Ribosomal proteins of sea urchin eggs

Summary Ribosomal proteins from unfertilized eggs of three sea urchin species, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, and Anthocidaris crassispina, were analyzed. Species-specific differences were observed in the profiles of large subunit proteins on two-dimensional slab gels, though the number of ribosomal proteins and the molecular weights of their counterparts were the same. The small subunit proteins revealed similarities in the electrophoretic profiles and in the phosphorylation patterns among these three species.     

A novel 15 kDa Ca2+-binding protein present in the eggs of the sea urchin, Hemicentrotus pulcherrimus

A novel Ca2+-binding protein, different from calmodulin, has been purified to homogeneity from the soluble cytoplasmic protein fraction of the egg of the sea urchin, Hemicentrotus pulcherrimus. This protein, designated as 15 kDa protein, shows a Ca2+-dependent mobility shift upon SDS-gel electrophoresis and has Ca2+-binding ability. This protein did not resemble the sea urchin egg calmodulin in either molecular mass or amino acid composition. The 15 kDa protein could not activate cyclic adenosine 3',5'-monophosphate-dependent phosphodiesterase from bovine brain and did not bind to fluphenazine-Sepharose 6B. Antibodies against the 15 kDa protein did not react with sea urchin egg calmodulin. These results suggest that the 15 kDa protein is a novel Ca2+-binding protein in the sea urchin egg.     

The centrosome cycle in the mitotic cycle of sea urchin eggs

When sea urchin eggs entering mitosis are exposed to an appropriate concentration of mercaptoethanol, the chromosome cycle is restrained while the centrosome cycle advances. The two poles of the mitotic apparatus separate into four poles, while the chromosomes remain in their metaphase arrangements until released by the removal of the mercaptoethanol. We follow the centrosomes through the stages of the generation of two poles by each original pole. In electron microscopic studies, the osmiophilic component of the centrosomes serves as an indicator of their changing forms as each pole generates two poles. In light microscopic studies, including observations of birefringence, the shapes of the polar ends of the spindles are taken as indicators of the shapes of the centrosomes. The successive stages of the centrosome cycle are (1) compact spherical centrosomes at the time of formation of the mitotic apparatus; (2) expansion and flattening of the centrosomes, leading to (3) formation of thin flat plates, perpendicular to the spindle axis. Corresponding to the extended flat shape of the centrosomes, the spindle poles are flat; microtubules 'point' to the centrosomal plate and not the centrioles. The centrioles are separated in the flattening of the centrosomes. (4) The flat plate divides into two and each of the two halves becomes more compact, defining two separate poles. Our findings resurrect and update Boveri's [5] observations and interpretations of the centrosome. Centrosomes have shapes. The shapes may be imparted to the microtubular structures that they generate. The formation of two separate centrosomes from one, in the formation of mitotic poles, is describable as a sequence of changes in shape.     

Immunocytochemical evidence for centrosomal phosphoproteins in mitotic sea urchin eggs

The change in distribution of centrosomal phosphoproteins was examined in sea urchin eggs from fertilization to the first cleavage by immunofluorescence staining with the anti-phosphoprotein antibodies, MPM-1 and MPM-2. The antibodies reacted with female pronuclei in unfertilized eggs as well as centriolar complexes located at the base of sperm flagella. After insemination, male and female pronuclei fused together to form a zygotic nucleus which was visualized by staining of fertilized eggs with the antiphosphoprotein antibodies. No major change in staining pattern was detected in extracted whole eggs until mitosis. As the fertilized eggs approached mitosis, however, the antigens started to redistribute from nuclei to the perinuclear position where the mitotic centrosomes were located. Detailed immunofluorescence observation of isolated spindles revealed that the phosphoantigens were retained in isolated structures. A major 225 kd polypeptide was recognized by the antibodies, suggesting that the 225 kd protein is a phosphocomponent of centrosomes. The area recognized by the antibody in mitotic poles enlarged with the progress of mitosis, suggesting that the antigens were apparently localized in the centrosphere. Centrospheres prepared from isolated spindles by salt extraction strongly reacted with the antibodies. One or two bright dots, which may represent centrioles, were visible in the isolated centrosphere. At the end of mitosis, the antigens again appeared in the newly formed daughter nuclei. Centriole-containing cytasters and centriole-free monasters were parthenogenetically induced in unfertilized eggs (Kuriyama and Borisy, (1983) J. Cell Sci. 61: 175-189). The antibodies stained centers of both the asters whether they contained centrioles or not, indicating that the antibodies recognizes the components of the pericentriolar material.     

