N-ethylmaleimide-sensitive protein(s) involved in cortical exocytosis in the sea urchin egg: localization to both cortical vesicles and plasma membrane

1990 ◽  
Vol 96 (2) ◽  
pp. 313-321
Author(s):  
R.C. Jackson ◽  
P.A. Modern
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Cellular Localization ◽  
Plasma Membranes ◽  
Protein S ◽  
Covalent Modification ◽  
Sea Urchin Egg ◽  
Contrast Microscopy ◽  
Cortical Vesicles ◽  
Secretory Products

The exocytotic release of secretory products from fragments of sea urchin egg cortex has been shown to be inhibited by covalent modification of membrane sulfhydryl groups with N-ethylmaleimide (NEM). Exocytotically competent preparations of reconstituted cortex, formed by recombination of purified cortical vesicles (CVs) with fragments of egg plasma membrane (PM) were also inhibited by treatment with NEM. The cellular localization of sulfhydryl-containing constituent(s) responsible for inhibition was investigated by treating CVs and/or PM with NEM prior to reconstitution. Both native cortex and cortex reconstituted with NEM-treated components were challenged with calcium-containing buffers. Exocytosis was monitored by phase-contrast microscopy, and quantitated by light scattering. Evidence for CV-PM fusion was obtained with an immunofluorescence-based assay that permits visualization of the transport of CV content proteins across the PM. Cortex reconstituted by recombination of NEM-treated CVs with untreated PM or by recombination of untreated CVs with NEM-treated PM was exocytotically competent, whereas cortex formed by recombination of NEM-treated CVs with NEM-treated PM was inactive. These results: (1) support the hypothesis that the mechanism of exocytosis in native and reconstituted cortex is the same; (2) provide evidence that both CV and plasma membranes participate in the release of CV contents from reconstituted cortex; and (3) suggest that sulfhydryl-containing protein(s) present on the surface of purified CVs and plasma membrane are involved in exocytosis.

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.

The mechanism of cell membrane repair

Zygote ◽  
1999 ◽  
Vol 8 (S1) ◽  
pp. S31-S32 ◽  
Author(s):  
Tatsuru Togo ◽  
Janet M. Alderton ◽  
Richard A. Steinhardt
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Plasma Membranes ◽  
Membrane Repair ◽  
Sea Urchin Egg ◽  
Calmodulin Kinase ◽  
Intracellular Ca ◽  
Plasma Membrane Repair ◽  
Botulinum Neurotoxins ◽  
Membrane Resealing

Disruption of plasma membranes is a widespread, common and normal event that occurs in many mechanically challenged tissues (McNeil & Steinhardt, 1997). After injury to the plasma membrane, rapid resealing of the membrane occurs with little loss of intracellular contents.Analysis of plasma membrane repair in the sea urchin egg and early embryos revealed a new model of the mechanism for plasma membrane repair. Resealing of disrupted plasma membranes required external Ca2+ that could be antagonised by Mg2+. Block of Ca2+/calmodulin kinase II, which regulates exocytotic vesicle availability at synapses (Llinás et al., 1991), inhibited membrane resealing. Resealing was also inhibited by botulinum neurotoxins A, B, C1, and tetanus toxin, which disrupt SNARE vesicle docking/fusion proteins. Confocal microscopic observations of exocytotic events in sea urchin eggs and embryos during membrane resealing showed that inhibition of kinesin or myosin motor activity, which are believed to be required for vesicle transport (Goodson et al., 1997), also inhibited membrane resealing and delivery of vesicles to sites of membrane disruption. This pattern of inhibition indicates that membrane repair of micrometre-sized lesions requires vesicle delivery, docking and fusion, similar to the exocytosis of neurotransmitter (Steinhardt et al., 1994; Bi et al., 1995, 1997).The mechanism of resealing in eggs and embyros was found to be a general property of all cells (Steinhardt et al., 1994; Togo et al., 1999). It is now known that elevated intracellular Ca2+ triggers exocytosis in various types of cells (Dan & Poo, 1992; Coorssen et al., 1996), and that endosomal compartments such as lysosomes can behave as Ca2+-regulated exocytotic vesicles (Rodríguez et al., 1997).

