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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 2 (1982), S. 103-113 
    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.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 241-249 
    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.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 6 (1986), S. 549-559 
    ISSN: 0886-1544
    Keywords: activation ; microinjection ; polar body ; sea urchin eggs ; starfish oocytes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Redistribution of alpha-actinin during fertilization was investigated by means of the microinjection of fluorescently labeled egg alpha-actinin in the sea urchin, Hemicentrotus pulcherrimus. Upon fertilization, labeled alpha-actinin accumulated locally around the sperm binding site, where the fertilization cone formed soon afterwards. The accumulation propagated all over the cortex within 10 sec after a latent period of 10-20 sec. When an egg in Na-free seawater was injected with both alpha-actinin and calcium buffer (intracellular free Ca2+ concentration = 9 μM), the accumulation of alpha-actinin was similar to that in normal seawater, which suggests that the accumulation did not depend on the increase in intracellular pH but only on the increase in the intracellular free Ca2+ concentration. In immature oocytes the accumulation was detected in the cortical region, including the huge protruding cytoplasm where the sperm entered. When labeled egg alpha-actinin was injected into starfish (Asterias amurensis) oocytes followed by insemination, it accumulated in the cortical layer in a manner similar to the case of sea urchin, except that the accumulation in fertilization cones of maturing oocytcs or reception cones of immature oocytes appeared ringlike and rodlike, respectively. Moreover, just after the arrival of the meiotic apparatus, egg alpha-actinin accumulated in the cortical region, where the formation of the polar body was expected. This suggests that the meiotic apparatus somehow induced the differentiation of the cortex so as to form a polar body. It is concluded that the cortical region where alpha-actinin accumulated coincided with the microfilament-rich region. This suggests that alpha-actinin plays a role in forming the cortical meshwork of actin filaments.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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