A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin

A one-to-one complex of a 45,000-mol-wt protein and actin was purified from unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, by means of DNase l-Sepharose affinity and gel filtration column chromatographies. Effects of the complex on the polymerization of actin were studied by viscometry, spectrophotometry, and electron microscopy. The results are summarized as follows: (a) The initial rate of actin polymerization is inhibited at a very low molar ratio of the complex to actin. (b) Acceleration of the initial rate of polymerization occurs at a relatively high, but still substoichiometric, molar ratio of the complex to actin. (c) Annealing of F-actin fragments is inhibited by the complex. (d) The complex prevents actin filaments from depolymerizing. (e) Growth of the actin filament is inhibited at the barbed end. In all cases except b, a molar ratio of less than 1:100 of the 45,000-mol-wt protein-actin complex to actin is sufficient to produce these significant effects. These results indicate that the 45,000-mol-wt protein-actin complex from the sea urchin egg regulates the assembly of actin by binding to the barbed end (preferred end or rapidly growing end) of the actin filament. The 45,000-mol-wt protein-actin complex can thus be categorized as a capping protein.     

A novel 24-kDa microtubule-associated protein purified from sea urchin eggs.

Chromatographic fractionation of a crude extract of sea urchin eggs on a hydrophobic column enabled us to find a new 24-kDa microtubule-associated protein (SU-MAP24) that bound tightly to the column and was eluted under alkaline conditions. Biochemical studies using the purified protein showed its direct binding to microtubules reconstituted from tubulin purified from starfish sperm outer fibers. SU-MAP24 promoted tubulin polymerization in a dose-dependent manner. Immunoblotting analysis showed that SU-MAP24 is present in a microtubule protein fraction obtained from a crude extract using taxol, and immunostaining of paraffin-sectioned metaphase eggs showed its localization in the mitotic apparatus. These results show that SU-MAP24 is a newly identified microtubule-associated protein.     

Identification of a calsequestrin-like protein from sea urchin eggs

Following studies on calcium transport by isolated smooth endoplasmic reticulum from unfertilized sea urchin eggs (Oberdorf, J. A., Head, J. F., and Kaminer, B. (1986) J. Cell Biol. 102, 2205-2210) we have purified and partially characterized a calsequestrin-like protein from this organelle isolated from eggs from Strongylocentrotus droebachiensis and Arbacia punctulata. Muscle calsequestrin from sarcoplasmic reticulum is well characterized as a calcium storage protein. The egg protein resembles calsequestrin in its behavior in purification steps, electrophoretic mobility, blue staining with Stains-all on polyacrylamide gels, and its calcium binding and amino acid composition. Purification was attained with DEAE-cellulose and hydroxyapatite chromatography. The egg protein Mr of 58,000 in the Laemmli gel system is reduced to 54,000 under Weber-Osborn (neutral) conditions, thus showing a pH dependence in its mobility, although less than occurs with muscle calsequestrins. 25% of its amino acids are acidic and 10% basic. It binds 309 nmol of Ca2+/mg of protein, within the range reported for cardiac calsequestrin. Antigenically, the sea urchin egg protein is related to cardiac calsequestrin capable of binding anti-cardiac calsequestrin antibody.     

Purification and properties of soluble actin from sea urchin eggs

Unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, were homogenized in a buffer containing 0.1 M KCl and 2 mM MgCl2 at pH 6.85. About 50% of the actin was recovered in the high-speed supernate of the homogenate. More than 80% of the actin in this supernate was found to be monomeric upon gel filtration chromatography through a Sephadex G-150 column or by a DNase I inhibition assay. The critical concentration for polymerization of this actin prior to further purification was 0.3-0.9 mg/ml under various conditions. Actin was purified to near homogeneity from the Sephadex G-150 pool with high yield. The purified actin had a critical concentration for polymerization of 0.02-0.03 mg/ml. The isoelectric point of the crude actin and the purified actin was the same. Indeed, we found that there is only one isoelectric focusing species of actin in the sea urchin egg, and it has an isoelectric point more basic than rabbit skeletal muscle actin. The discrepancy between the polymerizability of the crude and purified actin may be due to the presence of factors in the crude fraction which inhibit the polymerization of actin.     

