Exocytosis reconstituted from the sea urchin egg is unaffected by calcium pretreatment of granules and plasma membrane

1988 ◽  
Vol 8 (4) ◽  
pp. 335-343 ◽  
Author(s):  
Tim Whalley ◽  
Michael Whitaker
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Calcium Ions ◽  
Calcium Ion ◽  
Secretory Granules ◽  
Cortical Granules ◽  
Allosteric Activation ◽  
Ion Concentrations ◽  
Sea Urchin Egg ◽  
Reconstituted System

Micromolar calcium ion concentrations stimulate exocytosis in a reconstituted system made by recombining in the plasma membrane and cortical secretory granules of the sea urchin egg. The isolated cortical granules are unaffected by calcium concentrations up to 1 mM, nor do granule aggregates undergo any mutual fusion at this concentration. Both isolated plasma membrane and cortical granules can be pretreated with 1 mM Ca before reconstitution without affecting the subsequent exocytosis of the reconstituted system in response to micromolar calcium concentrations. On reconstitution, aggregated cortical granules will fuse with one another in response to micromolar calcium provided that one of their number is in contact with the plasma membrane. If exocytosis involves the generation of lipid fusogens, then these results suggest that the calcium-stimulated production of a fusogen can occur only when contiguity exists between cortical granules and plasma membrane. They also suggest that a substance involved in exocytosis can diffuse and cause piggy-back fusion of secretory granules that are in contact with the plasma membrane. Our results are also consistent with a scheme in which calcium ions cause a reversible, allosteric activation of an exocytotic protein.

Cortical Granule Exocytosis and Fertilization Coat Elevation is Reduced in Aging Sea Urchin Eggs

2000 ◽  
Vol 6 (S2) ◽  
pp. 966-967
Author(s):  
Amitabha Chakrabarti ◽  
Heide Schatten
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Sea Water ◽  
Secretory Granules ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Lytechinus Pictus ◽  
Sea Urchin Eggs ◽  
Cortical Granule Exocytosis ◽  
Granule Secretion

Cortical granules are specialized Golgi-derived membrane-bound secretory granules that are located beneath the plasma membrane in unfertilized sea urchin eggs. Upon fertilization cortical granules discharge in a reaction induced by calcium and release their contents between the plasma membrane and a thin vitelline layer that lines the plasma membrane. Microvilli at the plasma membrane elongate incorporting cortical granule membranes during elongation. The vitelline layer elevates and becomes the egg's fertilization coat that hardens and serves as physical block to polyspermy. While we do not understand the precise mechanisms that participate in cortical granule discharge it is believed that actin plays a role in this process. Because actin and calcium metabolism is affected in aging cells we investigated if cortical granule secretion is affected in aging sea urchin eggs.Lytechinus pictus eggs were obtained by intracoelomic injection of 0.5M KCI to release the eggs into sea water at 23°C.

scholarly journals THE MEMBRANE CAPACITANCE OF THE SEA URCHIN EGG

1957 ◽  
Vol 3 (1) ◽  
pp. 103-110 ◽  
Author(s):  
Lord Rothschild
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Sea Water ◽  
Unit Area ◽  
Membrane Capacitance ◽  
Percentage Reduction ◽  
Cortical Granules ◽  
Sea Urchin Egg

1. The surface of the unfertilized sea urchin egg is folded and the folds are reversibly eliminated by exposing the egg to hypotonic sea water. If the plasma membrane is outside the layer of cortical granules, unfolding may explain why the membrane capacitance per unit area decreases (and does not increase) when a sea urchin egg is put into hypotonic sea water. 2. The degree of surface folding markedly increases after fertilization, which provides an explanation for the increase in membrane capacitance per unit area observed after fertilization. 3. The percentage reduction in membrane folding in fertilized eggs after immersion in hypotonic sea water is probably sufficient to explain the decrease in membrane capacitance per unit area observed in these conditions.

scholarly journals Isolation and characterization of sea urchin egg cortical granules.

