Surface activity at the egg plasma membrane during sperm incorporation and its cytochalasin B sensitivity: Scanning electron microscopy and time-lapse video microscopy during fertilization of the sea urchin Lytechinus variegatus

Sperm incorporation and the formation of the fertilization cone with its associated microvilli were investigated by scanning electron microscopy of eggs denuded of their vitelline layers with dithiothreitol or stripped of their elevating fertilization coats by physical methods. The activity of the elongating microvilli which appear to engulf the entering spermatozoon was recorded in living untreated eggs with time-lapse video microscopy. Following the acrosome reaction, the elongated acrosomal process connects the sperm head to the egg surface. About 15 microvilli adjacent to the attached sperm elongate at a rate of 2.6 μm/min and appear to engulf the sperm head, midpiece, and sperm tail. These elongate microvilli swell to form the fertilization cone (average height, 6.7 ± 2.0 μm) and are resorbed as the sperm tail enters the egg cytoplasm 10 min after insemination. Cytochalasin B, an inhibitor of microfilament motility, completely inhibits the observed egg plasma membrane surface activity in both control and denuded eggs. These results argue for a role of the microfilaments found in the egg cortex and microvilli as necessary for the engulfment of the sperm during incorporation and indicate that cytochalasin interferes with the fertilization process at this site.     

The sperm entry site during fertilization of the zebrafish egg: localization of actin.

The sperm entry site (SES) of zebrafish (Brachydanio rerio) eggs was studied before and during fertilization by fluorescence, scanning, and transmission electron microscopy. Rhodamine phalloidin (RhPh), used to detect polymerized filamentous actin, was localized to microvilli of the SES and to cytoplasm subjacent to the plasma membrane in the unfertilized egg. The distribution of RhPh staining at the SES correlated with the ultrastructural localization of a submembranous electrondense layer of cortical cytoplasm approximately 500 nm thick and containing 5- to 6-nm filaments. Actin, therefore, was organized at the SES as a tightly knit meshwork of filaments prior to fertilization. Contact between the fertilizing sperm and the filamentous actin network was observed by 15-20 sec postinsemination or just before the onset of fertilization cone formation. Growing fertilization cones of either artificially activated or inseminated eggs exhibited intense RhPh staining and substantial increase in thickness of the actin meshwork. Collectively, TEM and RhPh fluorescence images of inseminated eggs demonstrated that the submembranous actin became rearranged in fertilization cones to form a thickened meshwork around the sperm nucleus during incorporation. The results reported here suggest that activation of the egg triggers a dramatic polymerization of actin beneath the plasma membrane of the fertilization cone. Furthermore, the actin involved in sperm incorporation is sensitive to the action of cytochalasins.     

Changes in actin organization in the living egg apparatus of Torenia fournieri during fertilization

Changes in actin organization in the living egg apparatus of Torenia fournieri from anthesis to post-fertilization have been investigated using microinjection and confocal microscopy. Our results revealed that the actin cytoskeleton displays dramatic changes in the egg apparatus and appears to coordinate the events of synergid degeneration, pollen tube arrival and gametic fusion during fertilization. Synergid degeneration occurs after anthesis and is accompanied by actin fragmentation and degradation. The actin cytoskeleton becomes organized with numerous aggregates in the chalazal end of the degenerating synergid, and some of the actin infiltrates into the intercellular gap between synergids, egg and central cell, forming a distinct actin band. An actin cap is present near the filiform apparatus after anthesis and disappears after pollen tube arrival. In the egg cell, actin filaments initially organize into a network and after pollination become fragmented into numerous patches in the cortex. These structures, along with the actin in the degenerating synergid and intercellular spaces form two distinct actin coronas during fertilization. The actin coronas vanish after gametic fusion. This is the first report of changes in actin organization in the living egg apparatus. The reorganization of the actin cytoskeleton in the egg apparatus and the presence of the actin coronas during fertilization suggest these events may be a necessary prelude to reception of the pollen tube and fusion of the male and female gametes. Received: 11 November 1999 / Accepted: 31 January 2000     

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.     

The binding of cytochalasin D to monomeric actin

The binding of cytochalasin D to monomeric actin has been measured directly. In the presence of 200 microM Ca2+, actin binds cytochalasin D in a 1:1 molar ratio with a KD of 18 microM. After incubation with 250 microM Mg2+ for 10 minutes, actin binds cytochalasin D with a KD of 2.6 microM but with one mole of cytochalasin D per 2 moles of actin. This suggests that cytochalasin D induces dimerization of Mg2+-induced actin monomers.     

The effect of cytochalasin B and D on the fertilization of sea urchins

Two electrical events occur across the plasma membrane of the sea urchin egg during the interaction with the fertilizing spermatozoon; the first, a step-like depolarization pecedes the fertilization potential (FP) by 11 sec at 20°C. Cytochalasins B and D alter these events in several ways depending upon dose and time of exposure of the gametes. When both the egg and the spermatozoon are exposed to these microfilament-active drugs, the former for minutes whilst the latter in the order of seconds, the delay between the two electrical events is increased twofold. We suggest that a microfilament-involved process is occurring during this delay period which, although itself not altering the electrical properties of the plasma membrane, determines the time of initiation of the FP. Pretreating spermatozoa with higher doses of either CB or CD, inhibits fertilization completely or lowers the successful collision rate (α); we suggest that the two drugs lower α by affecting the acrosomal region of the spermatozoon. Although both drugs increase the delay period to the same degree CD has fewer secondary effects than CB.     

