Initial analysis of the molecular image of pamlin, a sea urchin cell adhesion protein, by transmission electron microscopy

Pamlin, an important extracellular protein required early for sea urchin embryogenesis, is readily isolated from the embryos of Hemicentrotus pulcherrimus . A molecular image analysis of pamlin was conducted using immuno-electron microscopy, rotary shadowing and negative staining technique-applied electron microscopy. The electron microscopy showed that a monoclonal antibody to the pamlin α-subunit bound to a position 13.5 nm from one end of a purified 255 kDa pamlin molecule, which is a 132 nm long and 6.8 nm wide linear structure. The pamlin structure is composed of three subunits, a 47 nm long 52 kDa α-subunit that attaches to one end of a 105 nm long 180 kDa β-subunit, and a 15.6 nm diameter globular 23 kDa γ-subunit that binds to the middle of the β-subunit. The α- and β-subunits together form a 125–140 nm linear structure. Intermolecular aggregation frequently occurred between the free end of two β-subunits of the αβγ pamlin molecule, leaving the entire α-subunit surface free. Occasionally associations between the ends of α-subunits, or between an α-subunit and the middle of a β-subunit also occurred, but no aggregations of pamlin formed through the γ-subunit. These homophilic molecular aggregations of pamlin formed a large supramolecular network. In addition, the single pamlin molecule rounded at one end under high calcium ion concentration to form a 'loop', suggesting the presence of a calcium sensitive region in the molecule.     

Role of cell adhesion in the specification of pigment cell lineage in embryos of the sea urchin, Hemicentrotus pulcherrimus

To clarify the role of cell adhesion in the specification of pigment cell lineage in sea urchin embryos, cell contacts were inhibited by Ca²⁺-free artificial seawater (ASW) treatment, and the number of differentiated pigment cells was examined by the method devised for the present study. Obtained results showed that inhibition of cell contacts during mid-to-late blastula stage greatly affects the number of pigment cells. Treatment with Ca²⁺-free ASW during 7.5–10.5h of development drastically decreased the number of pigment cells, indicating that cell adhesion during this period is indispensable for the specification of pigment cell lineage. On the other hand, the number of pigment cells were increased by the treatment during 9.5–12.5 h of development. It was suggested that this increase was caused by excess divisions of the precursor cells, that is, the division schedule of the precursor cells was altered by inhibition of cell contacts at this period. Interestingly, the number of pigment cells was a multiple of four in a majority of embryos in which pigment cells were drastically decreased in number. These findings suggest that the founder blastomeres of the pigment cell lineage are specified during 7–10 h of development, and that these blastomeres divide twice before they differentiate into pigment cells.     

A new extracellular matrix protein of the sea urchin embryo with properties of a substrate adhesion molecule

Summary A new embryonic extracellular matrix protein has been purified from eggs of the sea urchin Paracentrotus lividus. The molecule is a 210 kD dimer consisting of two 105 kD subunits that are held together by S-S bridges. In the unfertilized egg, the protein is found within granules uniformly distributed throughout the cytoplasm. After the egg is fertilized, the antigen is polarized to the apical surface of ectodermal and endodermal cells during all of the developmental stages examined, until the pluteus larva is formed. The protein promotes the adhesion of blastula cells to the substrate and is antigenically distinct from echinonectin, a well characterized substrate adhesion molecule. This report adds a new candidate to the list of known extracellular matrix molecules for the regulation of differentiation and morphogenesis in the sea urchin embryo. Offprint requests to: V. Matranga     

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.     

Establishment of pigment cell lineage in embryos of the sea urchin, Hemicentrotus pulcherrimus

In an attempt to estimate the number of pigment precursor cells in sea urchin embryos, DNA synthesis and cell divisions were blocked with aphidicolin from various stages of development. Interestingly, pigment cells differentiated on a normal time schedule, even if the embryos were treated from late cleavage stages on. In most of the embryos treated from 10 h on, 10-15 pigment cells differentiated. Thereafter, the number of pigment cells in the aphidicolin-treated embryos further increased, as the initiation of the treatment was delayed. On the other hand, total cell volumes in the pigment lineage, calculated from the averaged number and diameter of differentiated pigment cells, were almost the same irrespective of the time of the initiation of aphidicolin treatment. This indicated that the increase in the number was caused by divisions of the pre-existing cells in the pigment lineage. Thus, the founder cells that exclusively produce pigment cells could be identified. They are nine times-cleaved blastomeres and specified by 10 h post-fertilization. The obtained results also clarified the division schedule in the pigment lineage; the founder cells divide once (10th) until hatching, and divide once more (11th) by the end of gastrulation.     

