Proteins In Sea Urchin Embryos Research Articles

A vesicular Na+/Ca2+ exchanger in coral calcifying cells.

The calcium carbonate skeletons of corals provide the underlying structure of coral reefs; however, the cellular mechanisms responsible for coral calcification remain poorly understood. In osteoblasts from vertebrate animals, a Na+/Ca2+ exchanger (NCX) present in the plasma membrane transports Ca2+ to the site of bone formation. The aims of this study were to establish whether NCX exists in corals and its localization within coral cells, which are essential first steps to investigate its potential involvement in calcification. Data mining identified genes encoding for NCX proteins in multiple coral species, a subset of which were more closely related to NCXs from vertebrates (NCXA). We cloned NCXA from Acropora yongei (AyNCXA), which, unexpectedly, contained a peptide signal that targets proteins to vesicles from the secretory pathway. AyNCXA subcellular localization was confirmed by heterologous expression of fluorescently tagged AyNCXA protein in sea urchin embryos, which localized together with known markers of intracellular vesicles. Finally, immunolabeling of coral tissues with specific antibodies revealed AyNCXA was present throughout coral tissue. AyNCXA was especially abundant in calcifying cells, where it exhibited a subcellular localization pattern consistent with intracellular vesicles. Altogether, our results demonstrate AyNCXA is present in vesicles in coral calcifying cells, where potential functions include intracellular Ca2+ homeostasis and Ca2+ transport to the growing skeleton as part of an intracellular calcification mechanism.

Hsp56 mRNA in Paracentrotus lividus embryos binds to a mitochondrial protein

We previously demonstrated that Paracentrotus lividus Hsp56 mitochondrial chaperonin is constitutively expressed during development, that it has a specific territorial distribution, both in normal and heat-shocked embryos, and that its amount increases after heat shock [Roccheri MC, Patti M, Agnello M, Gianguzza F, Carra E, Rinaldi AM. Localization of mitochondrial Hsp56 chaperonin during sea urchin development. Biochem Biophys Res Commun 2001;287:1093-98] and cadmium treatment [Roccheri MC, Agnello M, Boneventura R, Matranga V. Cadmium induces the expression of specific stress proteins in sea urchin embryos. Biochem Biophys Res Commun 2004;321:80-7]. In this study, we looked at Hsp56 mRNA during normal development and under stress conditions. The messenger is almost constantly expressed at all stages of development and its amount is steadily increased in stressed embryos. Moreover, we found, using T1 RNase protection assay, that the most proximal region of the 3'-UTR of the Hsp56 mRNA binds a 40 kDa protein: this factor is more abundant in the mitochondrial extract and, more specifically, in the outer membrane of the organelle.

Cadmium induces the expression of specific stress proteins in sea urchin embryos

Marine organisms are highly sensitive to many environmental stresses, and consequently, the analysis of their bio-molecular responses to different stress agents is very important for the understanding of putative repair mechanisms. Sea urchin embryos represent a simple though significant model system to test how specific stress can simultaneously affect development and protein expression. Here, we used Paracentrotus lividus sea urchin embryos to study the effects of time-dependent continuous exposure to subacute/sublethal cadmium concentrations. We found that, between 15 and 24 h of exposure, the synthesis of a specific set of stress proteins (90, 72–70, 56, 28, and 25 kDa) was induced, with an increase in the rate of synthesis of 72–70 kDa (hsps), 56 kDa (hsp), and 25 kDa, which was dependent on the lengths of treatment. Recovery experiments in which cadmium was removed showed that while stress proteins continued to be synthesized, embryo development was resumed only after short lengths of exposure.

Cloning and characterization of cDNA for syndecan core protein in sea urchin embryos.

