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  • 1
    ISSN: 0886-1544
    Keywords: actin ; cleavage ; fluorescein-labeled phalloidin ; microinjection ; phalloidin ; sand dollar eggs ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Effects of microinjection of phalloidin on fertilization and cleavage of sand dollar (Clypeaster japonicus and Scaphechinus mirabilis) eggs were studied. The drug, previously injected into unfertilized eggs, showed no effect on the elevation of the fertilization membrane upon insemination up to an intracellular concentration of 50 μM. However, the movement of the egg pronucleus to the sperm pronucleus was inhibited and the fusion of pronuclei did not occur. The subsequent development no longer took place. When phalloidin was injected into fertilized eggs, the thickness of the cortical layer increased and the microvilli became conspicuous. Both nuclear division and cleavage were inhibited at the intracellular concentration of more than 20 μM, though the latter seemed to be more sensitive to phalloidin than the former.Fluorescein-labeled phalloidin (FL-phalloidin) was injected into eggs in order to investigate F-actin localization by fluorescence microscopy. In both unfertilized and fertilized eggs, FL-phalloidin was localized in the cortical layer within 1 min after injection. It was also localized in the cortical layer as radially oriented rodlike structures when injected into fertilized eggs before the disappearance of the nuclear membrane. No distinct fluorescence was detected in the mitotic apparatus or in the cleavage furrow. FL-phalloidin redistributed gradually into egg cytoplasm. In unfertilized eggs, fluorescent rods were found especially in the egg pronucleus 30 min after injection.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0886-1544
    Keywords: birefringence ; polarizing microscope ; sea urchin egg ; cortex ; mitosis ; cleavage ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Birefringence (BR) at the cell surface of fertilized eggs of the sand-dollar, Clypeaster japonicus, during mitosis and cleavage was determined with a photoelectric BR detection apparatus [Hiramoto et al, 1981a]. The cortex of about 2 μm thickness is birefringent positive with respect to the normal to the cell surface. The hyaline layer is negatively birefringent. The halo-layer consisting of a row of microvilli surrounding the egg is positively birefringent in normal Ca-free sea water, while it is negatively birefringent in Ca-free sea water with high refractive index. The BR of the cortex gradually increases over the entire surface during mitosis until the onset of cleavage. The BR of the cortex at the polar region reaches a maximum shortly after the onset of cleavage and then decreases, while the BR of the cortex at the equatorial region begins to decrease shortly before the onset of cleavage, reaches a minimum shortly after the cleavage starts, and then increases again as the cleavage furrow advances. The coefficient of birefringence of the cortex is about 2.5 × 10-5 at the maximum. The BR change of the cortex during mitosis and cleavage is interpreted as a passive deformation caused by the constriction of the contractile ring as well as an active structural change of the cortex occurring in the dividing cell.
    Additional Material: 5 Ill.
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  • 3
    ISSN: 0886-1544
    Keywords: immunofluorescence ; microinjection ; mitotic apparatus ; monoclonal antibodies ; sand dollar egg ; tubulin isotypes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effect on fixation on the reactivities of mitotic microtubules with monoclonal anti-tubulin antibodies was investigated by the indirect immunofluorescence procedure. All of the seven antibodies used intensely stained mitotic microtubules in sea urchin eggs lysed and fixed with methanol at -20°C, whereas only two of them stained the stabilized microtubules in the lysed eggs before the fixation. The other five did not stain the mitotic microtubules even after microtubule components other than tubulin were removed by treating the lysed eggs with 0.4 M KCl solution containing taxol. These results exclude the possibility that the fixation affects proteins, which interact with microtubules including microtubule-associated proteins (MAPs) and interfere with the binding of monoclonal antibodies with tubulin, and strongly suggest that the fixation directly affects the three-dimensional conformation of tubulin Furthermore, microinjection of these antibodies indicated the results as follows [combining the results reported previously; Oka et al., 1990: Cell Struct. Funct. 15: 373-378]: The antibodies which stained mitotic microtubules stabilized in the lysed eggs induced disassembly of native mitotic microtubules in the living eggs, but those which did not stain the stabilized microtubules did not disassemble the native microtubules. From these results, it is suggested that the monoclonal antibodies which stain microtubules in the eggs lysed but not fixed are useful for microinjection experiments. © 1994 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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