Isolation and identification of novel sulfated and nonsulfated oligosialyl glycosphingolipids from sea urchin sperm

Novel sulfated and nonsulfated oligosialyglycosphingolipids were isolated from sperm of the sea urchin,Hemicentrotus pulcherrimus, and their structures were established as follows: ±HSO₃Neu5Ac2(8Neu5Ac2)_n6G1c11´Cer, wheren=0, 1, 2, 3. This provides the first evidence for the natural occurrence of a tetrasialic acid structure in glycosphingolipids. The finding of sulfated oligosialyl chains is especially noteworthy in that the sulfate group exclusively resides on the C-8 of the nonreducing terminal residues of oligo/polysialyl chains and that sulfation appears to be a termination signal for elongation of oligosialyl chains. Sulfation at the nonreducing terminal Neu5Ac residues of oligosialyl chains was also found to facilitate the formation of an inter-residue lactone between the carboxyl group at the nonreducing terminal sulfated Neu5Ac and the hydroxyl group at C-9 of the penultimate Neu5Ac residue. The long chain base was 4-hydroxysphinganine (t18:0) and the major fatty acid species were identified as C20:1, C21:1, and C22:1.Abbreviations C:M chloroform:methanol - FAB-MS fast atom bombardment mass spectrometry - GLC gas-liquid chromatography - GSL glycosphingolipid - HPTLC high performance thin-layer chromatography - Neu5Ac8HSO₃ or HSO₃ Neu5Ac 5-N-acetyl-8-O-sulforylneuraminic acid - Neu5Gc9HSO₃ 5-N-glycolyl-9-O-sulforylneuraminic acid - NMR nuclear magnetic resonance - PB sodium phosphate buffer, pH 7.2 - Sia sialic acid - HSO₃ Sia sulfated sialic acid - polySia polysialic acid - TFA trifluoroacetic acid Since the dividing line between oligo- and polysialic acids is not rigidly defined, we propose here that those containing more than penta-sialic acid chains can be referred to as the polysialic acid group. The rationale for this is that 2 8-linked pentasialic acid is the minimum chain length to exhibit the conformational property of polysialic acid [of Michon, F, Brisson, J-R, Jennings, H (1987)Biochemistry 26: 8399–405] and to act as a substrate for the endosialidase, Endo-N [Hallenbeck, PC, Vimr, ER, Yu, F, Bassler, B, Troy, FA (1987)J Biol Chem 262: 3553–61].     

Behavior and differentiation process of pigment cells in a tropical sea urchin Echinometra mathaei

The behavior and differentiation processes of pigment cells were studied in embryos of a tropical sea urchin Echinometra mathaei, whose egg volume was one half of those of well-known sea urchin species. Owing to earlier accumulation of pigments, pigment cells could be detected in the vegetal plate even before the onset of gastrulation, distributed dorsally in a hemi-circle near the center of the vegetal plate. Although some pigment cells left the archenteron during gastrulation, most of them remained at the archenteron tip. At the end of gastrulation, pigment cells left the archenteron and migrated into the blastocoele. Unlike pigment cells in typical sea urchins, however, they did not enter the ectoderm, and stayed in the blastocoele even at the pluteus stage. It is of interest that the majority of pigment cells were distributed in the vicinity of the larval skeleton. Aphidicolin treatment revealed that eight blastomeres were specific to pigment cell lineage after the eighth cleavage, one cell cycle earlier than that in well-known sea urchins. The pigment founder cells divided twice, and the number of pigment cells was around 32 at the pluteus stage. It was also found that the differentiation of pigment cells was blocked with Ni2+, whereas the treatment was effective only during the first division cycle of the founder cells.     

