Immunocytochemical evidence for the presence of two domains in the plasma membrane of sea urchin blastomeres

Summary The blastomeres of sea urchin embryos have two surface regions with different properties. Numerous microvilli are present in the apical surface region, while the baso-lateral surface region, either on adjoining adjacent cells or facing the blastocoel, is smooth. When blastomeres are isolated from embryos and stained with fluorescein-isothiocyanate-labelled anti-(egg surface) antibody (anti-ES) prepared against membranes isolated from fertilized eggs, the apical microvillous region fluoresces while the smooth region does not [Yazaki I (1984) Acta Embryol Morphol Exp 5∶3–22]. In order to study quantitatively the ‘bindability’ of the membrane in the two regions to anti-ES, immunoelectron microscopy was used. Blastomeres isolated from embryos ofHemicentrotus pulcherrimus at the eight-cell stage were treated with rabbit anti-ES serum or pre-immune serum and then with ferritin-conjugated goat anti-(rabbit IgG) for 10 min at 0°C, mainly before fixation. About 10 times (maximally 45 times) more ferritin particles were counted per contour length in the microvillous surface region than in the smooth surface region. These results suggest that the membrane of the blastomeres of sea urchin embryos is a mosaic of two different membrane territories: one represented by the microvillous surface originating from the unfertilized egg, which binds anti-ES, the other by the smooth surface newly organized after the first cleavage, which does not react with anti-ES. The mechanism of segregation of the membrane into these two regions is discussed.     

Accumulation of WGA Receptors in the Cleavage Furrow during Cytokinesis of Sea Urchin Eggs

Sea urchin eggs stained with fluorescein-conjugated wheat germ agglutinin (F-WGA) before or after fixation showed a marked accumulation of fluorescence at the cleavage furrow in the first and the second cell divisions. WGA receptors (WGA-binding membrane glycoproteins) were redistributed to the equatorial region through several steps in compressed eggs. Accumulated WGA receptors showed a distribution similar to that of contractile-ring microfilaments throughout most of the steps. Therefore, the former is probably associated with the latter directly or indirectly. Labeling with F-WGA provides a simple method to detect contractile-ring microfilaments in living eggs. Treatment of eggs with colcemid shortly before cytokinesis dispersed the ring-like accumulation of WGA receptors together with contractile-ring microfilaments. This result suggests that microtubule structures, probably asters, are involved in the redistribution of WGA receptors. Cytochalasin B prevented furrowing when it was applied shortly before cytokinesis. While contractile-ring microfilaments showed a spotty distribution in the expected furrow region, WGA receptors were normally redistributed. Furthermore, a higher concentration of the drug allowed the appearance of accumulated WGA receptors in compressed eggs although the development into a ring-like configuration was inhibited. These observations suggest the possibility that the redistribution of WGA receptors is involved in the formation of contractile ring.     

The role of calcium, polyamines and centrosomes in the formation and organization of cleavage furrows in amphibian eggs.

Cleavage furrows of amphibian eggs exhibit characteristic morphological features: the presence of finger-like microvilli (MV) along their outer edges, the formation of furrow walls from new plasma membrane lacking MV, and the subsequent retrieval of this membrane during the infolding of the furrow. A similar structure can be induced, specifically, by certain cytoplasmic components such as centrosomes, polyamines and calcium. Their respective roles in the events associated with the furrowing process have been investigated by injecting these agents into nucleated and enucleated Pleurodeles eggs and evaluating their effects using cytochemical labelling of the egg surface with a biotin-streptavidin system. The injection of polyamines (spermine or spermidine) and in some cases, calcium into enucleated eggs provoked MV elongation and the appearance of newly formed, smooth plasma membrane. In these eggs, this membrane was not incorporated into the furrows, and as a consequence, the blastomeres did not actually separate. In contrast, the injection of centrosomes into enucleated eggs induced both the incorporation and internalization of new membrane, resulting in the formation of furrows and a true cellularization of the eggs, identical to the cleavage process observed in fertilized eggs. The present results provide further evidence that the establishment of the furrow depends on two complementary interacting systems: the contractile elements of the egg cortex which regulate the insertion of new membrane and the mitotic center which is essential for the invagination of the furrow.     