The action of cytochalasin B during egg cleavage in Xenopus laevis: dependence on cell membrane permeability

By exposing Xenopus eggs during the first cleavage to cytochalasin B (CCB) for successive periods of 4 min, it has been shown that CCB sensitivity becomes manifest approximately 7 min after the onset of furrow formation. However, even before this time furrow regression can be induced by the injection of CCB under the membrane in the furrow. This shows that during the first 7 min of cleavage the operative contractile system is CCB sensitive. Using microelectrode techniques, electrical membrane characteristics (membrane potential and resistance) were measured continuously in normally cleaving eggs and in cleaving eggs injected with CCB. It was found that the onset of sensitivity to externally applied CCB coincides with a rapid alteration of the membrane potential and resistance. We have concluded that externally applied CCB can only enter the egg when the membrane permeability increases. No evidence has been found that CCB alters the ionic permeability of preexisting cell membrane.     

The kinetics of cytochalasin D binding to monomeric actin

It has been shown previously, using G-actin labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine, that Mg2+ induces a conformational change in monomeric G-actin as a consequence of binding to a tight divalent cation binding site (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886). Using the same fluorescent probe, we show that, subsequent to the Mg2+-induced conformational change, cytochalasin D induces a fluorescence decrease. The data are consistent with a mechanism which proposes that, after Mg2+ binding, cytochalasin D binds and induces a second conformational change which results in overall tight binding of the cytochalasin. The initial binding of cytochalasin D to monomeric actin labeled with the fluorescent probe was found to be 200 microM, and the forward and reverse rate constants for the subsequent conformational change were 350 s-1 and 8 s-1, respectively, with an overall dissociation constant to the Mg2+-induced form of 4.6 microM. The conformational change does not occur in monomeric actin in the presence of Ca2+ rather than Mg2+, but Ca2+ competes with Mg2+ for the tight binding site on the G-actin molecule. Direct binding studies show that actin which has not been labeled with the fluorophore binds cytochalasin D more tightly. The conformational change induced by Mg2+ and cytochalasin D precedes the formation of an actin dimer.     

Immunofluorescence demonstration of tubulin and actin in estrogen-induced rat prolactinoma cells in vitro : Alteration of their distribution after bromocriptine, colchicine and cytochalasin B treatments

Cultured cells in vitro from estrogen-induced rat prolactin-secreting adenomas (prolactinomas) were examined by indirect immunofluorescence microscopy for the distribution of cytoskeletal proteins and alterations of cytoskeleton after treatment with bromocriptine, colchicine and cytochalasin B (CB). After 8 days in culture, prolactinoma cells were well expanded and developed cytoplasmic processes were seen. The cytoplasmic microtubules were observed as fine reticular networks radiating from perinuclear portions toward the cell periphery when decorated with an antibody against tubulin. On the other hand, the actin filaments showed diffuse and spotty distribution when detected with an anti-actin antibody. Contaminated fibroblasts showed a reticular distribution of microtubules and a parallel array of actin cables which corresponds to "stress fibers" throughout the cytoplasm. After treatment with bromocriptine, the reticular distribution of microtubules in prolactinoma cells changed into a coarse and sparse pattern, which was identical with the changes in the distribution of tubulin after treatment with colchicine. On the other hand, distribution of actin was not affected by bromocriptine. Bromocriptine treatment did not alter the distribution of microtubules and actin filaments in fibroblasts, whereas colchicine changed the distribution of microtubules in both prolactinoma cells and fibroblasts. CB treatment changed the localization of actin filaments in both kinds of cells. These in vitro studies indicated bromocriptine would selectively affect the cytoplasmic microtubular system of prolactinoma cells.     

Effects of cytochalasin B on the cortex of the unfertilized sea urchin egg

The peripheral cytoplasm of the unfertilized sea urchin egg contains approximately 18,000 cortical granules. These granules remain monolayered within the normal boundaries of the cortex when the egg is centrifuged at forces sufficient to stratify other intracellular inclusions. Exposure of unfertilized eggs to the microfilament disrupting agent, cytochalasin B (CB) causes the granules to rearrange into several layers and occasionally to undergo exocytosis or break down in situ. When these eggs are centrifuged, the cortical granules are dislodged from the cortex and migrate centrifugally among the densest intracellular components. In addition, cytoplasmic inclusions, which normally are excluded from the cortex, impinge directly upon the egg plasma membrane in CB-treated, centrifuged eggs. These results are consistent with the existence of a microfilamentous network which confines the cortical granules within and excludes other intracellular inclusions from the cortex of the unfertilized egg.     

