Chromatin-associated proteins of the developing sea urchin embryo. II. Acid-soluble proteins

Of the five well-characterized histories, only the slightly lysine-rich histories F2a and F2b are present in sea urchin embryos before the 16-cell stage. At the 16-cell stage, the arginine-rich (F3) and lysine-rich (Fla) histones appear and all the major histones are then present in the same relative proportions until the pluteus stage except for a second lysine-rich protein, Flb, which is first detected at 12 to 16 hours of development and increases to the pluteus stage. From 16 cells to pluteus at 70 hours, all the histones are labeled by a 30-minute incubation with radioactive lysine, with the exception of the lysine-rich histone Fla which does not incorporate label after 20 to 30 hours of development and Fib which is labeled only after 20 to 30 hours. Fla is conserved, however, to the pluteus stage.The total acid-soluble protein content of chromatin remains constant to 22 hours of development. During the period of 22 to 45 hours, there is a slight loss of protein followed by a rapid loss from 45 to 70 hours such that at 70 hours only 20% remains.     

High mobility group nonhistone chromosomal proteins of the developing sea urchin embryo

The high mobility group or HMG proteins are nonhistone chromosomal proteins that have been found in relatively high amounts in nuclei of many tissues. A number of studies have shown that some of these proteins are preferentially associated with actively transcribed regions of the genome and may play a role in maintaining these regions in an active state. In this study, we undertook an investigation of the high mobility group proteins from the sea urchin, Stronglyocentrotus purpuratus. Initially the putative sea urchin HMGs were extracted from isolated nuclei of hatching blastula-stage embryos with 5% perchloric acid (PCA). The major proteins in this extract were characterized according to their electrophoretic mobility, amino acid composition, and association with isolated deoxyribonucleoprotein particles. The results indicate there is only one "major" sea urchin HMG protein, termed P2 in this paper. An estimate of the amount of P2 in relation to the inner histones, however, was low compared to what has been found for other HMG proteins. Of the other major 5% PCA-extractable proteins, one was identified as the cleavage stage H1. Another protein apparently resulted from H3 contamination in the 5% PCA extract, and the fourth major protein did not have all the characteristics of an HMG. In particular, it was not found associated with nucleosomal particles. The HMG proteins from other developmental stages were then examined. Five percent PCA extracts of nuclei from unfertilized eggs, 2-cell, 16-cell, hatching blastula, gastrula, and pluteus stages were analyzed on SDS- and acetic acid-urea gels. This analysis indicated that P2 exists in two different forms differing slightly in charge. The less basic form was found in the egg, 2-cell and 16-cell extracts. At the hatching blastula stage, both forms were present and by pluteus stage, the more basic form predominated. It appears that P2 is undergoing a developmental change from a less to more basic form. The presence of P2 in the 5% PCA extract of egg nuclei is proof that P2 does not initially appear sometime during embryogenesis but is already in the egg nucleus prior to fertilization.     

Fibronectin in the developing sea urchin embryo

The presence of fibronectin in developing sea urchin embryos was studied uing immunofluorescence staining. The fluorescence pattern indicates that fibronectin is found on the cell surfaces and between cells in the blastula and gastrula stages, indicating that it plays a role in cell adhesion. Its presence on invaginating cells also suggests its involvement in morphogenesis during early development.     

Matrix metalloproteases of the developing sea urchin embryo

Abstract. A distinct group of metalloproteases has been identified in the developing sea urchin embryo by gelatin substrate gel zymography, a highly sensitive protease detection assay. The developing Arbacia embryo exhibited four prominent bands of gelatinase activity with apparent molecular masses of 55, 50, 42 and 38 kDa. The activity of the 55, 42 and 38 kDa tissue gelatinases increased and that of the 50 kDa tissue gelatinase decreased during embryonic development. All four enzymes were EDTA- and 1,10-phenanthroline sensitive and phenyl methyl sulphonyl fluoride (PMSF) insensitive. None of the enzymes had detectable caseinolytic activity in casein substrate gels. Although the Arbacia enzymes possessed a number of properties that are characteristic of the mammalian matrix metalloprotease family, they did not appear to be converted to lower molecular weight forms by organomercurial treatment and are distinct in this aspect. The Arbacia metalloproteases are candidate enzymes for the tissue and matrix remodeling that occurs during sea urchin embryo development.     

