Abstract
At fertilization in sea urchin eggs, elevated cytosolic Ca2+ leads to the exocytosis of 15,000-18,000 1.3-microns-diam cortical secretory granules to form the fertilization envelope. Cortical granule exocytosis more than doubles the surface area of the egg. It is thought that much of the added membrane is retrieved by subsequent endocytosis. We have investigated how this is achieved by activating eggs in the presence of aqueous- and lipid-phase fluorescent dyes. We find rapid endocytosis of membrane into 1.5-microns-diam vesicles starting immediately after cortical granule exocytosis and persisting over the following 15 min. The magnitude of this membrane retrieval can compensate for the changes in the plasma membrane of the egg caused by exocytosis. This membrane retrieval is not stimulated by PMA treatment which activates the endocytosis of clathrin-coated vesicles. When eggs are treated with short wave-length ultraviolet light, cortical granule exocytosis still occurs, but granule cores fail to disperse. After egg activation, large vesicles containing semi-intact cortical granule protein cores are observed. These data together with experiments using sequential pulses of fluid-phase markers support the hypothesis that the bulk of membrane retrieval immediately after cortical granule exocytosis is achieved through direct retrieval into large endocytotic structures.
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- Alliegro M. C., McClay D. R. Storage and mobilization of extracellular matrix proteins during sea urchin development. Dev Biol. 1988 Jan;125(1):208–216. doi: 10.1016/0012-1606(88)90074-7. [DOI] [PubMed] [Google Scholar]
- Alvarez de Toledo G., Fernandez J. M. The events leading to secretory granule fusion. Soc Gen Physiol Ser. 1988;43:333–344. [PubMed] [Google Scholar]
- 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]
- Baker P. F., Knight D. E. Calcium control of exocytosis and endocytosis in bovine adrenal medullary cells. Philos Trans R Soc Lond B Biol Sci. 1981 Dec 18;296(1080):83–103. doi: 10.1098/rstb.1981.0174. [DOI] [PubMed] [Google Scholar]
- Baker P. F., Ravazzola M., Malaisse-Lagae F. Secretion-dependent uptake of extracellular fluid by the rat neurohypophysis. Nature. 1974 Jul 12;250(462):155–157. doi: 10.1038/250155a0. [DOI] [PubMed] [Google Scholar]
- Betz W. J., Bewick G. S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science. 1992 Jan 10;255(5041):200–203. doi: 10.1126/science.1553547. [DOI] [PubMed] [Google Scholar]
- Bisgrove B. W., Andrews M. E., Raff R. A. Fibropellins, products of an EGF repeat-containing gene, form a unique extracellular matrix structure that surrounds the sea urchin embryo. Dev Biol. 1991 Jul;146(1):89–99. doi: 10.1016/0012-1606(91)90449-d. [DOI] [PubMed] [Google Scholar]
- 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]
- Brown M. S., Anderson R. G., Goldstein J. L. Recycling receptors: the round-trip itinerary of migrant membrane proteins. Cell. 1983 Mar;32(3):663–667. doi: 10.1016/0092-8674(83)90052-1. [DOI] [PubMed] [Google Scholar]
- Chandler D. E. Multiple intracellular signals coordinate structural dynamics in the sea urchin egg cortex at fertilization. J Electron Microsc Tech. 1991 Mar;17(3):266–293. doi: 10.1002/jemt.1060170304. [DOI] [PubMed] [Google Scholar]
- Chandler D. E., 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. doi: 10.1083/jcb.109.3.1269. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen M. S., Obar R. A., Schroeder C. C., Austin T. W., Poodry C. A., Wadsworth S. C., Vallee R. B. Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis. Nature. 1991 Jun 13;351(6327):583–586. doi: 10.1038/351583a0. [DOI] [PubMed] [Google Scholar]
- Chernomordik L. V., Melikyan G. B., Chizmadzhev Y. A. Biomembrane fusion: a new concept derived from model studies using two interacting planar lipid bilayers. Biochim Biophys Acta. 1987 Oct 5;906(3):309–352. doi: 10.1016/0304-4157(87)90016-5. [DOI] [PubMed] [Google Scholar]
- Chernomordik L. V., Vogel S. S., Sokoloff A., Onaran H. O., Leikina E. A., Zimmerberg J. Lysolipids reversibly inhibit Ca(2+)-, GTP- and pH-dependent fusion of biological membranes. FEBS Lett. 1993 Feb 22;318(1):71–76. doi: 10.1016/0014-5793(93)81330-3. [DOI] [PubMed] [Google Scholar]
- Ciapa B., Crossley I., De Renzis G. Structural modifications induced by TPA (12-O-tetradecanoyl phorbol-13-acetate) in sea urchin eggs. Dev Biol. 1988 Jul;128(1):142–149. doi: 10.1016/0012-1606(88)90276-x. [DOI] [PubMed] [Google Scholar]
