The cortical contraction related to the ooplasmic segregation inCiona intestinalis eggs

Summary The egg cytoplasm of ascidian,Ciona intestinalis, segregates towards both the animal and vegetal poles within a few minutes of fertilization or parthenogetic activation with ionophore A23187. A constriction appears first on the egg surface near the animal pole and then moves to the vegetal pole. Carmine granules and spermatozoa attached to the egg surface move towards the vegetal pole with the movement of the constriction. Microvilli, which are distributed uniformly in unfertilized egg, disappear on the animal side of the constriction and became more dense on the vegetal side of the constriction. Transmission electron microscopy revealed that sub-cortical cytoplasm, containing numerous mitochondria and sub-cortical granules, moves towards the vegetal pole with the movement of the constriction and then concentrates into a cytoplasmic cap at the vegetal pole. An electron-dense layer appears in the cortex of the cap. The ooplasmic segregation and the cortical contraction were inhibited by cytochalasin B and induced by ionophore A23187. These observations suggest that ooplasmic segregation is caused by the cortical contraction which is characterised by a surface constriction and by the formation of an electron-dense layer.     

Response of the cortex to the mitotic apparatus during polar body formation in the starfish oocyte of Asterina pectinifera

In order to understand the mechanism of unequal division, polar body formation was investigated using the oocytes of the starfish, Asterina pectinifera. Cortical actin filaments were quantitatively measured after staining the maturing oocytes with fluorescently labeled phalloidin using a computer and image-processing software. Before polar body formation, at first the actin filaments at the animal pole decreased and subsequently the animal pole bulged. On the other hand, actin filaments surrounding the animal pole increased gradually and made a cleavage furrow around the animal pole as the bulge grew. Then the furrow ingressed and finally a polar body formed. When the surface force was calculated according to the cell shape, the surface force decreased at the animal pole but the force at the contractile ring increased. When by micromanipulation the mitotic apparatus was detached and translocated to the cortex other than the animal pole, polar body formation occurred all over the cortex of the oocyte, which indicates that the response of the whole cortex to the mitotic apparatus is equal. These results indicate that the decrease in the actin filaments and surface force near the centrosome of the mitotic apparatus as well as the increase in the actin filaments and surface force at some distance of the centrosome is important for cytokinesis.     

Unfertilized sea urchin eggs contain a discrete cortical shell of actin that is subdivided into two organizational states

Changes in the distribution and organizational state of actin in the cortex of echinoderm eggs are believed to be important events following fertilization. To examine the initial distribution and form of actin in unfertilized eggs, we have adapted immunogold-labeling procedures for use with eggs of Strongylocentrotus purpuratus. Using these procedures, as well as fluorescence microscopy, we have revealed a discrete 1-micron-thick concentrated shell of actin in the unfertilized egg cortex. This actin is located in the short surface projections of unfertilized eggs and around the cortical granules in a manner that suggests it is associated with the cortical granule surface. The actin in the short surface projections appears to be organized into filaments. However, most if not all of the actin surrounding the cortical granules is organized in a form that does not bind phalloidin, even though it is accessible to actin antibody. The lack of phalloidin binding is consistent with either the presence of nonfilamentous actin associated with the cortical granules or the masking of actin-filament phalloidin-binding sites by some cellular actin-binding component. In addition to the concentrated shell of actin found in the cortex, actin was also found to be concentrated in the nuclei of unfertilized eggs.     

Dynamic organization of cortical actin filaments during the ooplasmic segregation of ascidian Ciona eggs

Spatial reorganization of cytoplasm in zygotic cells is critically important for establishing the body plans of many animal species. In ascidian zygotes, maternal determinants (mRNAs) are first transported to the vegetal pole a few minutes after fertilization and then to the future posterior side of the zygotes in a later phase of cytoplasmic reorganization, before the first cell division. Here, by using a novel fluorescence polarization microscope that reports the position and the orientation of fluorescently labeled proteins in living cells, we mapped the local alignments and the time-dependent changes of cortical actin networks in Ciona eggs. The initial cytoplasmic reorganization started with the contraction of vegetal hemisphere approximately 20 s after the fertilization-induced [Ca²⁺] increase. Timing of the vegetal contraction was consistent with the emergence of highly aligned actin filaments at the cell cortex of the vegetal hemisphere, which ran perpendicular to the animal–vegetal axis. We propose that the cytoplasmic reorganization is initiated by the local contraction of laterally aligned cortical actomyosin in the vegetal hemisphere, which in turn generates the directional movement of cytoplasm within the whole egg.     

