Localization and possible function of 20 kDa actin-modulating protein (actolinkin) in the sea urchin egg

We have previously described a novel actin-capping protein, a 20,000-molecular weight protein (20K protein)-actin complex (20K-A) isolated from sea urchin eggs. In the present study, the localization and possible function of this 20K protein were investigated. The 20K protein was localized in the sea urchin egg cortex. Its distribution in the cortex as revealed by immunofluorescence microscopy did not change during or after fertilization up to the first mitosis, but it was concentrated to some extent in the cleavage furrow region. Exogenously added actin polymerized on the cortex isolated from unfertilized egg; however, actin did not polymerize on the cortex extracted with 0.6 M KCl, that is, the cell membrane, which lost the 20K protein. The cell membrane preincubated with 20K-A restored the activity to grow actin filaments. When decorated with myosin subfragment 1, almost all the actin filaments showed the arrowhead configuration pointing away from the membrane, indicating that they were connected to the membrane at their barbed ends. These results strongly suggest that the 20K protein connects actin filaments to the plasma membrane of sea urchin eggs. Because of this property we call this protein "actolinkin".     

A marker of animal-vegetal polarity in the egg of the sea urchin Paracentrotus lividus. The pigment band

We have examined the subequatorial accumulation of pigment granules (the so-called 'pigment band') in the egg of the sea urchin Paracentrotus lividus, which constitutes an unambiguous marker of animal-vegetal polarity. Most of the reddish pigment granules are situated at the periphery of the egg. They exhibit occasional saltatory movements and can aggregate into large patches. Pigment granules are retained as a band in the isolated cortex when the egg surface complex is isolated by shearing eggs attached to polylysine-coated surfaces with calcium-free isotonic solutions. Pigment granules remain as the main vesicular component of fertilized egg cortices or of unfertilized egg cortices perfused with calcium to provoke cortical granule exocytosis. They may be anchored to the isolated cortex through associations with the plasma membrane and with an extensive subsurface network of rough endoplasmic reticulum (rough ER). Pigment granules contain antimonate-precipitable calcium and, in this respect and many others, resemble acidic vesicles recently identified in the cortex of unpigmented sea urchin eggs. We discuss the similarities observed between granules and acidic vesicles in various urchin egg species and their possible functions.     

THE EFFECT OF PENICILLIN ON EGGS OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. Penicillin in the range of concentration from 250 U/ml. to approximately 2650 U/ml. inhibits the rate of cell division of the fertilized sea urchin egg from 0 to 100 per cent. 2. Penicillin in the same range of concentrations has no effect on the oxygen consumption of the unfertilized or the fertilized eggs. 3. Penicillin is bound by some component of the sea urchin egg in amounts sufficiently large to lower the initial concentration, this binding apparently not being related to the inhibitory action.     

Induction of the cortical reaction in isolated sea urchin egg cortices: effects of Ca2+ and ionophore A23187

The cortical reaction in isolated sea urchin (Strongylocentrotus purpuratus) egg cortices has been monitored with phase-contrast video microscopy. It was confirmed that the cortical reaction is induced by exposure to Ca2+. No induction was observed after exposure to the Ca2+-ionophore A23187, although the cortices remain sensitive to a subsequent exposure to Ca2+, and the cortical reaction in unfertilized eggs suspended in cortex isolation medium remains inducible by exposure to A23187. These results imply: (1) that A23187 does not induce the cortical reaction directly; (2) that the release of intracellular Ca2+, through which A23187 induces the cortical reaction, is not from storage sites localized entirely in the cortex; and (3) that intracellular storage sites for the Ca2+ involved in the cortical reaction are also present outside the cortex.     

Modulation of calcium sensitivity by a specific cortical protein during sea urchin egg cortical vesicle exocytosis

A study of the Ca2+ sensitivity of cortical vesicle (CV) discharge has been accomplished using isolated sea urchin egg cortices. Cortices isolated in a medium ionically similar to normal egg cytoplasm discharge 50% of their CVs at 1.6 microM Ca2+ (=[Ca2+]50). Alternatively, cortices isolated in a medium containing 500 mM chaotropic anions (Cl-, Br-, I-, or NO-3) discharge their CVs at 16 microM [Ca2+]50. Incubation with the 500 mM KCl extract of cortices restores high Ca2+ sensitivity and the mode of CV discharge characteristic of cortices before extraction. Fractionation of egg homogenates by differential centrifugation reveals that about 20% of the total restoring activity is associated with the cortex. In eggs of Hemicentrotus pulcherrimus, the factor responsible for this restorative function is a heat and protease labile protein with a molecular weight of 100,000. Similar activity is seen also in the eggs and sperm of other species of sea urchin.     