- Crossley I., Whalley T., Whitaker M. Guanosine 5'-thiotriphosphate may stimulate phosphoinositide messenger production in sea urchin eggs by a different route than the fertilizing sperm. Cell Regul. 1991 Feb;2(2):121–133. doi: 10.1091/mbc.2.2.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Curran M. J., Cohen F. S., Chandler D. E., Munson P. J., Zimmerberg J. Exocytotic fusion pores exhibit semi-stable states. J Membr Biol. 1993 Apr;133(1):61–75. doi: 10.1007/BF00231878. [DOI] [PubMed] [Google Scholar]
- Deits T., Farrance M., Kay E. S., Medill L., Turner E. E., Weidman P. J., Shapiro B. M. Purification and properties of ovoperoxidase, the enzyme responsible for hardening the fertilization membrane of the sea urchin egg. J Biol Chem. 1984 Nov 10;259(21):13525–13533. [PubMed] [Google Scholar]
- Faire K., Bonder E. M. Sea urchin egg 100-kDa dynamin-related protein: identification of and localization to intracellular vesicles. Dev Biol. 1993 Oct;159(2):581–594. doi: 10.1006/dbio.1993.1266. [DOI] [PubMed] [Google Scholar]
- 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]
- Fisher G. W., Rebhun L. I. Sea urchin egg cortical granule exocytosis is followed by a burst of membrane retrieval via uptake into coated vesicles. Dev Biol. 1983 Oct;99(2):456–472. doi: 10.1016/0012-1606(83)90295-6. [DOI] [PubMed] [Google Scholar]
- Fisher G. W., Summers R. G., Rebhun L. I. Analysis of sea urchin egg cortical transformation in the absence of cortical granule exocytosis. Dev Biol. 1985 Jun;109(2):489–503. doi: 10.1016/0012-1606(85)90474-9. [DOI] [PubMed] [Google Scholar]
- Foltz K. R., Partin J. S., Lennarz W. J. Sea urchin egg receptor for sperm: sequence similarity of binding domain and hsp70. Science. 1993 Mar 5;259(5100):1421–1425. doi: 10.1126/science.8383878. [DOI] [PubMed] [Google Scholar]
- Hall H. G. Hardening of the sea urchin fertilization envelope by peroxidase-catalyzed phenolic coupling of tyrosines. Cell. 1978 Oct;15(2):343–355. doi: 10.1016/0092-8674(78)90003-x. [DOI] [PubMed] [Google Scholar]
- Heuser J. E., Reese T. S. Structural changes after transmitter release at the frog neuromuscular junction. J Cell Biol. 1981 Mar;88(3):564–580. doi: 10.1083/jcb.88.3.564. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hylander B. L., Summers R. G. The effect of local anesthetics and ammonia on cortical granule-plasma membrane attachment in the sea urchin egg. Dev Biol. 1981 Aug;86(1):1–11. doi: 10.1016/0012-1606(81)90309-2. [DOI] [PubMed] [Google Scholar]
- Jaffe L. A., Hagiwara S., Kado R. T. The time course of cortical vesicle fusion in sea urchin eggs observed as membrane capacitance changes. Dev Biol. 1978 Nov;67(1):243–248. doi: 10.1016/0012-1606(78)90314-7. [DOI] [PubMed] [Google Scholar]
- Kay E. S., Shapiro B. M. Ovoperoxidase assembly into the sea urchin fertilization envelope and dityrosine crosslinking. Dev Biol. 1987 Jun;121(2):325–334. doi: 10.1016/0012-1606(87)90168-0. [DOI] [PubMed] [Google Scholar]
- Kirchhausen T., Harrison S. C. Protein organization in clathrin trimers. Cell. 1981 Mar;23(3):755–761. doi: 10.1016/0092-8674(81)90439-6. [DOI] [PubMed] [Google Scholar]
- Kline D., Stewart-Savage J. The timing of cortical granule fusion, content dispersal, and endocytosis during fertilization of the hamster egg: an electrophysiological and histochemical study. Dev Biol. 1994 Mar;162(1):277–287. doi: 10.1006/dbio.1994.1085. [DOI] [PubMed] [Google Scholar]
- Lane M. C., Koehl M. A., Wilt F., Keller R. A role for regulated secretion of apical extracellular matrix during epithelial invagination in the sea urchin. Development. 1993 Mar;117(3):1049–1060. doi: 10.1242/dev.117.3.1049. [DOI] [PubMed] [Google Scholar]
- Lee H. C., Epel D. Changes in intracellular acidic compartments in sea urchin eggs after activation. Dev Biol. 1983 Aug;98(2):446–454. doi: 10.1016/0012-1606(83)90374-3. [DOI] [PubMed] [Google Scholar]
- Malencik D. A., Anderson S. R. Dityrosine formation in calmodulin. Biochemistry. 1987 Feb 10;26(3):695–704. doi: 10.1021/bi00377a006. [DOI] [PubMed] [Google Scholar]
- Markin V. S., Kozlov M. M., Borovjagin V. L. On the theory of membrane fusion. The stalk mechanism. Gen Physiol Biophys. 1984 Oct;3(5):361–377. [PubMed] [Google Scholar]