The repetitive calcium waves in the fertilized ascidian egg are initiated near the vegetal pole by a cortical pacemaker.

Ascidian eggs respond to fertilization with a series of repetitive calcium waves that originate mostly from the vegetal/contraction pole region (J. E. Speksnijder, C. Sardet, and L. F. Jaffe, 1990, Dev. Biol. 142, 246-249), where the myoplasm is concentrated during the first phase of ooplasmic segregation. This suggests that the myoplasm may be involved in initiating these calcium waves. To test this possibility, the starting position of the calcium waves was determined in eggs that had the subcortical, mitochondria-rich part of the myoplasm displaced by centrifugation. Such centrifuged eggs display four cytoplasmic layers: a large centrifugal yolk zone, a narrow clear zone, a mitochondria-rich layer, and a small clear zone at the centripetal pole. Imaging of the cytosolic calcium in centrifuged eggs that were injected with the calcium-specific photoprotein aequorin reveals a series of repetitive calcium waves after fertilization. About 70% of these waves start in the vegetal/contraction pole area, which is similar to the number of waves previously found to start in this area in uncentrifuged eggs. In contrast, only about 25% of the waves start close to the displaced mitochondria-rich layer. From this result it is concluded that the main wave initiation site is not displaced by the centrifugal forces that displace the subcortical, mitochondria-rich part of the myoplasm. Moreover, the observation that the animal-vegetal polarity of cortical components such as actin filaments and the endoplasmic reticulum has been retained after centrifugation further suggests that a cortical component located in the vegetal hemisphere--most likely the endoplasmic reticulum network in the cortical region of the myoplasm--is involved in initiating the repetitive calcium waves in the fertilized ascidian egg.     

Reorganization of actin filaments by ADF/cofilin is involved in formation of microtubule structures during Xenopus oocyte maturation

We examined the reorganization of actin filaments and microtubules during Xenopus oocyte maturation. Surrounding the germinal vesicle (GV) in immature oocytes, the cytoplasmic actin filaments reorganized to accumulate beneath the vegetal side of the GV, where the microtubule-organizing center and transient microtubule array (MTOC-TMA) assembled, just before GV breakdown (GVBD). Immediately after GVBD, both Xenopus ADF/cofilin (XAC) and its phosphatase Slingshot (XSSH) accumulated into the nuclei and intranuclear actin filaments disassembled from the vegetal side with the shrinkage of the GV. As the MTOC-TMA developed well, cytoplasmic actin filaments were retained at the MTOC-TMA base region. Suppression of XAC dephosphorylation by anti-XSSH antibody injection inhibited both actin filament reorganization and proper formation and localization of both the MTOC-TMA and meiotic spindles. Stabilization of actin filaments by phalloidin also inhibited formation of the MTOC-TMA and disassembly of intranuclear actin filaments without affecting nuclear shrinkage. Nocodazole also caused the MTOC-TMA and the cytoplasmic actin filaments at its base region to disappear, which further impeded disassembly of intranuclear actin filaments from the vegetal side. XAC appears to reorganize cytoplasmic actin filaments required for precise assembly of the MTOC and, together with the MTOC-TMA, regulate the intranuclear actin filament disassembly essential for meiotic spindle formation.     

Irradiations of rabbit myofibrils with an ultraviolet microbeam. II. Phalloidin protects actin in solution but not in myofibrils from depolymerization by ultraviolet light

We tested whether phalloidin protects actin in myofibrils from depolymerization by ultraviolet light (UV). I bands in glycerinated rabbit psoas myofibrils were irradiated with a UV microbeam in the presence and absence of phalloidin. We used the retention of contractility of the irradiated I band as the assay for protection of actin by phalloidin, since previous experiments indicated that UV blocks contraction of an irradiated I band by depolymerizing the thin filaments. The I bands of myofibrils incubated in phalloidin were as sensitive to UV as control I bands, indicating that phalloidin did not protect the thin filaments. However, phalloidin did protect F-actin in solution from depolymerization by UV. This apparent contradiction between F-actin in myofibrils and F-actin in solution was resolved by observing unirradiated myofibrils that were stained with rhodamine-phalloidin. It was found that phalloidin does not bind uniformly to the thin filaments, though as the fluorescence image is observed over time the staining pattern changes until it does appear to bind uniformly. We conclude that phalloidin does not protect F-actin in myofibrils from depolymerization by UV because it does not bind uniformly to the filaments.     

