scholarly journals Rendezvin: An Essential Gene Encoding Independent, Differentially Secreted Egg Proteins That Organize the Fertilization Envelope Proteome after Self-Association

2006 ◽  
Vol 17 (12) ◽  
pp. 5241-5252 ◽  
Author(s):  
Julian L. Wong ◽  
Gary M. Wessel
Keyword(s):  
Extracellular Matrix ◽  
Sea Urchins ◽  
Essential Gene ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Gene Encoding ◽  
Self Association ◽  
Granule Secretion

Preventing polyspermy during animal fertilization relies on modifications to the egg's extracellular matrix. On fertilization in sea urchins, the contents of cortical granules are secreted and rapidly assemble into the egg's extracellular vitelline layer, forming the fertilization envelope, a proteinaceous structure that protects the zygote from subsequent sperm. Here, we document rendezvin, a gene whose transcript is differentially spliced to yield proteins destined for either cortical granules or the vitelline layer. These distinctly trafficked variants reunite after cortical granule secretion at fertilization. Together, they help coordinate assembly of the functional fertilization envelope, whose proteome is now defined in full.

Cortical granule biogenesis is active throughout oogenesis in sea urchins

Development ◽  
1994 ◽  
Vol 120 (5) ◽  
pp. 1325-1333 ◽  
Author(s):  
M. Laidlaw ◽  
G.M. Wessel
Keyword(s):  
Recombinant Proteins ◽  
Sea Urchins ◽  
Germinal Vesicle ◽  
Cortical Granule ◽  
Cdna Clones ◽  
Secretory Vesicles ◽  
Cortical Granules ◽  
Oocyte Growth ◽  
Peak Abundance ◽  
Cognate Protein

Cortical granules are secretory vesicles formed in the eggs of most animals and are essential for the prevention of polyspermy in these organisms. We have studied the biogenesis of cortical granules in sea urchin oocytes by identifying cDNA clones that encode proteins targeted selectively to the cortical granules. These cDNA clones were identified by an immunoscreen of a cDNA library using antibodies to proteins of the fertilization envelope. Four different mRNAs were identified, ranging from 4 kb to 13 kb in length, that encoded proteins targeted specifically to cortical granules. Accumulation of these mRNAs began very early in oogenesis, in oocytes approximately 10–15 microns in diameter, and continued throughout oogenesis. The mRNAs reached peak abundance (on a per cell basis) in germinal vesicle stage oocytes, and the accumulation of each mRNA was linear with respect to oocyte growth. During breakdown of the germinal vesicle these mRNAs were degraded so that in eggs the mRNA signals were at background levels. Antibodies generated to recombinant proteins made from each of these cDNA clones showed that in the oocyte each cognate protein appeared early in oogenesis. These proteins accumulated only in cortical granules: no accumulation was seen in the cytoplasm, in Golgi, or in other vesicles, and no heterogeneity of the contents was seen within the population of cortical granules. Using these antibodies we show that cortical granules accumulated linearly throughout oogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Regulated Proteolysis by Cortical Granule Serine Protease 1 at Fertilization

2004 ◽  
Vol 15 (5) ◽  
pp. 2084-2092 ◽  
Author(s):  
Sheila A. Haley ◽  
Gary M. Wessel
Keyword(s):  
Serine Protease ◽  
Protease Activity ◽  
Sea Urchins ◽  
Small Population ◽  
Structural Proteins ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Fluorescent Indicators ◽  
Seawater Ph ◽  
Protease Activation

Cortical granules are specialized organelles whose contents interact with the extracellular matrix of the fertilized egg to form the block to polyspermy. In sea urchins, the granule contents form a fertilization envelope (FE), and this construction is critically dependent upon protease activity. An autocatalytic serine protease, cortical granule serine protease 1 (CGSP1), has been identified in the cortical granules of Strongylocentrotus purpuratus eggs, and here we examined the regulation of the protease activity and tested potential target substrates of CGSP1. We found that CGSP1 is stored in its full-length, enzymatically quiescent form in the granule, and is inactive at pH 6.5 or below. We determined the pH of the cortical granule by fluorescent indicators and micro-pH probe measurements and found the granules to be pH 5.5, a condition inhibitory to CGSP1 activity. Exposure of the protease to the pH of seawater (pH 8.0) at exocytosis immediately activates the protease. Activation of eggs at pH 6.5 or lower blocks activation of the protease and the resultant FE phenotypes are indistinguishable from a protease-null phenotype. We find that native cortical granule targets of the protease are β-1,3 glucanase, ovoperoxidase, and the protease itself, but the structural proteins of the granule are not proteolyzed by CGSP1. Whole mount immunolocalization experiments demonstrate that inhibition of CGSP1 activity affects the localization of ovoperoxidase but does not alter targeting of structural proteins to the FE. The mistargeting of ovoperoxidase may lead to spurious peroxidative cross-linking activity and contribute to the lethality observed in protease-null cells. Thus, CGSP1 is proteolytically active only when secreted, due to the low pH of the cortical granules, and it has a small population of targets for cleavage within the cortical granules.