The cDNA for the core protein of the heparan sulfate proteoglycan, syndecan, of embryos of the sea urchin Anthocidaris crassispina was cloned and characterized. Reverse transcription-polymerase chain reaction (RT-PCR) was used with total ribonucleic acid (RNA) from late gastrula stage embryos and degenerate primers for conserved regions of the core protein, to obtain a 0.1 kb PCR product. A late gastrula stage cDNA library was then screened using the PCR product as a probe. The clones obtained contained an open reading frame of 219 amino acid residues. The predicted product was 41.6% identical to mouse syndecan-1 in the region spanning the cytoplasmic and transmembrane domains. Northern analysis showed that the transcripts were present in unfertilized eggs and maximum expression was detected at the early gastrula stage. Syndecan mRNA was localized around the nuclei at the early cleavage stage, but was then found in the ectodermal cells of the gastrula embryos. Western blotting analysis using the antibody against the recombinant syndecan showed that the proteoglycan was present at a constant level from the unfertilized egg stage through to the pluteus larval stage. Immunostaining revealed that the protein was expressed on apical and basal surfaces of the epithelial wall in blastulae and gastrulae.

EGTA Treatment Causes the Synthesis of Heat Shock Proteins in Sea Urchin Embryos

Paracentrotus lividus embryos, at post-blastular stage, when subjected to a rise in temperature from physiologic (20°C) to 31°C, synthesize a large group of heat shock proteins (hsps), and show a severe inhibition of bulk protein synthesis. We show, by mono- and two-dimensional electrophoresis, that also EGTA (ethylene glycol-bis[β-aminoethyl ether] tetraacetic acid) treatment induces in sea urchin embryos both marked inhibition of bulk protein synthesis and the synthesis of the entire set of hsps. Furthermore, EGTA-treated sea urchin embryos are able to survive at a temperature otherwise lethal (35°C) becoming thermotolerant. Because incubation with a different calcium-chelator, EDTA (ethylenediaminetetraacetic acid), or in calcium-free medium did not induce hsps synthesis we conclude that the stress response caused by EGTA is not related to its calcium chelator function.

Studies on heat shock proteins in sea urchin development.

Work on stress proteins in sea urchin embryos carried out over the last 20 years is reviewed and the following major results are described. Entire sea urchin embryos, if subjected to a rise in temperature at any postblastular stage undergo a wave of heat shock protein (hsp) synthesis and survive. If subjected to the same rise between fertilization and blastula formation, they are not yet able to synthesize hsp and die. Four clones coding for the major hsp, hsp70, have been isolated and sequenced; evidence for the existence of a heat shock factor has been provided, and a mechanism for the developmental regulation of hsp synthesis discussed. Intraembryonic and intracellular hsp location has been described; and a mechanism for achievement of thermotolerance proposed. A chaperonine role for a constitutive mitochondrial hsp56 has been suggested, as well as a role for the constitutive hsp70 in cell division. Heat shock, if preceded by 12-O-tetradecanoylphorbol-12-acetate (TPA) treatment causes apoptosis.

Arylsulfatase exists as non-enzymatic cell surface protein in sea urchin embryos.

The physiological role of arylsulfatase (Ars) and its function during development have yet to be satisfactorily defined in any species, though the proteins are widely distributed and the genes have been cloned from various organisms. Here we report the dual location of two types of Ars in sea urchin embryos. The majority of sea urchin Ars does not exhibit enzyme activity and is extracellularly distributed in aboral ectoderm cells (nonenzymatic Ars). Only a small portion has enzyme activity and is localized in lysosomal vesicles (enzymatic Ars). The elution pattern of Ars proteins processed by DEAE-cellulose or analytical gel-column chromatography reveals that although the molecular radius of enzymatic Ars differs from that of nonenzymatic Ars, they have the same charge. Furthermore, sedimentation analysis shows that purified Ars of sea urchin embryos is soluble in the absence of divalent cations but becomes insoluble in the presence of Ca2+ or Mg2+. Taken together, the present results suggest that non-enzymatic Ars is a new member of the cell surface component or extracellular matrix. It is possible that this cell surface Ars plays an important role in morphogenesis of sea urchin embryos.