A new G-stretch-DNA-binding protein, Unichrom, displays cell-cycle-dependent expression in sea urchin embryos

We report the identification and characterization of Unichrom, a gene encoding a new G-stretch-DNA-binding protein in the sea urchin embryo. The derived amino acid sequence of Unichrom contains plant homeodomain (PHD) finger and high mobility group (HMG) motifs as well as motifs required for cell-cycle-dependent degradation. The expression of a Unichrom-green fluorescent protein (GFP) fusion protein in sea urchin embryonic cells indicates that Unichrom protein accumulates in nuclei during interphase and disperses into the cytoplasm at mitosis. Overexpression of dominant negative Unichrom, which contains the DNA binding domain lacking the motif for cell-cycle-dependent degradation, causes impairment of chromosome segregation. These results suggest that Unichrom binds to genome DNA at G-stretch and that degradation of Unichrom is required for segregation of chromosomes.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.     

Sulfhydryl groups are involved in the activation of sea urchin eggs

Unfertilized sea urchin eggs exposed to the sulfhydryl reagents Ag⁺ or N-ethylmaleimide either elevated fertilizationlike membranes, formed surface protrusions, developed a clear cortical layer devoid of organelles, or cytolysed. The relative fraction of each modification varied from batch to batch and was also dose and time dependent. With Ag⁺ and higher doses of N-EMI (10^?3 M), the most common effect was the elevation of a membrane indicating cortical exocytosis, while at lower doses of N-EMI protrusions were predominant. Glutathione (GSH) protected eggs against these reagents also in a dose-dependent manner. Eggs exposed to equimolar amounts of N-EMI and GSH, which otherwise formed membranes, produced protrusions, while increasing GSH tenfold afforded complete protection. We suggest there are two targets for the sulfhydryl reagents–the first, SH groups on proteins that regulate the release of Ca²⁺ from the intracellular sequestering mechanism which subsequently triggers cortical exocytosis; the second, SH groups on the egg surface that may regulate cortical organization.     

The toposome, essential for sea urchin cell adhesion and development, is a modified iron-less calcium-binding transferrin

We describe the structure and function of the toposome, a modified calcium-binding, iron-less transferrin, the first member of a new class of cell adhesion proteins. In addition to the amino acid sequence of the precursor, we determined by Edman degradation the N-terminal amino acid sequences of the mature hexameric glycoprotein present in the egg as well as that of its derived proteolytically modified fragments necessary for development beyond the blastula stage. The approximate C-termini of the fragments were determined by a combination of mass spectrometry and migration in reducing gels before and after deglycosylation. This new member of the transferrin family shows special features which explain its evolutionary adaptation to development and adhesive function in sea urchin embryos: (i) a protease-inhibiting WAP domain, (ii) a 280 amino acid cysteine-less insertion in the C-terminal lobe, and (iii) a 240 residue C-terminal extension with a modified cystine knot motif found in multisubunit external cell surface glycoproteins. Proteolytic removal of the N-terminal WAP domain generates the mature toposome present in the oocyte. The modified cystine knot motif stabilizes cell-bound trimers upon Ca-dependent dissociation of hexamer-linked cells. We determined the positions of the developmentally regulated cuts in the cysteine-less insertion, which produce the fragments observed previously. These fragments remain bound to the hexameric 22S particle in vivo and are released only after treatment of the purified toposome with reducing agents. In addition, some soluble smaller fragments with possible signal function are produced. Sequence comparison of five sea urchin species reveals the location of the cell-cell contact site targeted by the species-specific embryo dissociating antibodies. The evolutionary tree of 2-, 1-, and 0-ferric transferrins implies their evolution from a basic cation-activated allosteric design modified to serve multiple functions.     