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 Effects of Dithiothreitol on Fertilization and Early Development in Sea Urchin

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3573
Author(s):  
Nunzia Limatola ◽  
Jong Tai Chun ◽  
Sawsen Cherraben ◽  
Jean-Louis Schmitt ◽  
Jean-Marie Lehn ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Early Development ◽  
Sea Urchin ◽  
Actin Filaments ◽  
Cortical Granule ◽  
Cortical Actin ◽  
Phenotypic Changes ◽  
Sea Urchin Egg ◽  
Cortical Granule Exocytosis

The vitelline layer (VL) of a sea urchin egg is an intricate meshwork of glycoproteins that intimately ensheathes the plasma membrane. During fertilization, the VL plays important roles. Firstly, the receptors for sperm reside on the VL. Secondly, following cortical granule exocytosis, the VL is elevated and transformed into the fertilization envelope (FE), owing to the assembly and crosslinking of the extruded materials. As these two crucial stages involve the VL, its alteration was expected to affect the fertilization process. In the present study, we addressed this question by mildly treating the eggs with a reducing agent, dithiothreitol (DTT). A brief pretreatment with DTT resulted in partial disruption of the VL, as judged by electron microscopy and by a novel fluorescent polyamine probe that selectively labelled the VL. The DTT-pretreated eggs did not elevate the FE but were mostly monospermic at fertilization. These eggs also manifested certain anomalies at fertilization: (i) compromised Ca2+ signaling, (ii) blocked translocation of cortical actin filaments, and (iii) impaired cleavage. Some of these phenotypic changes were reversed by restoring the DTT-exposed eggs in normal seawater prior to fertilization. Our findings suggest that the FE is not the decisive factor preventing polyspermy and that the integrity of the VL is nonetheless crucial to the egg’s fertilization response.

Isolated Plasma Membranes of Sea Urchin Eggs

1971 ◽  
Vol 29 ◽  
pp. 534-535
Author(s):  
S. Inoue ◽  
E. C. Preddie ◽  
P. Guerrier
Keyword(s):  
Electron Microscope ◽  
Sea Urchin ◽  
Plasma Membranes ◽  
Membrane System ◽  
Vitelline Membrane ◽  
Thin Sections ◽  
Sea Urchin Eggs ◽  
Sea Urchin Egg ◽  
Discontinuous Sucrose Gradient ◽  
Fertilization Membrane

From electron microscope studies of thin sections the sea urchin egg is known to be surrounded by the peripheral membrane system which is made up of the outer coat (vitelline membrane), which elevates from an egg surface after fertilization and becomes a part of the fertilization membrane, and the plasma membrane. In these experiments an effort has been made to isolate plasma membranes of sea urchin eggs and these isolated membranes were observed in the electron microscope.The vitelline membrane of the eggs from the sea urchin Strongylocentrotus purpuratus was at first digested away by the treatment with 0.02% trypsin in 0.01 M Tris-HCl buffer (pH 8.0) for 5 minutes at 28°C. The plasma membranes were then isolated according to the method of Song et al. which was used for the isolation of rat liver plasma membranes. The vitelline membrane-free eggs were gently homogenized in 10-3 M NaHC03 (pH 7.5) and freed membranes were collected by centrifugation over a discontinuous sucrose gradient preparation.

scholarly journals Hierarchies of protein cross-linking in the extracellular matrix: involvement of an egg surface transglutaminase in early stages of fertilization envelope assembly.

1988 ◽  
Vol 107 (6) ◽  
pp. 2447-2454 ◽  
Author(s):  
D E Battaglia ◽  
B M Shapiro
Keyword(s):  
Cell Surface ◽  
Sea Urchin ◽  
Physiological Function ◽  
Strongylocentrotus Purpuratus ◽  
Cross Linking ◽  
Early Step ◽  
Transglutaminase Activity ◽  
Sea Urchin Egg ◽  
A Cell ◽  
Secretory Products

The involvement of transglutaminase activity in fertilization envelope (FE) formation was investigated using eggs from the sea urchin, Strongylocentrotus purpuratus. Eggs fertilized in the presence of the transglutaminase inhibitors, putrescine and cadaverine, had disorganized and expanded FEs with inhibition of the characteristic I-T transition. The permeability of the FE was increased by these agents, as revealed by the loss of proteins from the perivitelline space and the appearance of ovoperoxidase activity in supernates from putrescine-treated eggs. [3H]putrescine was incorporated into the FE during fertilization in a reaction catalyzed by an egg surface transglutaminase that could also use dimethylcasein as a substrate in vitelline layer-denuded eggs. Egg secretory products alone had no transglutaminase activity. The cell surface transglutaminase activity was transient and maximal within 4 min of activation. The enzyme was Ca2+ dependent and was inhibited by Zn2+. We conclude that sea urchin egg surface transglutaminase catalyzes an early step in a hierarchy of cross-linking events during FE assembly, one that occurs before ovoperoxidase-mediated dityrosine formation (Foerder, C. A., and B. M. Shapiro. 1977. Proc. Natl. Acad. Sci. USA. 74:4214-4218). Thus it provides a graphic example of the physiological function of a cell surface transglutaminase.

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