Effects of 6-dimethylaminopurine on the length of the cell cycle and on the state of phosphorylation of putative intermediate filament proteins in sea urchin embryos

The effects of 6-dimethylaminopurine (6-DMAP) on the length of the cell cycle and on the state of phosphorylation of a putative intermediate filament protein, p117, have been studied in sea urchin embryos. Embryos were transferred into sea water containing 600 microM 6-DMAP at 0.5, 2 or 5 min after insemination, and incubated for 30 or 90 min. The effects of 6-DMAP on cell cycle length were studied by determining the time required for completion of mitosis upon return of the embryos in normal sea water. In all instances, except for the embryos transferred 0.5 min after insemination (AI) and incubated for 30 min, the duration of the M phase was shortened compared to controls, being faster in the embryos incubated for 90 minutes compared to the 30 min incubation period. However, embryos transferred 0.5 min AI have a longer M-phase than those transferred 2 minutes or later after fertilization, suggesting that between 0.5 and 2 min after fertilization, critical phosphorylating events occur which affect the commitment of the cells to enter M-phase. To study the pattern of p117 phosphorylation during the cell cycle, the eggs were transferred 2 minutes after fertilization in presence of 600 microM 6-DMAP and with 200 microCi/ml of 32P-orthophosphate. Analyses of 32P-labelled proteins after exposure of SDS-PAGE gels and their corresponding blots suggested that phosphorylation of p117 greatly increases at the time of pronuclear fusion, and then declines slightly at prophase-metaphase. This decrease is markedly enhanced when the cells are treated with 6-DMAP during metaphase in order to induce a premature breakdown of the mitotic apparatus. A causal link is suggested between the level of phosphorylation of p117 and its state of assembly.

SpOct, a Gene Encoding the Major Octamer-Binding Protein in Sea Urchin Embryos: Expression Profile, Evolutionary Relationships, and DNA Binding of Expressed Protein

We have characterized a sea urchin gene, SpOct, that encodes a 78-kDa POU-domain protein related to mammalian Oct-1 and Oct-2. The SpOct protein binds octamer elements in the promoters of the α H2B (Bell et al., 1992, Dev. Biol. 150, 363-371) and CylIIa actin genes, and it closely resembles the major octamer-binding activity obtained from sea urchin blastula nuclear lysates in the size of its DNase I footprint on a canonical octamer element and in its relative binding affinity (Kr) for the octamer element versus poly(dAT) (1.4 × 104). Moreover, partial protein sequences obtained from affinity-purified octamer-binding protein match sequences present in SpOct. These data suggest that SpOct is closely related to, if not identical with, the major octamer-binding activity in blastula nuclear extracts. RNA gel blots reveal four forms of SpOct mRNA, ranging in size from 4 to 12 kb. They are regulated coordinately in the embryo: all are present in the unfertilized egg, increase 28-fold in amount by the 8-hr blastula stage, and decline 6-fold by the 12-hr blastula stage. The same four size classes of SpOct mRNAs are present in several adult tissues, although their relative amounts vary. The temporal profile of SpOct mRNA expression in embryos closely resembles that of the α histone H2B gene. Our previous work (Bell et al., 1992) showed that expression of the α H2B gene in blastula-stage embryos was entirely dependent on an octamer element. Together, these data strongly suggest that SpOct may be the key regulator of the a H2B gene.

Cyclic appearance of cytoplasmic NOR–silver‐stained particles in sea urchin embryos

