Echinonectin: a new embryonic substrate adhesion protein

An extracellular matrix molecule has been purified from sea urchin (Lytechinus variegatus) embryos. Based on its functional properties and on its origin, this glycoprotein has been given the name "echinonectin." Echinonectin is a 230-kD dimer with a unique bow tie shape when viewed by electron microscopy. The molecule is 12 nm long, 8 nm wide at the ends, and narrows to approximately 4 nm at the middle. It is composed of two 116-kD U-shaped subunits that are attached to each other by disulfide bonds at their respective apices. Polyclonal antibodies were used to localize echinonectin in paraffin-embedded, sectioned specimens by indirect immunofluorescence. The protein is stored in vesicles or granules in unfertilized eggs, is released after fertilization, and later becomes localized on the apical surface of ectoderm cells in the embryo. When used as a substrate in a quantitative in vitro assay, echinonectin is highly effective as an adhesive substrate for dissociated embryonic cells. Because of the quantity, pattern of appearance, distribution, and adhesive characteristics of this protein, we suggest that echinonectin serves as a substrate adhesion molecule during sea urchin development.     

The structure and activities of echinonectin: a developmentally regulated cell adhesion glycoprotein with galactose-specific lectin activity.

The extracellular matrix of the sea urchin embryo contains a 230 kD homodimeric glycoprotein known as echinonectin (EN). EN contains a cell attachment domain as well as a galactose-specific lectin activity. Cell attachment to EN is differentially regulated in the three primary germ layers, endoderm, ectoderm and mesoderm. Prior to gastrulation all embryonic cells adhere equally to EN-coated substrates, but during gastrulation primary mesenchyme cells lose affinity for EN, ectoderm cells increase their binding to the molecule, and cells of the endoderm maintain a similar or slightly lowered level of binding. The mechanisms governing these adhesive changes and the specific functions they serve in development are not currently understood. They are timed to coincide with distinct morphogenetic events such as primary mesenchyme cell ingression and archenteron formation, suggesting that regulated adhesion to EN plays at least a permissive role in early morphogenesis.     

Sea urchin arylsulfatase,an extracellular matrix component,is involved in gastrulation during embryogenesis

Arylsulfatases (Arses) have been regarded as lysosomal enzymes because of their hydrolytic activities on synthetic aromatic substrates and their lysosomal localization of their enzymatic activities. Using sea urchin embryos, we previously demonstrated that the bulk of Hemicentrotus Ars (HpArs) does not exhibit enzyme activity and is located on the apical surface of the epithelial cells co-localizing with sulfated polysaccharides. Here we show that HpArs strongly binds to sulfated polysaccharides and that repression of the synthesis by HpArs-morpholino results in retardation of gastrulation in the sea urchin embryo. Accumulation of HpArs protein and sulfated polysaccharides on the apical surface of the epithelial cells in sea urchin larvae is repressed by treatment with β-aminopropionitrile (BAPN), suggesting that deposition of HpArs and sulfated polysaccharides is dependent on the crosslinking of proteins such as collagen-like molecules. We suggest that HpArs functions by binding to components of the extracellular matrix.     

Biochemical and immunological relationships among fibronectin-like proteins from different sea urchin species

Fibronectin-like proteins were purified from ovaries of the sea urchin species, Paracentrotus lividus (PI), Sphaerechinus granularis (Sg), Arbacia lixula (Al), Pseudocentrotus depressus (Pd), and Anthocidaris crassispina (Ac), by gelatin-Sepharose affinity chromatography. The major component had a molecular mass of 180 kDa and was eluted by 1 M NaCl or 8 M urea, depending on the species used. By substrate adhesion assay, we tested the biological activity of the 180 kDa protein purified from Paracentrotus lividus (P1-180K) and showed that it promotes the adhesion of homologous embryonic cells to the substrate. An antiserum, developed against Temnopleurus hardwickii fibronectin-like protein (Th-180K), was used in Western blots of the proteins purified from the five species. The antibody cross-reacted with Pl-180K, Pd-180K and Ac-180K. A peptide map of P1-180K, obtained by V8 protease partial digestion, was compared with those obtained from the other four proteins and showed an homology between 40 and 56%. This report confirms that fibronectin-like proteins can be purified from sea urchins on the basis of their binding to gelatin-Sepharose; the proteins differ for their binding affinity to gelatin and share different epitopes, suggesting that they are members of a sea urchin fibronectin super family.     