Structure of actin filament bundles from microvilli of sea urchin eggs

The detailed substructure of actin filament bundles in microvilli of fertilized sea urchin eggs has been studied by analysing electron microscope images of negatively stained specimens. Transverse stripes which repeat about every 130 Å along the axis of a bundle, as previously observed by Burgess & Schroeder (1977), reflect the positions of cross-bridges that connect the filaments into a bundle. Analysis of optical transforms of the micrographs reveals that there are approximately 14 actin monomers between cross-overs of the two long-pitch helical strands of the actin filaments, with three cross-bridges in this interval. The structure is basically similar to that of the hexagonally packed bundles prepared in vitro from high speed supernatants of sea urchin eggs by Kane (1975) and analyzed by DeRosier et al. (1977). One clear difference, however, is that the in vivo microvillar filament bundles are supercoiled, giving rise to long axial repeats of 1500 to 2000 Å.Computationally filtered images of regions that were only slightly supercoiled reveal the relative alignment of filaments within the bundles and show that crossbridges appear to interact with four actin monomers, apparently linking two actin monomers on one strand of one filament to the nearest two monomers on a neighbouring filament. However, the cross-bridges are not spaced at equal intervals corresponding to four actin subunits, presumably because of the lack of hexagonal symmetry in the individual filaments, which have about 14 actin monomers between cross-overs. Instead, the cross-bridges are arranged quasiequivalently along the longitudinal axis of the bundles, in steps of four or five actin subunit spacings (28 Å each).     

The 45K molecular weight actin-modulating protein from sea urchin eggs forms a complex with actin in the presence of calcium ions

The 45K protein from sea urchin eggs forms a complex with actin in the presence of Ca2+. The fraction was not readily dissociated on depletion of Ca2+ by gel filtration, but was dissociated on dialysis against an EGTA-containing solution. The free 45K protein and the 45K protein-actin complex affect F-actin in different manners in the presence of Ca2+. The former severs F-actin in the presence of Ca2+ but not in its absence, while the latter caps the barbed end of F-actin in a Ca2+-independent manner thereby lowering the viscosity of F-actin slowly.     

A 45,000-mol-wt protein from unfertilized sea urchin eggs severs actin filaments in a calcium-dependent manner and increases the steady-state concentration of nonfilamentous actin

A 45,000-mol-wt protein has been purified from unfertilized sea urchin (Strongylocentrotus purpuratus) eggs. The isolation scheme includes DEAE cellulose ion-exchange chromatography, gel filtration, and hydroxylapatite chromatography. The homogeneity of the isolated protein is greater than 90% by SDS PAGE. The 45,000-mol-wt protein reduces the viscosity of actin filaments in a Ca2+-dependent manner. The free calcium concentration required for the activity of this protein is in the micromolar range. Electron microscopic studies reveal that the formation of short filaments parallels the decrease in viscosity. Energy transfer and sedimentation experiments indicate a net disassembly of actin filaments and an increase in the steady-state nonfilamentous actin concentration in the presence of Ca2+ ions and the 45,000-mol-wt protein. The increase in the steady-state nonfilamentous actin concentration is proportional to the amount of 45,000-mol-wt protein added. The actin molecules disassembled by the addition of the 45,000-mol-wt protein are capable of polymerization.     

Gelsolin as a calcium-regulated actin filament-capping protein.

Various concentrations of gelsolin (25-100 nM) were added to 2 microM polymerized actin. The concentrations of free calcium were adjusted to 0.05-1.5 microM by EGTA/Ca2+ buffer. Following addition of gelsolin actin depolymerization was observed that was caused by dissociation of actin subunits from the pointed ends of treadmilling actin filaments and inhibition by gelsolin of polymerization at barbed ends. The time course of depolymerization revealed an initial lag phase that was followed by slow decrease of the concentration of polymeric actin to reach the final steady state polymer and monomer concentration. The initial lag phase was pronounced at low free calcium and low gelsolin concentrations. On the basis of quantitative analysis the kinetics of depolymerization could be interpreted as capping, i.e. binding of gelsolin to the barbed ends of actin filaments and subsequent inhibition of polymerization, rather than severing. The main argument for this conclusion was that even gelsolin concentrations (100 nM) that exceed the concentration of filament ends ( approximately 2 nM), cause the filaments to depolymerize at a rate that is similar to the rate of depolymerization of the concentration of pointed ends existing before addition of gelsolin. The rate of capping is directly proportional to the free calcium concentration. These experiments demonstrate that at micromolar and submicromolar free calcium concentrations gelsolin acts as a calcium-regulated capping protein but not as an actin filament severing protein, and that the calcium binding sites of gelsolin which regulate the various functions of gelsolin (capping, severing and monomer binding), differ in their calcium affinity.     