The changes in structural organization of actin in the sea urchin egg cortex in response to hydrostatic pressure

We have used hydrostatic pressure to study the structural organization of actin in the sea urchin egg cortex and the role of cortical actin in early development. Pressurization of Arbacia punctulata eggs to 6,000 psi at the first cleavage division caused the regression of the cleavage furrow and the disappearance of actin filament bundles from the microvilli. Within 30 s to 1 min of decompression these bundles reformed and furrowing resumed. Pressurization of dividing eggs to 7,500 psi caused both the regression of the cleavage furrow and the complete loss of microvilli from the egg surface. Following release from this higher pressure, the eggs underwent extensive, uncoordinated surface contractions, but failed to cleave. The eggs gradually regained their spherical shape and cleaved directly into four cells at the second cleavage division. Microvilli reformed on the egg surface over a period of time corresponding to that required for the recovery of normal egg shape and stability. During the initial stages of their regrowth the microvilli contained a network of actin filaments that began to transform into bundles when the microvilli had reached approximately 2/3 of their final length. These results demonstrate that moderate levels of hydrostatic pressure cause the reversible disruption of cortical actin organization, and suggest that this network of actin stabilizes the egg surface and participates in the formation of the contractile ring during cytokinesis. The results also demonstrate that actin filament bundles are not required for the regrowth of microvilli after their removal by pressurization. Preliminary experiments demonstrate that F-actin is not depolymerized in vitro by pressures up to 10,000 psi and suggest that pressure may act indirectly in vivo, either by changing the intracellular ionic environment or by altering the interaction of actin binding proteins with actin.     

Cortical differentiation of the animal pole during maturation division in fertilized eggs of Tubifex (Annelida,Oligochaeta): II. Polar body formation

Changes in the cortical organization at the animal pole are examined by scanning and transmission electron microscopy in the Tubifex egg undergoing second polar body formation. At very early anaphase of the second meiosis, the egg surface overlying the meiotic apparatus is undulated, but its neighboring surface appears to be smooth. Although a microfilamentous cortical layer is found in the smooth area, the cortical layer of the undulating area is thin and devoid of filamentous structures except for its central part where some filaments are observed. This local differentiation takes place normally in colchicine-treated eggs where the meiotic apparatus is destroyed. Along with the progression of the anaphase movement, the egg surface of the undulating area is, first, uplifted into a cone-shaped cytoplasmic bulge (presumptive polar body); then the height and surface area of the bulge gradually increase. The distal surface of the growing bulge appears to be undulated whereas the sides of the bulge are relatively smooth. Transmission electron microscopy reveals that a thick microfilamentous cortical layer is always localized at the proximal region of this bulge; other regions of the bulge are characterized by a thin cortical layer which is devoid of filamentous structure except for the apical portion of the bulge. Microfilaments at the base of the bulge are perpendicular or oblique to the egg surface. The cortical layer of the egg which is continuous to that of the proximal region of the bulge comprises microfilaments running parallel to the surface. The attainment of the bulge to its full size is followed by the development of the cleavage furrow along its base. The cleavage furrow appears to bisect the spindle midway between its poles. In cytochalasin B-treated eggs, where some cortical microfilaments are detected at the animal pole, a cytoplasmic bulge lower in height and wider in the diameter of its base than the normal one forms at the animal pole; however, it is subsequently resorbed into the egg. The formation of a cleavage furrow is not observed in these eggs. The mechanism of the polar body formation is discussed in the light of the present observations.     

Simulation of density gradients of astral microtubules at cell surface in cytokinesis of sea urchin eggs

