Sea Urchin Cells Research Articles

Rapid identification of lectin receptors and their possible function in sea urchin cell systems.

An assay using lectin derivatized agarose beads to rapidly and inexpensively identify cell surface lectin receptors was recently described by Latham et al. (1995). In this earlier study, the assay was tested on large, early stage sea urchin embryo cells. In this study this assay was used to examine lectin receptors on small, later stage sea urchin embryo cells that are more typical of cells that most investigators deal with, to ascertain if cell size is a determining factor in the assay's validity. The results indicated that the assay is a valid method to identify lectin receptors on small as well as large cells. Twenty-three hour Strongylocentrotus purpuratus embryo cells strongly bound Triticum vulgaris, concanavalin A, Artocarpus integrifolia and Vicia villosa using both the agarose bead and fluorescence assays, while three other lectins, Ulex europaeus I, Lotus tetragonolobus and Lens culinaris did not strongly bind to the cells using these two assays. As in earlier studies agglutinability results did not correlate well with results using the two other assays. In all cases where lectin bead binding, fluorescent lectin binding or lectin-mediated agglutination occurred, specific sugars reduced the observed binding. The second part of this study examined the putative role of concanavilin A receptors in a specific cellular interaction: sperm-egg binding. Concanavalin A inhibited fertilization of dejellied sea urchin eggs when their vitelline layers were intact and to a lesser extent when their vitelline layers were removed. This effect was counteracted by alpha methyl glucose. The major differences between these studies and previous work is that here concanavalin A was washed out after incubation with eggs, making it more likely that results reflect binding to cell surface lectin receptors rather than toxicity. In addition, performing the experiments on eggs with or without vitelline layers provided information on the location of concanavalin A receptors that may function in sperm-egg interaction.

Purification of an oligo(dG).oligo(dC)-binding sea urchin nuclear protein, suGF1: a family of G-string factors involved in gene regulation during development.

Contiguous deoxyguanosine residues (G strings) have been implicated in regulation of gene expression in several organisms via the binding of G-string factors. Regulation of expression of the chicken adult beta-globin gene may involve the interplay between binding of an erythrocyte-specific G-string factor, BGP1, and the stability of a positioned nucleosome (C. D. Lewis, S. P. Clark, G. Felsenfeld, and H. Gould, Genes Dev. 2:863-873, 1988). We have purified a 59.5-kDa nuclear protein (suGF1) from sea urchin embryos by DNA affinity chromatography. suGF1 has high binding affinity and specificity for oligo(dG).oligo(dC). The identity of the purified protein was confirmed by renaturation of sequence-specific DNA-binding activity from a sodium dodecyl sulfate-polyacrylamide gel slice and by Southwestern (DNA-protein) blotting. suGF1 binds in vitro to a G11 string present in the H1-H4 intergenic region of a sea urchin early histone gene battery. This suGF1 DNA recognition site occurs within a homopurine-homopyrimidine stretch previously shown to be incorporated into a positioned nucleosome core in vitro. DNase I footprinting shows that suGF1 protects the same base pairs on the promoter of the chicken beta A-globin gene as does BGP1. We show that a G-string cis-regulatory element of a sea urchin cell lineage-specific gene LpS1 (M. Xiang, S.-Y. Lu, M. Musso, G. Karsenty, and W. H. Klein, Development 113:1345-1355, 1991) also represents a high-affinity recognition site for suGF1. suGF1 may be a member of a family of G-string factors involved in the regulation of expression of unrelated genes during development of a number of different organisms.

Purification of an oligo(dG).oligo(dC)-binding sea urchin nuclear protein, suGF1: a family of G-string factors involved in gene regulation during development.

Contiguous deoxyguanosine residues (G strings) have been implicated in regulation of gene expression in several organisms via the binding of G-string factors. Regulation of expression of the chicken adult beta-globin gene may involve the interplay between binding of an erythrocyte-specific G-string factor, BGP1, and the stability of a positioned nucleosome (C. D. Lewis, S. P. Clark, G. Felsenfeld, and H. Gould, Genes Dev. 2:863-873, 1988). We have purified a 59.5-kDa nuclear protein (suGF1) from sea urchin embryos by DNA affinity chromatography. suGF1 has high binding affinity and specificity for oligo(dG).oligo(dC). The identity of the purified protein was confirmed by renaturation of sequence-specific DNA-binding activity from a sodium dodecyl sulfate-polyacrylamide gel slice and by Southwestern (DNA-protein) blotting. suGF1 binds in vitro to a G11 string present in the H1-H4 intergenic region of a sea urchin early histone gene battery. This suGF1 DNA recognition site occurs within a homopurine-homopyrimidine stretch previously shown to be incorporated into a positioned nucleosome core in vitro. DNase I footprinting shows that suGF1 protects the same base pairs on the promoter of the chicken beta A-globin gene as does BGP1. We show that a G-string cis-regulatory element of a sea urchin cell lineage-specific gene LpS1 (M. Xiang, S.-Y. Lu, M. Musso, G. Karsenty, and W. H. Klein, Development 113:1345-1355, 1991) also represents a high-affinity recognition site for suGF1. suGF1 may be a member of a family of G-string factors involved in the regulation of expression of unrelated genes during development of a number of different organisms.