Filamentous actin organization in the unfertilized sea urchin egg cortex

We have investigated the organization of filamentous actin in the cortex of unfertilized eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus variegatus. Rhodamine phalloidin and anti-actin immunofluorescent staining of isolated cortices reveal a punctate pattern of fluorescent sources. Comparison of this pattern with SEM images of microvillar morphology and distribution indicates that filamentous actin in the cortex is predominantly localized in the microvilli. Thin-section TEM and quick-freeze deep-etch ultrastructure of isolated cortices demonstrates that this microvillar-associated actin is in a novel organizational state composed of very short filaments arranged in a tight network and that these filament networks form mounds that extend beyond the plane of the plasma membrane. Actin filaments within the networks do not exhibit free ends and make end-on attachments with the membrane only within the region of the evaginating microvilli. Myosin S-1 dissociable crosslinks, 2-3 nm in diameter, are observed between network filaments and between network filaments and the membrane. A second population of long, individual actin filaments is observed in close lateral association with the plasma membrane and frequently complexes with the microvillar actin networks. The filamentous actin of the unfertilized egg cortex may participate in establishing the mechanical properties of the egg surface and may function in nucleating the assembly of cortical actin following fertilization.     

Polar effects of concanavalin A on the cortical cytoskeleton of a molluscan egg (Nassarius reticulatus,Gastropoda)

Summary The organization of the surface of fertilizedNassarius reticulatus eggs was probed by investigating the effects of treatment with concanavalin A (Con A). This lectin causes abnormal polar lobe formation as well as inhibition of cleavage. At low concentrations of Con A (0.3–1.0 μg/ml) the polar lobe constriction becomes considerably elongated, whereas at higher concentrations (2.5–50 μ/ml) the position of the constriction undergoes an extreme shift towards the animal pole. In the latter case, the surface of the animal part of the egg forms large protrusions and folds. Con A also causes resorption of microvilli and disappearance of the extracellular layer covering these villi; this process starts at the vegetal pole and propagates towards the animal pole. These changes in surface architecture are associated with profound changes in the organization of filamentous (F-) actin as assessed by confocal laser scanning microscopy of NBD-phallacidin-labelled eggs. Divalent succinyl-Con A has the same effects on polar lobe formation and surface architecture as tetravalent Con A, but only at very high concentrations (100–200 μg/ml), indicating that Con A exerts its effects by cross-linkage of its binding sites. Experiments with cytoskeleton inhibitors (cytochalasin D, colchicine, and nocodazole) reveal that in Con A-treated eggs — as in untreated eggs — microfilaments, but not microtubules, are involved in the formation of the polar lobe constriction. The calcium ion channel blocker D600 affects neither normal nor Con A-induced abnormal polar lobe formation, which suggests that influx of external calcium is not required. In contrast, treatment with TMB-8, an antagonist of internal calcium release, prevents the formation of a polar lobe in both normal and Con A-treated eggs. Finally, eggs from which the polar lobe has been removed prior to Con A treatment show none of the effects described, whereas isolated polar lobes react similarly to intact eggs. These results suggest that binding of Con A to sites present at the vegetal pole of the egg is responsible for the observed effects of the lectin.     

