Reassociation of cortical secretory vesicles with sea urchin egg plasma membrane: Assessment of binding specificity

Summary An assay has been developed for quantitating the reassociation of cortical secretory vesicles (CVs) with fragments of sea urchin egg plasma membrane attached to glass slides (PM lawns). Binding ofS. pupuratus CVs to homologous PM lawns increased with time and CV concentration. The observation that CV binding was blocked by chymotrypsin digestion of the PM fragments suggested that a PM protein(s) is required for reassociation. The possibility that the extent of CV lysis that occurred during CV preparation (15.4±3.8% as assessed by ovoperoxidase assay) influenced reassociation was investigated by determining the effect of CV content proteins (isolated as fertilization product) on binding. Various concentrations of fertilization product (up to equivalent amounts of fertilization product and CV protein) had no effect on CV binding. The specificity of binding was investigated by assessing the ability of CVs to bind to PM lawns prepared from human red blood cells, and by determining the ability of heterologous vesicles to bind to egg PM fragments. PM lawns from HRBCs did not support CV binding; however, PM lawns prepared from the eggs of several species of sea urchin did bindS. pupuratus CVs. Vesicles from a partially purified preparation of yolk platelets bound to egg PM lawns with low efficiency (1/7 that of CVs), but immunofluorescence analysis with an anti-hyalin monoclonal antibody demonstrated that 74±9% of the bound vesicles were CVs that contaminated the yolk platelet preparation. Dioleoylphosphatidyl choline liposomes were also unable to bind to egg PM lawns. These results are consistent with hypothesis that CV binding to egg PM lawns is a specific, protein-mediated event.     

Calcium-induced fusion of sea urchin egg secretory vesicles with planar phospholipid bilayer membranes.

The fusion of sea urchin egg secretory vesicles to planar phospholipid bilayer membranes was studied by differential interference contrast (DIC) and fluorescent microscopy, in combination with electrical recordings of membrane conductance. A strong binding of vesicles to protein-free planar membranes was observed in the absence of calcium. Calcium-induced fusion of vesicles was detected using two independent assays: loss of the contents of individual vesicles visible by DIC microscopy: and vesicle content discharge across the planar membrane detected by an increase in the fluorescence of a dye. In both cases, no increase in the membrane conductance was observed unless vesicles were incubated with either Amphotericin B or digitonin prior to applying them to the planar membrane, an indication that native vesicles are devoid of open channels. Pre-incubation of vesicles with n-ethylmaleimide (NEM) abolished calcium-induced fusion. Fusion was also detected when vesicles were osmotically swollen to the point of lysis. In contrast, no fusion of vesicles to planar bilayers was seen when vesicles on plasma membrane (native cortices) were applied to a phospholipid membrane, despite good binding of vesicles to the planar membrane and fusion of vesicles to plasma membrane. It is suggested that cortical vesicles (CVs) have sufficient calcium-sensitive proteins for fusion to lipid membranes, but in native cortices granular fusion sites are oriented toward the plasma membrane. Removal of vesicles from the plasma membrane may allow fusion sites on vesicles access to new membranes.     

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.     

Characterization of sea urchin egg endoplasmic reticulum in cortical preparations

The cortical endoplasmic reticulum (ER) of sea urchin eggs was localized on isolated egg cortices by staining with aqueous suspensions of the dicarbocyanine "DiI." Immunofluorescence localization of a calsequestrin-like protein was essentially identical; this is consistent with a role for the ER in calcium regulation. The ER often encircles cortical granules, making it well-suited for initiating fusion and propagating the calcium wave. Thiazole orange and Hoechst dye 33258 at pH 2 stain ribosomes bound to the ER, providing evidence that the cortical ER is rough ER. High chloride concentrations were found to disrupt ER continuity.     

Evidence that the Mg-ATPase in the cortical layer of sea urchin egg is dynein

Indirect immunofluorescence staining of cleaving sea urchin eggs with an antiserum against a tryptic fragment of dynein 1 (fragment 1A) from sea urchin sperm flagella suggested the presence of dynein in the cortex as well as in the mitotic apparatus. In the present study, we found that the Mg²⁺-ATPase activity of the isolated cortices from sea urchin eggs, which exhibited similar characteristics to those of flagellar dynein, was inhibited by 60–80% with the anti-fragment 1A serum. Faintly stained bands corresponding to the A-band (dynein 1) and the B-band of the sperm flagella was detected on sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis of the isolated cortices. Furthermore, the SDS-gel electrophoresis revealed the presence of a polypeptide band corresponding to dynein 1 in the antigen-antibody complex precipitated from the KCl-extract of the cortices with the antiserum.     