TAME stabilizes the cortex and mitotic apparatus of the sea urchin egg during isolation

The synthetic substrate p-tosyl-L-arginine methyl ester (TAME) has been included in buffered EGTA media used for the isolation of the mitotic apparatus from clam eggs and also for the isolation of the cortex from sea urchin eggs. In the course of an investigation of the role of actin-fascin and actin-myosin interactions in cytokinesis, the isolation of the sea urchin egg cortex was re-examined and the stability of the cortex to lysis in a buffered EGTA medium near neutrality found to depend directly on the presence of TAME. Lysis of eggs at metaphase in this medium yielded a mixture of cortices and mitotic apparatuses (MA); MA stability under these conditions also required the presence of TAME, although a reduced pH allowed MA isolation in its absence. The action of TAME in stabilizing the actin-based structure of the cortex and the microtubule-based structure of the MA is not duplicated by other proteolysis inhibitors and this compound will also induce actin polymerization and gelation in extracts of the soluble cytoplasmic proteins of the egg under conditions where these are normally inhibited.     

A protein factor extracted from murine brains confers physiological Ca2+ sensitivity to exocytosis in sea urchin eggs.

Exocytosis in sea urchin eggs can be reconstituted in vitro using the cell ghosts (the isolated cortices). When the isolated cortices were handled in the medium primarily composed of non-chaotropic ions, exocytosis can be induced by a micromolar level of Ca2+. However, when the cortices are exposed to chaotropic anions such as Cl-, it is induced only at higher Ca2+ concentrations of 10(-5) to 10(-4) M, due to the chaotropic anionic effect, by which a specific protein(s) is dissociated from the cortex. The dissociated protein can be added back to the cortex to restore the original Ca2+ sensitivity [(1984) Dev. Biol. 101, 125-135]. A protein which has the similar effect on the isolated cortex was also found in the extract of murine brain. This protein was neither calmodulin, a G-protein or a kinase. The data suggest the general regulatory mechanism of the Ca2+ sensitivity of exocytosis by a protein factor widely distributed among cells.     

EFFECTS OF THE SURFACTANTS ON THE CLEAVAGE AND FURTHER DEVELOPMENT OF THE SEA URCHIN EMBRYOS II. DISTURBANCE IN THE ARRANGEMENT OF CORTICAL VESICLES AND CHANGE IN CORTICAL APPEARANCE

After fertilization, two types of cortical vesicles ware examined under the electron microscope (the cortical vesicle I and II) and the light microscope (pigment granules and another kind of vesicles). The cortical vesicle I corresponds to the pigment granule and the cortical vesicle II does to the other vesicle.
The unequal division of the sea urchin embryo which occurs at the fourth cleavage was modified to an equal cleavage pattern by the treatment with sodium lauryl sulfate (SLS) or cetyl trimethyl ammonium bromide (CTAB). But other surfactants such as sodium deoxycholate, Tween 80, Lubrol PX did not have such an effect. The cell surface of the embryo which had been treated either SLS or CTAB became rough or smooth. Cortical vesicles and pigment granules disappeared and/or were dislocated from the cortex. However, cell organelles were as normal as the control. On the other hand, the cortical appearance of other surfactant-treated embryos showed no disturbance and cell organelles were also more or less normal. Therefore, the equalization of unequal cleavage is caused by the disturbance in the cortex and thus the cortex plays a major role on the micromere formation at the 16-cell stage and on the further sea urchin development.     

ANTIGENS IN EGGS AND DEVELOPMENTAL STAGES OF THE SEA URCHIN : II. Localization

Homogenates of fertilized eggs of the sea urchin Paracentrotus lividus were fractionated by differential centrifugation. In addition, whole eggs were fragmented, on a preparative scale, by centrifugation in sea water-sucrose gradients. The fractions and fragments were subsequently assayed for their content of soluble protein antigens described in an earlier publication. Relative concentrations of antigen present in quantitatively isolated cell fractions were estimated by graded antiserum absorption in combination with agar-diffusion technique. Two of six antigens were found to be associated mainly with the low speed sediments. Treatment of the various sediments with hypotonic medium and results obtained with fragmented eggs suggested that these two antigens and possibly a third were probably located in the yolk granules. The other antigens were more evenly distributed among the low speed sediments and the non-sedimented part of the cytoplasm. Only one of the antigens was consistently associated with the microsomal fraction.     

