Eizo Nakano

Polymerization of actin free from nucleotide and divalent cations

Abstract EDTA-Dowex treatment of G-actin in sucrose was found to remove nucleotides and divalent cations from G-actin without loss of polymerizability. This actin could be polymerized into F-actin by the addition of a small amount of simple monovalent salts; that is, polymerization of actin was realised in the sucrose solution without participation of nucleotides and divalent cations. The bond between G-actins to form F-actin induced neither nucleotides nor divalent cations. The polymerization was accelerated by the addition of ATP or ITP, which were bound to G-actin and dephosphorylated during polymerization. The polymerizable G-actin free from nucleotides, could also be obtained by depolymerization in sucrose of the nucleotide-free F-actin prepared by a long dialysis.

Fibronectin from the ovary of the sea urchin,Pseudocentrotus depressus

SummaryFibronectin, with a subunit molecular weight of 220,000 daltons, was isolated from the ovary of the sea urchin,Pseudocentrotus depressus, using affinity chromatography on heat-denatured mammalian collagen coupled to Sepharose 4B. The distribution of fibronectin in the sea urchin ovary was examined by indirect immunofluorescence using antifibronectin serum. The basement membrane and the connective tissues exhibited strong fluorescence. The fibronectin was localized closely together with collagen bundles in the sea urchin ovary. Biochemical and immunological examinations indicate that sea urchin fibronectin has similar properties as those of mammalian fibronectin.

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

SummaryA 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.

Regulative specification of ectoderm in skeleton disrupted sea urchin embryos treated with monoclonal antibody to Pl‐nectin

Pl‐nectin is a glycoprotein first discovered in the extracellular matrix (ECM) of Paracentrotus lividus sea urchin embryo, apically located on ectoderm and endoderm cells. The molecule has been described as functioning as an adhesive substrate for embryonic cells and its contact to ectoderm cells is essential for correct skeletogenesis. The present study was undertaken to elucidate the biochemical characteristics of Pl‐nectin and to extend knowledge on its in vivo biological function. Here it is shown that the binding of mesenchyme blastula cells to Pl‐nectin‐coated substrates was calcium dependent, and reached its optimum at 10 m M Ca2+. Perturbation studies using monoclonal antibody (McAb) to Pl‐nectin, which prevent ectoderm cell‐Pl‐nectin contact, show that dorsoventral axis formation and ectoderm differentiation were retarded. At later stages, embryos recovered and, even if growth and patterning of the skeleton was greatly affected, the establishment of dorsoventral asymmetry was reached. Similarly, the expression of specific ectoderm and endoderm territorial markers was achieved, although occurring with some delay. Endoderm differentiation and patterning was not obviously affected. These results suggest that both endoderm and ectoderm cells have regulative capacities and differentiation of territories is restored after a lag period. On the contrary, failure of inductive differentiation of the skeleton cannot be rescued, even though the ectoderm has recovered.

DIFFERENTIATION OF THE RETINA AND RETINAL LACTATE DEHYDROGENASE ISOENZYMES IN THE TELEOST, ORYZIAS LATIPES

The activity of lactate dehydrogenase in the teleost Oryzias latipes, shows a gradual increase during embryonic development, followed by an abrupt rise on hatching, which coincides with the appearance of the retinal isoenzymes. By the time of hatching, the retina has differentiated, and no significant change is observed in the retinal structure until 2 days after hatching. It was assumed that metabolic changes which occur on hatching may trigger the synthesis of retinal lactate dehydrogenase isoenzymes.

Adhesion of sea-urchin embryonic cells to substrata coated with cell adhesion molecules

Summary— A cell‐to‐substratum adhesion assay is developed to study the adhesion of sea‐urchin embryonic cells to coated substrata. The involvement in this process of both carbohydrate and protein molecules is reported. Concanavalin A (Con A) increases the attachment of cells to the substratum in a dose‐dependent manner and this effect is completely abolished when the incubation is carried out in the presence of the specific monocarbohydrate Con A‐inhibitor, α‐methyl‐d‐mannoside. A Con A‐mediated enhancement of cell‐to‐substratum adhesion was also detected on cells deprived of toposome, a glycoprotein complex responsible for cell‐to‐cell adhesion. The involvement of other molecules as well as toposome in the process of cell‐to‐substratum adhesion is also investigated. Results of these in vitro experiments indicate that all the molecules tested contribute to the process of cell‐to‐substratum adhesion.

Enhancement of spicule formation and calcium uptake by monoclonal antibodies to fibronectin-binding acid polysaccharide in cultured sea urchin embryonic cells

The effect of monoclonal antibodies to fibronectin-binding acid polysaccharide (anti-FAPS) on differentiation of primary mesenchyme cells and spicule formation was examined in cultured embryonic cells isolated from the sea urchin, Hemicentrotus pulcherrimus. Spicule formation of micromere-derived cells was enhanced by anti-FAPS. The increase of spicule formation correlated with the increase of calcium uptake into micromere-derived cells and spicules. Furthermore, both spicule formation and calcium uptake were inhibited by calcium-channel blockers (verapamil, diltiazem and nicardipine) and divalent ions (manganese and cobalt). These results suggest that FAPS, a component of the blastocoelic extracellular matrix surrounding the primary mesenchyme cells, may regulate the level of calcium uptake and spicule formation.

Fibronectin-induced migration of melanophores in vitro in scales of medaka, Oryzias latipes

SummaryThe effects of fibronectin on melanophores were examined in two mutant strains of medaka, Oryzias latipes: mm (BmmR), which has condensed melanophores and normal dendritic melanophores; and cm (BcmR), which has condensed melanophores. When medaka scales were cultured in the presence of fibronectin, melanophores of the wild type and dendritic melanophores of the mm mutant changed their shape and migrated, whereas melanophore migration was rarely seen in the absence of fibronectin. Melanophores of the cm mutant and condensed melanophores of the mm mutant did not migrate even in the presence of fibronectin. When melanophores of the wild type and mm mutant were condensed by adrenalin, they did not migrate. On the other hand, when melanophores of the cm mutant were dispersed by theophylline, they were able to migrate. These results indicate that fibronectin induces the migration of melanophores and that dispersion of melanin granules may be requisite for such migration.

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.

A large calcium-binding protein associated with the larval spicules of the sea urchin embryo

A tissue-specific, high molecular weight, calcium-binding protein from the sea urchin embryo is described. This protein, designated as CBP 180, has a molecular weight of 180,000 under reducing conditions, and is extractable with 1% Triton X-100. It accumulates rapidly during development, starting roughly at the onset of spiculogenesis. When embryos are cultured in the presence of inhibitors of spicule formation, such as tunicamycin and zinc ions, accumulation of CBP180 is depressed or stopped. By immunofluorescence technique and by using an antibody specifically generated against this protein, CBP180 is mainly localized in primary mesenchyme cells and spicular syncytium of the pluteus larva. Little or none is detectable in ectoderm, endoderm or blastocoelar extracellular matrix. These results suggest that the protein is involved in calcium sequestration in the differentiation of larval spicules.