Demonstration of calcium uptake and release by sea urchin egg cortical endoplasmic reticulum

The calcium indicator dye fluo-3/AM was loaded into the ER of isolated cortices of unfertilized eggs of the sea urchin Arbacia punctulata. Development of the fluorescent signal took from 8 to 40 min and usually required 1 mM ATP. The signal decreased to a minimum level within 30 s after perfusion with 1 microM InsP3 and increased within 5 min when InsP3 was replaced with 1 mM ATP. Also, the fluorescence signal was lowered rapidly by perfusion with 10 microM A23187 or 10 microM ionomycin. These findings demonstrate that the cortical ER is a site of ATP-dependent calcium sequestration and InsP3-induced calcium release. A light-induced wave of calcium release, traveling between 0.7 and 2.8 microns/s (average speed 1.4 microns/s, N = 8), was sometimes observed during time lapse recordings; it may therefore be possible to use the isolated cortex preparation to investigate the postfertilization calcium wave.     

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.     

Degradation of sperm histones in vitro by cytoplasm of the sea urchin egg

Sperm-specific histone variants in the sea urchin Paracentrotus lividus are replaced early after fertilization with a specific embryonic set of histone variants. A possible in vitro model for the involvement of a degradation mechanism in the replacement of sperm-specific histones is presented. Soluble sperm histones are shown to be degraded quickly by egg cytoplasm. The proteolytic activity is maximal at pH 3.0; H1 and H2A histones are the most sensitive while H3 and H4 are the most resistant. H2B histones have an intermediate sensitivity. Histone degradation by egg cytoplasm or by purified fractions of it can be inhibited by chymostatin and leupeptin and, to a lesser degree, by pepstatin.     

Sea urchin egg cortical granule exocytosis is followed by a burst of membrane retrieval via uptake into coated vesicles

Using improved fixation procedures we have found that extensive endocytotic activity is turned on at fertilization in eggs of three species of sea urchins. Beginning after completion of cortical granule exocytosis and after exocytotic pits have completely smoothed over, the entire activated egg surface engages in a limited period of extensive removal of membrane via uptake into coated vesicles. This “burst phase” lasts about 3–5 min after which the number of invaginating coated vesicles decreases rapidly. At the end of this burst phase all the patches of cortical granule membranes have disappeared, and the egg surface is left uniformly covered by microvilli. For the remainder of the first cell cycle coated pits continue to form at a slower but steady rate. Endocytotic activity continues past the time of first cleavage. There is distinct overlap in onset and duration of the burst phase of endocytosis with the period of medium acidification during normal development. However, activation of eggs in choline sea water, which inhibits acid secretion, results in an endocytic burst whose timing and duration are similar to those in normal eggs. The endocytic burst is, therefore, independent of cytoplasmic alkalinization. These results suggest, in accord with the two-step model of egg activation (D. Epel, R. A. Steinhardt, and R. A. Humphreys, 1974; Dev. Biol.40, 245–255; D. Epel, 1978, Curr. Top. Dev. Biol.12, 185–246) that initiation of endocytosis is most likely a Ca²⁺-dependent event.     

Gel electrophoretic isolation, in the hundred microgram range, of recombinant SDS-syntaxin from sea urchin egg cortical vesicles.

Recombinant urchin syntaxin [Xa cut], electrophoresed at pH 9.0 (25 degrees C) or 10.2 (0 degrees C) in a discontinuous Tris-chloride-glycinate buffer system in the presence of 0.03% SDS in the catholyte, exhibits a multicomponent pattern in gels of a polyacrylamide concentration of 12% and 3% crosslinking. The position in the pattern of the syntaxin band was identified by reference to electropherograms of a previous study (P. Backlund, pers. comm.). The complexity of the protein composition of the preparation was reduced by selective stacking of proteins with mobilities greater than that of syntaxin. This provides a gel pattern consisting of two bands with mobilities close to that identified as syntaxin, as well as a minor, more slowly migrating, contaminant. The two major components are designated as S1 and S2, the latter being the larger species. In the absence of SDS, the preparation exhibits two pairs of protein components. Three of the proteins are charge isomers, i.e., of equal size, differing only in net charge, assumed to be forms of S1, while the fourth component is larger and is assumed to be S2. Aliquots of the preparation, containing 150 microg of protein were loaded on a cylindrical polyacrylamide gel of 18 mm diameter, and separated S1 and S2 were excised in a position defined by their characteristic values of relative mobility (Rf). Two or three gel slices, corresponding in Rf to S1 or S2, were pooled and loaded onto a Stacking Gel (5% polyacrylamide, 20% cross-linked) of 18 mm diameter, equipped with a collection chamber of 200 microL volume. The protein was electroeluted from the gel slices and concentrated into a stack by electrophoresis. The stack, marked by bromphenolblue, was allowed to migrate into the collection chamber, was collected and analyzed by protein assay and re-electrophoresis. Re-electrophoresis of S1 shows that it consists of at least three components. Recovered S1 constitutes 47% of the preparation, based on protein assay, S2 4%. S1, isolated from SDS-PAGE, exhibits an apparent Mr of 22.7 kDa, S2 one of 34.5 kDa, similar to the value of 32.6 kDa expected from the structure of syntaxin. The absence of S2 from the electroeluate re-electrophoresed at 0 degrees C and their molecular weight relationship suggest a proteolytic transformation of S2 to S1.     