Astral microtubules extend close to the cell surface just before cytokinesis in sea urchin eggs. At this time, a small region with a constant area is considered around a point on the egg surface. To calculate the number of microtubules that reach the surface region, i.e. the microtubule density at the point, a simple mathematical model was set up. The density was estimated at many surface points in multipolar and distorted eggs by using the model. A contour map was drawn to investigate the density gradients. The gradient patterns were compared with the distributions of contractile-ring microfilaments. The simulated cases were: (1) an unusual distribution of contractile-ring microfilaments in an egg that had polyasters and was compressed by a coverslip; (2) formation of contractile-ring microfilaments at the equatorial region in compressed eggs with a centrally-located mitotic apparatus; (3) normal furrowing in the plane of the spindle midpoint in eggs inserted into a glass loop or confined in a capillary; (4) failure of furrow formation in spherical eggs treated with ethyl urethane and revival of furrowing by pushing the equatorial surface close to the spindle. These simulations proposed the hypothesis that contractile-ring microfilaments form at surface regions where the microtubule density has a local minimum, not a local maximum. In addition, it was suggested that the probability of the formation of the contractile-ring microfilaments is dependent on how abruptly the density gradient changes at the local-minimum point. These results support the idea that the gradient pattern of the microtubule density determines whether and where contractile-ring microfilaments appear.     

Holoblastic early cleavage of Tetrodontophora bielanensis (Collembola) eggs, with special reference to its irregularity.

The fertilized eggs of Tetrodontophora bielanensis start to cleave 6 to 8 days after oviposition and initially only karyokineses occur. The cytokinesis begins after two karyokineses, when four nuclei are observed in the ooplasm. Two cleavage furrows, perpendicular to each other, appear simultaneously at the egg poles where polar bodies are located and gradually the furrows encompass the whole egg diameter. The furrow formation is initiated by the bundle of microfilaments that contract and pull superficial fragments of the oolemma into the yolk and subsequently new membranes, separating the daughter cells, start to form. However, they do not grow towards the egg centre but bifurcate, leaving the central part of the ooplasm outside of the newly formed blastomeres. Starting from the fourth or fifth cleavage division, the bifurcations permanently occur and multiple cleavage furrows are formed on the embryo surface. Moreover, fragments of the ooplasm, enclosed within the cell membrane but devoid of cell nucleus are observed. During further development such cell fragments become reincorporated into the embryo. This mode of cleavage leads eventually to the formation of cellular blastoderm on the embryo surface. The results presented in the paper suggest that the control of cleavage in T. bielanensis acts not at the level of cytoplasmic determinants but rather at the level of positional information of blastomeres.     

Absence of furrowing activity following regional cortical tension reduction in sand dollar blastomere and fertilized egg fragment surfaces

The purpose of the present investigation was to test experimentally the possibility that division mechanism establishment at the equator of sand dollar eggs may be a consequence of cortical tension gradients between the equator and the poles. Cytochalasin has been shown to decrease tension at the sea urchin egg surface. The concave ends of cytochalasin D-containing agarose cylinders were held against regions of the surface of Echinarachnius parma blastomeres and enucleated fertilized egg fragments. The ability to interfere with normal furrowing activity was used as a biological indicator of the effectiveness of cytochalasin. When agarose containing 2 microg/mL cytochalasin contacted the equatorial region of the blastomeres resulting from the first cleavage, or the equatorial surfaces of nucleated fertilized egg halves, furrowing was blocked, stalled or delayed, indicating that the concentration of cytochalasin was effective. When the same concentration of cytochalasin was applied to the poles, the cells and nucleated fertilized egg fragments divided in the same way as the controls, indicating that the effectiveness of the cytochalasin did not spread from the poles to the equator and that bisection did not interfere with the division of nucleated fertilized egg fragments. When the same concentration of cytochalasin was applied to diametrically opposed surfaces of enucleated, spherical egg fragments, there was no evidence of furrowing activity between the areas that contacted the cytochalasin or in any other part of the surface. Because of the tension-reducing effect of cytochalasin, a tension gradient existed between the regions affected and unaffected by cytochalasin. The results strongly suggest that establishment of the division mechanism by simple gradients of tension at the surface is unlikely.     

Localized calcium signals along the cleavage furrow of the Xenopus egg are not involved in cytokinesis

It has been proposed that a localized calcium (Ca) signal at the growing end of the cleavage furrow triggers cleavage furrow formation in large eggs. We have examined the possible role of a Ca signal in cleavage furrow formation in the Xenopus laevis egg during the first cleavage. We were able to detect two kinds of Ca waves along the cleavage furrow. However, the Ca waves appeared after cleavage furrow formation in late stages of the first cleavage. In addition, cleavage was not affected by injection of dibromoBAPTA or EGTA into the eggs at a concentration sufficient to suppress the Ca waves. Furthermore, even smaller classes of Ca release such as Ca puffs and Ca blips do not occur at the growing end of the cleavage furrow. These observations demonstrate that localized Ca signals in the cleavage furrow are not involved in cytokinesis. The two Ca waves have unique characteristics. The first wave propagates only in the region of newly inserted membrane along the cleavage furrow. On the other hand, the second wave propagates along the border of new and old membranes, suggesting that this wave might be involved in adhesion between two blastomeres.     