Matrix metalloproteases of the developing sea urchin embryo

Abstract. A distinct group of metalloproteases has been identified in the developing sea urchin embryo by gelatin substrate gel zymography, a highly sensitive protease detection assay. The developing Arbacia embryo exhibited four prominent bands of gelatinase activity with apparent molecular masses of 55, 50, 42 and 38 kDa. The activity of the 55, 42 and 38 kDa tissue gelatinases increased and that of the 50 kDa tissue gelatinase decreased during embryonic development. All four enzymes were EDTA- and 1,10-phenanthroline sensitive and phenyl methyl sulphonyl fluoride (PMSF) insensitive. None of the enzymes had detectable caseinolytic activity in casein substrate gels. Although the Arbacia enzymes possessed a number of properties that are characteristic of the mammalian matrix metalloprotease family, they did not appear to be converted to lower molecular weight forms by organomercurial treatment and are distinct in this aspect. The Arbacia metalloproteases are candidate enzymes for the tissue and matrix remodeling that occurs during sea urchin embryo development.     

Purification and characterization of the sea urchin embryo hatching enzyme

The sea urchin hatching enzyme provides an interesting model for the control of gene expression during early development. In order to study its properties and developmental regulation, the hatching enzyme of the species Paracentrotus lividus has been purified. The fertilization envelopes of the embryos were digested before hatching by a crude culture supernatant previously made. The enzyme was then solubilized by 1 M NaCl and 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and purified by hydrophobic chromatography on Procion-agarose. A 470-fold increase in specific activity was obtained. The kinetic parameters of the proteolytic activity using dimethylcasein as substrate are: Km = 120 micrograms x ml-1, Vm = 200 mumol x min-1 x mg-1, and kcat = 180 s-1 at 500 mM NaCl, 10 mM CaCl2, pH 8.0, at 35 degrees C. The purified enzyme is highly active on fertilization envelopes: at 20 degrees C and 500 mM NaCl, 10 mM CaCl2, pH 8.0, 100 ng of enzyme completely denudes embryos in about 20 min under standard conditions. The molecular mass of the enzyme was estimated as 57 kDa by gel filtration, 51 kDa by gel electrophoresis, and 52 kDa by amino acid analysis. The hatching enzyme was shown to be a glycoprotein which autolyzes to a 30-kDa inactive form. Antibodies raised against the 51- or 30-kDa forms reacted with both these forms. Immunoblotting experiments showed that the hatching supernatants contain important amounts of the autolyzed species.     

Glycoprotein synthesis in developing sea urchin embryos

Surface membrane glycoproteins have been postulated in many mammalian cells to be involved in external surface membrane functions such as cell adhesion, cell-cell recognition, and cell movement. In developing echinoderm embryos, cell adhesion, recognition, and movement of individual cell types have been attributed to differences in the external surface membranes of these cells. Results reported here suggest that the three cell types of 16-cell sea urchin embryos have a mechanism that could establish differences in the carbohydrate portion of glycoproteins located in the external surface membrane. The results demonstrate 1) that glycoproteins are synthesized during early sea urchin development and 2) that slightly different rates of glycoprotein synthesis exist for the three types of blastomeres from 16-cell sea urchin embryos.     

Pool sizes of the deoxynucleoside triphosphates in the sea urchin egg and developing embryo.

The four deoxynucleoside triphosphate pools in unfertilized eggs of L. pictus and S. purpuratus were measured and found to be very large, ranging from 10^?3 to 10^?2 pmoles per egg. The high levels of the individual dNTP pools are sufficient for one to eight rounds of DNA synthesis. During the first division cycle these pools fluctuate with the highest levels being attained prior to DNA synthesis. The pools then decrease just preceding or during the S period. There is a large reduction in the total cellular dNTP in later stages of development when DNA synthesis is reduced relative to the cleavage stages.     

Ontogeny and characterization of mesenchyme antigens of the sea urchin embryo

A monoclonal antibody, Sp12, binds to cortical granules, the hyaline layer, and skeletogenic, chromogenic, and blastocoelar mesenchyme of sea urchin eggs and embryos. Adult urchins also express Sp12 antigens in the dermal layer of the test and spines. Antigen is expressed on the surface of primary mesenchyme cells after they have entered the blastocoel, and by two secondary mesenchyme derivatives--the blastocoelar cells after they have been released from the tip of the archenteron, and the pigment cells in prism stage embryos. Immunogold localizations show antigen on the surfaces of mesenchyme, within membrane bounded vesicles, and associated with the Golgi apparatus. Western blots of antigens immunoprecipitated from seven developmental stages reveal twelve antigens ranging in Mr from 35 k to 240 k. Most of these antigens appear, disappear or change Mr over the first five days of development. Characterizations of this complex array of antigens show that the epitope recognized by Sp12 is eliminated by proteolytic enzymes and endoglycosidase F, while immunoreactivity is only reduced by periodate oxidation. As well, calcium magnesium free seawater extracts a subset of antigens different from that retained by crude membrane preparations. It is proposed that the mesenchyme of sea urchin embryos produces a family of developmentally regulated cell surface and extracellular matrix glycoproteins which all exhibit a carbohydrate epitope recognized by Sp12.     