Immunocytochemical evidence suggesting heterogeneity in the population of sea urchin egg cortical granules

Unfertilized eggs of many species of animals contain cortical granules, which are specialized secretory granules that upon fertilization release their contents from the egg. The unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, contain cortical granules that all display an identical and elaborate internal morphology. It has been assumed that they all contain identical components. In this report we present immunocytochemical data which indicate that the cortical granule population of S. purpuratus eggs is heterogeneous. Two monoclonal antibodies are shown to react to the spiral lamellae region of approximately 20% of the cortical granules, implying that the contents of the reactive granules differ from the contents of the majority of the population. An egg protein of greater than 320 kDa is recognized by the antibody. These antibodies also stain a 130-kDa protein expressed on the surface of primary mesenchyme cells in later development. Both antibodies recognize a post-translational modification of this protein. This suggests that an antigenically similar epitope is present both on the 130-kDa primary mesenchyme cell-specific protein and in the cortical granules. To determine if the primary mesenchyme and cortical granule proteins are related, a fusion protein antibody specific for a region of the 130-kDa protein was used to stain unfertilized eggs. This antibody did not stain cortical granules. Thus, 20% of the cortical granules contain a molecule that has an epitope antigenically similar to the post-translational modification recognized in primary mesenchyme cells by the monoclonal antibodies.     

The appearance of a type of cortical vesicles subsequent to fertilization of the sea urchin egg,their character and possible function

Summary Three different types of vesicles present before fertilization in the sea urchin egg were examined. The a-type corresponds to rough endoplasmic vesicles; the vesicles of b-type are rather smooth but may have vestigial granules within their membranes; the c-type belongs to the multivesicular bodies. Upon fertilization, vesicles appear in the outer cortical zone (vesicles of d-type). The majority of them arises by budding from the vesicles ofb-type. The budding occurs mainly at the basis of or within ridges of the cell surface; they may also be present in broader microvilli. The distance between the ridges was varied by early transfer of the eggs to calcium-free sea water. An inducing effect of the ridges on location of theb-vesicles and on the formation ofd-vesicles by budding could thus be demonstrated.Thed-vesicles appearing upon fertilization resemble in size and structure Golgi vesicles formed by budding from Golgi bodies present in the interior cortex or below it. Also theb-vesicles have their counterpart in the Golgi bodies. Theb andd-vesicles are therefore regarded as a Golgi system in which theb-vesicles represent dispersed Golgi bodies and thed- vesicles Golgi vesicles. Thed-vesicles may be designated as cortical Golgi vesicles, (c.G.v.).In thec-vesicles i.e. the multivesicular bodies (m.v.b.), a nucleid was observed which may be subdivided. A compartmentation of m.v.b. was observed which may lead to a detachment of vesicles of about the same size as thed-vesicles (c.G.v.) but probably of a different character.The differentiation of the fertilization membrane of the sea urchin egg occurs in two stages: the assembly and the solidification stage. The tentative conclusion is drawn that a secretion from the c.G.v. functions as an agent in the solidification process. The c.G.v. may also act upon the hyaline layer, both in its early formation and its maintenance during the course of embryonic development.The material for the present investigation was collected at Kristineberg Zoological Station and at Stazione Zoologica, Naples. The writer is indebted to the authorities of these stations for generous helop. Financial support was received from the Swedish Natural Sciences Research Council and the Swedish Cancer Society for which I express my gratitude.I also express my gratitude to Dr.Björn Afzelius for surveying the electron microscopic part of the work, to Dr.Jane Baxandall for permission to study her original electron micrographs and to Dr.Elsa Wicklund and Dr. L.Contoli for contribution to the experiments on the effect of calcium-free sea water. I thank Mrs.Astri Runnwström and Mrs.AnneMarie Ede for their able assistance.This research was carried out in association with the Research group of embryology for the study of cellular differentiation of the Department of Zoology, University of Rome (Director Professor P.Pasquini).     