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.

The extracellular matrix of echinoderm and amphibian eggs: Visualization in quick-frozen, deep-etched, and rotary-shadowed specimens

1990 ◽  
Vol 48 (3) ◽  
pp. 60-61
Author(s):  
Barry Bonnell ◽  
Carolyn Larabell ◽  
Douglas Chandler
Keyword(s):  
Extracellular Matrix ◽  
Plasma Membrane ◽  
Sea Urchin ◽  
Crystalline Structure ◽  
Cortical Granule ◽  
Two Dimensional ◽  
Small Peptides ◽  
Deep Etching ◽  
Quick Freezing ◽  
Acrosomal Reaction

Eggs of many species including those of echinoderms, amphibians and mammals exhibit an extensive extracellular matrix (ECM) that is important both in the reception of sperm and in providing a block to polyspermy after fertilization.In sea urchin eggs there are two distinctive coats, the vitelline layer which contains glycoprotein sperm receptors and the jelly layer that contains fucose sulfate glycoconjugates which trigger the acrosomal reaction and small peptides which act as chemoattractants for sperm. The vitelline layer (VL), as visualized by quick-freezing, deep-etching, and rotary-shadowing (QFDE-RS), is a fishnet-like structure, anchored to the plasma membrane by short posts. Orbiting above the VL are horizontal filaments which are thought to anchor the thicker jelly layer to the egg. Upon fertilization, the VL elevates and is transformed by cortical granule secretions into the fertilization envelope (FE). The rounded casts of microvilli in the VL are transformed into angular peaks and the envelope becomes coated inside and out with sheets of paracrystalline protein having a quasi-two dimensional crystalline structure.

scholarly journals The Novel ALG-2 Target Protein CDIP1 Promotes Cell Death by Interacting with ESCRT-I and VAPA/B

2021 ◽  
Vol 22 (3) ◽  
pp. 1175
Author(s):  
Ryuta Inukai ◽  
Kanako Mori ◽  
Keiko Kuwata ◽  
Chihiro Suzuki ◽  
Masatoshi Maki ◽  
...  
Keyword(s):  
Cell Death ◽  
Essential Gene ◽  
Susceptibility Gene ◽  
Target Protein ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Endosomal Sorting ◽  
Cell Death Pathways ◽  
Apoptotic Protein

Apoptosis-linked gene 2 (ALG-2, also known as PDCD6) is a member of the penta-EF-hand (PEF) family of Ca2+-binding proteins. The murine gene encoding ALG-2 was originally reported to be an essential gene for apoptosis. However, the role of ALG-2 in cell death pathways has remained elusive. In the present study, we found that cell death-inducing p53 target protein 1 (CDIP1), a pro-apoptotic protein, interacts with ALG-2 in a Ca2+-dependent manner. Co-immunoprecipitation analysis of GFP-fused CDIP1 (GFP-CDIP1) revealed that GFP-CDIP1 associates with tumor susceptibility gene 101 (TSG101), a known target of ALG-2 and a subunit of endosomal sorting complex required for transport-I (ESCRT-I). ESCRT-I is a heterotetrameric complex composed of TSG101, VPS28, VPS37 and MVB12/UBAP1. Of diverse ESCRT-I species originating from four VPS37 isoforms (A, B, C, and D), CDIP1 preferentially associates with ESCRT-I containing VPS37B or VPS37C in part through the adaptor function of ALG-2. Overexpression of GFP-CDIP1 in HEK293 cells caused caspase-3/7-mediated cell death. In addition, the cell death was enhanced by co-expression of ALG-2 and ESCRT-I, indicating that ALG-2 likely promotes CDIP1-induced cell death by promoting the association between CDIP1 and ESCRT-I. We also found that CDIP1 binds to vesicle-associated membrane protein-associated protein (VAP)A and VAPB through the two phenylalanines in an acidic tract (FFAT)-like motif in the C-terminal region of CDIP1, mutations of which resulted in reduction of CDIP1-induced cell death. Therefore, our findings suggest that different expression levels of ALG-2, ESCRT-I subunits, VAPA and VAPB may have an impact on sensitivity of anticancer drugs associated with CDIP1 expression.

scholarly journals Essential and nonessential histone H2A variants in Tetrahymena thermophila.

1996 ◽  
Vol 16 (8) ◽  
pp. 4305-4311 ◽  
Author(s):  
X Liu ◽  
B Li ◽  
GorovskyMA
Keyword(s):  
Tetrahymena Thermophila ◽  
Essential Gene ◽  
Histone Variants ◽  
Gene Replacement ◽  
Histone H2a ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Simple Mass ◽  
Essential Function ◽  
High Degree