Polarized distribution of membrane components on two-cell mouse embryos

Summary In two-cell mouse embryos, membrane components detected by a variety of antisera, lectins and lipid analogues and covalent labeling were found to be localized in the poles of the two blastomeres opposite the cleavage furrow. The proportion of polarized blastomeres increased rapidly during the first 4–5 h following first cleavage and then diminished approximately two-fold over the remaining period before second cleavage. Concurrent with this decrease in percent polarization, observed poles were found to be less spatially restricted. This polarization is not the result of a ligand induced capping or a manifestation of differences in surface topography. In light of recent measurements of lateral diffusion, the polarization of membrane components may be significant for the formation of morphogenetic gradients during cleavage.     

Electrical Signs of New Membrane Production during Cleavage of Rana pipiens Eggs

Rana pipiens eggs dividing normally in diluted Ringer's solution show an increase in transmembrane potential inside negative, a decrease in resistance, and no change in total surface membrane capacitance at the appearance of a division furrow. Furrows of eggs in solutions with the tonicity of full Ringer develop partially, then regress so that the surface is again spherical. The potential and resistance changes are greater and substantial increases in capacitance occur when furrowing is so inhibited. It is proposed that the electrical changes at division are due to the introduction of new plasma membrane, between the blastomeres, having selective permeability to K and a low resistance compared to the outer spherical membrane. A narrow gap between blastomeres limits current flow through new membrane during normal division. A direct exposure of new membrane to the bathing medium when furrowing is disrupted results in larger changes in potential and resistance and permits the capacitance of new membrane to be detected.     

ON PROPAGATION OF CORTICLA FACTOR AND CYTOPLASMIC FACTOR PARTICIPATING IN CLEAVAGE FURROW FORMATION OF THE NEWT'S EGG*

From Cynops pyrrhogaster eggs just after the start of the first cleavage, a fragment of cortical layer with a small entire cleavage furrow was cut out. In the fragment, the cortex had already acquired susceptibility to and the subcortical cytoplasm had already accquired inducibility for furrow formation. The fragment was transplanted to the animal hemisphere of uncleaved fertilized eggs or eggs immediately after the onset of the first cleavage, from which a portion of the host cortex was removed. Observation was made on division of the graft, and on propagation of the cortical susceptibility and the cytoplasmic inducibility of the graft onto the host egg. The transplant divided succesively on the host egg in many cases, but the furrow of the graft never advanced to the surface of the host egg. Neither the cortical factor nor the cytoplasmic factor was transmitted across the graft to the recipient egg.     

Microfilaments during sea urchin fertilization: Fluorescence detection with rhodaminyl phalloidin

Rhodaminyl-labeled phalloidin is used to demonstrate the distribution of microfilaments during fertilization and early development in eggs of the sea urchins Arbacia punctulata and Lytechinus variegatus. The surface of unfertilized eggs have numerous punctate fluorescence sites at which rhodaminyl phalloidin binds, indicating the presence of actin oligomers or polymers. During fertilization this punctate pattern of fluorescence begins to change. Within thirty seconds of insemination, the fertilization cone is first detectable with this technique as an erect structure on the surface of the egg. The fertilization cone grows to a maximum size by 8–9 minutes, and is resorbed by 16 minutes after insemination. The surface of the fertilized egg displays numerous fluorescent fibers by 10 minutes after insemination rather than the punctate fluorescence observed in unfertilized eggs, indicative of the burst of microfilament assembly resulting in microvillar elongation. The elongated microfilaments persist through cytokinesis. Staining is also detected throughout the cortices of unfertilized, fertilized, and cleaving eggs. Cytochalasin E (10 μM, 30 min) prevents microfilament elongation and cytokinesis and reduces the cortical staining intensity after fertilization. At cleavage, contractile rings, appearing as narrow equatorial bundles of fibers, have been detected in Lytechinus variegatus as transient structures.     