Cold shock induces actin reorganization and polyspermy in sea urchin eggs

The effect of low temperature on the cortical actin system and polyspermy in sea urchin eggs was studied. Eggs cold-shocked at 3 degrees C for 1 hour formed a cortical system of polymerized actin and were polyspermic when fertilized. These effects were completely reversible following a 7-20 minute recovery period at room temperature. The present data raises the possibility that actin, or components of the egg cytoskeleton, regulate sperm entry in sea urchin eggs.     

Protein kinase C from sea urchin eggs

1. Protein kinase C is considered to be ubiquitous in tissues and organs; however, its isolation and characterization have been principally with adult mammalian tissues. 2. There is increasing evidence for the importance of this enzyme during early development. 3. In this study, protein kinase C has been identified and partially characterized in cytosolic fraction from sea urchin eggs. 4. The enzyme was resolved from other protein kinase activities by ion exchange chromatography. 5. Phosphatidylserine and Ca2+ were required for protein kinase C to be active. 6. Diacylglycerol and phorbol ester enhanced the activation of the enzyme.     

The 50 kDa protein-actin complex from unfertilized sea-urchin (Strongylocentrotus purpuratus) eggs. Interaction with actin.

In the preceding paper [Golsteyn & Waisman (1989) Biochem. J. 257, 809-815] an EGTA-stable, Ca2+-binding heterodimer comprised of a 50 kDa protein and actin called '50K-A' was identified in the unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. In the present paper we have documented the binding of 50K-A to DNAase I and the effect of 50K-A on the kinetics of actin polymerization. When 50K-A was added to pyrene-labelled rabbit skeletal-muscle actin and the salt concentration increased, the initial rate of actin polymerization was inhibited by a very low molar ratio of 50K-A to actin. Furthermore, the steady-state level of G-actin was increased in the presence of 50K-A, suggesting that 50K-A caps the preferred end of actin polymer, shifting the steady-state concentration to that of the non-preferred end. Dilution of F-actin to below its critical concentration into 50K-A resulted in a much slower rate of depolymerization, consistent with capping of the preferred end. In contrast with the Ca2+-dependent binding to DNAase, the effect of 50K-A on the kinetics of actin assembly and disassembly was Ca2+-independent. These results suggest that 50K-A is a novel actin-binding protein with some similarities to the severin/fragmin/gelsolin family of F-actin-capping proteins.     

Polarized bundles of actin filaments within microvilli of fertilized sea urchin eggs

We report on the internal ultrastructure of long, finger-like microvilli which cover the surface of the fertilized sea urchin egg. Eggs were attached to polylysine-coated surfaces; their upper portions were sheared away with a stream of buffer which left behind only their plasma membranes and adjacent cytoplasmic structures. Scanning electron microscopy (EM) of such fragments revealed intact thin protoplasmic projections radiating away from the body of the cortex. By transmission EM of cortices similarly prepared on grids, small bundles of microfilaments appear as cores within the thin cytoplasmic projections. These microfilaments are shown to be composed of actin by their ability to interact with muscle heavy meromyosin (HMM). HMM-decorated microfilaments possess repeating arrowheads which uniformly point toward the cell interior. Actin bundles in the microvilli of sea urchin eggs may mediate microvillus support and elongation.     

A novel ceramide trihexoside from the eggs of the sea urchin, Hemicentrotus pulcherrimus.

Glucosylceramide (Glc beta 1-1Cer) and a novel ceramide trihexoside (Gal beta 1-6Gal beta 1-6Glc beta 1-1Cer) were purified from the eggs of the sea urchin, Hemicentrotus pulcherrimus. Their chemical structures were determined by gas-liquid chromatography, methylation analysis, chromic acid oxidation, enzymatic hydrolysis, enzyme-linked immunosorbent assay, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The ceramide trihexoside has a novel carbohydrate structure, and its core structure, Gal beta 1-6Glc, is also novel. The ceramide moieties of these glycolipids are almost identical. Two fatty acids, 22:1 and 22h:1, constitute more than 80% of the total acids. Long-chain bases are all phytosphingosine, approximately 90% of which is n-t18:0. The finding of melibiosylceramide (Gal alpha 1-6Glc beta 1-1Cer) from the eggs of another sea urchin species [Kubo, H. et al. (1988) J. Biochem. 104, 755-760] and the present finding of the novel ceramide trihexoside suggest that there are a variety of unique sugar structures in sea urchin glycosphingolipids.     