Preliminary observations on the synthesis of glycosaminoglycans in the sea urchin embryo.

A method based on the degradation by enzymes and nitrous acid of isotopically labelled glycosaminoglycans has been employed to study the synthesis of these compounds in normal, animalized and vegetalized sea urchin embryos. According to standard criteria, these organisms synthesize dermatan sulfate, heparan sulfate, hyaluronate and keratan sulfate. The hyaluronate seems to be slightly sulfated, it may thus be mucoitin sulfate. The preliminary results obtained suggest a conspicuous difference between animalized and vegetalized embryos: the synthesis of dermatan sulfate is suppressed in the former, while proceeding normally in the latter. The synthesis of heparan sulfate is not affected by our experimental conditions, but the isotope incorporation in hyaluronate and in keratan sulfate is decreased, more in the vegetalized than in the animalized embryos.     

Automated sequential affinity chromatography of sea urchin embryo DNA binding proteins.

An automated method of running a tandem sequence of oligonucleotide affinity columns was used to purify factors that interact specifically with cis-regulatory sites of the CyIIIa cytoskeletal actin gene of the sea urchin embryo (Strongylocentrotus purpuratus). The method allows quantitative enrichment in a single chromatographic run of up to 12 different sequence-specific DNA binding proteins, each of which may then be readily purified to homogeneity by methods such as preparative gel electrophoresis. The affinity chromatography and identification of six different CyIIIa-regulatory factors is described, and the general utility of the method is discussed.     

Cell movements in the sea urchin embryo.

Recent studies show that gastrulation in the sea urchin embryo involves movement of cells over the blastopore lip (involution). Some cells in the vegetal plate of the late blastula become bottle-shaped but they play a limited role in gastrulation. The functions of specific integrins, regulators of cell-cell adhesion, and extracellular matrix components in gastrulation are currently being analyzed. In addition, light-microscopic studies continue to provide a unique picture of dynamic cell behavior in vivo.     

Soluble microtubule proteins of the sea urchin embryo: partial characterization of the proteins and behavior of the pool in early development

A partial characterization of the soluble microtubule proteins of sea urchin eggs and embryos is presented. Vinblastine precipitation yielded a pellet with a high colchicine binding activity. This precipitate when electrophoresed on an alkaline SDS/urea gel system yields two protein bands which correspond to molecular weights of 57,000 ± 2000 and 52,000 ± 2000. These values are very close to our values and to the published values for axonemal microtubule proteins. Electrophoresis of the vinblastine precipitated proteins on a neutral SDS system without urea yielded only one band with an apparent molecular weight of 52,000 ± 2000. The amino acid composition of the vinblastine-precipitated microtubule protein was determined to be similar to that of axonemal protein.The pool of microtubule proteins was found to remain constant in size throughout early development in both control and actinomycin-treated embryos. Soluble microtubule proteins comprise about 0.37% of the total protein of the sea urchin (Arbacia) egg. Approximately 20% of the total microtubule protein in the egg appears to be particle bound.     

Differential distribution of spicule matrix proteins in the sea urchin embryo skeleton

Spicule matrix proteins are the products of primary mesenchyme cells, and are present in calcite spicules of the sea urchin embryo. To study their possible roles in skeletal morphogenesis, monoclonal antibodies against SM50, SM30 and another spicule matrix protein (29 kDa) were obtained. The distribution of these proteins in the embryo skeleton was observed by immunofluorescent staining. In addition, their distribution inside the spicules was examined by a 'spicule blot' procedure, direct immunoblotting of proteins embedded in crystallized spicules. Our observations showed that SM50 and 29 kDa proteins were enriched both outside and inside the triradiate spicules of the gastrulae, and also existed in the corresponding portions of growing spicules in later embryos and micromere cultures. The straight extensions of the triradiate spicules and thickened portions of body rods in pluteus spicules were also rich in these proteins. The SM30 protein was only faintly detected along the surface of spicules. By examination using the spicule blot procedure, however, SM30 was clearly detectable inside the body rods and postoral rods. These results indicate that SM50 and 29 kDa proteins are concentrated in radially growing portions of the spicules (normal to the c-axis of calcite), while SM30 protein is in the longitudinally growing portions (parallel to the c-axis). Such differential distribution suggests the involvement of these proteins in calcite growth during the formation of three-dimensionally branched spicules.