scholarly journals Comparison of quick-frozen and chemically fixed sea-urchin eggs: structural evidence that cortical granule exocytosis is preceded by a local increase in membrane mobility

1984 ◽  
Vol 72 (1) ◽  
pp. 23-36 ◽  
Author(s):  
D.E. Chandler
Keyword(s):  
Sea Urchin ◽  
Large Scale ◽  
Osmium Tetroxide ◽  
Plasma Membranes ◽  
Strongylocentrotus Purpuratus ◽  
Cortical Granule ◽  
Granule Exocytosis ◽  
Purple Sea Urchin ◽  
Membrane Blebs ◽  
Cortical Granule Exocytosis

Eggs of the purple sea-urchin, Strongylocentrotus purpuratus, were fertilized and fixed with 2% glutaraldehyde at various stages during cortical granule exocytosis. Fixation resulted in membrane blebs being formed precisely at the point of incipient granule fusion. These blebs pinched off to form the membranous vesicles frequently seen in exocytic pockets and in the perivitelline space. In contrast, eggs that were fixed with osmium tetroxide or were quick-frozen without chemical fixation, showed no signs of bleb or vesicle formation. Rather, fusion of each granule appeared to begin at a single minute pore, 30–50 nm in diameter, which then enlarged. We suggest that formation of blebs during glutaraldehyde fixation is an artifact that is caused by a highly localized and transient increase in membrane mobility. Normally, this increased mobility facilitates fusion of granule and plasma membranes, but in the presence of glutaraldehyde it leads to large-scale distortions of these fusing membranes.

scholarly journals Poisson-distributed active fusion complexes underlie the control of the rate and extent of exocytosis by calcium.

1996 ◽  
Vol 134 (2) ◽  
pp. 329-338 ◽  
Author(s):  
S S Vogel ◽  
P S Blank ◽  
J Zimmerberg
Keyword(s):  
Poisson Process ◽  
Sea Urchin ◽  
Physiological Responses ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Granule Exocytosis ◽  
Calcium Dependence ◽  
Sea Urchin Egg ◽  
High Calcium ◽  
Cortical Granule Exocytosis

We have investigated the consequences of having multiple fusion complexes on exocytotic granules, and have identified a new principle for interpreting the calcium dependence of calcium-triggered exocytosis. Strikingly different physiological responses to calcium are expected when active fusion complexes are distributed between granules in a deterministic or probabilistic manner. We have modeled these differences, and compared them with the calcium dependence of sea urchin egg cortical granule exocytosis. From the calcium dependence of cortical granule exocytosis, and from the exposure time and concentration dependence of N-ethylmaleimide inhibition, we determined that cortical granules do have spare active fusion complexes that are randomly distributed as a Poisson process among the population of granules. At high calcium concentrations, docking sites have on average nine active fusion complexes.

scholarly journals Phosphoprotein inhibition of calcium-stimulated exocytosis in sea urchin eggs.

1991 ◽  
Vol 113 (4) ◽  
pp. 769-778 ◽  
Author(s):  
T Whalley ◽  
I Crossley ◽  
M Whitaker
Keyword(s):  
Sea Urchin ◽  
Okadaic Acid ◽  
Cortical Granule ◽  
Granule Exocytosis ◽  
Sea Urchin Eggs ◽  
Transduction Mechanisms ◽  
Egg Signal ◽  
Cortical Granule Exocytosis ◽  
Gamma S

We have investigated the role of protein phosphorylation in the control of exocytosis in sea urchin eggs by treating eggs with a thio-analogue of ATP. ATP gamma S (adenosine 5'-O-3-thiotriphosphate) is a compound which can be used as a phosphoryl donor by protein kinases, leading to irreversible protein thiophosphorylation (Gratecos, D., and E.H. Fischer. 1974. Biochem. Biophys. Res. Commun. 58:960-967). Microinjection of ATP gamma S inhibits cortical granule exocytosis, but has no effect on the sperm-egg signal transduction mechanisms which normally cause exocytosis by generating an increase in [Ca2+]i. ATP gamma S requires cytosolic factors for its inhibition of cortical granule exocytosis: it does not affect exocytosis when applied directly to the isolated exocytotic apparatus. Our data suggest that ATP gamma S irreversibly inhibits exocytosis via thiophosphorylation of proteins associated with the egg cortex. We have identified two thiophosphorylated proteins (33 and 27 kD) that are associated with the isolated exocytotic apparatus. They may mediate the inhibition of exocytosis by ATP gamma S. In addition, we show that okadaic acid, an inhibitor of phosphoprotein phosphatases, prevents cortical granule exocytosis at fertilization without affecting calcium mobilization. Like ATP gamma S, okadaic acid has no effect on exocytosis in vitro. Our results suggest that an inhibitory phosphoprotein can obstruct calcium-stimulated exocytosis in sea urchin eggs; on the other hand, they do not readily support the idea that a protein phosphatase is an essential component of the mechanism controlling exocytosis.