When sea urchin embryos were subjected to nucleolar organizer region (NOR)-silver staining, densely stained particles were observed in the cytoplasm. The appearance of these cytoplasmic particles (CPs) was cell-cycle dependent. During early development, the CPs were detected at interphase, but not during mitosis; they disappeared at metaphase and reappeared at telophase. The CPs appeared periodically even when embryos were treated with cytochalasin B or aphidicolin, which inhibits the progression of cytokinesis and nuclear division, respectively. By contrast, CPs were not detected in the colchicine-treated embryos in which both cytokinesis and nuclear divisions were prevented. The CPs were observed only in the embryos whose stage was early blastula (about 6th to 7th cleavage) or earlier; no CPs were detected even at interphase in the embryos at late blastula (about 8th to 9th cleavage) or later. Electron microscopic evaluation showed CPs to be granular structures, similar to heavy bodies. Also, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) showed that 95-kDa and 38-kDa proteins were the NOR-silver-staining proteins in sea urchin embryos. These proteins existed during the course of the cell cycles. These results suggest that (1) the cyclic appearance of the CPs or heavy bodies is closely related to the cell cycle as well as the programming of the embryogenesis, but independent of the cycle of cytokinesis and nuclear division; (2) 95-kDa and 38-kDa proteins are the major NOR-silver-staining proteins in sea urchin embryos.

Immunohistochemical localization of a tenascin-like extracellular matrix protein in sea urchin embryos.

We have used polyclonal antisera raised against vertebrate tenascin to identify and localize tenascin-like proteins in the developing sea urchin. These antisera recognize high-molecular weight proteins on immunoblots of sea urchin embryo homogenates that are similar in size and appearance to tenascin from vertebrates. These proteins appear as a doublet with an apparent molecular weight of 150 kDa and a larger, broad band with an apparent molecular weight of 350 kDa. Whole mounts of sea urchin embryos and larvae were stained with one of these antisera. The anti-tenascin stained the surface of primary mesenchyme cells during their phase of active migration. This staining was sensitive to detergent, suggesting that the protein recognized by the antiserum was associated with the cell surface. During later stages of development, the bulk of the antitenascin staining was found dispersed throughout the blastocoel matrix, and was no longer sensitive to detergent. We conclude that sea urchins express tenascin-like proteins during early stages of development, and that these proteins may play a role associated with primary mesenchyme cell morphogenesis.

Immunological evidence for the presence in sea urchin embryos of a cell adhesion protein similar to mouse uvomorulin (E-cadherin)

A tryptic fragment (88 kDa), obtained from external digestion of sea urchin embryos carried out in the presence of calcium, shows immunological cross-reactivity with polyclonal and monoclonal antibodies (DECMA-1) against mouse teratocarcinoma uvomorulin. Fab fragments obtained from anti-mouse terato-carcinoma uvomorulin mono- and polyclonal antibodies, and from polyclonal antibodies against the partially purified 88-kDa tryptic fragment, decompact early sea urchin embryos and block reaggregation of dissociated sea urchin blastula cells. These data indicate the presence of an uvomorulin-like protein in sea urchin embryos and suggest an important role for this protein in embryonic development.

Unusual pattern of accumulation of mRNA encoding EGF-related protein in sea urchin embryos.

A sea urchin (Strongylocentrotus purpuratus) messenger RNA encoding a protein (SpEGF2) related to epidermal growth factor (EGF) was identified. The full-length complementary DNA sequence predicts a protein with an unusually simple structure, including four tandem EGF-like repeats and a hydrophobic leader, but lacking a potential transmembrane domain. Sequence similarities suggest that the peptides are homologous to two peptides from a different sea urchin species, which cause a classic developmental defect, exogastrulation, when added to the seawater outside of embryos. The SpEGF2 messenger RNA begins to accumulate at blastula stage, and in pluteus larvae it is distributed in discrete regions of ectoderm that are not congruent with known histological borders. One region corresponds to that expressing the homeodomain-containing protein, SpHbox1. The structure of the SpEGF2 protein and the pattern of accumulation of its messenger RNA suggest that it may have important functions as a secreted factor during development of sea urchin embryos.