1982 ◽  
Vol 95 (3) ◽  
pp. 924-932 ◽  
Author(s):  
G S Kopf ◽  
G W Moy ◽  
V D Vacquier
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Marker Enzyme ◽  
Cortical Granules ◽  
Electrophoretic Patterns ◽  
Isolation And Characterization ◽  
Sea Urchin Egg

A method has been developed to isolate cortical granules (CG) free in suspension. It involves the mechanical disruption of the CG from CG lawns (CGL; Dev. Biol. 43:62-74, 1975) and concentration of the CG by low speed centrifugation. The isolated CG are intact and are a relatively pure population as judged by electron microscopy. Granule integrity is confirmed by the fact that isolated intact CG are radioiodinated to only 0.05% of the specific activity of hypotonically lysed CG. Purity of the CG preparation is assessed by the enrichment (four- to sevenfold) of CG marker enzymes and the absence or low activity of plasma membrane, mitochondrial, cytoplasmic, and yolk platelet marker enzyme activities. CG isolated from 125I-surface-labeled eggs have a very low specific radioactivity, demonstrating that CG contamination by the plasma membrane-vitelline layer (PM-VL) is minimal. CG yield is approximately 1% of the starting egg protein. The CG isolation method is simple and rapid, 4 mg of CG protein being obtained in 1 h. Isolated CG and PM-VL display distinct electrophoretic patterns on SDS gels. Actin is localized to the PM-VL, and all bands present in the CGL are accounted for in the CG and PM-VL. Calmodulin is associated with the CGL, CG, and PM-VL fractions, but is not specifically enriched in these fractions as compared with whole egg homogenates. This method of isolating intact CG from unfertilized sea urchin eggs may be useful for exploring the mechanism of Ca2+-mediated CG exocytosis.

scholarly journals Mechanism for Increase in Intracellular Concentration of Free Calcium in Fertilized Sea Urchin Egg

1974 ◽  
Vol 63 (3) ◽  
pp. 374-388 ◽  
Author(s):  
Masahisa Nakamura ◽  
Ikuo Yasumasu
Keyword(s):  
Dissociation Constant ◽  
Sea Urchin ◽  
Calcium Ion ◽  
Calcium Binding ◽  
Intracellular Concentration ◽  
Sodium Ion ◽  
Free Calcium ◽  
Binding Ability ◽  
Sea Urchin Egg ◽  
Fertilized Egg

Intracellular free calcium concentration in the sea urchin egg was calculated to increase from 0.1 mM in an unfertilized egg to 1 mM in a fertilized egg 10 min after fertilization, based on measurement of the dissociation constant between free calcium and sea urchin egg homogenate. The dissociation constant between free calcium (dialyzable calcium) and homogenate of sea urchin eggs was measured by means of dialysis equilibrium. The dissociation constant of the unfertilized egg was about 10–4 M and that of the fertilized egg was about 10–3 M in three species of sea urchin, Hemicentrotus pulcherrimus, Anthocidaris crassispina, and Pseudocentrotus depressus. An increase in the dissociation constant of the unfertilized egg homogenate was observed after the addition of calcium ion at a concentration above 0.3 mM, the dissociation constant becoming the same as that observed in the fertilized egg homogenate after the administration of CaCl2 at a concentration above 1 mM. Sodium ion also caused a decrease in the calcium-binding ability of the unfertilized egg homogenate. Therefore, penetration of calcium ion or sodium ion upon fertilization might induce an increase in the dissociation constant and then intracellular concentration of free calcium would increase at fertilization. Almost all calcium-binding ability of the egg homogenate was found in the microsomal fraction, and the substance which bound calcium was thought to be protein in nature, since trypsin could decrease the level of calcium-binding substance in the homogenate of the eggs.

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 Isolation and partial characterization of the plasma membrane of the sea urchin egg.

