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

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.

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.

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

Cortical granule exocytosis in Bufo arenarum oocytes matured in vitro

Zygote ◽  
2001 ◽  
Vol 9 (3) ◽  
pp. 251-259 ◽  
Author(s):  
J. Oterino ◽  
G. Sánchez Toranzo ◽  
L. Zelarayán ◽  
J.N. Valz-Gianinet ◽  
M.I. Bühler
Keyword(s):  
Cortical Granule ◽  
Granule Exocytosis ◽  
Cortical Reaction ◽  
Progesterone Treatment ◽  
Sperm Entry ◽  
Bufo Arenarum ◽  
Chronological Sequence ◽  
Cortical Granule Exocytosis

Denuded Bufo arenarum oocytes matured in vitro by progesterone treatment exhibited abnormal segmentation due to the penetration of more than one sperm. These oocytes were able to respond to activation stimuli and exhibited the external signs characteristic of activation. However, the prevention of polyspermy was not effective in these oocytes, which exhibited numerous sperm in their cytoplasm. The aim of this work was to analyse the cortical reaction in polyspermic Bufo arenarum oocytes matured in vitro. The result indicate that the cortical reaction of these oocytes seems to occur with a chronological sequence similar to that described for ovoposited oocytes of this species. In addition, when, 1 min after pricking, cortical granule exocytosis occurred, the oocytes became refractory to sperm entry, suggesting that they are able to establish a slow block to polyspermy.

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