Yukihisa Hamaguchi

A rho-like protein is involved in the organisation of the contractile ring in dividing sand dollar eggs

Sand dollar eggs were microinjected with botulinum C3 exoenzyme, an ADP-ribosyltransferase from Clostridium botulinum that specifically ADP-ribosylates and inactivates rho proteins. C3 exoenzyme microinjected during nuclear division interfered with subsequent cleavage furrow formation. No actin filaments were detected in the equatorial cortical layer of these eggs by rhodamine-phalloidin staining. When microinjected into furrowing eggs, C3 exoenzyme rapidly disrupted the contractile ring actin filaments and caused regression of the cleavage furrows. C3 exoenzyme had no apparent effect on nuclear division, however, and multinucleated embryos developed from the microinjected eggs. By contrast, C3 exoenzyme did not affect the organisation of cortical actin filaments immediately after fertilisation. Only one protein (molecular weight 22,000) was ADP-ribosylated by C3 exoenzyme in the isolated cleavage furrow. This protein co-migrated with ADP-ribosylated rhoA derived from human platelets when analysed by two-dimensional gel electrophoresis. These results strongly suggest that a rho-like, small GTP-binding protein is selectively involved in the organisation and maintenance of the contractile ring.

Activation of sea urchin eggs by microinjection of calcium buffers.

Abstract The effects of changing the intracellular Ca 2+ concentration on unfertilized sea urchin eggs were investigated by microinjecting calcium buffers, i.e., aqueous solutions for stabilizing concentrations of free calcium ions (Ca 2+ ) containing calcium salt and calcium-chelating substances (ethyleneglycol bis (β-aminoethylether)- N,N′ -tetraacetic acid (EGTA) or N -hydroxyethyl-ethylenediaminetriacetic acid (HEDTA)) at various ratios. When the intracellular Ca 2+ concentration was raised to more than 0.2 μM, the elevation of fertilization membrane was observed in more than 50% of eggs in normal sea water. The threshold Ca 2+ concentration inducing membrane-elevation was 0.5 μM in eggs in Ca 2+ -free sea water. Activation accompanying the formation of a monaster was induced by raising the intracellular Ca 2+ concentration to 3–4 times the above threshold levels in both eggs in normal sea water and eggs in Ca 2+ -free sea water. Eggs were not fertilized when the intracellular Ca 2+ concentration had been clamped below 0.1 μM by microinjecting calcium buffers before insemination. When the egg was put into sea water containing 5 mM procaine, the threshold Ca 2+ concentration for inducing the membrane-elevation increased. It was concluded that the rise of Ca 2+ concentration in the cytoplasm to a definite level is the primary cause of the activation of the sea urchin egg at fertilization.

MICROINJECTION OF COLCHICINE INTO SEA URCHIN EGGS

Inhibition of cleavage by colchicine was examined by microinjecting colchicine solution into one of the blastomeres of a sea urchin egg at the two‐cell stage. Cleavage was inhibited if the microinjection was made before a critical point prior to the cleavage, whereas cleavage occurred in spite of the destruction of the mitotic apparatus if the microinjection was made after the critical point. The critical point was 10 min before the mid‐stage of the cleavage in Clypeaster japonicus and 8 min before the mid‐stage in Temnopleurus toreumaticus at 20 ± 1°C, corresponding to the beginning of anaphase. The threshold for the cleavage inhibition of colchicine was estimated to be 3 × 10−5 M to 3 × 10−6 M in final concentration in the cell.