Although variants have been identified for every class of histone, their functions remain unknown. We have been studying the histone H2A variant hv1 in the ciliated protozoan Tetrahymena thermophila. Sequence analysis indicates that hv1 belongs to the H2A.F/Z type of histone variants. On the basis of the high degree of evolutionary conservation of this class of histones, they are proposed to have one or more distinct and essential functions that cannot be performed by their major H2A counterparts. Considerable evidence supports the hypothesis that the hv1 protein in T. thermophila and hv1-like proteins in other eukaryotes are associated with active chromatin. In T. thermophila, simple mass transformation and gene replacement techniques have recently become available. In this report, we demonstrate that either the HTA1 gene or the HTA2 gene, encoding the major H2As, can be completely replaced by disrupted genes in the polyploid, transcriptionally active macronucleus, indicating that neither of the two genes is essential. However, only some of the HTA3 genes encoding hv1 can be replaced by disrupted genes, indicating that the H2A.F/Z type variants have an essential function that cannot be performed by the major H2A genes. Thus, an essential gene in T. thermophila can be defined by the fact that it can be partially, but not completely, eliminated from the polyploid macronucleus. To our knowledge, this study represents the first use of gene disruption technology to study core histone gene function in any organism other than yeast and the first demonstration of an essential gene in T. thermophila using these methods. When a rescuing plasmid carrying a wild-type HTA3 gene was introduced into the T. thermophila cells, the endogenous chromosomal HTA3 could be completely replaced, defining a gene replacement strategy that can be used to analyze the function of essential genes.

scholarly journals Structure, expression and phylogenetic analysis of the gene encoding actin I in Pneumocystis carinii.

Genetics ◽  
1994 ◽  
Vol 137 (3) ◽  
pp. 743-750 ◽  
Author(s):  
L D Fletcher ◽  
J M McDowell ◽  
R R Tidwell ◽  
R B Meagher ◽  
C C Dykstra
Keyword(s):  
Phylogenetic Analysis ◽  
Essential Gene ◽  
Pneumocystis Carinii ◽  
Comparative Sequence Analysis ◽  
Cdna Clones ◽  
Actin Gene ◽  
Gene Encoding ◽  
Actin Protein ◽  
Relationship Of ◽  
High Degree

Abstract Actin is a major component of the cytoskeleton and one of the most abundant proteins found in eukaryotic cells. Comparative sequence analysis shows that this essential gene has been highly conserved throughout eukaryotic evolution making it useful for phylogenetic analysis. Complete cDNA clones for the actin-encoding gene were isolated and characterized from Pneumocystis carinii purified from immunosuppressed rat lungs. The nucleotide sequence encodes a protein of 376 amino acids. The predicted actin protein of P. carinii shares a high degree of conservation to other known actins. Only one major actin gene was found in P. carinii. The P. carinii actin sequence was compared with 30 other actin sequences. Gene phylogenies constructed using both neighbor-joining and protein parsimony methods places the P. carinii actin sequence closest to the majority of the fungi. Since the phylogenetic relationship of P. carinii to fungi and protists has been questioned, these data on the actin gene phylogeny support the grouping of P. carinii with the fungi.

scholarly journals RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

1992 ◽  
Vol 12 (10) ◽  
pp. 4314-4326 ◽  
Author(s):  
C Mann ◽  
J Y Micouin ◽  
N Chiannilkulchai ◽  
I Treich ◽  
J M Buhler ◽  
...  
Keyword(s):  
Cell Division ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Essential Gene ◽  
Temperature Sensitive ◽  
G1 Arrest ◽  
Restrictive Temperature ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Carboxy Terminal

RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.

scholarly journals OOCYTE DIFFERENTIATION IN THE SEA URCHIN, ARBACIA PUNCTULATA, WITH PARTICULAR REFERENCE TO THE ORIGIN OF CORTICAL GRANULES AND THEIR PARTICIPATION IN THE CORTICAL REACTION

1968 ◽  
Vol 37 (2) ◽  
pp. 514-539 ◽  
Author(s):  
Everett Anderson
Keyword(s):  
Sea Urchin ◽  
Golgi Complex ◽  
Morphological Evidence ◽  
Cortical Granule ◽  
Perivitelline Space ◽  
Unit Membrane ◽  
Cortical Granules ◽  
Arbacia Punctulata ◽  
Cortical Reaction ◽  
Oocyte Differentiation

This paper presents morphological evidence on the origin of cortical granules in the oocytes of Arbacia punctulata and other echinoderms. During oocyte differentiation, those Golgi complexes associated with the production of cortical granules are composed of numerous saccules with companion vesicles. Each element of the Golgi complex contains a rather dense homogeneous substance. The vesicular component of the Golgi complex is thought to be derived from the saccular member by a pinching-off process. The pinched-off vesicles are viewed as containers of the precursor(s) of the cortical granules. In time, they coalesce and form a mature cortical granule whose content is bounded by a unit membrane. Thus, it is asserted that the Golgi complex is involved in both the synthesis and concentration of precursors utilized in the construction of the cortical granule. Immediately after the egg is activated by the sperm the primary envelope becomes detached from the oolemma, thereby forming what we have called the activation calyx (see Discussion). Subsequent to the elaboration of the activation calyx, the contents of cortical granules are released (cortical reaction) into the perivitelline space. The discharge of the constituents of a cortical granule is accomplished by the union of its encompassing unit membrane, in several places, with the oolemma.

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.

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