DNA synthesis in vitro with an endoplasmic-reticulum-DNA-polymerase complex from unfertilized sea urchin eggs

An endoplasmic-reticulum-DNA-polymerase complex was prepared from unfertilized sea urchin eggs and its DNA-synthesizing activity was examined using single-stranded DNA of bacteriophage fd as a template. The complex catalyzed the ribonucleotide-dependent DNA synthesis which required dNTPs, NTPs, Mg2+ and single-stranded DNA. The DNA synthesis was sensitive to aphidicolin and N-ethylmaleimide but was resistant to 2',3'-dideoxyribosylthymine 5'-triphosphate (ddTTP) and alpha-amanitin, suggesting the involvement of DNA polymerase alpha. In parallel with the DNA synthesis, a small amount of RNA was synthesized in the presence of 100 micrograms/ml alpha-amanitin. The Km value of ribonucleotides for the RNA synthesis coincided with that for the DNA synthesis, suggesting a correlation between the DNA and RNA syntheses. Labelling of the products with [gamma-32P]ATP followed by DNA digestion with pancreatic DNase I revealed the attachment of an oligoribonucleotide (7-11 bases in length) at the 5' ends of the DNA products. These observations suggest that in DNA synthesis, primer RNA synthesis occurs first, followed by DNA chain elongation. During 1-90-min incubation, the amount of the DNA synthesized increased but the length was not significantly increased. Over 80% of the number of synthesized DNA molecules comprised a single population of short DNA fragments (60-200 bases, on average 120 bases in length) and the number of fragments increased, depending on the incubation time. However, DNA fragments of various sizes (about 100-6000 bases) were synthesized with DNA polymerase alpha solubilized from the endoplasmic-reticulum-DNA-polymerase complex. All this evidence suggests that in vitro, the complex preferentially synthesizes a particular size of short DNA fragments. The significance of the fragments is discussed.     

Kinetics of actin assembly attending fertilization or artificial activation of sea urchin eggs

Changes in the state of actin assembly triggered by fertilization or by artificial activation of sea urchin eggs were quantified using the DNase I inhibition assay. Insemination of Lytechinus pictus or Strongylocentrotus purpuratus eggs induces a cyclic variation in the level of G-actin as follows: between 0 and 30 s after insemination, the G-actin content decreases. This is followed by an increase in the amount of monomeric actin between 30 and 60 s, and then from 60 s to 5 min postinsemination there is a progressive decrease in the egg's level of G-actin. This latter decrease is more pronounced in S. purpuratus eggs than in L. pictus eggs. Using sperm mimetics that trigger an increase in intracellular calcium concentration (A23187 in sodium-free seawater), a cytoplasmic alkalinization (NH4Cl), a plasma membrane depolarization (seawater enriched with potassium ions), or all three of these phenomena (A23187 in normal seawater), each phase depicted at fertilization correlates with the following metabolic events accompanying egg awakening: phase 1, of uncertain origin (possibly related to plasma membrane depolarization); phase 2, elevation of intracellular calcium concentration; phase 3, alkalinization of the intracellular milieu but only if the transient intracellular calcium rise has taken place.     

Distribution of fluorescently labeled actin in living sea urchin eggs during early development

Rabbit skeletal muscle actin was labeled with 5-iodoacetamidofluorescein (5-IAF) and purified by gel filtration, ion-exchange chromatography, and polymerization-depolymerization. The resultant fluorescent conjugates retained full biochemical activities. The labeled actin was incorporated into unfertilized eggs of Lytechinus pictus by direct microinjection and the distribution of fluorescence was investigated after fertilization through the first division cycle. The results were interpreted by comparing the images with those of control eggs injected with fluorescein isothiocyanate (FITC)-labeled ovalbumin. After fertilization of eggs containing IAF actin, the membrane-cortical regions showed dramatic increases in fluorescence intensity which were not observed in FITC ovalbumin controls. During the first division, spindle regions of both IAF-actin-injected eggs and control eggs became distinctly fluorescent. However, no distinctly fluorescent contractile ring was detected in the cleavage furrow. After cytokinesis, the surface between blastomeres containing IAF actin exhibited an increase in fluorescence intensity. These observations have been compared with those of previous studies using different methods, and the possible implications have been discussed in relation to cellular functions.