Microinjected Polystyrene Beads Move Along Astral Rays in Sand Dollar Eggs

Movements of polystyrene beads along astral rays of the sperm aster and the mitotic aster were investigated in eggs of the sand dollars, Clypeaster japonicus and Scaphechinus mirabilis. Polystyrene beads injected into the unfertilized egg were at a standstill in the protoplasm. After fertilization, these beads exhibited movements toward the center of the sperm aster along the rays, and finally gathered around the astral center. They were distributed in blastomeres together with the mitotic centers during successive cleavages. When injected into eggs during mitosis, beads moved to the centers of the mitotic asters along astral rays. The injected beads did not move when the aster was disorganized by treatment with Colcemid, and moved when it formed after UV‐irradiation. These results indicate that microtubules of astral rays are essential to the movement of polystyrene beads. The movement of small polystyrene beads (0.2–0.3 μm in diameter) resembled the saltatory movement of endogenous cytoplasmic granules, and the movement of large beads (ca. 1 μm in diameter) resembled the female pronuclear migration. All of these movements observed in fertilized eggs were demonstrated to be microtubule‐dependent, perhaps sharing the same basic mechanisms.

Aster formation in sand dollar eggs by micro-injection of calcium buffers and centriolar complexes isolated from starfish sperm☆

Abstract Centriolar complexes isolated from sperm of the starfish, Asterina pectinifera , could initiate irregular cleavage by injecting them into the fertilized eggs of the sand dollar, Scaphechinus mirabilis . Unfertilized sand dollar eggs could be activated when Ca 2+ concentration in the egg cytoplasm increased by means of microinjecting calcium buffers in which the concentration of free Ca 2+ was controlled by mixing calcium salt and calcium chelator at various proportions. When the centriolar complexes were injected into Ca 2+ -activated eggs, aster formation was induced in 25 out of the 55 eggs injected with calcium buffers at an intracellular Ca 2+ level above 2.9 μM. The number of asters detected in one egg was quite variable, ranging from 2 to 20. The pronucleus in injected eggs was seen to migrate towards the induced asters. Eight of those 25 eggs cleaved.

Displacement of cleavage plane in the sea urchin egg by locally applied taxol.

Taxol enhanced assembly and stability of microtubules in the mitotic apparatus and subsequently inhibited chromosome movement and cleavage when injected in the sea urchin egg as reported previously [Y. Hamaguchi et al., 1987: Cell Struct. Funct. 12:43-52]. In this study, to examine the local effect of taxol on cleavage, taxol was injected in small doses. When taxol was injected into the cortical region of the equatorial plane, the birefringence (BR) of the mitotic apparatus near the injection site increased, chromosome movement became slow near this site, and then cleavage furrow formation was inhibited in the cortex near the site, although chromosome movement and cleavage furrow formation were apparently normal in the other side of the cell. When taxol was injected in the polar cortex, BR of the mitotic apparatus near the injection site was enhanced and then the cleavage furrow was displaced toward this site from the equator. Accordingly, resultant blastomeres were unequal in size. The enhancement of microtubule assembly and stabilization of microtubule dynamics, which were detected as the increase in BR were confirmed by immunofluorescence microscopy with anti-tubulin antibody. Consequently, it was found that taxol injection caused local suppression of dynamics of microtubules in the cell, thereby modifying cleavage furrow formation.

Skeletogenic potential of induced secondary mesenchyme cells derived from the presumptive ectoderm in echinoid embryos

Abstract During the normal development of echinoids, an animal cap consisting of 8 mesomeres in a 16-cell stage embryo differentiates exclusively into ectoderm. Micromeres in an embryo at the same stage differentiate into primary mesenchyme cells (PMC) and coelomic pouch constituents. An animal cap and a quartet of micromeres were isolated from a 16-cell stage embryo and recombined to make a chimeric embryo devoid of presumptive endoderm and secondary mesenchyme cells (SMC). The PMC in the chimeric embryo were completely removed at the mesenchyme blastula stage. The PMC-depleted chimeric embryos formed an archenteron derived from the mesomeres. Some secondary mesenchyme-like cells (induced SMC) were released from the archenteron tip. A considerable fraction of the induced SMC formed the typical mesenchyme pattern after migrating into the vegetal region, synthesized skeletogenic mesenchyme cell-surface protein (msp130) and produced the larval skeleton. These findings indicate that induced SMC derived from the presumptive ectoderm have the same nature as natural SMC in both the timing of their release and their skeletogenic potential expressed in the absence of PMC.