In vitro reactivation of anaphase B in isolated spindles of the sea urchin egg

Spindles may be isolated from sea urchin eggs so that some mitotic processes can be reactivated in vitro. The isolation media allow spindles to remain stable for days. Transfer of the spindles to reactivation media results in loss of birefringence and breakdown of the matrix within which the microtubules function. If, however, tubulin and either guanosine triphosphate or adenosine triphosphate are present in these media so that tubulin can cycle, the spindles do not break down but grow in size and birefringence and show some of the movements of in vivo spindles. The most prominent is that of anaphase B if the mitotic apparatuses (MAs) have been isolated at a time when anaphase was initiated. When isolated during metaphase, MAs either do not show chromosome movement or, if they do, it is a random movement which causes redistribution of the chromosomes on the spindle surface. In either case, such metaphase spindles grow in size and birefringence. Thus under the proper conditions, cycling microtubules can interact with the spindle matrix to induce chromosome movements which resemble those seen in in vivo cells in the case of anaphase B and show some aspects of anaphase A in at least half the spindles isolated at metaphase, although such movements are not coordinated to show a true anaphase movement.     

The sea urchin egg jelly coat is a three-dimensional fibrous network as seen by intermediate voltage electron microscopy and deep etching analysis

The egg jelly (EJ) coat which surrounds the unfertilized sea urchin egg undergoes extensive swelling upon contact with sea water, forming a threedimensional network of interconnected fibers extending nearly 50 μm from the egg surface. Owing to its solubility, this coat has been difficult to visualize by light and electron microscopy. However, Lytechinus pictus EJ coats remain intact, if the fixation medium is maintained at pH 9. The addition of alcian blue during the final dehydration step of sample preparation stains the EJ for visualization of resin embedded eggs by both light and electron microscopy. Stereo pairs taken of thick sections prepared for intermediate voltage electron microscopy (IVEM) produce a threedimensional image of the EJ network, consisting of interconnected fibers decorated along their length by globular jelly components. Using scanning electron microscopy (SEM), we have shown that before swelling, EJ exists in a tightly bound network of jelly fibers, 50–60 nm in diameter. In contrast, swollen EJ consists of a greatly extended network whose fibrous components measure 10 to 30 nm in diameter. High resolution stereo images of hydrated jelly produced by the quick-freeze/deep-etch/rotary-shadowing technique (QF/DE/RS) show nearly identical EJ networks, suggesting that dehydration does not markedly alter the structure of this extracellular matrix. © 1993 Wiley-Liss, Inc.     

The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratas sea urchin egg and is secreted during fertilization

The sea urchin egg has thousands of secretory vesicles known as cortical granules. Upon fertilization, these vesicles undergo a Ca2+-dependent exocytosis. G-protein-linked mechanisms may take place during the egg activation. In somatic cells from mammals, GTP-binding proteins of the Rho family regulate a number of cellular processes, including organization of the actin cytoskeleton. We report here that a crude membrane fraction from homogenates of Strongylocentrotus purpuratus sea urchin eggs, incubated with C3 (which ADP-ribosylates specifically Rho proteins) and [32P]NAD, displayed an [32P]ADP-ribosylated protein of 25 kDa that had the following characteristics: i) identical electrophoretic mobility in SDS-PAGE gels as the [32P]ADP-ribosylated Rho from sea urchin sperm; ii) identical mobility in isoelectro focusing gels as human RhoA; iii) positive cross-reactivity by immunoblotting with an antibody against mammalian RhoA. Thus, unfertilized S. purpuratus eggs contain a mammalian RhoA-like protein. Immunocytochemical analyses indicated that RhoA was localized preferentially to the cortical granules; this was confirmed by experiments of [32P]ADP-ribosylation with C3 in isolated cortical granules. Rho was secreted and retained in the fertilization membrane after insemination or activation with A23187. It was observed that the Rho protein present in the sea urchin sperm acrosome was also secreted during the exocytotic acrosome reaction. Thus, Rho could participate in those processes related to the cortical granules, i.e., in the Ca2+-regulated exocytosis or actin reorganization that accompany the egg activation.     

A spiral array of microtubules in the fertilized sea urchin egg cortex examined by indirect immunofluorescence and electron microscopy

A transient spiral system of fibers in the cortex of fertilized eggs of the sea urchin Strongylocentrotus purpuratus was examined with indirect immunofluorescence microscopy and found to contain tubulin. Electron microscopy identified the tubulin-containing bands as bundles of up to 40 or more microtubules. These cortical microtubules, which are initially radial, form a spiral array about the time of pronuclear fusion. This basket-like structure, at a depth of 10–15 μm below the cell surface, reaches a peak of development about 45 min after fertilization and disappears before the streak stage at 70 min, in a division cycle of slightly more than 2 h. Possible functions of the cortical microtubules, which appear to be independent of the interphase asters, are discussed.     

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.