Change in the adhesive properties of blastomeres during early cleavage stages in sea urchin embryo

Blastomeres of sea urchin embryo change their shape from spherical to columnar during the early cleavage stage. It is suspected that this cell shape change might be caused by the increase in the adhesiveness between blastomeres. By cell electrophoresis, it was found that the amount of negative cell surface charges decreased during the early cleavage stages, especially from the 32-cell stage. It was also found that blastomeres formed lobopodium-like protrusions if the embryos were dissociated in the presence of Ca2+. Interestingly, a decrease in negative cell surface charges and pseudopodia formation first occurred in the descendants of micromeres and then in mesomeres, and last in macromeres. By examining the morphology of cell aggregates derived from the isolated blastomeres of the 8-cell stage embryo, it was found that blastomeres derived from the animal hemisphere (mesomere lineage) increased their adhesiveness one cell cycle earlier than those of the vegetal hemisphere (macromere lineage). The timing of the initiation of close cell contact in the descendants of micro-, meso- and macromeres was estimated to be 16-, 32- and 60-cell stage, respectively. Conversely, the nucleus-to-cell-volume ratios, which are calculated from the diameters of the nucleus and cell, were about 0.1 when blastomeres became adhesive, irrespective of the lineage.     

Characterization of a membrane-associated glycoprotein complex implicated in cell adhesion to fibronectin

We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.     

Isolation of sea urchin embryo cell surface membranes on polycationic beads

Summary Blastula cell surface membranes of the sea urchin, Strongylocentrotus purpuratus, were isolated on polycationic beads by a method modified from Jacobson and Branton (1977) and Jacobson (1980). This study represents the first application of this procedure to an embryonic system. Embryo cells were attached to polylysine-coated polyacrylamide beads and lysed, leaving the embryo cell surface membranes still attached to the beads, and cytoplasmic particles were washed free of the exposed inner surfaces of the membranes. Cell surface membrane sheets were desorbed from the beads and collected by centrifugation. Approximately 8% and 5% of the cell surface membranes of dissociated embryo cells were recovered on the beads and in the membrane pellet, respectively. Specific activities of [³H]concanavalin A-binding and of the cell surface marker enzymes, alkaline phosphatase and Na⁺/K⁺ ATPase, were 16-, 19-, and 32-fold higher, respectively, in the cell surface membrane fraction than in the embryo cell homogenate. Membranes were relatively free of cytoplasmic contaminants as judged from electron micrographs and enzyme analysis. Activities in the membrane fraction of the cytoplasmic marker enzymes, cytochrome c oxidase, catalase, acid phosphatase, NADP- and NADPH-cytochrome c reductase, and acetylcholinesterase, were substantially less than homogenate levels. The entire procedure can be completed in 4 h. Since this cell surface membrane isolation technique relies only on the tendency of a negatively charged cell to adhere to a positively charged surface, it is less likely than most other methods to exhibit species and developmental stage specificity and should prove useful in the study of the developmental role of embryonic stage-specific membrane components.     

Mass isolation and culture of sea urchin micromeres

Summary A procedure is described for large-scale isolation of micromeres from 16-cell stage sea urchin embryos. One to two grams of >99% pure, viable micromeres (2.3 to 4.6 × 10⁸ cells) are routinely isolated in a single preparation. In culture, these cells uniformly proceed through their normal development, in synchrony with micromeres in whole embryos, ultimately differentiating typical larval skeletal structures. The attributes of this procedure are: (a) the very early time of isolation of the cells, directly after the division that establishes the cell line; (b) the large yield of cells; (c) the purity of the preparation of cell; and (d) their synchronous development in culture through skeletogenesis. The procedure greatly aids in making sea urchin micromeres a favorable material for molecular analysis of development. This work was supported in part by the following grants from the National Institutes of Health: Grant HL-10312 to A.H.W., Grant GM-20784 to Helen R. Whiteley, Grant ES-02190 to N. Karle Mottet, M.D., and Training Grants ES-07032 and HD-00266.     

Isolation of nine nuclear microsatellites in the common Mediterranean sea urchin, Paracentrotus lividus (Lamarck)

Nine polymorphic microsatellite loci were isolated and characterized for the edible common sea urchin Paracentrotus lividus. Loci were obtained from two genomic libraries enriched with different di-, tri- and tetranucleotides. Most microsatellites obtained were imperfect dinucleotides. Allelic variation was screened for a total of 56 individuals from two populations from North-Western Mediterranean. Microsatellites showed a number of alleles ranging from 13 to 35. Linkage disequilibrium was not detected between any pair of loci, and all loci showed a significant departure from Hardy-Weinberg equilibrium which is not likely to be the result of null alleles, suggesting that demographic features may be acting upon this commercially interesting species.