Sea urchin egg hyaline layer: evidence for the localization of hyalin on the unfertilized egg surface

The sea urchin embryo hyaline layer is an extracellular investment which develops within 20 min postinsemination of Strongylocentrotus purpuratus eggs and contains a single calcium-precipitable subunit termed hyalin. Other ultrastructural and biochemical studies have suggested that hyalin is localized in the cortical granules. We have examined the hypothesis that hyalin is a cell surface protein of the unfertilized egg using vectorial lactoperoxidase-catalyzed radioiodination. Extracts of labeled unfertilized eggs contained several labeled proteins, one of which was electrophoretically indistinguishable from authentic hyalin isolated by each of three different procedures. Pronase digestion of labeled unfertilized eggs removed 75% of the label, but the labeled hyalin-like molecule was still present in whole cell extracts. Upon insemination, pronase-digested, labeled eggs formed an apparently normal hyaline layer and whole cell extracts contained the labeled hyalin-like molecule. Denuded, labeled eggs were inseminated and the hyaline layer was selectively solubilized in calcium- and magnesium-free artificial seawater. Labeled hyalin was purified from this crude hyalin preparation to constant specific radioactivity and apparent homogeneity as shown by gel electrophoresis. These data strongly suggest that hyalin or a precursor is a cell surface protein of the unfertilized sea urchin egg.     

Synaptotagmin I is involved in the regulation of cortical granule exocytosis in the sea urchin

Cortical granules are stimulus-dependent secretory vesicles found in the egg cortex of most vertebrates and many invertebrates. Upon fertilization, an increase in intracellular calcium levels triggers cortical granules to exocytose enzymes and structural proteins that permanently modify the extracellular surface of the egg to prevent polyspermy. Synaptotagmin is postulated to be a calcium sensor important for stimulus-dependent secretion and to test this hypothesis for cortical granule exocytosis, we identified the ortholog in two sea urchin species that is present selectively on cortical granules. Characterization by RT-PCR, in-situ RNA hybridization, Western blot and immunolocalization shows that synaptotagmin I is expressed in a manner consistent with it having a role during cortical granule secretion. We specifically tested synaptotagmin function during cortical granule exocytosis using a microinjected antibody raised against the entire cytoplasmic domain of sea urchin synaptotagmin I. The results show that synaptotagmin I is essential for normal cortical granule dynamics at fertilization in the sea urchin egg. Identification of this same protein in other developmental stages also shown here will be important for interpreting stimulus-dependent secretory events for signaling throughout embryogenesis.     

The effect of nicotine on sperm-Egg interaction in the sea urchin: Polyspermy and electrical events

We have studied some of the effects of nicotine on sea urchin eggs, spermatozoa, and their interaction using electrical recording techniques and fertilization-rate experiments. Pretreating eggs with nicotine enhances the fertilization rate, whereas this drug has an inhibitory effect on spermatozoa. Pulse-treated eggs or eggs fertilized in the presence of nicotine give rise to attenuated step depolarizations, which may be attributed to a decrease in membrane resistance (Rm) of the egg or, in the latter case, to an alteration to the spermatozoon. Concurrently, with the change in the step depolarization there is a reduction in amplitude of the fertilization potential (FP) suggesting that the cortical reaction is in some way altered. Nicotine has no effect on the Rm of fertilized eggs or oocytes, where there are no cortical granules. We suggest that nicotine alters the cortex of sea urchin eggs–possibly by causing a partial dissolution of cortical granules–which renders the eggs more receptive to spermatozoa. The reductions in amplitude of the step depolarization and the FP are consequences of this alteration.     

THE ISOLATION OF A MAJOR STRUCTURAL ELEMENT OF THE SEA URCHIN FERTILIZATION MEMBRANE

Procedures for isolating the contents of the cortical granules from the ova of the sea urchin, Strongylocentrotus purpuratus, are reported. Dithiothreitol is used to remove the vitelline coat; the "demembranated" eggs are then subsequently activated with butyric acid. By means of these procedures, the hyaline protein and crystalline or paracrystalline material have been isolated from the cortical granules. The crystalline material consists of sheets of cylinders or tubules 150–200 A in diameter. This material is believed to be a major structural element of the fertilization membrane which, in the absence of the vitelline coat, does not form.     

SELECTIVE EXTRACTION OF ISOLATED MITOTIC APPARATUS : Evidence That Typical Microtubule Protein Is Extracted by Organic Mercurial

Mitotic apparatus isolated from sea urchin eggs has been treated with meralluride sodium under conditions otherwise resembling those of its isolation. The treatment causes a selective morphological disappearance of microtubules while extracting a major protein fraction, probably consisting of two closely related proteins, which constitutes about 10% of mitotic apparatus protein. Extraction of other cell particulates under similar conditions yields much less of this protein. The extracted protein closely resembles outer doublet microtubule protein from sea urchin sperm tail in properties considered typical of microtubule proteins: precipitation by calcium ion and vinblastine, electrophoretic mobility in both acid and basic polyacrylamide gels, sedimentation coefficient, molecular weight, and, according to a preliminary determination, amino acid composition. An antiserum against a preparation of sperm tail outer doublet microtubules cross-reacts with the extract from mitotic apparatus. On the basis of these findings it appears that microtubule protein is selectively extracted from isolated mitotic apparatus by treatment with meralluride, and is a typical microtubule protein.     

