scholarly journals Activation of protein kinase Cη triggers cortical granule exocytosis in Xenopus oocytes

2002 ◽  
Vol 115 (6) ◽  
pp. 1313-1320
Author(s):  
Cameron B. Gundersen ◽  
Sirus A. Kohan ◽  
Qian Chen ◽  
Joseph Iagnemma ◽  
Joy A. Umbach
Keyword(s):  
Protein Kinase ◽  
Xenopus Oocytes ◽  
Phorbol Esters ◽  
Subcellular Fractionation ◽  
Cortical Granule ◽  
The Novel ◽  
Cortical Granules ◽  
Granule Exocytosis ◽  
The Family ◽  
Cortical Granule Exocytosis

Previous work has shown that phorbol esters or diacylglycerol trigger cortical granule exocytosis in Xenopus oocytes. We sought to identify the isoform(s) of protein kinase C (PKC) that mediate(s) this regulated secretory event. Because this process is initiated by lipid activators of PKC but is independent of calcium ions, we focused on the family of novel(calcium-independent) PKCs. Pharmacological investigations using Gö6976 and Gö6983 tended to exclude PKCδ, ϵ and μ as secretory triggers. Subcellular fractionation and immunoblot data revealed that these oocytes expressed all five members of the novel PKC family, but it was only PKCη that colocalized with cortical granules. Finally, expression of wild type or constitutively active forms of PKCδ and η strongly supported the conclusion that it is PKCη that initiates cortical granule exocytosis in these cells. These observations represent an important step in identifying the mechanism of secretory triggering in this system.

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.

Weak bases partially activate Xenopus eggs and permit changes in membrane conductance whilst inhibiting cortical granule exocytosis

1987 ◽  
Vol 87 (2) ◽  
pp. 205-220
Author(s):  
M. Charbonneau ◽  
D.J. Webb
Keyword(s):  
Polar Body ◽  
Membrane Conductance ◽  
Transient Increase ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Weak Base ◽  
Granule Exocytosis ◽  
Weak Bases ◽  
Prolonged Contact ◽  
Cortical Granule Exocytosis

At extracellular pH values close to their pKa values the weak bases, ammonia and procaine, elicited a series of events in non-activated Xenopus eggs, some of which resembled those normally occurring at fertilization. These included: (1) a transient increase in membrane conductance; (2) modification of the microvilli; (3) thickening of the cortical cytoplasm and displacement of the cortical granules; (4) pigment accumulation; (5) contractions and shape changes. However, these eggs did not undergo the cortical reaction nor emit the second polar body. Cortical granule exocytosis of inseminated or artificially stimulated eggs was inhibited if the eggs had been previously treated for 15 min with the weak base and subsequently rinsed. Multiple sperm-entry sites were exhibited by the inseminated eggs, suggesting polyspermy. However, such eggs did not cleave and although sperm had fused with the egg membrane, they were not incorporated. Nevertheless, a transient increase in membrane conductance was evoked, which was longer in duration and had a slightly different form from that normally accompanying fertilization. In these eggs cortical granules were intact but displaced away from the plasma membrane. Prolonged contact with the weak base rendered eggs totally unresponsive to sperm or artificial stimuli but eggs recovered when rinsed sufficiently. These effects of weak bases on unfertilized Xenopus eggs or during fertilization were completely absent at pH 7.4. Although changes in intracellular pH or Ca2+ may be involved in these phenomena, direct action by the weak base itself cannot be ruled out.

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)

Cortical granule exocytosis is triggered by different thresholds of calcium during fertilisation in sea urchin eggs

Zygote ◽  
1998 ◽  
Vol 6 (1) ◽  
pp. 55-63 ◽  
Author(s):  
John C. Matese ◽  
David R. McClay
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Calcium Release ◽  
Threshold Concentration ◽  
Calcium Wave ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Granule Exocytosis ◽  
Sea Urchin Eggs ◽  
Cortical Granule Exocytosis

SummaryIn sea urchin eggs, fertilisation is followed by a calcium wave, cortical granule exocytosis and fertilisation envelope elevation. Both the calcium wave and cortical granule exocytosis sweep across the egg in a wave initiated at the point of sperm entry. Using differential interference contrast (DIC) microscopy combined with laser scanning confocal microscopy, populations of cortical granules undergoing calcium-induced exocytosis were observed in living urchin eggs. Calcium imaging using the indicator Calcium Green-dextran was combined with an image subtraction technique for visual isolation of individual exocytotic events. Relative fluorescence levels of the calcium indicator during the fertilisation wave were compared with cortical fusion events. In localised regions of the egg, there is a 6s delay between the detection of calcium release and fusion of cortical granules. The rate of calcium accumulation was altered experimentally to ask whether this delay was necessary to achieve a threshold concentration of calcium to trigger fusion, or was a time-dependent activation of the cortical granule fusion apparatus after the ‘triggering’ event. Calcium release rate was attenuated by blocking inositol 1,4,5-triphospate (InsP3)-gated channels with heparin. Heparin extended the time necessary to achieve a minimum concentration of calcium at the sites of cortical granule exocytosis. The data are consistent with the conclusion that much of the delay observed normally is necessary to reach threshold concentration of calcium. Cortical granules then fuse with the plasma membrane. Further, once the minimum threshold calcium concentration is reached, cortical granule fusion with the plasma membrane occurs in a pattern suggesting that cortical granules are non-uniform in their calcium sensitivity threshold.

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