Association of actin with chromaffin granule membranes and the effect of cytochalasin B on the polarity of actin filament elongation

Membranes of chromaffin granules isolated from bovine adrenal medulla are shown to bind dihydrocytochalasin B with high affinity. These membranes also bound [3H]actin in a time- and Mg2+-dependent manner and electron microscopy showed the presence of membrane-attached actin filaments following addition of exogenous actin. Binding of [3H]actin was partially inhibited by cytochalasin B. Electron microscopic analysis of heavy meromyosin-decorated, membrane-attached filaments showed terminally (end-on) attached filaments with both possible polarities (i.e., filaments with arrowheads pointing both towards and away from the membranes). Treatment of samples with cytochalasin B preferentially inhibited growth of filaments with their 'barbed' ends pointing away from membranes. These results are discussed with respect to the role of actin in secretory granule function and the mechanism of cytochalasin action.     

Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface. © 1995 Wiley-Liss, Inc.     

Ligand-dependent redistribution of the IgA receptor on cultured rat hepatocytes and its disturbance by cytochalasin B

The topography and dynamics of IgA-secretory component (SC) complexes on the surface of cultured hepatocytes and its disturbance by cytochalasin B were investigated using the colloidal gold technique in conjunction with surface replication. The distribution of IgA-gold conjugates after incubation at 4 degrees C was similar in normal and cytochalasin B-treated hepatocytes and was characterized by diffusely scattered single and clustered particles, the latter often associated with coated pits. After raising the temperature to 37 degrees C, redistribution of particles and their gradual uptake into coated vesicles was observed in control cultures. This ligand-induced redistribution led to a progressive gathering of single and grouped particles in larger clusters (50-200 particles), which appeared to be the site of the most intensive endocytotic activity. In contrast, huge patches of IgA-gold conjugates were formed at the cell periphery of cytochalasin B-treated hepatocytes within 20-60 min at 37 degrees C, while central areas were cleared. Patch formation was triggered by binding of both unlabeled and labeled IgA, but could not be observed with the unoccupied receptor as demonstrated by gold-labeled antibodies against SC. These results show that the topography of SC is markedly changed by binding of its ligand, IgA, and suggest that the dynamics of the IgA-SC complexes in hepatocyte plasma membrane are affected by microfilaments.     

Proliferation characteristics of multinucleate cells during prolonged exposure to cytochalasin B

The growth of the Chinese hamster cell cultures for 7 days after the stopping of the prolonged cell cultivation with cytochalasin B (5 micrograms/ml, 7 days), on the one hand, results in the decrease in the amount of multinucleate cells from 75 to 25%. On the other hand, it leads to the appearance of a new class of multinucleate cells with more than 7 nuclei. By the 2nd day a rapid increase in the proliferation rate is observed both in uninucleate and multinucleate cells. Then, by the 7th day, the rate of proliferation activity of multinucleate cells decrease more quickly than that of uninucleate ones. It is shown that during a prolonged cell cultivation after the action of cytochalasin B all classes of multinucleate cells are able to DNA reproduction. A question on proliferation abilities of multinucleate cells is discussed.     

Intracellular sodium activity in the sea urchin egg during fertilization

Fertilization of the sea urchin egg triggers a sequence of events that are necessary for metabolic derepression and stimulation of proliferation. Changes in intracellular Ca2+ and H+ activities regulate the sequence of events. Intracellular sodium activity is important in the regulation of the intracellular activities of these ions and may directly regulate metabolic events. Using Na+-sensitive microelectrodes we continuously measured the intracellular Na+ activity during fertilization. The results show an increase in intracellular sodium activity medicated by two pathways of Na+ entry: Na+ permeability increase during the fertilization potential and initiation of Na+-H+ exchange activity. Intracellular Na+ activity returned to unfertilized levels by 20 min after fertilization. This decrease was inhibited by ouabain, which suggests the activation of Na+, K+ ATPase during fertilization.     

Effect of cytochalasin A on actin distribution in the fission yeastSchizosaccharomyces pombe studied by fluorescent and electron microscopy

Summary Actin distribution and ultrastructure of the fission yeastSchizosaccharomyces pombe treated with cytochalasin A (CA) were investigated by fluorescence microscopy using rhodamine-conjugated phalloidin (rh-ph) and freeze substitution electron microscopy. Among the cytochalasins tested, CA was most effective and at 5 g/ ml inhibited the appearance of the actin ring at the cell equator at the stage prior to septum formation and the accumulation of actin dots at the septum-forming site both in wild-type cells and the mutantcdc 11, which is defective in septum formation at restrictive temperature. Freeze substitution electron microscopy of CA-treated cells revealed the displacement and morphological alteration of cytoplasmic vesicles and dictyosomes within 30 min and the appearance of dense bodies in the cytoplasm. A sub-population of cytoplasmic vesicles and dictyosomes were insensitive to CA and maintained their original structure. An electron less dense layer containing filamentous material was noted beneath the plasma membrane and thought to be the area of heavy actin patches stained with rh-ph at the cells ends. These results suggest that CA disrupted an actin network that normally maintains the organization of the secretory pathway involving dictyosomes and vesicles.