Developmental time, cell lineage, and environment regulate the newly synthesized proteins in sea urchin embryos

Strongylocentrotus purpuratus embryos were fractionated into two cell populations of defined lineages at times corresponding to two critical developmental events: determination (16-cell stage) and early differentiation (mesenchyme blastula). The 16-cell stage blastomeres, labeled with [ 35S]methionine, exhibited identical protein synthesis patterns by fluorography, and this pattern was not significantly altered by cell separation. In comparing the proteins of the mesenchyme blastula to the 16-cell stage, differences (increases and decreases) were seen by fluorography of newly synthesized proteins. The synthesis of 2.9% of the mesenchyme blastula proteins is specific to or enriched in primary mesenchyme cells and 8.2% is specific to or enriched in endoderm/ectoderm cells. Additionally, in contrast to the earlier stage, the pattern of protein synthesis in the mesenchyme blastula embryos is substantially altered by cell separation. The ability to alter protein synthesis in response to environmental factors may be a further demonstration of the differentiation of these cells.

Heat-Shock Proteins in Sea Urchin Embryos: Transcriptional and Posttranscriptional Regulation

The production of heat-shock proteins in sea urchin embryos is accompanied by the appearance at the polysomal level of their relative mRNAs, as shown by their translation in a cell-free system; thus suggesting that the regulation of their production occurs at a transcriptional level. The mechanism for the inhibition of the bulk protein synthesis and for its reversal on the other hand should be looked for at a posttranscriptional level, since both these phenomena occur also in the presence of actinomycin D. The heat-shock proteins produced as early as at the mesenchyme blastula stage persist within the embryo at least till the pluteus stage.

Synthesis of “heat shock” proteins in sea urchin embryos

Heating at 31 °C produces in sea urchin gastrulae a general and severe inhibition of the protein synthesis together with a marked increase in the rate of synthesis of one or very few classes of specific proteins.

Microtubule protein pools in early development.

Microtubule protein pools have been demonstrated to exist in unfertilized eggs and the early embryonic stages of several organisms. The microtubule pool of the sea urchin embryo is constant in size (about 0.4% of the total embryo protein) throughout early development. Protein withdrawn from this pool for organelle assembly is replaced by new synthesis. Eggs and embryos of Drosophila similarly contain a pool of microtubule proteins (larger than or equal to 0.4% of the total embryo protein, congruent to 3% of the soluble protein), which is constant in size throughout early development. The Drosophila egg microtubule proteins are easily purified by self-assembly in vitro of microtubules, and are similar to microtubule proteins from other organisms in molecular weight and other properties. Synthesis of microtubule proteins in sea urchin embryos is supported by oogenetic mRNA. This appears also to be the case in molluscan (Ilyanassa) embryos. It is not known whether Drosophila embryos synthesize microtubule proteins during the early stages of development.

Fractionation and analysis of nuclear proteins in sea urchin embryos

Acid-soluble protein from the nuclei of three sea urchin species ( Strongylocentrotus purpuratus, Sphaerechinus granulosus and Paracentrotus lividus) were analyzed during early embryonic development. No histone-like proteins could be detected in unfertilized eggs or in embryos before the blastula stage. Electrophoretic patterns showing five major bands, similar to the mammalian histones, were found for acid-soluble proteins extracted from the blastula nuclei. This pattern did not change qualitatively during further development. There were no differences in histone patterns from the three sea urchin species. Lysine-rich histones from sperm differed electrophoretically from histones in embryos. Electrophoretic patterns of acid-soluble nuclear proteins in unfertilized eggs and embryos before blastula strongly resembled patterns obtained for proteins extracted with acid from cytoplasmic components of the same embryos. The amino acid composition of acid-soluble proteins from unfertilized eggs was not basic. In blastula and especially in gastrula and later stages the amino acid composition of acid-soluble nuclear proteins became basic, resembling the composition of histones from vertebrate nuclei. Nuclear acidic proteins (NaOH-soluble) and nuclear residual proteins increased their content of dicarboxylic amino acids during blastulation. In gastrula and later stages the dicarboxylic amino acid contents of these proteins returned to values seen in unfertilized eggs.

Differences in isotopic labelling of nucleic acid and protein in sea urchin embryos developing from animal and vegetal egg halves

Differences in isotopic labelling of nucleic acid and protein in sea urchin embryos developing from animal and vegetal egg halves