Sea urchin egg hyaline layer: evidence for the localization of hyalin on the unfertilized egg surface

The sea urchin embryo hyaline layer is an extracellular investment which develops within 20 min postinsemination of Strongylocentrotus purpuratus eggs and contains a single calcium-precipitable subunit termed hyalin. Other ultrastructural and biochemical studies have suggested that hyalin is localized in the cortical granules. We have examined the hypothesis that hyalin is a cell surface protein of the unfertilized egg using vectorial lactoperoxidase-catalyzed radioiodination. Extracts of labeled unfertilized eggs contained several labeled proteins, one of which was electrophoretically indistinguishable from authentic hyalin isolated by each of three different procedures. Pronase digestion of labeled unfertilized eggs removed 75% of the label, but the labeled hyalin-like molecule was still present in whole cell extracts. Upon insemination, pronase-digested, labeled eggs formed an apparently normal hyaline layer and whole cell extracts contained the labeled hyalin-like molecule. Denuded, labeled eggs were inseminated and the hyaline layer was selectively solubilized in calcium- and magnesium-free artificial seawater. Labeled hyalin was purified from this crude hyalin preparation to constant specific radioactivity and apparent homogeneity as shown by gel electrophoresis. These data strongly suggest that hyalin or a precursor is a cell surface protein of the unfertilized sea urchin egg.     

Identification and characterization of PlAlix,the Alix homologue from the Mediterranean sea urchin Paracentrotus lividus

The sea urchin provides a relatively simple and tractable system for analyzing the early stages of embryo development. Here, we use the sea urchin species, Paracentrotus lividus, to investigate the role of Alix in key stages of embryogenesis, namely the egg fertilization and the first cleavage division. Alix is a multifunctional protein involved in different cellular processes including endocytic membrane trafficking, filamentous (F)‐actin remodeling, and cytokinesis. Alix homologues have been identified in different metazoans; in these organisms, Alix is involved in oogenesis and in determination/differentiation events during embryo development. Herein, we describe the identification of the sea urchin homologue of Alix, PlAlix. The deduced amino acid sequence shows that Alix is highly conserved in sea urchins. Accordingly, we detect the PlAlix protein cross‐reacting with monoclonal Alix antibodies in extracts from P. lividus, at different developmental stages. Focusing on the role of PlAlix during early embryogenesis we found that PlAlix is a maternal protein that is expressed at increasingly higher levels from fertilization to the 2‐cell stage embryo. In sea urchin eggs, PlAlix localizes throughout the cytoplasm with a punctuated pattern and, soon after fertilization, accumulates in larger puncta in the cytosol, and in microvilli‐like protrusions. Together our data show that PlAlix is structurally conserved from sea urchin to mammals and may open new lines of inquiry into the role of Alix during the early stages of embryo development.     

Ontogeny of the basal lamina in the sea urchin embryo

The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen types I, III, and IV; and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 micron) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de novo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.     

Sea urchin egg-cortical granule protease has arginyl proteolytic specificity

Sea urchin eggs secrete esteroproteolytic activity at fertilization. This enzyme has been shown to be proteolytic toward embryo protein and casein, but a systematic study of its substrate specificity has not been done. In this communication we present data that demonstrates for the first time that the cortical granule protease from Strongylocentrotus purpuratus eggs cleaves arginyl residues in a protein substrate, lysozyme. We have developed a sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) assay that detects femtomole levels of trypsin and chymotrypsin protease activity [Green, 1986: Anal Biochem 152:83–88]. In the sea urchin system, we have detected protease activity from as few as 50 eggs. Correlating the RP-HPLC analysis with a spectrophotometric Nα-benzoyl-L-arginine ethyl ester assay, we have found that each egg secretes approximately 40 attomoles of trypsin-like activity. This general method should be quite useful in investigations into the natural substrate of the egg protease.     