1980 ◽  
Vol 87 (1) ◽  
pp. 248-254 ◽  
Author(s):  
W H Kinsey ◽  
G L Decker ◽  
W J Lennarz
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Sea Urchin ◽  
Plasma Membranes ◽  
Binding Property ◽  
Marker Enzyme ◽  
Surface Complex ◽  
Sea Urchin Egg ◽  
Peripheral Proteins ◽  
Cortical Vesicles

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.

Purification of cortical granules from unfertilized sea urchin egg homogenates by zonal centrifugation

1972 ◽  
Vol 29 (3) ◽  
pp. 307-320 ◽  
Author(s):  
H. Schuel ◽  
W.L. Wilson ◽  
R.S. Bressler ◽  
J.W. Kelly ◽  
J.R. Wilson
Keyword(s):  
Sea Urchin ◽  
Cortical Granules ◽  
Sea Urchin Egg

scholarly journals DIRECT ISOLATION OF THE HYALINE LAYER PROTEIN RELEASED FROM THE CORTICAL GRANULES OF THE SEA URCHIN EGG AT FERTILIZATION

1970 ◽  
Vol 45 (3) ◽  
pp. 615-622 ◽  
Author(s):  
R. E. Kane
Keyword(s):  
Protein Content ◽  
Sea Urchin ◽  
Insoluble Fraction ◽  
Glycerol Solution ◽  
Cortical Granules ◽  
Simple Method ◽  
Quantitative Measurements ◽  
Hawaiian Species ◽  
Sea Urchin Egg

Treatment of the eggs of the sea urchin with a 1 M solution of glycerol at fertilization allows the recovery from this solution of the protein released from the cortical granules, including that which would normally give rise to the hyaline layer. The calcium-gelable protein previously extracted from whole eggs and from isolated cortical material was found to be present in the glycerol solution, confirming its localization in the cortical granules and its role in the hyaline layer. Quantitative measurements on the eggs of two Hawaiian species, Colobocentrotus atratus and Pseudoboletia indiana, which have the widest variation in the gel protein content, demonstrated that a proportionate amount of this material was released at fertilization in these species, which correlates with the thickness of the hyaline layer in the two cases. In addition, the calcium-insoluble fraction of Sakai can be extracted from these eggs after removal of the hyaline protein by glycerol, showing that this is a different material. A simple method for the separation of the hyaline protein from the calcium-insoluble fraction in solution is provided.

scholarly journals A COMPARATIVE STUDY OF THE ISOLATION OF THE CORTEX AND THE ROLE OF THE CALCIUM-INSOLUBLE PROTEIN IN SEVERAL SPECIES OF SEA URCHIN EGG

1969 ◽  
Vol 41 (1) ◽  
pp. 133-144 ◽  
Author(s):  
R. E. Kane ◽  
R. E. Stephens
Keyword(s):  
Comparative Study ◽  
Sea Urchin ◽  
Protein Composition ◽  
Cortical Region ◽  
Cortical Granules ◽  
Divalent Ions ◽  
Sea Urchin Egg ◽  
Protein Gelation ◽  
Isolated Cortex

A comparative study was made of the isolation of the cortex in the eggs of several sea urchin species. Since the isolation method developed by Sakai depends on the presence of magnesium in the medium, the protein composition of the cortex was investigated to determine whether the protein component of the egg described by Kane and Hersh which is gelled by divalent ions, is present in these cortices. Isolation of the cortex was found to require the same divalent ions at the same concentrations as protein gelation, and in the eggs of some species much of the gel protein of the cell was found in the isolated cortical material. In the eggs of other species a smaller fraction of this protein was found in the isolated cortex, although it was more concentrated there than in the endoplasm, and in one species this protein appeared to be uniformly distributed throughout the cell. These results indicate that this protein is localized in the cortical region of the eggs of some species of sea urchin, possibly in the cortical granules, but also point up the fact that results from one species cannot be uncritically extrapolated to others.

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