Polarization of the Surface Membrane and Cortical Layer of Sea Urchin Blastomeres, and its Inhibition by Cytochalasin B

Sea-urchin blastomeres have two domains of the plasma membrane which can be distinguished immunocytochemically. An egg-surface antibody (anti-ES), which binds to the membrane of the entire surface region of eggs before cleavage, binds to the membrane of the outer surface region of blastomeres after cleavage, but not to that of the cleavage furrow region or interblastomeric surface region.
The anti-ES binding sites on the egg membrane were chased after cleavage by labeling the egg plasma membrane with FITC conjugated monovalent anti-ES (FITC-Fab anti-ES) before the first cleavage, and then allowing the eggs to cleave. The surface fluorescence increased in intensity in the cleavage furrow region with progress of furrowing, but after completion of the furrowing, the fluorescence became uniform and finally decreased in the interblastomeric surface region.
The distributions of pigment granules and NBD-phallacidin stainable microfilaments in the cortex after completion of furrowing were polarized in the same way as the anti-ES binding area. As cytochalasin B completely inhibited the polarization in both the surface and cortical layer but colchicine did not, polarization of the anti-ES binding area was concluded to be due to the post-cleavage polarized distribution of submembranous microfilaments in the cortical layer.     

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.     

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.     

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.     

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.     

Membrane protein redistribution during Xenopus first cleavage

A large increase in surface area must accompany formation of the amphibian embryo first cleavage furrow. The additional membrane for this areal expansion has been thought to be provided entirely from cytoplasmic stores during furrowing. We have radioiodinated surface proteins of fertilized, precleavage Xenopus laevis embryos and followed their redistribution during first cleavage by autoradiography. Near the end of first cleavage, membrane of the outer, pigmented surface of the embryo and a short band of membrane at the leading edge of the furrow displayed a high silver grain density, but the remainder of the furrow membrane was lightly labeled. The membrane of the cleavage furrow is thus mosaic in character; the membrane at the leading edge originates in part from the surface of the zygote, but most of the membrane lining the furrow walls is derived from a source inaccessible to surface radioiodination. The furrow membrane adjacent to the outer, pigmented surface consistently showed a very low silver grain density and was underlain by large membranous vesicles, suggesting that new membrane derived from cytoplasmic precursors is inserted primarily in this location, at least during the later phase of cleavage. Radioiodinated membrane proteins and surface-attached carbon particles, which lie in the path of the future furrow, contract toward the animal pole in the initial stages of cleavage while markers in other regions do not. We suggest that the domain of heavily labeled membrane at the leading edge of the definitive furrow contains the labeled elements that are gathered at the animal pole during the initial surface contraction and that they include membrane anchors for the underlying contractile ring of microfilaments.     

Cleavage in a saponin model of the sea urchin egg

A cell model, in which cleavage could be induced, was obtained from fertilized sea urchin eggs by putting eggs that were in the first cleavage into a solution containing 3 X 10(-5) g/ml saponin and suitable amounts of ATP and Ca2+. The cell membrane became freely permeable to ATP and Ca2+ within 1 minute. The respective optimal concentrations of ATP and Ca2+ that advanced the cleavage furrow in this model were 2 mM and 10(-8) M. With the optimal ATP and Ca2+ concentrations, the cleavage furrow of the model advanced at a rate that differed little from that in living eggs. The cleavage furrow soon receded, however, when the concentration of ATP was decreased to less than 1 mM or increased to more than 3 mM, as well as when the concentration of Ca2+ was increased to more than 10(-7) M.     

Intracellular distribution of calcium and phosphorus during the first cell division of the sea urchin egg

The intracellular distribution of calcium and phosphorus during metaphase and anaphase of the first cleavage in sea urchin eggs was studied with the electron-probe microanalyzer. This study allowed a comparison of the relative concentrations of both elements on the polar and cleavage furrow regions of the membrane and on the mitotic asters and cytoplasm. The results show that in most eggs, both calcium and phosphorus are more highly concentrated in the mitotic asters than in surrounding cytoplasm during both anaphase and metaphase. Calcium is more concentrated at the furrow region than at the polar region during metaphase but not anaphase. The role of calcium during mitosis was reviewed with special reference to the theories on the formation of the cleavage furrow along the equatorial zone between two mitotic centers.     

Regional Difference in Mechanical Properties of the Cell Surface in Dividing Echinoderm Eggs*

Stiffness of the cell was surface was determined in fertilized sea urchin and starfish eggs by measuring the mechanical resistivity of the cell surface against negative pressure applied to a restricted part with a micropipette in contact with the cell surface at its tip (elastimetry). In both sea urchin and starfish eggs, the stiffness of the cell surface changed almost in parallel between the presumptive furrow and polar surfaces before the onset of the first cleavage, and the stiffness of the furrow surface became larger than that of the polar surface when cleavage started, although temporal changes in the stiffness were different between sea urchin and starfish eggs. The stiffness of the cell surface changed almost in parallel between the surfaces at the equator and at the animal pole in starfish eggs before the onset of polar body formation. The stiffness of the cell surface around the forming polar body increased during the formation of the polar body and remained at a high level after the polar body formation. It seems that the stiffness difference responsible for the formation of the contractile ring develops simultaneously with rather than prior to the formation of the cleavage furrow.     