Pl-nectin,a discoidin family member,is a ligand for βC integrins in the sea urchin embryo

Pl-nectin is a component of the extracellular matrix that surrounds embryos of the sea urchin Paracentrotus lividus. Pl-nectin mediates adhesion of dissociated embryonic cells to substrates and interfering with ectodermic cells contacting Pl-nectin results in defects in skeleton growth and morphogenesis. Recently, we reported that Pl-nectin is a new member of the discoidin family, in agreement with the notion that many discoidin-containing proteins are involved in cell adhesion processes as integrin ligands. To better understand the molecular basis for the interaction of Pl-nectin with ectoderm, we investigated the hypothesis that Pl-nectin is an integrin ligand in sea urchin embryos. We show that in P. lividus embryos, βC-containing integrins localize to the apical surface of ectodermic cells, which are in contact with Pl-nectin. Immunoprecipitation experiments indicate that the two proteins are part of a complex in vivo and affinity chromatography indicates that βC-containing integrin receptors bind purified Pl-nectin. These data support a model in which ectodermic integrins binding to Pl-nectin mediate cellular adhesion to the hyaline layer. Regulated adhesion of cells to the hyaline layer is a critical component of several morphogenetic processes and the identification of the receptors and ligands involved provides new opportunities to investigate the underlying molecular mechanisms of ECM adhesion and morphogenesis.     

Detection of two immunochemically identical forms of mannan-binding lectin in the sea urchin <Emphasis Type="Italic">Strongylocentrotus nudus</Emphasis>

This study revealed a new lectin (MBL-SN) in the coelomic fluid of the sea urchin Strongylocentrotus nudus. Based on the peculiarities of molecular structure and carbohydrate specificity, MBL-SN can be assigned to the mannan-binding lectin family. Using polyclonal monospecific rabbit antibodies against MBL-SN, the presence of MBL-SN in the sea urchin was detected in two forms: a soluble form dissolved in the coelomic fluid and an extracellular matrix-bound form. The biosynthesis site of this lectin may be one of the subpopulations of morula cells-coelomic fluid cells that perform heterosynthesis. Our results demonstrate the similarity of the sea urchin lectin MBL-SN to the previously investigated MBLs of the holothurians Cucumaria japonica and Apostichopus japonicus, and suggest a similarity to MBLs of vertebrates, which also have soluble and bound forms.     

Cell-to-substratum adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus

Summary Some plant lectins, Concanavalin agglutinin (Con A), succinyl Con A and wheat germ agglutinin (WGA) increased the adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus, to the substratum (plastic and glass surface) in vitro. Other plant lectins,Ulex europeus agglutinin (UEA) andDolichos biflorus agglutinin (DBA) had no effect on the cell-to-substratum interaction. A specific monocarbohydrate inhibitor of lectins, -methyl-d-mannoside, inhibited the Con A-induced cell-to-substratum adhesion of dissociated embryonic cells. This observation suggests that the Con A-induced cell-to-substratum adhesion may be attributed to the Con A-carbohydrate interaction. In Millipore-filtered sea water (MPFSW) containing Con A (0.1 mg/ml), dissociated embryonic cells adhered to the substratum for more than 6 h at 18°C, while in MPFSW as control, almost all the dissociated cells were released from the substratum after 1 h. A scanning electron microscopic study showed that dissociated embryonic cells adhered to the substratum were surrounded by an extracellular fibrous material, when the cells were cultured in MPFSW containing Con A. The induction of the extracellular fibrous material by Con A was inhibited by -methyl-d-mannoside. The appearance of this material may be related to the cell-to-substratum adhesion of dissociated cells. Sequential extractions of Con A-treated dissociated cells with Triton X 100 and urea solubilized most of the cellular components, leaving the fibrous material on the surface. Biochemical conponents of the isolated fibrous material included sea urchin fibronectin, Con A and minor components (88 and 140 kilodalton proteins). Fibronectin preformed in the cells was excreted after the dissociation, while the 88 and 140 kilodalton proteins were synthesized and released to the extracellular space.     