Direct isolation of the hyaline layer protein released from the cortical granules of the sea urchin egg at fertilization

Treatment of the eggs of the sea urchin with a 1 M solution of glycerol at fertilization allows the recovery from this solution of the protein released from the cortical granules, including that which would normally give rise to the hyaline layer. The calcium-gelable protein previously extracted from whole eggs and from isolated cortical material was found to be present in the glycerol solution, confirming its localization in the cortical granules and its role in the hyaline layer. Quantitative measurements on the eggs of two Hawaiian species, Colobocentrotus atratus and Pseudoboletia indiana, which have the widest variation in the gel protein content, demonstrated that a proportionate amount of this material was released at fertilization in these species, which correlates with the thickness of the hyaline layer in the two cases. In addition, the calcium-insoluble fraction of Sakai can be extracted from these eggs after removal of the hyaline protein by glycerol, showing that this is a different material. A simple method for the separation of the hyaline protein from the calcium-insoluble fraction in solution is provided.     

Cortical localization of a calcium release channel in sea urchin eggs

We have used an antibody against the ryanodine receptor/calcium release channel of skeletal muscle sarcoplasmic reticulum to localize a calcium release channel in sea urchin eggs. The calcium release channel is present in less than 20% of immature oocytes, where it does not demonstrate a specific cytoplasmic localization, while it is confined to the cortex of all mature eggs examined. This is in contrast to the cortical and subcortical localization of calsequestrin in mature and immature eggs. Immunolocalization of the calcium release channel reveals a cortical reticulum or honeycomb staining network that surrounds cortical granules and is associated with the plasma membrane. The network consists of some immunoreactive electron-dense material coating small vesicles and elongate cisternae of the endoplasmic reticulum. The fluorescent reticular staining pattern is lost when egg cortices are treated with agents known to affect sarcoplasmic reticulum calcium release and induce cortical granule exocytosis (ryanodine, calcium, A-23187, and caffeine). An approximately 380-kD protein of sea urchin egg cortices is identified by immunoblot analysis with the ryanodine receptor antibody. These results demonstrate: (a) the presence of a ryanodine-sensitive calcium release channel that is located within the sea urchin egg cortex; (b) an altered calcium release channel staining pattern as a result of treatments that initiate the cortical granule reaction; and (c) a spatial and functional dichotomy of the ER which may be important in serving different roles in the mobilization of calcium at fertilization.     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.     

Wave of cortical actin polymerization in the sea urchin egg

The distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm-attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discussed.     

AN ACTIN-LIKE PROTEIN OF THE SEA URCHIN EGGS II. DIRECT ISOLATION PROCEDURE

A direct extraction procedure for isolation of an actin-like protein from fresh sea urchin eggs was tried. The actin-like protein was found in the 10,000g supernatant of the egg homogenate. The crude extract obtained from the egg homogenate was purified by ammonium sulfate fractionation and dialysis against an ATP-cysteine solution. The purified actin-like protein occurred as 2.8S globular protein units, which transformed into heavy components of 7–8S and 12–13S on addition of salts. This change was accompanied by a rise in viscosity.     

Alpha-actinin from sea urchin eggs: biochemical properties, interaction with actin, and distribution in the cell during fertilization and cleavage

A protein similar to alpha-actinin has been isolated from unfertilized sea urchin eggs. This protein co-precipitated with actin from an egg extract as actin bundles. Its apparent molecular weight was estimated to be approximately 95,000 on an SDS gel: it co-migrated with skeletal-muscle alpha-actinin. This protein also co-eluted with skeletal muscle alpha-actinin from a gel filtration column giving a Stokes radius of 7.7 nm, and its amino acid composition was very similar to that of alpha-actinins. It reacted weakly but significantly with antibodies against chicken skeletal muscle alpha-actinin. We designated this protein as sea urchin egg alpha-actinin. The appearance of sea urchin egg alpha-actinin as revealed by electron microscopy using the low-angle rotary shadowing technique was also similar to that of skeletal muscle alpha-actinin. This protein was able to cross-link actin filaments side by side to form large bundles. The action of sea urchin egg alpha-actinin on the actin filaments was studied by viscometry at a low-shear rate. It gelled the F-actin solution at a molar ratio to actin of more than 1:20, at pH 6-7.5, and at Ca ion concentration less than 1 microM. The effect was abolished by the presence of tropomyosin. Distribution of this protein in the egg during fertilization and cleavage was investigated by means of microinjection of the rhodamine-labeled protein in the living eggs. This protein showed a uniform distribution in the cytoplasm in the unfertilized eggs. Upon fertilization, however, it was concentrated in the cell cortex, including the fertilization cone. At cleavage, it seemed to be concentrated in the cleavage furrow region.