Effects of intracellular ph on the mitotic apparatus and mitotic stage in the sand dollar egg

The effect of change in intracellular pH (pHi) on mitosis was investigated in the sand dollar egg. The pHi in the fertilized egg of Scaphechinus mirabilis and Clypeaster japonicus, which was 7.34 and 7.31, respectively, changed by means of treating the egg at nuclear envelope breakdown with sea water containing acetate and/or ammonia at various values of pH. The mitotic apparatus at pHi 6.70 became larger than that of normal fertilized eggs; that is, the mitotic spindle had the maximal size, especially in length at pHi 6.70. The spindle length linearly decreased when pHi increased from 6.70 to 7.84. By polarization microscopy, the increase in birefringence retardation was detected at slightly acidic pHi, suggesting that the increase in size of the spindle is caused by the increase in the amount of microtubules in the spindle. At pHi 6.30, the organization of the mitotic apparatus was inhibited. Furthermore, slightly acidic pHi caused cleavage retardation or inhibition. By counting the number of the eggs at various mitotic stages with time after treating them with the media, it is found that metaphase was persistent and most of the S. mirabilis eggs were arrested at metaphase under the condition of pHi 6.70. It is concluded that at slightly acidic pH, the microtubules in the spindle are stabilized and more microtubules assembled than those in the normal eggs.

Chromosomal behavior in starfish (Asterina pectinifera) zygotes under the effect of aphidicolin, an inhibitor of DNA polymerase

When calf thymus histones were labeled fluorescently and microinjected into oocytes of the starfish, Asterina pectinifera, the labeled histones visualized chromosomes during maturation division and cleavage. In doing so, we confirmed the previously reported phenomenon that chromosomes became incompetent at the first cleavage in the aphidicolin-treated egg, although cleavage itself took place. Moreover, we found that chromosomes were aligned at the equator of the metaphase spindle of the first cleavage and that they did not separate into two groups at all, but made a lump in the middle of the spindle. Chromosomes finally entered one blastomere, although they did not participate in the following karyokinesis. DNA and microtubules were examined by cytochemistry and immunofluorescence in order to investigate the relation between chromosome movement and the microtubular cytoskeleton. The mitotic apparatus developed and grew in the aphidicolin-treated cells in the same manner as those in normal cells without normal chromatin condensation or chromosome movement during the first cleavage. However, the mitotic apparatus consisted of two asters without the spindle formed at subsequent cleavages. Electron microscopic study revealed that chromosomes did not condense normally and kinetochores were not detected during the first cleavage. These results indicate that the dynamic changes in microtubular structures during mitosis have poor relation with the chromosome behavior such as prophase chromosome condensation and anaphase chromosome movement.

Asymmetry in the mitotic spindle induced by the attachment to the cell surface during maturation in the starfish oocyte

In order to study the dynamic behavior of the mitotic apparatus leading to unequal cleavage, we investigated the distribution of mitotic microtubules (MTs) during maturation division of starfish oocytes. When the mitotic apparatus attached to the cell surface at metaphase, in both the first and second meiotic division, it is revealed, by immunofluorescence, that the MT distribution in the spindle, as well as in the aster, became asymmetric. MTs in the peripheral half spindle increased in number compared with those in the inner half spindle. Furthermore, these results were confirmed in the living cell by polarization microscopy; shortly after the attachment, the birefringence retardation of the peripheral half spindle became greater than that of the inner one, and the difference increased with time during anaphase. By inhibiting the attachment of the mitotic apparatus by means of centrifugation, the MT distribution maintained a symmetrical pattern through mitosis. These results suggest that the attachment of the mitotic apparatus to the cell surface induces the asymmetrical distribution of MTs not only in the aster but also in the spindle. Such a rich distribution of MTs in the peripheral half spindle appears to ensure chromosome exclusion into the polar body by anchoring them firmly to the cell surface of the animal pole.