Transmembrane effects of lectins : I. Effects of wheat germ agglutinin and soybean agglutinin on furrow formation and cortical wound healing in Xenopus laevis eggs

Studies on the binding of FITC-WGA and FITC-SBA to normal and cytochalasin (CB)-treated Xenopus eggs have demonstrated the presence of lectin receptors all over the egg surface; there are aggregates of receptors in the region where microvilli are numerous and microfilaments well organized. Addition of WGA or SBA to the incubation medium provokes inhibition of furrow formation provided that the lectins are added before the streak stage, or arrest of contraction when added during furrow formation. This arrest of contraction is followed by furrow regression. The lectins also affect wound healing. All these effects may be explained by an inhibition of membrane proteins movement and an alteration of the relations between the surface and the cytoskeleton.     

CHANGES IN PROTOPLASMIC CONSISTENCY AND THEIR RELATION TO CELL DIVISION

1. The development of the amphiaster is associated with the formation of two semisolid masses within the more fluid egg substance. 2. The elongation of the egg during cleavage is possibly produced as a consequence of the mutual pressure of these two growing semisolid masses. 3. The division of the egg into two blastomeres consists essentially in a growth, within the egg, of two masses of material at the expense of the surrounding cytoplasm. When all the cytoplasm of the egg is incorporated in these two masses cleavage occurs. 4. After a certain period of time the semisolid masses revert to a more fluid state. In the eggs studied this normally occurs after the cleavage furrow has completed the separation of the two blastomeres. The formation of the furrow, however, may be prevented in various ways, upon which the egg reverts to a single spherical semifluid mass containing two nuclei. 5. An egg mutilated during its semisolid state (amphiaster stage) may or may not revert to a more fluid state. If the more solid state is maintained, the cleavage furrow persists and proceeds till cleavage is completed. If the mutilation causes the egg to revert to the more fluid state the furrow becomes obliterated and a new cleavage plane is subsequently adopted. 6. The nuclei of eggs in the semifluid state are able to alter their positions. In semifluid mutilated eggs the nuclei tend to move to positions which may assure symmetry in aster formation and cleavage.     

Ultrastructural Basis of the Tension Increase in Sea-Urchin Eggs Prior to Cytokinesis

Cortices of sea-urchin eggs were studied by electron microscopy to identify the structure responsible for the rise in tension at the egg surface prior to cleavage. During anaphase the tension increased and fine filaments of 70–90 Å in diameter appeared in the cell cortex forming a thin mesh-work beneath the cell membrane. The meshwork spread all around the egg cortex without reference to the mitotic axis and the number of filaments seemed to increase up to telophase. Immediately before appearance of the cleavage furrow, the meshwork in the anticipated furrow region became dense. As the furrow appeared the tension began to decrease and the meshwork disappeared. In the progressing furrow region fine filaments of the same size as that of the meshwork-filament were oriented in a bundle to form a contractile ring. Treatment with cytochalasin B suppressed both the tension increase and the formation of the filamentous meshwork. These results suggest that the component filament of the meshwork is an actin microfilament, and that the tension increase at anaphase is due to formation of a meshwork of actin microfilaments from which a contractile ring is subsequently derived at late telophase.     

Temporal change in local forces and total force all over the surface of the sea urchin egg during cytokinesis

We determined the tension over the entire surface of the sea urchin eggs during cytokinesis, on the basis of the intracellular pressure and cell shape. This allowed us to determine the temporal changes in both the distribution of local forces and the total force produced in the whole cell cortex. A spike-like peak at anaphase and a broader peak at the onset of furrowing were observed in the time-course of the total force. Treatment of the eggs with cytochalasin D, blebbistatin, ML-9, or ML-7 significantly lowered the total force when they inhibited cytokinesis, suggesting that the tension results mainly from the interaction between intact actin filaments and activated myosin II. Myosin II would function as a motor, not only in the furrow region, but over a wide area of the cell surface, because the sum of the tensions outside the furrow region was larger than that inside the furrow region throughout cytokinesis. The distribution of the local force revealed that a global increase in the cortical force started well before the onset of furrowing, and that the force inside the furrow region continued to increase despite the decrease in the force outside the furrow region after the onset of furrowing. The spatial and temporal patterns of the force over the entire surface support the hypothesis that there are two separate but coordinated actomyosin activation mechanisms, one of which induces global activation of the cortex and the other of which then maintains the contractility only inside the furrow region.     