The cleavage plane will bend when one aster of the mitotic apparatus stops growing in compressed sea urchin eggs

Astral microtubules are elongated greatly during anaphase and telophase in sea urchin eggs. The surface density of microtubules reaching the cell surface can be defined at each surface point. Gradients of the surface-density function were assumed to drive membrane proteins whose accumulation causes the formation of contractile-ring microfilaments. An equation was constructed to calculate the movements of the membrane proteins on a curved surface. The equation was applied to eggs compressed between a coverslip and a glass slide by regarding the egg shape as an oblate spheroid. The simulations explained the observations that contractile-ring microfilaments locally appeared and then developed into a complete ring in compressed eggs. When one aster in the mitotic apparatus stopped growing during anaphase, the equation predicted that the zone of contractile-ring microfilaments is displaced toward the inactivated aster, curves in the view from above and tapers off toward the cell edge. The curve gets sharper as eggs are compressed more greatly and as microtubules from the growing aster penetrate more deeply into the opposite hemisphere. The predictions were compared with the observations by Ishii and Shimizu in 1995 and by Hamaguchi in 1998 regarding the furrow formation by the asymmetric mitotic apparatus.     

225-Kilodalton phosphoprotein associated with mitotic centrosomes in sea urchin eggs

Protein phosphorylation during development of sea urchin eggs from fertilization to first cleavage was examined by labeling cells with specific antiphosphoprotein antibodies. Indirect immunofluorescence staining with monoclonal antithiophosphoprotein antibody (Gerhart et al.: Cytobios 43:335-347, 1985) has revealed that nuclei as well as centrosomes, kinetochores, and midbodies were specifically thiophosphorylated in developing eggs incubated with adenosine 5'-O (3-thiotriphosphate) (ATP-gamma-S). The phosphorylation reaction required Mg2+ but was not dependent on cAMP or calmodulin in detergent-extracted models. Centrosomes were purified by fractionation of isolated mitotic spindles with 0.5 M KCl extraction. The thiophosphoproteins were retained in the purified centrosomes and the antibody recognized a major 225-Kd polypeptide on immunoblots. In an independent preparation, a monoclonal antiphosphoprotein antibody (CHO3) was found also to react with mitotic poles and stained a 225-Kd polypeptide, confirming the centrosome specificity of this protein. Immunoelectron microscopy showed that the 225-Kd thiophosphoprotein was found at mitotic poles associated with granules to which mitotic microtubules were directly attached. Unlike centrosomes in permeabilized eggs, those in isolated spindles could not be thiophosphorylated, possibly due to inactivation or loss of either phosphorylation enzymes or cofactors, or both, during isolation. The immunofluorescence labeling of thiophosphate could be inhibited by ATP and AMP.PNP in a concentration-dependent manner. Exogenous ATP could abolish thiophosphate-staining more effectively when added with phosphatase inhibitors, suggesting a dynamic state in which centrosomal proteins are being phosphorylated and dephosphorylated in rapid succession by the action of protein kinase(s) and phosphatase(s).     

Interaction of ruthenium red with Ca2(+)-binding proteins.

The interaction of ruthenium red, [(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5]Cl6.4H2O, with various Ca2(+)-binding proteins was studied. Ruthenium red inhibited Ca2+ binding to the sarcoplasmic reticulum protein, calsequestrin, immobilized on Sepharose 4B. Furthermore, ruthenium red bound to calsequestrin with high affinity (Kd = 0.7 microM; Bmax = 218 nmol/mg protein). The dye stained calsequestrin in sodium dodecyl sulfate-polyacrylamide gels or on nitrocellulose paper and was displaced by Ca2+ (Ki = 1.4 mM). The specificity of ruthenium red staining of several Ca2(+)-binding proteins was investigated by comparison with two other detection methods, 45Ca2+ autoradiography and the Stains-all reaction. Ruthenium red bound to the same proteins detected by the 45Ca2+ overlay technique. Ruthenium red stained both the erythrocyte Band 3 anion transporter and the Ca2(+)-ATPase of skeletal muscle sarcoplasmic reticulum. Ruthenium red also stained the EF hand conformation Ca2(+)-binding proteins, calmodulin, troponin C, and S-100. This inorganic dye provides a simple, rapid method for detecting various types of Ca2(+)-binding proteins following electrophoresis.     