Membrane phospholipid dynamics during cytokinesis: regulation of actin filament assembly by redistribution of membrane surface phospholipid

To study molecular motion and function of membrane phospholipids, we have developed various probes which bind specifically to certain phospholipids. Using a novel peptide probe, RoO9-0198, which binds specifically to phosphatidylethanolamine (PE) in biological membranes, we have analyzed the cell surface movement of PE in dividing CHO cells. We found that PE was exposed on the cell surface specifically at the cleavage furrow during the late telophase of cytokinesis. PE was exposed on the cell surface only during the late telophase and no alteration in the distribution of the plasma membranebound peptide was observed during the cytokinesis, suggesting that the surface exposure of PE reflects the enhanced transbilayer movement of PE at the cleavage furrow. Furthermore, cell surface immobilization of PE induced by adding of the cyclic peptide coupled with streptavidin to prometaphase cells effectively blocked the cytokinesis at late telophase. The peptide-streptavidin complex bound specifically to cleavage furrow and inhibited both actin filament disassembly at cleavage furrow and subsequent plasma membrane fusion. Binding of the peptide complex to interphase cells also induced immediate disassembly of stress fibers followed by assembly of cortical actin filaments to the local area of plasma membrane where the peptide complex bound. The cytoskeletal reorganizations caused by the peptide complex were fully reversible; removal of the surface-bound peptide complex by incubating with PE-containing liposome caused gradual disassembly of the cortical actin filaments and subsequent formation of stress fibers. These observations suggest that the redistribution of plasma membrane phospholipids act as a regulator of actin cytoskeleton organization and may play a crucial role in mediating a coordinate movement between plasma membrane and actin cytoskeleton to achieve successful cell division.     

A subcortical, pigment-containing structure in Xenopus eggs with contractile properties

An accumulation of insoluble, finely granular material has been observed under the pigmented surface of Xenopus eggs by a specialized "dry fracture" technique and scanning electron microscopy. Cortical granules and pigment granules can be recognized with the techniques and can be seen to be embedded in the material. Thin sections show that the region also contains mitochondria and membranous vesicles or reticula. Yolk platelets are largely excluded from the heaviest accumulations of the material. The substance is most dense just under the cortex and grades off gradually into the more diffuse, yolk-containing network of the endoplasm. The accumulation of material is much thicker in the animal hemisphere of the egg than in the vegetal hemisphere, and the pigment embedded in it defines the pigmented area of the animal hemisphere. In the pigmented area the material excludes yolk for a thickness of 3-7+ microns from the surface. In the vegetal hemisphere there is no such accumulation and yolk platelets can be found almost touching the plasmalemma. Cortical contractions have been experimentally induced in eggs. Their relative strength correlates with the relative thickness of the finely granular, subcortical material. During contraction the material accumulates to much greater thicknesses, excluding yolk from thicknesses of 15-30+ microns from the surface. The contracting entity is, or is in, the finely granular material. Injection of cytochalasins into the eggs inhibits cleavage furrow operation but does not inhibit the induced cortical contractions. The thus do not seem to be dependent on actin microfilamentogenesis as is the operation of the contractile ring of the cleavage furrow. The differential sensitivity to cytochalasins of the contractile ring and the system responding in the induced cortical contractions, suggests a two-component system for cortical contractions in the egg. A model is presented which accommodates the available data.     

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.     

Cytological and biochemical analyses of the maternal-effect mutant embryos with abnormal cleavage furrow formation in Xenopus laevis

We describe an embryonic lethal mutation in Xenopus laevis that provokes regression of cleavage furrow formation. The mutant females (designated as af) were obtained by the back-cross of a female with one of her sons. All the fertilized eggs laid by the mutant females, regardless of the wild-type male used in the mating, failed to cleave although each furrow ran at a proper position superficially. Light and electron microscopic observations of the embryos revealed that the cleavage furrows stayed on the surface and cytoplasmic divisions did not take place at all, while nuclear divisions did. Two-dimensional gel-electrophoretic comparisons of af and wild-type embryos demonstrated that two proteins, having estimated molecular masses of about 38 kDa (pI 6.6) and 78 kDa (pI 7.6), were missing in af embryos. Microinjection of clear cytoplasm from a wild-type egg into fertilized af eggs provoked partial surface contraction and cleavage furrow formation in recipient af eggs. The results showed that the af females carry a lethal maternal-effect mutation which causes cleavage furrow regression by being deficient in a few proteins, and that cytoplasm of wild-type eggs can partially rescue the cleavage furrow formation of af eggs by furnishing the corrective material, presumably a product of the normal allele of af.     