Fluorescence staining of living cells with fluorescamine

Summary The interaction of fluorescamine with living plant and animal cells was investigated to determine which subcellular structures and molecular species might react with the dye and to assess its effects on cell viability and function.Plasma and nuclear membranes ofXenopus erythrocytes, mitochondria of sea urchin sperm, growing apices of Timothy root hairs, and various organelles ofNitella andEuglena were labelled as judged by fluorescence microscopy. Cytoplasmic fluorescence was particulate inNitella and easily displaced by moderate centrifugal fields in sea urchin eggs. Chloroplasts and nuclei isolated from cells labelledin vivo exhibited fluorescamine dependent fluorescence.Reaction seemed to have little or no effect on cell viability (Euglena) photoautotrophic growth (Euglena), cell motility (sperm), fertilizability (sperm or egg), embryonic development (sea urchin), or cytoplasmic streaming (Nitella, Timothy).Quantitative fluorometric analysis of thein vivo reactants in sperm indicated a reaction preference for phospholipid over protein compared to control cells dissociated in SDS prior to labelling. The bulk of labelled lipid was phosphatidylethanolamine.These results suggest that fluorescamine is a true vital dye which can label the cell surface as well as penetrate deeply within cells to label a variety of organelles. The distribution of fluorescence and results of chemical analysis suggest thatin vivo the dye may preferentially react with membrane.     

A directed miniscreen for genes involved in the Drosophila anti-parasitoid immune response

Drosophila larvae react against eggs from the endoparasitoid wasp Leptopilina boulardi by surrounding them in a multilayered cellular capsule. Once a wasp egg is recognized as foreign, circulating macrophage-like cells, known as plasmatocytes, adhere to the invader. After spreading around the wasp egg, plasmatocytes form cellular junctions between the cells, effectively separating the egg from the hemocoel. Next, a second sub-type of circulating immunosurveillance cell (hemocyte), known as lamellocytes, adhere to either the wasp egg or more likely the plasmatocytes surrounding the egg. From these events, it is obvious that adhesion and cell shape change are an essential part of Drosophila's cellular immune response against parasitoid wasp eggs. To date, very few genes have been described as being necessary for a proper anti-parasitization response in Drosophila. With this in mind, we performed a directed genetic miniscreen to discover new genes required for this response. Many of the genes with an encapsulation defect have mammalian homologues involved in cellular adhesion, wound healing, and thrombosis, including extracellular matrix proteins, cellular adhesion molecules, and small GTPases.     

Extracellular matrix triggers a directed cell migratory response in sea urchin primary mesenchyme cells

The role of extracellular matrix in cell migration has generally been considered in terms of a substratum. However, when thin cell processes from migrating sea urchin primary mesenchyme cells contact small latex beads coated with extracellular matrix from the blastocoel, the cells migrate directly to the coated beads. Since the beads are not anchored, this result indicates that highly localized contact with the extracellular matrix can stimulate movement independently of any change in cell adhesion.     

The sea urchin egg receptor for sperm: isolation and characterization of the intact, biologically active receptor

The species-specific binding of sea urchin sperm to the egg is mediated by an egg cell surface receptor. Although earlier studies have resulted in the cloning and sequencing of the receptor, structure/function studies require knowledge of the structure of the mature cell surface protein. In this study, we report the purification of this glycoprotein to homogeneity from a cell surface complex of Strongylocentrotus purpuratus eggs using lectin and ion exchange chromatography. Based on the yield of receptor it can be calculated that each egg contains approximately 1.25 x 10(6) receptor molecules on its surface. The receptor, which has an apparent M(r) of 350 kD, is a highly glycosylated transmembrane protein composed of approximately 70% carbohydrate. Because earlier studies on the partially purified receptor and on a pure, extracellular fragment of the receptor indicated that the carbohydrate chains were important in sperm binding, we undertook compositional analysis of the carbohydrate in the intact receptor. These analyses and lectin binding studies revealed that the oligosaccharide chains of the receptor are sulfated and that both N- and O-linked chains are present. Functional analyses revealed that the purified receptor retained biological activity; it inhibited fertilization in a species-specific and dose-dependent manner, and polystyrene beads coated with it bound to acrosome-reacted sperm in a species-specific manner. The availability of biochemical quantities of this novel cell recognition molecule opens new avenues to studying the interaction of complementary cell surface ligands in fertilization.     