Mastoparan induces Ca2+-independent cortical granule exocytosis in sea urchin eggs

2003 ◽  
Vol 301 (1) ◽  
pp. 13-16 ◽  
Author(s):  
Juana López-Godı́nez ◽  
Teresa I. Garambullo ◽  
Guadalupe Martı́nez-Cadena ◽  
Jesús Garcı́a-Soto
Keyword(s):  
Sea Urchin ◽  
Cortical Granule ◽  
Granule Exocytosis ◽  
Sea Urchin Eggs ◽  
Cortical Granule Exocytosis

scholarly journals The N-ethylmaleimide-sensitive protein thiol groups necessary for sea-urchin egg cortical-granule exocytosis are highly exposed to the medium and are required for triggering by Ca2+

1994 ◽  
Vol 302 (2) ◽  
pp. 391-396 ◽  
Author(s):  
T Whalley ◽  
A Sokoloff
Keyword(s):  
Sea Urchin ◽  
High Molecular Mass ◽  
Cortical Granule ◽  
Topological Properties ◽  
Thiol Groups ◽  
Protein Thiol ◽  
Granule Exocytosis ◽  
Sea Urchin Egg ◽  
Cortical Granule Exocytosis ◽  
Related Proteins

It is known that sea-urchin egg cortical-granule exocytosis is inhibited by agents such as N-ethylmaleimide (NEM) which modify thiol groups. The fusion-related proteins modified by these agents have yet to be identified, nor is there information regarding the topography of these thiol groups. Furthermore, the step in cortical-granule exocytosis at which these thiol groups participate is unknown. In this study we have investigated the topological properties of, and the temporal requirement for the function of, the fusion-related thiol groups by treating the isolated exocytotic apparatus with high-molecular-mass dextrans and BSA carrying thiol-reactive 3-(2-pyridyldithio)propionate groups. The dextran derivatives inhibited exocytosis. The BSA derivative was much less inhibitory. Inhibition was reversed by treatment with dithiothreitol. When NEM was added to the dextran-derivative-treated exocytotic apparatus, treatment with dithiothreitol completely reversed inhibition, indicating that the dextran derivatives inhibit by reacting at the NEM-sensitive sites. A pulse of Ca2+ applied in the presence of inhibitors did not trigger any fusion following the removal of the inhibitor by dithiothreitol. These data show that the thiol groups, the modification of which by NEM inhibits exocytosis, are exposed to the medium in terms of their accessibility to macromolecules. They also show that the fusion-related thiol groups are required during the Ca(2+)-dependent stage of exocytosis.

Using Caged Calcium to Study Sea Urchin Egg Cortical Granule Exocytosis in Vitro

Methods ◽  
1994 ◽  
Vol 6 (1) ◽  
pp. 82-92 ◽  
Author(s):  
Nadeem I. Shafi ◽  
Steven S. Vogel ◽  
Joshua Zimmerberg
Keyword(s):  
Sea Urchin ◽  
Cortical Granule ◽  
Granule Exocytosis ◽  
Caged Calcium ◽  
Sea Urchin Egg ◽  
Cortical Granule Exocytosis

Visualization of exocytosis during sea urchin egg fertilization using confocal microscopy

1995 ◽  
Vol 108 (6) ◽  
pp. 2293-2300 ◽  
Author(s):  
M. Terasaki
Keyword(s):  
Confocal Microscopy ◽  
Sea Urchin ◽  
Sea Water ◽  
Fluorescent Labeling ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Granule Exocytosis ◽  
New Methods ◽  
Cortical Granule Exocytosis ◽  
Fluorescent Dextran

A Ca2+ wave at fertilization triggers cortical granule exocytosis in sea urchin eggs. New methods for visualizing exocytosis of individual cortical granules were developed using fluorescent probes and confocal microscopy. Electron microscopy previously provided evidence that cortical granule exocytosis results in the formation of long-lived depressions in the cell surface. Fluorescent dextran or ovalbumin in the sea water seemed to label these depressions and appeared by confocal microscopy as disks. FM 1–43, a water-soluble fluorescent dye which labels membranes in contact with the sea water, seemed to label the membrane of these depressions and appeared as rings. In double-labeling experiments, the disk and ring labeling by the two types of fluorescent dyes were coincident to within 0.5 second. The fluorescent labeling is coincident with the disappearance of cortical granules by transmitted light microscopy, demonstrating that the labeling corresponds to cortical granule exocytosis. Fluorescent labeling was simultaneous with an expansion of the space occupied by the cortical granule, and labeling by the fluorescent dextran was found to take 0.1-0.2 second. These results are consistent with, and reinforce the previous electron microscopic evidence for, long-lived depressions formed by exocytosis; in addition, the new methods provide new ways to investigate cortical granule exocytosis in living eggs. The fluorescence labeling methods were used with the Ca2+ indicator Ca Green-dextran to test if Ca2+ and cortical granule exocytosis are closely related spatially and temporally. In any given region of the cortex, Ca2+ increased relatively slowly.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Direct membrane retrieval into large vesicles after exocytosis in sea urchin eggs.

1995 ◽  
Vol 131 (5) ◽  
pp. 1183-1192 ◽  
Author(s):  
T Whalley ◽  
M Terasaki ◽  
M S Cho ◽  
S S Vogel
Keyword(s):  
Sea Urchin ◽  
Fluorescent Dyes ◽  
Secretory Granules ◽  
Wave Length ◽  
Fluid Phase ◽  
Cortical Granule ◽  
Lipid Phase ◽  
Granule Exocytosis ◽  
Sea Urchin Eggs ◽  
Cortical Granule Exocytosis

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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