Centrosomes are phosphorylated in sea urchin eggs throughout the cell cycle, during artificial activation, and when microtubule growth is inhibited

In most animal systems, microtubules are nucleated and organized by centrosomes which undergo considerable modifications during the cell cycle. Typically, centrosomes are phoshorylated at the transition from interphase to mitosis as shown with MPM2, an antibody directed against phosphoproteins. Using the MPM2 antibody, we show in this paper with transmission electron microscopy (TEM) and immunofluorescence microscopy (IFM) that in the sea urchin system centrosomes are phosphorylated in the sperm before fertilization and at every stage of the first cell cycle. MPM2 exhibits identical staining patterns as Ah-6 and 5051 previously shown to reliably identify centrosomal material in sea urchin cells. After centrosomal material is brought into the egg by the sperm, it spreads around the zygote nucleus where it gets distributed and becomes bipolar and compacted to form the mitotic apparatus. Typically, at these mitotic stages, centrosomal material exhibits the brightest staining with MPM2, Ah-6 and 5051 Since in this system phoshorylated centrosomal material is contributed by the sperm, the egg's competence for centrosome phosphorylation was analyzed by activating centrosomal material in the unfertilized egg by treatment with either A23187, ammonia, D2O, or taxol.

The carboxyl-terminal domain of kinesin heavy chain is important for membrane binding.

Sea urchin kinesin is a plus end-directed microtubule-based motor consisting of two heavy chains and two light chains and is proposed to be responsible (a) for the transport of membranous organelles along microtubules in sea urchin mitotic spindles (Wright, B. D., Henson, J. H., Wedaman, K. P., Willy, P. J., Morand, J. N., and Scholey, J. M. (1991) J. Cell Biol. 113, 817-833) and (b) for the radial dispersion of endoplasmic reticulum and endosomal membranes in non-mitotic cultured coelomocytes (Henson, J. H., Nesbitt, D., Wright, B. D., and Scholey, J. M. (1992) J. Cell Sci. 103, 309-320). We report here that sea urchin kinesin is indeed able to bind in a concentration-dependent and saturable manner to microsomal membranes isolated from sea urchin eggs in the presence of MgATP. The kinesin light chains may not be essential for membrane binding since kinesin containing negligible amounts of light chains binds as well as kinesin containing stoichiometric amounts of light chains. Finally, we propose that kinesin binds to membranes with the carboxyl-terminal domain of the heavy chain (amino acid residues 858-1031) since the bacterially expressed and then isolated stalk-tail fragment of kinesin heavy chain, in contrast to the stalk fragment, is able (a) to bind membranes in a concentration-dependent and saturable manner and (b) to compete with native kinesin for membrane binding. Our results support the hypothesis that the carboxyl-terminal domains of the heavy chains attach kinesin molecules to their membranous cargo in mitotic and interphase sea urchin cells.

Sulfhydryl bond formation is a prerequisite for proper cycling of centrosomes and chromosomes in mammalian tissue culture cells

To study the role of sulfhydryl group formation during cell cycle progression, mammalian tissue culture cells (PTK2) were exposed to 100¼M 2-mercaptoethanol for 2 to 6 h during their exponential phase of growth. The effects of 2-mercaptoethanol on centrosomes, chromosomes, microtubules, membranes and intermediate filaments were analyzed by transmission electron microscopy (TEM) and by immunofluorescence microscopy (IFM) methods using a human autoimmune antibody directed against centrosomes (SPJ), and a mouse monoclonal antibody directed against tubulin (E7). Chromosomes were affected most by this treatment: premature chromosome condensation was detected in interphase nuclei, and the structure in mitotic chromosomes was altered compared to control cells. This would support previous findings in dividing sea urchin cells in which chromosomes are arrested at metaphase while the centrosome splitting cycle continues. It might also support findings that certairt-sulfhydryl-blocking agents block cyclin destruction. The organization of the microtubule network was scattered probably due to a looser organization of centrosomal material at the interphase centers and at the mitotic poles.