Effect of Microtubular Poisons on Cleavage Furrow Formation and Induction of Furrow-like Dent in Amphibian Eggs

The effects of the microtubular poisons colchicine, vinblastine and nocodazole, on cleavage furrow formation and induction of furrow-like dents in eggs of the newt, Cynops pyrrhogaster , were examined.
Solutions of the poisons were injected beneath the cortex around the small initial furrow, or around the advancing tip of the furrow of eggs during the first cleavage. This resulted in prompt block of the progress of the furrow at the injection site, and subsequent total regression of the furrow or incomplete cleavage.
The ability of the cortex of a cleavage-arrested blastomere to form a furrow-like dent was tested by inhibiting furrow formation of one blastomere of two-cell embryos by injection of the microtubular poisons, and then transplantation of the blastomere under the cortex of the animal half with furrow-inducing cytoplasm (FIC) taken from normally cleaving eggs. No dent was formed. Moreover, FIC from eggs treated with a poison had no ability to induce a dent on the surface of normally cleaving eggs.
These results show that microtubule structures are directly involved in formation of a cleavage furrow.     

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.     

Microtuble organization in Xenopus eggs during the first cleavage and its role in cytokinesis

It has been suggested that the organization of microtubules during mitosis plays an important role in cytokinesis in animal cells. We studied the organization of microtubules during the first cleavage and its role in cytokinesis of Xenopus eggs. First, we examined the immunofluorescent localization of microtubules in Xenopus eggs at various stages during the first cleavage. The astral microtubules that extend from each of the two centrosomes towards the division plane meet and connect with each other at the division plane as cytokinesis proceeds. The microtubular connection thus advances from the animal pole to the vegetal pole, and its leading edge is located approximately beneath the leading edge of the cleavage furrow. Furthermore, an experiment using nocodazole suggests that microtubules have an essential role in advancement of the cleavage furrow, but neither in contraction nor maintenance of the already formed contractile ring which underlies the cleavage furrow membrane. These results suggest that the astral microtubules play an important role in controlling the formation of the contractile ring in Xenopus eggs.     

NEW MEMBRANE FORMATION DURING CYTOKINESIS IN NORMAL AND CYTOCHALASIN B-TREATED EGGS OF XENOPUS LAEVIS : I. Electron Microscope Observations

A method is described for measuring and calculating the preexisting surface in uncleaved Xenopus eggs and the rate of surface growth in cleaving eggs. Surface-marking experiments with cytochalasin B-treated eggs show that the unpigmented surface grows by de novo formation and not by expansion of preexisting pigmented surface. The onset of new surface formation during first cleavage was studied by using transmission electron microscope and scanning electron microscope techniques. At 3–4 min and at 7–8 min after the onset of cleavage the eggs were fixed in the presence of ruthenium red (RR). Evidence is presented that unpigmented surface representing new membrane comes into appearance between four and eight min. This surface has a selective binding capacity for RR. Concomitantly with the appearance of new membrane, endoplasmic reticulum (ER) cisternae are in continuity with, and dense cytoplasmic inclusions coalesce with, the membrane along the furrow. The latter give rise to liposome-like bodies. The possibility is discussed that the ER cisternae transport a surface exudate (a carbohydrate complex), that the dense cytoplasmic inclusions represent pools of membrane precursor, and that membranogenesis takes place by direct insertion of pooled precursors into the cell surface. In a second paper, these findings will be correlated with electrophysiological measurements.     

Why does a cleavage plane develop parallel to the spindle axis in conical sand dollar eggs? A key question for clarifying the mechanism of contractile ring positioning

Three types of models have been proposed about how the mitotic apparatus determines the position of the cleavage furrow in animal cells. In the first and second types, the contractile ring appears in a cortical region that least and most astral microtubules reach, respectively. The third type is that the spindle midzone positions the contractile ring. In the previous study, a new model was proposed through analyses of cytokinesis in sand dollar and sea urchin eggs. Gradients of the surface density of microtubule plus ends are assumed to drive membrane proteins whose accumulation causes the formation of contractile-ring microfilaments. In the present study, the validity of each model is examined by simulating the furrow formation in conical sand dollar eggs with the mitotic apparatus oriented perpendicular to the cone axis. The new model predicts that unilateral furrows with cleavage planes roughly parallel to the spindle axis appear between the mitotic apparatus and the vertex besides the normally positioned furrow. The predictions are consistent with the observations by Rappaport & Rappaport (1994, Dev. Biol.164, 258-266). The other three types of models do not predict the formation of the ectopic furrows. Furthermore, it is pointed out that only the new model has the ability to explain the geometrical relationship between the mitotic apparatus and the contractile ring under various experimental conditions. These results strongly suggest the real existence of the membrane proteins postulated in the model.     

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.     

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.