Ca2+ influx and stimulation of protein synthesis in sea urchin eggs

Acid release, Ca2+ influx and stimulation of protein synthesis were investigated with sea urchin eggs submitted to an excess of KCl, to NH4Cl, and to a combination of both. KCl, though unable to promote any acid release, triggers a large 45Ca uptake by eggs and slightly stimulates protein synthesis, provided that external Ca2+ is present. NH4Cl, which induces an intracellular pH increase, triggers a late and small 45Ca uptake but highly stimulates protein synthesis. The combined use of NH4Cl + KCl allows a large 45Ca uptake to occur but the level of protein synthesis is similar to that obtained with NH4Cl alone and is identical whether external Ca2+ is present or not. In contrast to previous works, our results show that the large stimulation of protein synthesis triggered by an intracellular pH increase, as after NH4Cl activation, cannot be enhanced by a Ca2+ influx. This suggests that the Ca2+ influx occurring after fertilization has only a minimal effect on the overall stimulation of protein synthesis.     

Two acid phosphatases in sea urchin eggs and embryos

• 1.1. Two types of acid phosphatases from sea urchin eggs and embryos were studied in three Japanese species, Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus.
• 2.2. Acid phosphatase type 1, designated AcP-1, hydrolysed only flavin mononucleotide besides p-nitrophenylphosphate. The activity of AcP-1 was not inhibited by NaF and tartrate. This enzyme showed molecular weight between 14,000 and 16,000 by gel filtration through Sephadex G-75.
• 3.3. The higher molecular weight type of acid phosphatase, designated AcP-2, showed relatively high substrate specificity toward ADP and ATP. Molecular weight of AcP-2 ranged from 42,000 to 48,000 by gel filtration through Sephadex G-100.
• 4.4. Some properties of AcP-2 from Sphaerechinus granularis embryos are also described.

     

Mastoparan induces Ca2+-independent cortical granule exocytosis in sea urchin eggs

In most species, cortical granule exocytosis is characteristic of egg activation by sperm. It is a Ca(2+)-mediated event which results in elevation of the vitelline coat to block permanently the polyspermy at fertilization. We examined the effect of mastoparan, an activator of G-proteins, on the sea urchin egg activation. Mastoparan was able to induce, in a concentration-dependent manner, the egg cortical granule exocytosis; mastoparan-17, an inactive analogue of mastoparan, had no effect. Mastoparan, but not sperm, induced cortical granule exocytosis in eggs preloaded with BAPTA, a Ca(2+) chelator. In isolated egg cortical lawns, which are vitelline layers and membrane fragments with endogenously docked cortical granules, mastoparan induced cortical granule fusion in a Ca(2+)-independent manner. By contrast, mastoparan-17 did not trigger fusion. We conclude that in sea urchin eggs mastoparan stimulates exocytosis at a Ca(2+)-independent late site of the signaling pathway that culminates in cortical granule discharge.     

Solubility changes of proteins in sea urchin eggs upon fertilization

The results presented in this paper give evidence of changes in solubility occurring in a protein fraction of the eggs of Arbacia lixula upon fertilization. The electrophoretic analysis indicates that it is only one part of one of the components of the KCl fraction that undergoes the change. However, under some experimental conditions (freezing and thawing of the KCl fraction or extraction of the whole eggs with water at room temperature) a larger portion of the KCl fraction, namely the whole group of components a and b, may be involved and undergoes coagulation. Therefore assuming that the results obtained on the extracts of frozen-dried fertilized eggs do reflect what actually occurs under natural conditions, we must also assume the existence of mechanisms controlling the extent of this change in the living eggs. The fact that in many cases one part or the whole of the sensitive fraction has been found to undergo an increase in solubility may suggest that the process of coagulation discovered by Mirsky is a two-step process. In the first step the sensitive fraction undergoes a change that makes it more soluble and then, when certain conditions are fulfilled, coagulation occurs. An alternative explanation could also be that the coagulated or coagulating fraction is attacked by the proteolytic enzyme that, as shown by Lundblad (1949, 1950), is activated on fertilization. This, however, seems to be less probable, as extraction was always carried out at 0° C. and in as short a time as possible. However, further experiments are needed to decide whether the coagulation of the sensitive fraction is an actual occurrence under natural conditions. The results obtained with the eggs of Arbacia punctulata may cast some doubt on this assumption.