Spatial distribution of two maternal messengers encoding two sea urchin cell surface proteins

We have localized, byin situ hybridization, two messengers, called bepl and bep4 (butanol extracted proteins), coding for cell surface proteins, involved in cell interaction, in sections of eggs of the sea urchinP. lividus. These maternal RNAs are spatially distributed in a gradient with a maximum at one pole of the egg. These results represent the first demonstration of the existence of a gradient of informational molecules in the sea urchin egg,i.e., the biological material for which the gradient theory was first proposed.

Separation and enrichment of cell populations by centrifugal elutriation

Centrifugal elutriation can be a powerful tool for the separation of cells by their size and density. This article discusses the practical aspects of how the elutriation rotor functions and considers the possible factors that can influence the cell separation in such a rotor. Several examples of diverse applications from the author's laboratory are presented. The first example describes the isolation of large quantities of two different kinds of red pigmented sea urchin cell populations separated from the non-pigmented cells as well as from each other. The second application demonstrates the utility of cell cycle enrichment for the isolation of rare developmental intermediates in the mouse thymus.

Monoclonal antibody against the centrosome.

A monoclonal antibody, MPM-13, raised against mitotic HeLa cell extracts recognized a perinuclear area in interphase cells and spindle poles in mitotic cells of human, mouse, Chinese hamster and sea urchin. On immunoblots MPM-13 recognized a major protein band at 43 X 10(3) Mr and a variable minor band at 56 X 10(3) Mr in both mitotic and interphase HeLa cells. These antigens were detectable in a variety of mammalian cells as well as in the unicellular ciliate Tetrahymena. In cells arrested in mitosis by colcemid and stained with MPM-13 by indirect immunofluorescence, numerous fluorescent speckles were seen throughout the cytoplasm. Reversal of colcemid block in Chinese hamster ovary (CHO) cells by washing resulted in the reappearance of a fluorescent patch at the poles of the re-formed spindles. In HeLa cells arrested in mitosis by the microtubule stabilizing drug taxol, MPM-13 stained a large fluorescent patch encircled by dark metaphase chromosomes. This pattern indicated the failure of centrioles to move to the opposite poles in the presence of taxol. These data indicate that the MPM-13 antigens are associated with the colcemid-sensitive pericentriolar material from which microtubules originate but not with the centrioles themselves. It is also clear that these antigens are highly conserved during evolution.

Specific binding of lectins with the nucleus of the sea urchin embryo and changes in the lectin affinity of the embryonic chromatin during the course of development

(1) Embryonic cells of sea urchins were made permeable by treating them with glycerol solution for the purpose of allowing penetration of macromolecules into the cell. With the use of such permeabilized cells, several kinds of fluorescent dye-labeled lectins were introduced into the cell, and it was found that some lectins showed notable affinity with the nucleus as compared with cytoplasmic structures. (2) Isolated chromatin was incubated with several kinds of fluorescent dye-labeled lectins in vitro, and the amount of lectins bound with the chromatin was measured by fluorometry. By means of this method, the lectin-binding capacity of chromatin was estimated and compared at various stages of development. It was found that lectins could be classified into three groups according to the mode of binding with the chromatin: (a) Extent of binding increased notably at the gastrula stage (Con A and RCA-120); (b) extent of binding showed a temporary decrease at the gastrula stage (TTA); and (c) very low level of binding was maintained throughout all stages, and no particular change was observed at any stage of development (WGA, SBA, and UEA-I). (3) These facts seem to suggest that lectin-binding components are contained in sea urchin chromatin, and that drastic changes occur in these components of chromatin at the stage of gastrulation. It was proposed that the lectin-binding components such as proteoglycans and glycoproteins may play regulatory roles in embryonic chromatin at early stages of development.

