hochmuth R. M. Electro-mechanical permeabilization of lipid vesicles. Role of membrane tension and compressibility. Biophys J. 1989 May;55(5):1001–1009. doi: 10.1016/S0006-3495(89)82898-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Oberhauser A. F., Fernandez J. M. Hydrophobic ions amplify the capacitive currents used to measure exocytotic fusion. Biophys J. 1995 Aug;69(2):451–459. doi: 10.1016/S0006-3495(95)79918-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Oberhauser A. F., Monck J. R., Balch W. E., Fernandez J. M. Exocytotic fusion is activated by Rab3a peptides. Nature. 1992 Nov 19;360(6401):270–273. doi: 10.1038/360270a0. [DOI] [PubMed] [Google Scholar]
  34. Okano K., Monck J. R., Fernandez J. M. GTP gamma S stimulates exocytosis in patch-clamped rat melanotrophs. Neuron. 1993 Jul;11(1):165–172. doi: 10.1016/0896-6273(93)90280-5. [DOI] [PubMed] [Google Scholar]
  35. Ornberg R. L., Reese T. S. Beginning of exocytosis captured by rapid-freezing of Limulus amebocytes. J Cell Biol. 1981 Jul;90(1):40–54. doi: 10.1083/jcb.90.1.40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Parpura V., Fernandez J. M. Atomic force microscopy study of the secretory granule lumen. Biophys J. 1996 Nov;71(5):2356–2366. doi: 10.1016/S0006-3495(96)79483-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pusch M., Neher E. Rates of diffusional exchange between small cells and a measuring patch pipette. Pflugers Arch. 1988 Feb;411(2):204–211. doi: 10.1007/BF00582316. [DOI] [PubMed] [Google Scholar]
  38. Rothman J. E., Orci L. Molecular dissection of the secretory pathway. Nature. 1992 Jan 30;355(6359):409–415. doi: 10.1038/355409a0. [DOI] [PubMed] [Google Scholar]
  39. Setoguchi M., Ohya Y., Abe I., Fujishima M. Stretch-activated whole-cell currents in smooth muscle cells from mesenteric resistance artery of guinea-pig. J Physiol. 1997 Jun 1;501(Pt 2):343–353. doi: 10.1111/j.1469-7793.1997.343bn.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sokabe M., Sachs F., Jing Z. Q. Quantitative video microscopy of patch clamped membranes stress, strain, capacitance, and stretch channel activation. Biophys J. 1991 Mar;59(3):722–728. doi: 10.1016/S0006-3495(91)82285-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Spruce A. E., Breckenridge L. J., Lee A. K., Almers W. Properties of the fusion pore that forms during exocytosis of a mast cell secretory vesicle. Neuron. 1990 May;4(5):643–654. doi: 10.1016/0896-6273(90)90192-i. [DOI] [PubMed] [Google Scholar]
  42. Söllner T. SNAREs and targeted membrane fusion. FEBS Lett. 1995 Aug 1;369(1):80–83. doi: 10.1016/0014-5793(95)00594-y. [DOI] [PubMed] [Google Scholar]
  43. Söllner T., Whiteheart S. W., Brunner M., Erdjument-Bromage H., Geromanos S., Tempst P., Rothman J. E. SNAP receptors implicated in vesicle targeting and fusion. Nature. 1993 Mar 25;362(6418):318–324. doi: 10.1038/362318a0. [DOI] [PubMed] [Google Scholar]
  44. Südhof T. C. The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature. 1995 Jun 22;375(6533):645–653. doi: 10.1038/375645a0. [DOI] [PubMed] [Google Scholar]
  45. White S. H. Studies of the physical chemistry of planar bilayer membranes using high-precision measurements of specific capacitance. Ann N Y Acad Sci. 1977 Dec 30;303:243–265. [PubMed] [Google Scholar]
  46. Wightman R. M., Jankowski J. A., Kennedy R. T., Kawagoe K. T., Schroeder T. J., Leszczyszyn D. J., Near J. A., Diliberto E. J., Jr, Viveros O. H. Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10754–10758. doi: 10.1073/pnas.88.23.10754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zimmerberg J., Curran M., Cohen F. S., Brodwick M. Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1585–1589. doi: 10.1073/pnas.84.6.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zimmerberg J., Vogel S. S., Chernomordik L. V. Mechanisms of membrane fusion. Annu Rev Biophys Biomol Struct. 1993;22:433–466. doi: 10.1146/annurev.bb.22.060193.002245. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES