Extracts Of Sea Urchin Eggs Research Articles

Evidence for an association between U1 RNA and interspersed repeat single-copy RNAs in the cytoplasm of sea urchin eggs†

Psoralen crosslinking of RNA-RNA intermolecular duplexes in sea urchin egg extracts reveals that some maternal poly(A)+ RNA molecules are complexed with U1 RNA, a cofactor in somatic nuclear pre-mRNA splicing. Reaction of egg extracts with a monoclonal antibody specific for U1 snRNP selects, in addition to U1, RNAs that contain repeated sequences interspersed with single-copy elements. Antibody-selection experiments with nucleate and anucleate egg halves demonstrate that most of the U1 RNA-interspersed RNA complexes are cytoplasmic, as is the egg's store of total U1 snRNP. These results raise the possibility that maternal interspersed RNAs include unprocessed pre-messenger RNA molecules in arrested complexes with splicing cofactors.

Low ionic strength solubility of myosin in sea urchin egg extracts is mediated by a myosin-binding protein.

We identify a novel myosin-binding protein, designated 53K, which appears to mediate the low ionic strength solubility of myosin in extracts of unfertilized sea urchin eggs. The protein possesses a subunit molecular mass on SDS-PAGE of 53 kD, an S value of 7, may be organized into disulfide-linked oligomers, and is associated with myosin in egg extracts. Both myosin and 53K co-precipitate from extract upon the addition of nucleoside triphosphates and co-sediment with an S value of 24 by sedimentation velocity centrifugation. Myosin in extracts not associated with 53K has an S value of 10. Further, myosin can be immunoprecipitated from extract with antibody to 53K and the 53K in extracts binds to a myosin affinity column. When extract is depleted of 53K, a majority of the myosin precipitates out of extract in a nucleotide-independent manner. Whereas purified myosin precipitates in the absence of nucleotide when recombined with dialysis buffer or myosin-depleted extract, reconstituting 53K and myosin before addition to buffer or myosin-depleted extract partially restores the low ionic strength solubility demonstrated by myosin in fresh egg extracts. The 53-kD protein may represent a new class of authentic myosin-binding proteins that may regulate the supramolecular organization of myosin in nonmuscle cells.

Characterization of the microtubule movement produced by sea urchin egg kinesin.

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.

Temperature and pH govern the self-assembly of microtubules from unfertilized sea-urchin egg extracts.

A new method for microtubule purification from unfertilized sea-urchin eggs was developed in order to obtain large quantities of calcium- and cold-labile microtubules that contained microtubule-associated components important for mitosis. By taking into consideration the pH, ionic composition of egg cytoplasm, and the physiological temperature for growth of the Pacific coast sea-urchin Strongylocentrotus purpuratus, methods were developed for the assembly of intact microtubules directly from unfertilized egg extracts. The microtubules obtained by cycles of temperature-dependent assembly and disassembly are composed of tubulin and abundant microtubule-associated proteins. These microtubules are cold- and calcium-labile and assemble at a critical protein concentration of 0.11 mg ml-1 at 24 degrees C. The yield of microtubule protein obtained by this new method is equivalent to that obtained with taxol (6-8 mg/20 ml packed eggs). Microtubules that have been fixed and prepared for electron microscopy are decorated with large, globular projections that are attached to the microtubule by thin stalks.

Localization of fodrin during fertilization and early development of sea urchins and mice

Fodrin, a spectrin-like protein, is localized in gametes, zygotes, and embryos from sea urchins and mice. Mammalian fodrin comprises two polypeptides with molecular weights of approximately 240 kDa (alpha) and 235 kDa (beta). An antibody specific for mammalian α-fodrin cross-reacted with a 240-kDa polypeptide from sea urchin egg extracts. This indicates that sea urchins contain a protein of similar electrophoretic mobility and immunological properties to mammalian α-fodrin. When this antibody was used to stain the sea urchin gametes with indirect immunofluorescence, fodrinspecific fluorescence was localized to the acrosome of the sperm and was distributed over the entire egg near the surface in a punctate pattern similar to the distribution of polymeric actin. During sperm incorporation, the fodrin-specific fluorescence is found at the site of sperm incorporation, in the fertilization cone. After fertilization, the intensity of fodrin fluorescence increases. During mitosis and cytokinesis in sea urchins, the entire surface of the egg remains stained; the cleavage furrow also was stained but no more intensely than was the rest of the egg surface. Antibody labeling with colloidal gold followed by electron microscopy showed that fodrin was loated in the cytoplasm immediately beneath the plasma membrane. In unfertilized mouse oocytes, both actin and fodrin were stained most intensely beneath the membrane adjacent to the meiotic spindle. After insemination, the cell surfaces of the pronucleate egg and the second polar body were stained; however, the actin matrix surrounding the apposed pronuclei did not bind the fodrin antibody. During cytokinesis in the mouse, the cleavage furrow stained more intensely than did the rest of the egg cortex, and in embryos the cell borders were delineated. These results indicate that organisms as unrelated to mammals as sea urchins have fodrin-like proteins; the rearrangements of such proteins suggest that they participate in the actin-mediated events at the cell surface during fertilization and early development in both mice and sea urchins.

Purification and characterization of a sea urchin egg Ca2+-calmodulin-dependent kinase with myosin light chain phosphorylating activity.

The crude actomyosin precipitate from sea urchin (Arbacia punctulata) egg extracts contains Ca2+-sensitive myosin light chain kinase activity. Activity can be further increased by exogenous calmodulin (CaM). Egg myosin light chain kinase activity is purified from total egg extract by fractionating on three different chromatographic columns: DEAE ion exchange, gel filtration on Sephacryl-300, and Affi-Gel-CaM affinity. The purified egg kinase depends totally on Ca2+ and CaM for activity. Unphosphorylated egg myosin has very little actin-activated ATPase. After phosphorylation of the phosphorylable light chain by either egg kinase or gizzard myosin light chain kinase, the actin-activated ATPase of egg myosin is enhanced several fold. However, the egg kinase bears some unique characteristics which are very different from conventional myosin light chain kinases of differentiated tissues. The purified egg kinase has a native molecular mass of 405 kDa, while on sodium dodecyl sulfate-polyacrylamide electrophoresis it shows a single subunit of 56 kDa. The affinity of egg kinase for CaM (Ka = 0.4 microM) is relatively weaker than that of the gizzard myosin light chain kinase. The egg kinase autophosphorylates in the presence of Ca2+ and CaM and has a rather broad substrate specificity. The possible relationship between this egg Ca2+-CaM-dependent kinase and the Ca2+-CaM-dependent kinases from brain and liver is discussed.

An EHNA‐senstive ATPase in unfertilized sea urchin eggs

AbstractIn current purification strategies, affinity for microtubules or calmodulin is used to identify and purify cytoplasmic dynein‐like ATPase from cell‐free extracts of unfertilized sea urchin eggs. However, affinity purification procedures, though they define dynein‐like ATPase activity, have not yet proven to be quantitative. An alternative purification strategy capable of producing a high yield of enzyme would require a specific assay in order to monitor cytoplasmic dynein purity at each step.In this study, we make a detailed comparison of the effects of EHNA on 22 different ATP‐metabolizing enzyme activities, including 13 Mg++‐ATPases. We isolate cytoplasmic dynein‐like ATPase activity from three species of sea urchin eggs and sperm and show by means of dose‐response curves that their sensitivities to inhibition by EHNA are very similar to one another. We demonstrate further that the EHNA dose‐response characteristics of fourteen other ATP‐metabolizing enzyme activities, including seven nondynein Mg++‐ATPases, differ quantitatively from those of dynein‐like ATPases.In studies of three other agents (vanadate, Ca++/calmodulin, and Triton X‐100), we find that dynein‐like ATPases vary by two orders of magnitude in their sensitivities to inhibition by vanadate, and little or no stimulation by either Ca++/calmodulin or Triton X‐100 is seen. Our results suggest that inhibition by EHNA is a universal and specific property of dynein‐like ATPases, which ultimately should prove useful in the quantitative purification and characterization of cytoplasmic dynein‐like ATPase (s).

Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs.

Taxol was used to prepare microtubules from unfertilized eggs of sea urchins Lytechinus pictus, Strongylocentrotus droebachiensis , and Strongylocentrotus purpuratus. By electron microscopy, these microtubules possessed normal morphology and were decorated with projections. The polypeptides present were tubulin plus microtubule-associated proteins (MAPs) which included various high molecular weight polypeptides, and a Mr = 80,000 polypeptide. These MAPs were extracted from the microtubules by differential centrifugation in high ionic strength buffers, yielding a pellet of microtubules which were not decorated with projections. The Mr = 80,000 and high molecular weight MAPs were separated using Bio-Gel A-1.5 m chromatography, and shown to bind taxol-stabilized microtubules assembled from purified bovine brain tubulin. A dynein-like MgATPase activity is present in sea urchin egg extracts. 10-20% of this MgATPase co-pelleted with the taxol-assembled microtubules, under conditions where greater than 90% of the tubulin pelleted. During subsequent fractionation of the microtubules, by (i) high salt extraction followed by gel filtration or sucrose density gradient fractionation or (ii) ATP extraction, the MgATpase co-purified with high Mr MAPs. The MgATPase which remained in the microtubule-depleted egg extract was partially purified by (NH4)2SO4 fractionation, followed by Bio-Gel A-5 m and hydroxylapatite chromatography. The high Mr MAP MgATPase and the hydroxylapatite MgATPase both contained a prominent polypeptide (Mr approximately 350,000), which co-migrated on sodium dodecyl sulfate gels with the major heavy chain of dynein extracted from sperm axonemes. Our data suggest that this Mr approximately 350,000 polypeptide is cytoplasmic dynein.

Chapter 9 Actin Gelation in Sea Urchin Egg Extracts

Publisher Summary This chapter describes the different aspects of actin gelation in sea urchin egg extracts. Sea urchin eggs are not usually considered to be highly motile cells, and they do display a striking extension of microvilli following fertilization and classical material for studying cell division. Eggs were obtained from the Hawaiian sea urchin Tripneustes grutillu by injection of isotonic KCI into the body cavity. The jelly coat was removed by rapidly titrating a 10% egg suspension to pH 5 with HCl, then washing the eggs several times with Millipore-filtered seawater. The eggs were sedimented by hand centrifugation and the packed egg volume estimated. The formation of a structured actin gel consisting of linear aggregates of actin filaments can be induced by warming the cytoplasmic extracts. Actin has been isolated from acetone powders of sea urchin eggs and partially characterized. The procedure depends on the ability of this invertebrate actin to form large bundles in the presence of high salt and appears to be generally applicable to echinoderms. It is suggested that fascin functions in sea urchin eggs and other cells to organize actin filaments into the bundles that form the cores of filopodia and microvilli. A type of phagocytic coelomocyte from sea urchins was used to study the role of this actin-cross-linking protein.

Analysis of microtubule polymerization inhibitors in sea urchin egg extracts: Evidence for a protease

Inhibitors of microtubule polymerization have been found in extracts of unfertilized sea urchin eggs using neural tubulin polymerization assays without glycerol. The inhibitory activity is partially destroyed by boiling or by reduction and carboxymethylation and is nondialyzable. When chromatographed on DEAE-cellulose, the inhibitory activity is eluted over a broad NaCl gradient and is in association with several peaks. This partially purified inhibitor is not destroyed by incubation with RNase A. When the partially purified inhibitor is incubated with brain microtubule protein under conditions which support microtubule polymerization, both high molecular weight-microtubule associated proteins and tubulin appear to be digested when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteolytic digestion as well as inhibition of microtubule polymerization depend upon similar concentrations of partially purified inhibitor present in the polymerization reaction. It appears as though at least part of the microtubule polymerization inhibitory activity present in unfertilized sea urchin eggs is due to this protease.

Induction of either contractile or structural actin-based gels in sea urchin egg cytoplasmic extract.

The gel formed by warming the 100,000 g supernate of isotonic extracts of sea urchin eggs to 40 degrees C is made up of actin and two additional proteins of mol wt of 58,000 and 220,000. Actin and 58,000 form a characteristic structural unit which has now been identified in the microvilli of the urchin egg and in the filopods of urchin coelomocytes. However, egg extract gels did not contract as those from other cell types do, and the aim of these experiments was to determine the reason for this lack of contraction. Although the extracts are dialyzed to a low ionic strength, myosin is present in soluble form and makes up approximately 1% of the protein of the extract. It becomes insoluble in the presence of high ATP concentrations at 0 degrees C, and the precipitate formed under these conditions consists almost entirely of myosin. This procedure provides a simple method of isolating relatively pure myosin without affecting other extract components and functions. Contraction will follow gelation in these extracts if the temperature and time of incubation used to induce actin polymerization are reduced to minimize myosin inactivation. At the optimal ATP and KCl concentration for contraction, the contracted material has an additional 250,000 component and contains very little 58,000. The conditions found to provide maximum gel yields favor the formation of the actin-58,000-220,000 structural gel, while reduced temperature and increase in KCl concentration results in a contractile gel whose composition is similar to those reported from amoeboid cell types. Both the structural protein cores found in the egg microvilli and a gel contraction related to the amoeboid motion which is seen in later urchin embryonic development can thus be induced in vitro in the same extract.

Separation and interaction of the major components of sea urchin actin gel.

Abstract Warming of low calcium, isotonic extracts of sea urchin eggs causes gelation by inducing the polymerization of F-actin which then interacts with other proteins in the cytosol to form the gel. Two major protein components with molecular weights of 58,000 and 220,000 have been identified. F-actin has previously been purified from such preparations and shown to interact with a mixture of these proteins to form a gel composed of F-actin aggregates displaying a unique 11 nm banding pattern. The gelation is calcium-sensitive; half-maximal inhibition is at 1 to 2 μ m free calcium in the extract. Solubilization of the gel in 0.6 m -KI and chromatography on A-15m agarose in 0.6 m -KCI resolves the 220K protein (Mr = 220,000) from G-actin and the 58K protein (Mr = 58,000). Further purification on DEAE-cellulose yields pure 58K protein. When combined, the repolymerized actin and 58K protein form microscopic needles composed of linear aggregates of F-actin with the characteristic 11 nm banding. Saturation of the 58K bindingsites of F-actin occurs at ratios of one 58K protein/4 to 5 actin molecules. This actin—58K protein interaction does not show calcium sensitivity. Addition of the 220K protein to pure F-actin produces no detectable aggregation and this protein therefore appears to be distinct from the high molecular weight, actin-binding proteins isolated from vertebrates. The addition of the 220K protein to solutions of needles causes aggregation to a material morphologically indistinguishable from the original gel, but this gelation is not sensitive to calcium. These results show that the 58K protein links F-actin filaments to form cross-banded needles with a 11 nm periodicity, which are then linked into a three-dimensional gel by the 220K protein. Theso results suggest that the initial calcium sensitivity may be due to the presence of regulatory proteins which are removed during the purification.

Glutathione reductase in the sea urchin egg. III. Activation of the complex form by proteinases.

The G-200 flow-through fraction of the extract of sea urchin eggs contained a complex form of glutathione reductase (GR) [EC 1.6.4.2]. The complex was unstable and gradually dissociated with ain increase in GR activity. The activation was facilitated by high concentrations of EDTA, KCI or (NH4)2SO4. The rate of activation by salts was apparently dependent on the ionic strength. The complex form was also activated rather quickly by treatment with proteinases such as trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1] or subtilisin [EC 3.4.21.14]. Trypsin caused the complex to release the free form of GR. Even after trypsin treatment, little change was observed in the dependence of the GR activity on GSSG or NADPH concentration. The GR activity of the complex form was not inhibited at all by 0.2 mM N-ethylmaleimide (NEM) in the presence of GSSG, but was reduced to 3% in the presence of NADPH. When excess NEM was sequestered with GSH, the NEM-treated complex form was strikingly activated by trypsin, while no activation was detected with the free form of enzyme pretreated with NEM. These results suggest that the active site of GR in the complex form is largely masked by a polypeptide moiety of theinhbitiory component.

Actin polymerization and interaction with other proteins in temperature-induced gelation of sea urchin egg extracts.

The gel which forms on warming the extracts of the cytoplasmic proteins of sea urchin eggs has been separated into two fractions, one containing F-actin and the other containing two proteins of 58,000 and 22,000 mol wt. When combined in 0.1 M KCl, even at 0 degrees C, these components will form gel material identical to that formed by warming extracts. This gel is a network of laterally aggregated F-actin filaments which are in register and which display a complex cross-banding pattern generated by the presence of the other two proteins. Low concentrations of calcium block the assembly of these proteins to form this complex structure, which may play some cytoskeletal role in the cytoplasm. This association of F-actin with the other proteins to form a gel is very likely the last step fo the process occurring in warmed extracts. At low temperatures, gelation of extracts is limited by the relative absence of F-actin, as demonstrated by the inability to sediment it at 100,000 g and also by the fact that gelation occurs immediately if exogenous F-actin is added to cold extracts. The transformation of the G-actin present in the extract to the F-form is apparently repressed at low temperatures. This is shown directly by the failure of added G-actin to polymerize at low temperatures in the presence of extract. These observations resemble those which have been reported on preparations from amoeboid cells and may be significant in the involvement of actin and these other proteins in cell division and later developmental processes.

Pyrimidine biosynthesis and its regulation in embryos of the sea urchin, Arbacia punctulata

1. 1. Evidence for the occurrence of the orotate pathway for the de novo biosynthesis of pyrimidines in the developing sea urchin embryo was provided by the incorporation of ( 14C) NaHCO 3, ( 14C) carbamoylaspartate, and (6- 14C) orotic acid into the pyrimidines of RNA, in vivo. 2. 2. This interpretation was supported by the observation that the radio-label from ( 14C) NaHCO 3 appeared in the C-2 position of the uracil ring and by the demonstration of the activity of all of the enzymes of the orotate pathway in cell-free extracts of sea urchin eggs. 3. 3. Evidence was obtained for the regulation of the de novo pathway by end-product inhibition.

Preparation and purification of polymerized actin from sea urchin egg extracts.

Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F-actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when 1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.

Partial Purification and Characterization of Nicotinamide Adenine Dinucleotide Kinase from Sea Urchin Eggs

Abstract Nicotinamide adenine dinucleotide kinase (ATP:NAD+2'-phosphotransferase, EC 2.7.1.23) has been purified 20-fold from extracts of unfertilized sea urchin eggs. The pH optimum for activity was 6.5. The enzyme was specific for ATP, and a divalent cation was required for activity. At 1:1 cation/ATP ratios, relative activities were Mn2+ g Mg2+ g Ca2+. Free Mn2+ was inhibitory, whereas activation by free Ca2+ was observed. Complex kinetic behavior was observed when the concentration of one substrate was varied in the presence of a high level of the second substrate. Activation was observed with increasing concentrations of NAD+ and ATP. Substrate concentrations giving one-half maximum velocities were 0.83 mm ATP and 0.68 mm NAD+. No activation was seen with a variety of metabolites. NADPH, NADH, ADP, AMP, and cyclic 3',5'-AMP were inhibitors. The molecular weight estimated by gel filtration and density gradient centrifugation was 310,000. The relationship between the properties of the enzyme and the changes in pH, cation content, and substrate levels in eggs at fertilization were considered.

Reversible inactivation of nicotinamide adenine dinucleotide kinase in extracts of unfertilized sea urchin eggs

NAD kinase activity in extracts of unfertilized sea urchin eggs is lost on dialysis against dilute buffer and can be recovered by incubation with NAD and dithiothreitol.

Thymidine kinase, thymidylate kinase and 32P i and [ 14C]thymidine incorporation into DNA during early embryogenesis of the sea urchin

In the early embryogenesis of the sea urchin, the relationship between the activity of thymidine kinase (ATP:thymidine 5′-phosphotransferase, EC 2.7.1.21) and thymidylate kinase (ATP:thymidinemonophosphotransferase, EC 2.7.4.9) and of DNA synthesis was investigated. Thymidine kinase and thymidylate kinase activities were found in the homogenate and in the crude extract of unfertilized sea-urchin eggs and embryos. Maximal activity of the kinases was found in unfertilized eggs and the activity was decreased after fertilization. Thereafter, the activities changed periodically in parallel during the cell cycle and their maxima appeared at a defined stage of the cell cycle. [2- 14C]Thymidine and 32P i incorporation into the DNA fraction was found usually just after each division, with the exception of the first division cycle, which occurred at the stage when pronuclear fusion may take place. DNA synthesis was observed after the appearance of the peak of activity of these two kinases in the division cycles. The DNA synthesis system was not included in one division cycle (the division took place between the time of the appearance of the activity peak of DNA synthesis and that of the two kinases). In the early embryogenesis of the sea urchin, the mechanism of regulation of thymidine kinase activity was investigated and the following results were obtained. Puromycin and ethionine depressed thymidine kinase activity in vivo. Thus, the increase of thymidine kinase activity may be due mainly to enzyme synthesis de novo. Although thymidine kinase was observed to have a low stability, the enzyme had a similar lability in the homogenates of unfertilized eggs and of embryos. Thymidine protected thymidine kinase activity from heat inactivation. dTTP was a feedback inhibitor, but dCDP was not an activator.

Sulfhydryl groups and the retardation of cleavage by extracts of sea urchin eggs

Sulfhydryl groups and the retardation of cleavage by extracts of sea urchin eggs