On the role of calcium in the adhesion of embryonic sea urchin cells

Calcium is shown to have several roles in the adhesion of embryonic sea urchin cells. Using an assay that subdivides a cell interaction into sequential steps it is shown that Ca 2+ has distinct roles in at least two separate steps. The initial binding step between blastomeres is Ca 2+-sensitive, and is shown to require divalent cations themselves rather than replacement of a macromolecular material that might be removed in the absence of Ca 2+. The secondary steps of adhesion involve a strengthening of the initial contacts. Ca 2+ is shown to be required for at least one and probably more than one of these steps. In addition, the extracellular matrix molecule called hyalin is known to be sensitive to Ca 2+ for its polymerization. Hyalin may be involved in the initial steps of cell binding measured by the assay in that cell-cell binding is partially blocked by a Fab antibody fragment directed against hyalin. Finally, two extracts, known from other studies to enhance cell aggregation, were examined for their possible relationship to the Ca 2+-sensitive binding of cells. A butanol extract was shown to enhance initial binding in a Ca 2+-independent manner. Cells extracted with butanol were greatly reduced in adhesiveness even in the presence of Ca 2+. The second extract was obtained from cells by treatment of embryos with divalent cation-free saline. This Ca 2+-sensitive factor is shown to contain hyalin which may account for some of its observed activity, though not necessarily all of it. Divalent cation-free-extracted cells regain full adhesiveness immediately upon restoration of Ca 2+. Thus the extract is not essential for cell adhesiveness. Together these data show that divalent cations have several roles in the multistepped process called adhesion.

Adhesive and migratory behavior of normal and sulfate-deficient sea urchin cells in vitro

Dissociated cells from different stage embryos of the sea urchin Lytechinus pictus were compared in their adhesion to various substrates. Micromeres from 16-cell stage embryos bind to tissue culture and Petri dishes but not to Petri dishes coated with human plasma fibronectin. Other cell types did not adhere to any of the substrates tested. By hatched blastula stage, about 28% of the cells adhered to fibronectin as well as to tissue culture dishes. By the mesenchyme blastula stage, there was a further increase in the proportion of cells adhering to these substrates. At no stage did cells adhere to native rat tail collagen. Primary mesenchymal cells were isolated by their selective adhesion to tissue culture dishes in the presence of horse serum. These cells were then examined for their migratory capacity. Cell spreading and migration followed adhesion and occurred on fibronectin but not on the other substrates tested. Based on analysis of video tapes, greater than 60% of these cells moved faster than 1 μm/min. On the other hand, cells from sulfate-deprived embryos, in which primary mesenchyme migration is blocked in situ, failed to spread and migrated little on the same substratum. This defect was reversed by a 6 h pretreatment of the cells in normal sea water. Thus, the in vitro migratory behavior parallels that observed in vivo. These results support the hypothesis that the primary mesenchymal cells produce a sulfate-dependent component that is required for cell spreading and migration.

Effects of emetine on initiation of DNA synthesis in embryonic cells of sea urchin

The effects of the inhibition of protein synthesis by emetine on the initiation of DNA synthesis and mitosis in the fertilized eggs of the sea urchin, Anthocidaris crassispina, were studied. The initiation of DNA synthesis was completely prevented, and no delayed initiation was observed in eggs in which protein synthesis was completely inhibited by emetine, while delayed initiation of DNA synthesis occurred after prolonged culture in eggs in which protein synthesis was incompletely inhibited by emetine. In both cases, neither mitosis nor cleavage was observed. Synthesis of some protein factor seems to be necessary for the initiation of DNA synthesis.

Microinjection of fluorescent tubulin into dividing sea urchin cells.

To follow the dynamics of microtubule (MT) assembly and disassembly during mitosis in living cells, tubulin has been covalently modified with the fluorochrome 5-(4,6-dichlorotriazin-2-yl)aminofluorescein and microinjected into fertilized eggs of the sea urchin Lytechinus variegatus. The changing distribution of the fluorescent protein probe is visualized in a fluorescence microscope coupled to an image intensification video system. Cells that have been injected with fluorescent tubulin show fluorescent linear polymers that assemble very rapidly and radiate from the spindle poles, coincident with the position of the astral fibers. No fluorescent polymer is apparent in other areas of the cytoplasm. When fluorescent tubulin is injected near the completion of anaphase, little incorporation of fluorescent tubulin into polymer is apparent, suggesting that new polymerization does not occur past a critical point in anaphase. These results demonstrate that MT polymerization is very rapid in vivo and that the assembly is both temporally and spatially regulated within the injected cells. Furthermore, the microinjected tubulin is stable within the sea urchin cytoplasm for at least 1 h since it can be reutilized in successive daughter cell spindles. Control experiments indicate that the observed fluorescence is dependent on MT assembly. The fluorescence is greatly diminished upon treatment of the cells with cold or colchicine agents known to cause the depolymerization of assembled MT. In addition, cells injected with fluorescent bovine serum albumin or assembly-incompetent fluorescent tubulin do not exhibit fluorescence localized in the spindle but rather appear diffusely fluorescent throughout the cytoplasm.