Bipolar segregation of mitochondria,actin network,and surface in the Tubifex egg: Role of cortical polarity

The role of the cortex in ooplasmic segregation of the yolky eggs of Tubifex has been studied by epifluorescence microscopy. Living eggs labeled with rhodamine 123 and fine carbon particles placed on the surface showed that, following the second polar body formation, the egg surface cosegregates with subcortical mitochondria in a bipolar fashion, viz. toward the animal and vegetal poles in the animal and vegetal hemispheres, respectively. The egg surface of each pole moves spirally while the equatorial surface appears to remain stationary during this process. The rhodamine-phalloidin staining of whole eggs reveals that actin networks cosegregate with mitochondria. Isolated cortices which were stained with rhodamine-phalloidin demonstrated that cortical actin is organized bipolarly and that, during ooplasmic segregation, it undergoes reorganization directed toward both poles of the egg. The cortical polarity expressed as actin organization is not disrupted by centrifugal force sufficient to stratify the egg cytoplasm into five layers. The surface of a centrifuged egg moves according to the original cortical polarity. This surface movement is accompanied by the reorganization of cortical actin which appears to be identical to that in intact eggs. Other centrifugation experiments have demonstrated that the connection of the subcortical cytoplasm to the cortex is resistant to a centrifugal force of up to 650g. The nature of cortical polarity and its role in ooplasmic segregation are discussed in the light of the present results.     

Sarcomere structures in the rabbit psoas muscle as revealed by fluorescent analogs of phalloidin

Summary The fluorescent analogs of phalloidin (rhodamine-and fluorescein-phalloidin) bind tightly to the skinned fibres of rabbit psoas muscle at essentially the same sites as phalloidin and mainly stain the known regions of actin localization in the sarcomere: the thin filaments and Z bands. On both sides of the Z bands, unstained zones were observed, suggesting the presence of proteins tightly bound to the thin filaments. In myofibrils which are stretched to such an extent that the actin and myosin filaments do not overlap, stained bands could also be seen at the myosin-band border, which suggests the localization of actin at these sites.     

Ectoderm exerts the driving force for gastrulation in the sand dollar Scaphechinus mirabilis

How the ectodermal layer relates to the invagination processes was examined in the sand dollar Scaphechinus mirabilis. When the turgor pressure of blastocoele was increased, invagination was completely blocked. In contrast, an increase in turgor pressure did not affect elongation of the gut rudiment in the regular echinoid Hemicentrotus pulcherrimus. Rhodamine-phalloidin staining showed that the distribution of actin filaments was different between two species of embryos. In S. mirabilis gastrulating embryos, abundant actin filaments were seen at the basal cortex of ectoderm in addition to archenteron cells, while the intense signal was restricted to the archenteron in H. pulcherrimus. To investigate whether actin filaments contained in the ectodermal layer exert the force of invagination, a small part of the ectodermal layer was aspirated with a micropipette. If S. mirabilis embryos were aspirated from the onset of gastrulation, invagination did not occur at all, irrespective of the suction site. Even after the archenteron had invaginated to one-half of its full length, further elongation of the archenteron was severely blocked by suction of the lateral ectoderm. In contrast, suction of the ectodermal layer did not affect the elongation processes in H. pulcherrimus. These results strongly suggest that the ectodermal layer, especially in the vegetal half, exerts the driving force of invagination in S. mirabilis.     

Dynamics of the actin microfilament system in the Tubifex egg during ooplasmic segregation

Following the second polar body formation (PBF), the Tubifex egg undergoes ooplasmic segregation consisting of two steps, i.e., centrifugal migration of membranous organelles forming a subcortical ooplasmic layer and then movements of these organelles along the egg surface. The present investigation was undertaken to examine the microfilament organization in eggs during these ooplasmic rearrangements. Microfilaments throughout the egg are identified as actin by their reversible heavy meromyosin binding. Before the second PBF, a distinct network of actin filaments is present in the endoplasmic region. It is disorganized during the second PBF; short actin filaments are caused to aggregate with membranous organelles. Following the second PBF, similar short filaments become localized in the subcortical layer but not in the underlying yolky region. However, it is not until 50-60 min after the second PBF that an elaborate actin network is established in the subcortical layer. The cortex contains a sheet-like lattice of actin filaments. It is thickest around the animal pole, and tapes toward the equator of the egg. At about 90 min after the second PBF, this polarized distribution of cortical filaments becomes more pronounced as the result of their movements. Chronologically, subcortical actin network formation and cortical reorganization correspond to the later portion of the first step and the earlier portion of the second step of ooplasmic segregation, respectively. These findings are discussed in terms of ooplasmic movements and rearrangements.     