Simulation of the mechanism of determining the position of the cleavage furrow in cytokinesis of sea urchin eggs

In cytokinesis of sea urchin eggs, the numerical density of astral microtubules extending close to the cell surface has been thought to determine the position of the cleavage furrow. In the present study, a new model was constructed to simulate the relationship between the microtubule density and the furrow formation. In the model, gradients of the microtubule density drive fluid membrane proteins whose accumulation triggers the formation of contractile-ring microfilaments. The model could explain the behavior of the cleavage furrow under various experimental conditions. These simulations revealed two aspects of furrow formation. One is that in some cases, the cleavage furrow appears in a surface region where the microtubule density has neither a minimum nor a maximum. In all furrow regions, however, the second derivative of the microtubule-density function has large positive values. Membrane proteins greatly slow down to accumulate in such a region. The other is that the cleavage furrow is mobile, not fixed in one position, because of the fluidity of membrane proteins. These results strongly suggested that the mitotic apparatus determines the position of the cleavage furrow by redistributing membrane proteins through gradients of the microtubule density at the cell surface.     

Polarity of the ascidian egg cortex before fertilization.

The unfertilized ascidian egg displays a visible polar organization along its animal-vegetal axis. In particular, the myoplasm, a mitochondria-rich subcortical domain inherited by the blastomeres that differentiate into muscle cells, is mainly situated in the vegetal hemisphere. We show that, in the unfertilized egg, this vegetal domain is enriched in actin and microfilaments and excludes microtubules. This polar distribution of microfilaments and microtubules persists in isolated cortices prepared by shearing eggs attached to a polylysine-coated surface. The isolated cortex is further characterized by an elaborate network of tubules and sheets of endoplasmic reticulum (ER). This cortical ER network is tethered to the plasma membrane at discrete sites, is covered with ribosomes and contains a calsequestrin-like protein. Interestingly, this ER network is distributed in a polar fashion along the animal-vegetal axis of the egg: regions with a dense network consisting mainly of sheets or tightly knit tubes are present in the vegetal hemisphere only, whereas areas characterized by a sparse tubular ER network are uniquely found in the animal hemisphere region. The stability of the polar organization of the cortex was studied by perturbing the distribution of organelles in the egg and depolymerizing microfilaments and microtubules. The polar organization of the cortical ER network persists after treatment of eggs with nocodazole, but is disrupted by treatment with cytochalasin B. In addition, we show that centrifugal forces that displace the cytoplasmic organelles do not alter the appearance and polar organization of the isolated egg cortex. These findings taken together with our previous work suggest that the intrinsic polar distribution of cortical membranous and cytoskeletal components along the animal-vegetal axis of the egg are important for the spatial organization of calcium-dependent events and their developmental consequences.     

Propranolol, a beta-adrenergic receptor blocker, affects microfilament organization, but not microtubules, during the first division in sea urchin eggs

Propranolol, a beta-adrenergic receptor blocker, blocks the formation of the cleavage furrow, while karyokinesis is unaffected during first division in the sea urchins Paracentrotus lividus or Lytechinus pictus. This effect is reversed by adrenalin, indicating that it is mediated by an adrenergic mechanism. The staining of F-actin microfilaments by rhodamine phalloidin in eggs in which the cleavage is blocked by the drug has revealed that propranolol affects both the distribution and the organization of actin microfilaments. A low-voltage scanning electron microscopy (LVSEM) study of microvilli in these eggs shows an extensive rearrangement of the egg surface. Anti-tubulin immunofluorescence microscopy of eggs treated with propranolol shows that they form normal mitotic asters. This indicates that while cleavage is affected, mitotic spindle formation is not. These results suggest that neurotransmitter monoamines known to be present in the sea urchin egg might be involved in the reorganization of the actin cytoskeleton underlying the formation of the cleavage furrow.