Permeable embryonic cells of sea urchins as a model for studying nucleus-cytoplasm interactions.

Sea urchin embryonic cells were made permeable by treating them with glycerol solution for the purpose of exchanging cytoplasmic components. When proteoglycans prepared from embryos of advanced stages of development permeated into these glycerinated cells, these substances accumulated rapidly in the nucleus, being bound with chromatin.

The internal clock of reaggregating embryonic sea urchin cells

AbstractThe presence of micromere “a clock” during early cleavage in sea urchin embryos has been shown by several investigators. The suggestion that an internal clock may be operative in later developmental stages up to the pluteus is presented in this study.The development of reaggregating Arbacia cells was followed from dissociation at the 16‐cell stage to pluteus formation, using scanning electron microscopy. After 7 hr of development, the reaggregates were dissociated a second time and were again able to develop into plutei. The morphological development of both reaggregations appears to be identical. In addition, embryos of both reaggregations developed at the same rate as control embryos. Differences in the cells of the first and second dissociations as compared to controls are described, as is the ability of reaggregating cells to catch up to controls during early development. Possible reasons for their ability to make up for lost time are discussed, including non‐randomness of cell behavior, lack of dedifferentiation and the presence of an internal clock.

Phagocytosis and resorption during the reassembly of dissociated embryonic cells of sea urchins.

Time-lapse and electron microscopic observations were made on both epithelial and mesenchymal cells during the reassembly of embryos from dissociated cells of Strongylocentrotus purpuratus. In epithelial cells, where lysosomes are produced through the fusion of saccules formed from Golgi bodies, both phagocytosis of cell debris and resorption of differentiated cell structures were observed. In these cells, the lysosomes migrate and fuse with both autosomes and phagosomes. On the other hand, in the mesenchyme cells, where lysosomes are produced through the direct enlargement of the Golgi body's cisterna, neither phagocytosis nor resorption was observed. The migration of the lysosomes to the epithelial cell margins is the first indication of a re-establishment of cellular polarity after dissociation.

Chapter 9 Surface Antigens Involved in Interactions of Embryonic Sea Urchin Cells

This chapter explores the specificity hypothesis, with the intent to show the way the cell surface might contribute to the process of gastrulation. It discusses two lines of research—namely, (1) data on the formation of cell–cell contacts is presented to demonstrate an array of age-specific and tissue-specific recognition capabilities available to embryonic cells, (2) immunochemical data shows age-specific and tissue-specific differences in cell surface antigens during the early development of the sea urchin. A species-specific pattern of cellular recognition is identified experimentally between the embryonic cells of Lytechinus variegatus and Tripneustes esculentus . Hybrid embryos, formed as a cross between these two species, are studied to determine their pattern of cellular recognition. Prior to gastrulation, hybrid cells adhered to the cells of maternal genotype only. After gastrulation had begun in the hybrid embryos, hybrid cells adhered to the cells of both maternal and paternal genotype. A serological study identified a similar pattern of cell surface antigen expression; paternal antigens are first observed on hybrid cell surfaces at the gastrula stage. The study further identified four classes of antigens present on embryonic cell surfaces—namely, (1) maternal antigens present on cleavage-stage cells, (2) embryonic antigens appearing at gastrulation and specified by embryonic templates, (3) ectoderm-specific antigens, and (4) endoderm-specific antigens.

A model for cell division

We present a model of cell division of a spherical cell which relates the deformation of the cell and the cell membrane to the forces in the contractile ring or furrow region of a dividing cell. The spherical cell of a sea urchin egg is considered as a spherical nonlinear elastic membrane enclosing an incompressible fluid. The results of the analysis obtained by prescribing equatorial displacements agree well with the published experimental values and related calculations on cell division. The results add additional support to the contractile ring theory of cell division and further suggest that a general description of the elastic properties of cell membranes by a constitutive relation due to Skalak et al. (1973), may be applicable to sea urchin cell membranes.

Block of histone synthesis in isolated sea urchin cells actively synthesizing DNA

The synthesis of DNA and histones are normally coupled. Only a few exceptions have been found. Dissociated cells from sea urchin embryos stop DNA synthesis, but resume it after trypsin treatment. It was of interest, therefore, to examine whether the synthesis of histones is similarly affected. Sea urchin embryos were dissociated from swimming blastulae and the cells so obtained were divided into two batches, one of which was treated with trypsin. Normal embryos from the same culture were used as controls. It was found that, in the dissociated cells, histone synthesis ceases, but unlike DNA, its synthesis is not resumed after treatment with trypsin.