A protein of the basal lamina of the sea urchin embryo

The purification, biochemical characterization and functional features of a novel extracellular matrix protein are described. This protein is a component of the basal lamina found in embryos from the sea urchin species Paracentrotus lividus and Hemicentrotus pulcherrimus . The protein has been named PI-200 K or Hp-200 K, respectively, because of the species from which it was isolated and its apparent molecular weight in SDS-PAGE under reducing conditions. It has been purified from unfertilized eggs where it is found packed within cytoplasmic granules, and has different binding affinities to type I collagen and heparin, as assessed by affinity chromatography columns. By indirect immunofluorescence experiments it was shown that, upon fertilization, the protein becomes extracellular, polarized at the basal surface of ectoderm cells, and on the surface of primary mesenchyme cells at the blastula and gastrula stages. The protein serves as an adhesive substrate, as shown by an in vitro binding assay where cells dissociated from blastula embryos were settled on 200K protein-coated substrates. To examine the involvement of the protein in morphogenesis of sea urchin embryo, early blastula embryos were microinjected with anti-200K Fab fragments and further development was followed. When control embryos reached the pluteus stage, microinjected embryos showed severe abnormalities in arms and skeleton elongation and patterning. On the basis of current results, it was proposed that 200K protein is involved in the regulation of sea urchin embryo skeletogenesis.     

Endo16, a lineage-specific protein of the sea urchin embryo, is first expressed just prior to gastrulation

We have isolated and characterized a new endoderm-specific gene, designated Endo16, from a sea urchin gastrula stage cDNA library. Northern blot analysis and in situ hybridization experiments indicate that this gene is first expressed in the vegetal plate, a group of endodermal and mesenchymal precursor cells that are poised to invaginate in the first movement of gastrulation. Expression becomes progressively restricted to a subset of endodermal cells as development proceeds. To study the Endo16 gene product, a polyclonal antiserum was raised against bacterially expressed Endo16 protein. Indirect immunofluorescence experiments in midgastrula stage embryos reveal that the Endo16 protein is localized to the surface of endoderm and secondary mesenchyme cells. In Western blot experiments, the antiserum detects a small set of high molecular weight proteins ranging from 180 to greater than 300 kDa. Analysis of the nucleotide-derived amino acid sequence from a partial Endo16 cDNA clone reveals a highly repetitive, extremely acidic protein segment that includes the Arg-Gly-Asp (RGD) tripeptide known to be important in cell binding domains of a number of extracellular proteins. Taken together, these data suggest that the Endo16 protein may be an adhesion molecule involved in gastrulation of the sea urchin embryo.     

Guanine nucleotide-binding protein in sea urchin eggs serving as the specific substrate of islet-activating protein, pertussis toxin

A GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was partially purified from Lubrol extract of sea urchin egg membranes. The partially purified protein possessed two polypeptides of 39 and 37 kDa; the 39 kDa polypeptide was specifically ADP-ribosylated by IAP and the 37 kDa protein cross-reacted with the antibody prepared against purified beta gamma-subunits of alpha beta gamma-heterotrimeric IAP substrates from rat brain. Incubation of this sea urchin IAP substrate with a non-hydrolyzable GTP analogue resulted in a reduction of the apparent molecular mass on a column of gel filtration as had been the case with purified rat brain IAP substrates, suggesting that the sea urchin IAP substrate was also a heterooligomer dissociable into two polypeptides in the presence of GTP analogues. Thus, the 39 and 37 kDa polypeptides of the sea urchin IAP substrate correspond to the alpha- and beta-subunits, respectively, of mammalian IAP substrates which are involved in the coupling between membrane receptor and effector systems.