- Mohri T., Hamaguchi Y. Quantitative analysis of the process and propagation of cortical granule breakdown in sea urchin eggs. Cell Struct Funct. 1990 Oct;15(5):309–315. doi: 10.1247/csf.15.309. [DOI] [PubMed] [Google Scholar]
- 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]
- Monck J. R., Fernandez J. M. The exocytotic fusion pore. J Cell Biol. 1992 Dec;119(6):1395–1404. doi: 10.1083/jcb.119.6.1395. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nagasawa J., Douglas W. W., Schulz R. A. Micropinocytotic origin of coated and smooth microvesicles ("synaptic vesicles") in neurosecretory terminals of posterior pituitary glands demonstrated by incorporation of horseradish peroxidase. Nature. 1971 Jul 30;232(5309):341–342. doi: 10.1038/232341a0. [DOI] [PubMed] [Google Scholar]
- Nordmann J. J., Artault J. C. Membrane retrieval following exocytosis in isolated neurosecretory nerve endings. Neuroscience. 1992 Jul;49(1):201–207. doi: 10.1016/0306-4522(92)90088-j. [DOI] [PubMed] [Google Scholar]
- Obar R. A., Collins C. A., Hammarback J. A., Shpetner H. S., Vallee R. B. Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins. Nature. 1990 Sep 20;347(6290):256–261. doi: 10.1038/347256a0. [DOI] [PubMed] [Google Scholar]
- Rosenboom H., Lindau M. Exo-endocytosis and closing of the fission pore during endocytosis in single pituitary nerve terminals internally perfused with high calcium concentrations. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5267–5271. doi: 10.1073/pnas.91.12.5267. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothman J. H., Raymond C. K., Gilbert T., O'Hara P. J., Stevens T. H. A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting. Cell. 1990 Jun 15;61(6):1063–1074. doi: 10.1016/0092-8674(90)90070-u. [DOI] [PubMed] [Google Scholar]
- Ryan T. A., Reuter H., Wendland B., Schweizer F. E., Tsien R. W., Smith S. J. The kinetics of synaptic vesicle recycling measured at single presynaptic boutons. Neuron. 1993 Oct;11(4):713–724. doi: 10.1016/0896-6273(93)90081-2. [DOI] [PubMed] [Google Scholar]
- Sardet C. The ultrastructure of the sea urchin egg cortex isolated before and after fertilization. Dev Biol. 1984 Sep;105(1):196–210. doi: 10.1016/0012-1606(84)90275-6. [DOI] [PubMed] [Google Scholar]
- Schroeder T. E. Surface area change at fertilization: resorption of the mosaic membrane. Dev Biol. 1979 Jun;70(2):306–326. doi: 10.1016/0012-1606(79)90030-7. [DOI] [PubMed] [Google Scholar]
- Smythe E., Warren G. The mechanism of receptor-mediated endocytosis. Eur J Biochem. 1991 Dec 18;202(3):689–699. doi: 10.1111/j.1432-1033.1991.tb16424.x. [DOI] [PubMed] [Google Scholar]
- Swann K., Whitaker M. The part played by inositol trisphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs. J Cell Biol. 1986 Dec;103(6 Pt 1):2333–2342. doi: 10.1083/jcb.103.6.2333. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Terasaki M. Visualization of exocytosis during sea urchin egg fertilization using confocal microscopy. J Cell Sci. 1995 Jun;108(Pt 6):2293–2300. doi: 10.1242/jcs.108.6.2293. [DOI] [PubMed] [Google Scholar]
- Thilo L. Selective internalization of granule membrane after secretion in mast cells. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1711–1715. doi: 10.1073/pnas.82.6.1711. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas P., Lee A. K., Wong J. G., Almers W. A triggered mechanism retrieves membrane in seconds after Ca(2+)-stimulated exocytosis in single pituitary cells. J Cell Biol. 1994 Mar;124(5):667–675. doi: 10.1083/jcb.124.5.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ungewickell E., Branton D. Assembly units of clathrin coats. Nature. 1981 Jan 29;289(5796):420–422. doi: 10.1038/289420a0. [DOI] [PubMed] [Google Scholar]
- Vogel S. S., 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. doi: 10.1111/j.1749-6632.1991.tb36479.x. [DOI] [PubMed] [Google Scholar]
- Vogel S. S., Leikina E. A., Chernomordik L. V. Lysophosphatidylcholine reversibly arrests exocytosis and viral fusion at a stage between triggering and membrane merger. J Biol Chem. 1993 Dec 5;268(34):25764–25768. [PubMed] [Google Scholar]
- Watts C., Marsh M. Endocytosis: what goes in and how? J Cell Sci. 1992 Sep;103(Pt 1):1–8. doi: 10.1242/jcs.103.1.1a. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- von Gersdorff H., Matthews G. Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals. Nature. 1994 Feb 24;367(6465):735–739. doi: 10.1038/367735a0. [DOI] [PubMed] [Google Scholar]