Gustatory receptors and neighbouring cells in the surface layer of an amphibian taste disc: in situ relationships and response to cell isolation

Summary The outermost epithelial cell layer of frog taste buds consists of (1) mucus-secreting cells; (2) thin, sheet-like processes of wing cells, which enclose the basal and lateral aspects of the mucus cells; (3) slim apical processes (sensory dendrites) of taste receptor cells; the latter ascend within wing-cell processes and each process terminates apically in one villus-like rod, a dense, highly ordered bundle of 500–1500 actin filaments supporting the chemoreceptive membrane. Staining with fluorescent phalloidin indicates that there are about 760 receptor terminals in a taste disc of 200 m in diameter, or 200000 per tongue. Viable spindle-shaped taste receptor cells were isolated using a lowcalcium / collagenase / mechanical agitation protocol. Following fluorescent labelling of apical membranes prior to cell separation, the label remained at the dendritic tip of these cells during isolation. The tip was still stainable with phalloidin. Thus the sensory dendrite, including its apical pole, had been completely removed from the encasing wing-cell process. However, scanning electron microscopy and staining by lectin and phalloidin indicated that many taste receptor cells had undergone cytoskeletal reorganization in response to cell separation; they became flask-shaped following the shortening and thickening of their sensory dendrite. The cells also began to disassemble the actin filament bundle at the chemoreceptive apical cell pole.     

Manipulation of cytokinesis affects polar ionic current around the egg of Lymnaea stagnalis

Summary The fertilized egg of the mollusc Lymnaea stagnalis generates a polarized current pattern as measured with the vibrating probe. Here we investigated the basis of these polar ionic currents. Ionic currents were measured around eggs during the second meiotic division after interference with cytokinesis. Cytokinesis was either displaced by centrifugation or inhibited with cytochalasin or nocodazole. Furthermore, ectopic constrictions were induced with lectin treatment. It appeared that the inward current of the animal pole can be displaced by centrifugation and remains associated with the position of the meiotic apparatus. The influence of the meiotic apparatus on the polar current pattern seems to be directly related to membrane constrictions rather than to karyokinesis. This was demonstrated by a change in current density after induction of an ectopic constriction at the vegetal pole and by the abolishment of currents after cytochalasin treatment. Since the location of the outward current was not sensitive to centrifugation, it may be concluded that the vegetal outward current depends upon properties of the vegetal cortex. On the basis of these results, we conclude that the Lymnaea egg generates two types of ionic currents during the second meiotic division. The first is an inward current activated at the site of membrane constrictions. The second is an outward current associated with the vegetal cortex.     

Identification of a membrane skeleton in platelets

Platelets have previously been shown to contain actin filaments that are linked, through actin-binding protein, to the glycoprotein (GP) Ib-IX complex, GP Ia, GP IIa, and an unidentified GP of Mr 250,000 on the plasma membrane. The objective of the present study was to use a morphological approach to examine the distribution of these membrane-bound filaments within platelets. Preliminary experiments showed that the Triton X-100 lysis buffers used previously to solubilize platelets completely disrupt the three-dimensional organization of the cytoskeletons. Conditions were established that minimized these postlysis changes. The cytoskeletons remained as platelet-shaped structures. These structures consisted of a network of long actin filaments and a more amorphous layer that outlined the periphery. When Ca2+ was present, the long actin filaments were lost but the amorphous layer at the periphery remained; conditions were established in which this amorphous layer retained the outline of the platelet from which it originated. Immunocytochemical experiments showed that the GP Ib-IX complex and actin-binding protein were associated with the amorphous layer. Analysis of the amorphous material on SDS-polyacrylamide gels showed that it contained actin, actin-binding protein, and all actin-bound GP Ib-IX. Although actin filaments could not be visualized in thin section, the actin presumably was in a filamentous form because it was solubilized by DNase I and bound phalloidin. These studies show that platelets contain a membrane skeleton and suggest that it is distinct from the network of cytoplasmic actin filaments. This membrane skeleton exists as a submembranous lining that, by analogy to the erythrocyte membrane skeleton, may stabilize the plasma membrane and contribute to determining its shape.     

