The Journal of Membrane Biology

, Volume 112, Issue 3, pp 223–232 | Cite as

Hyperosmolality inhibits exocytosis in sea urchin eggs by formation of a granule-free zone and arrest of pore widening

  • Carrie J. Merkle
  • Douglas E. Chandler


Hyperosmolality is known to inhibit membrane fusion during exocytosis. In this study cortical granule exocytosis in sea urchin eggs is used as a model system to determine at what step this inhibition occurs.Strongylocentrotus purpuratus eggs were incubated in hyperosmotic seawater (Na2SO4, sucrose or sodium HEPES used as osmoticants), the eggs activated with 20 μm A23187 to trigger exocytosis, and then quick frozen or chemically fixed for electron microscopy. Thin sections and freeze-fracture replicas show that at high osmolality (2.31 osmol/kg), there is a decrease in cortical granule size, a 90% reduction in granule-plasma membrane fusion, and formation of a granulefree zone between the plasma membrane and cortical granules. This zone averages 0.64 μm in thickness and prevents the majority of granules from docking at the plasma membrane. The remaining granules (∼10%) exhibit early stages of fusion which appear to have been stabilized; the matrix of these granules remains intact. We conclude that exocytosis is blocked by two separate mechanisms. First, the granule-free zone prevents granule-plasma membrane contact required for fusion. Second, in cases where fusion does occur, opening of the pocket and dispersal of the granule contents are slowed in hyperosmotic media.

Key Words

membrane fusion exocytosis hyperosmolality cortex sea urchin egg freeze fracture 


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  1. Begg, D.A., Rebhun, L.I. 1979. pH regulates the polymerization of actin in the sea urchin egg cortex.J. Cell. Biol. 83:241–248Google Scholar
  2. Bilinski, M., Plattner, H., Matt, H. 1981. Secretory protein decondensation as a distinct, Ca2+-mediated event during the final stages of exocytosis inParamecium cells.J. Cell Biol. 88:179–188Google Scholar
  3. Breckenridge, L.J., Almers, W. 1987. Final steps in exocytosis in a cell with giant secretory granules.Proc. Natl. Acad. Sci. USA 84:1945–1949Google Scholar
  4. Chandler, D.E., Bennett, J.P., Gomperts, B. 1983. Freeze-fracture studies of chemotactic peptide-induced exocytosis in neutrophils: Evidence for two patterns of secretory granule fusion.J. Ultrastruct. Res. 82:221–232Google Scholar
  5. Chandler, D.E., Heuser, J.E. 1980. Arrest of membrane fusion events in mast cells by quick-freezing.J. Cell. Biol. 86:666–674Google Scholar
  6. Chandler, D.E., Whitaker, M., Zimmerberg, J. 1989. High molecular weight polymers block cortical granule exocytosis in sea urchin eggs at the level of granule matrix disassembly.J. Cell Biol. (in press) Google Scholar
  7. Cohen, F.S., Akabas, M.H., Zimmerberg, J., Finkelstein, A. 1984. Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes.J. Cell. Biol. 98:1054–1062Google Scholar
  8. Cohen, F.S., Zimmerberg, Z., Finkelstein, A. 1980.Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane.J. Gen. Physiol. 75:251–270Google Scholar
  9. Finkelstein, A., Zimmerberg, J., Cohen, F.S. 1986. Osmotic swelling of vesicles: Its role in the fusion of vesicles with planar phospholipid bilayer membranes and its possible role in exocytosis.Annu. Rev. Physiol. 48:163–174Google Scholar
  10. Gilligan, D.M., Satir, B. 1983. Stimulation and inhibition inParamecium: Role of divalent cations.J. Cell. Biol. 97:224–234Google Scholar
  11. Grinstein, S., Cohen, S., Goetz, J.D., Rothstein, A. 1985. Osmotic and phorbol ester-induced activation of Na+/H+ exchange: Possible role of protein phosphorylation in lymphocyte volume regulation.J. Cell. Biol. 101:269–276Google Scholar
  12. Hampton, R.Y., Holz, R.W. 1983. Effects of changes in osmolality on the stability and function of cultured chromaffin cells and the possible role of osmotic forces in exocytosis.J. Cell Biol. 96:1082–1088Google Scholar
  13. Henson, J.H., Begg, D.A. 1988. Filamentous actin organization in the unfertilized sea urchin egg cortex.Dev. Biol. 127:338–348Google Scholar
  14. Heuser, J.E., Reese, T.S., Dennis, M.J., Jan, Y., Jan, L., Evans, L. 1979. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.J. Cell Biol. 81:275–300Google Scholar
  15. Holz, R.W. 1986. The role of osmotic forces in exocytosis from adrenal chromaffin cells.Annu. Rev. Physiol. 48:175–189Google Scholar
  16. Kazilek, C.J., Merkle, C.J., Chandler, D.E. 1988. Hyperosmotic inhibition of calcium signals and exocytosis in rabbit neutrophils.Am. J. Physiol. 254:C709-C718Google Scholar
  17. Mabuchi, I., Spudich, J.A. 1980. Purification and properties of soluble actin from sea urchin eggs.J. Biochem. 87:785–802Google Scholar
  18. Parker, J.C., Castranova, V. 1984. Volume-responsive sodium and proton movements in dog red blood cells.J. Gen. Physiol. 84:379–401Google Scholar
  19. Payan, P., Girard, J.P., Ciapa, B. 1983. Mechanisms regulating intracellular pH in sea urchin eggs.Dev. Biol. 100:29–38Google Scholar
  20. Plattner, H., Reichel, K., Matt, H. 1977. Bivalent-cation-stimulated ATPase activity at preformed exocytosis sites inParamecium coincides with membrane-intercalated particle aggregates.Nature (London) 267:702–704Google Scholar
  21. Pollard, H.B., Pazoles, C.J., Creutz, C.E., Scott, J.H., Zinder, O., Hotchkiss, A. 1984. An osmotic mechanism for exocytosis from dissociated chromaffin cells.J. Biol. Chem. 259:1114–1121Google Scholar
  22. Pollard, H.B., Tack-Goldman, K., Pazoles, C.J., Creutz, C.E., Shulman, N.R. 1977. Evidence for control of serotonin secretion in human platelets by hydroxyl ion transport and osmotic lysis.Proc. Natl. Acad. Sci. USA 74:5295–5299Google Scholar
  23. Reynolds, E.S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy.J. Cell Biol. 17:208–212Google Scholar
  24. Whitaker, M., Zimmerberg, J. 1987. Inhibition of secretory granule discharge during exocytosis in sea urchin eggs by polymer solutions.J. Physiol. (London) 389:527–539Google Scholar
  25. Zimmerberg, J., Curran, M., Cohen, F.S., Brodwick, M. 1987. Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells.Proc. Natl. Acad. Sci. USA 84:1585–1589Google Scholar
  26. Zimmerberg, J., Sardet, C., Epel, D. 1985. Exocytosis of sea urchin egg cortical vesicles in vitro is retarded by hyperosmotic sucrose: Kinetics of fusion monitored by quantitative light-scattering microscopy.J. Cell Biol. 101:2398–2410Google Scholar
  27. Zimmerberg, J., Whitaker, M. 1985. Irreversible swelling of secretory granules during exocytosis caused by calcium.Nature (London) 315:581–584Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1989

Authors and Affiliations

  • Carrie J. Merkle
    • 1
  • Douglas E. Chandler
    • 1
  1. 1.Department of ZoologyArizona State UniversityTempe

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