Effect of phalloidin and viroisin on Acanthamoeba castellanii after permeabilization of the cell

We have developed a new technique for the permeabilization of the membrane of Acanthamoeba castellanii. This technique involves the use of digitonin which alters neither the morphology nor the motility of the cell, but favours the penetration of phalloidin and viroisin. Treatment of permeabilized cells with phalloidin or viroisin induces, in the cortex of the cell, an intensive proliferation of filaments which have been identified as actin. This cortical filamentous layer detaches from the membrane and slowly contracts, acting as a fine mesh sieve which concentrates the organelles in the middle of the cell, causing therefore the formation of a central granuloplasm and a cortical hyaloplasm. During this process, cell motility is irreversibly lost. The results indicate that extensive proliferation and reorganization of actin filaments cannot support cell motility and they are discussed in terms of a general understanding of amoeboid movement.     

Mechanism of the formation of contractile ring in dividing cultured animal cells. I. Recruitment of preexisting actin filaments into the cleavage furrow

Cytokinesis of animal cells involves the formation of the circumferential actin filament bundle (contractile ring) along the equatorial plane. To analyze the assembly mechanism of the contractile ring, we microinjected a small amount of rhodamine-labeled phalloidin (rh-pha) or rhodamine-labeled actin (rh-actin) into dividing normal rat kidney cells. rh-pha was microinjected during prometaphase or metaphase to label actin filaments that were present at that stage. As mitosis proceeded into anaphase, the labeled filaments became associated with the cortex of the cell. During cytokinesis, rh-pha was depleted from polar regions and became highly concentrated into the equatorial region. The distribution of total actin filaments, as revealed by staining the whole cell with fluorescein phalloidin, showed a much less pronounced difference between the polar and the equatorial regions. The sites of de novo assembly of actin filaments during the formation of the contractile ring were determined by microinjecting rh-actin shortly before cytokinesis, and then extracting and fixing the cell during mid-cytokinesis. Injected rhodamine actin was only slightly concentrated in the contractile ring, as compared to the distribution of total actin filaments. Our results indicate that preexisting actin filaments, probably through movement and reorganization, are used preferentially for the formation of the contractile ring. De novo assembly of filaments, on the other hand, appears to take place preferentially outside the cleavage furrow.     

Cytoplasmic actin and cochlear outer hair cell motility

Summary Isolated outer hair cells of the guinea pig lacking a cuticular plate and its associated infracuticular network retain the ability to shorten longitudinally and become thinner. Membrane ghosts lacking cytoplasm retain the cylindrical shape of the hair-cell, and although they do not shorten, they retain the ability to constrict and become thinner. These data suggest that cytoplasmic components are associated with outer hair-cell longitudinal shortening and that the lateral wall is responsible for maintaing cell shape and for constriction. Actin, a protein associated with the cytoskeleton and cell motility, is thought to be involved in outer hair-cell motility. To study its role, actin was localized in isolated outer hair cells by use of phalloidin labeled with fluorescein and antibodies against actin coupled to colloidal gold. In permeabilized guinea-pig hair cells stained with phalloidin, actin filaments are found along the lateral wall. In frozen-fixed hair cells actin filaments are distributed uniformly throughout the cytoplasm. Electron-microscopic studies show that antibodies label actin throughout the outer hair-cell body. Thus cytoplasmic actin filaments may provide the structural basis for the contraction-like events.     

Nuclear migration and spindle formation in the fourth cleavage of sea urchin eggs under the influence of inhibitors.

Mitosis of sea urchin eggs was inhibited when exposed to 3 micrograms/ml aphidicolin from the 2-cell stage onwards. Nevertheless the nuclei migrated to the vegetal pole at the time of the fourth unequal division in control eggs. Two or four equal or unequal asters developed. Asters in proximity to the vegetal pole were always considerably smaller than those close to the center of the two blastomeres. In contrast to colchicine, cytokinesis but not migration of the nuclei in the vegetal half was prevented by treatments with 5 microM cytochalasin B or D. Various mitotic figures were formed in the vegetal blastomeres of eggs treated with 0.4 mM colchicine or 3 microM griseofulvin after the third cleavage. In some eggs a centrally localized monaster with chromosomes in sphere-like arrangement was formed in others a monopolar mitotic figure pushed the chromosomes in bowl-like arrangements to the most vegetal cortex. In anaphase one set of chromatids migrated to the monopole leaving the scattered sister-chromatids behind. The mechanism of migration of the nuclei and of chromosome arrangement in the metaphase plate is discussed.