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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 7 (1987), S. 272-281 
    ISSN: 0886-1544
    Keywords: intranuclear mitosis ; spindle formation ; cell cycle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Tubulin synthesis in the naturally synchronous plasmodium of Physarum polycephalum is a markedly periodic event restricted to the late G2 period of the cell cycle. Mitosis in the plasmodium is intranuclear, and there are no cytoplasmic microtubules at any stage of the cell cycle. We have combined a biochemical investigation of the synthesis of the plasmodial tubulin isotypes and their participation in the mitotic spindle with a microscopic study (immunofluorescence) of the development of spindle microtubules throughout the cell cycle.We have shown that all four tubulin isotypes identified in the plasmodium (α1, α2, β1 and β2) are present in the mitotic spindle. The stoichiometry of isotype usage in the mitotic spindle generally reflects the overall abundance of isotypes in the plasmodium as a whole: β2 〉 α1 〉 α2 〉 β1. We have also shown that tubulins synthesized in the G2 period of one cell cycle can be incorporated into the spindles of the immediately ensuing mitosis and have sufficient biological longevity to allow participation in the mitotic divisions of future cell cycles. Thus, the phenomenon of periodic tubulin synthesis does not reflect a restricted use of tubulin to the cell cycle in which it was synthesized. The major polymerization of tubulin in the nucleus occurred less than 30 min before metaphase. A novel tubulin-containing structure was, however, present in the nucleus approximately 60 min before metaphase. Polymerized tubulin is rapidly removed from the nucleus following nucleokinesis.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 8 (1987), S. 182-189 
    ISSN: 0886-1544
    Keywords: Golgi apparatus ; microtubule-organizing center ; G-glycoprotein ; cytochalasin D ; monensin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: This paper is concerned with the proposition that the insertion of membrane mass into the leading edge of a motile cell plays a critical role in directed cell migration. We show by immunofluorescence, with cells transfected with a cloned cDNA encoding the G-protein of a temperature-sensitive mutant of vesicular stomatitis virus, that the first cell surface appearance of the G-protein is indeed at the leading edge of the motile cell. Two drugs capable of inhibiting directed cell migration, cytochalasin D and monensin, appear to function independently, the former by affecting the actin cytoskeleton without affecting the polarized insertion of membrane mass into the cell surface and the latter by abrogating membrane mass insertion without affecting the actin cytoskeleton.
    Additional Material: 5 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 8 (1987), S. 193-209 
    ISSN: 0886-1544
    Keywords: growth cone ; microtubules ; actin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Neurite elongation involves two distinct cytoskeletal functions the “push” of anterograde transport of the cytoskeleton and associated organelles to the neurite tip, and the “pull” exerted by protrusion and generation of tensions in the growth cone. We investigated the roles of these two activities in neurite elongation via the drugs taxol and cytochalasin B (CB), which act on the key cytoskeletal components, microtubules and actin filaments, respectively. When neurons are treated with concentrations of CB below 0.2 μg/ml, neurite elongation, growth cone protrusion, and neurite tension are all inhibited in a similar concentration dependent manner. Protrusive activity and tensions are absent at CB concentrations above 0.3 μg/ml, yet neurite elongation continues at a plateau level. Thus, “pull” does modulate, but it is not required for neurite elongation. Surprisingly, the inhibitory effects of taxol on neurite elongation are removed by the addition of CB at levels that substantially disrupt the actin filaments of neurites. The neurites extended by taxol-CB neurons are unbranched and curiously unattached to the substratum. When CB is added to taxol-treated neurons, neurite extension begins rapidly, even if protein synthesis is severely reduced. We propose that taxol inhibits microtubule transport in neurites, and this inhibition of “push” is reversed by the disruptive effects of CB on the cytoplasmic matrix, allowing taxol-induced microtubule bundles to be transported distally.
    Additional Material: 13 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 7 (1987), S. 31-38 
    ISSN: 0886-1544
    Keywords: microtubule assembly ; proleolysis ; Vinca drugs ; Zn2+-induced assembly ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Limited proteolysis of tubulin with subtilisin results in cleavage of both the α and β subunits, releasing small peptides from the C-terminal ends. At 37°C the digested tubulin assembles into polymorphic structures: microtubules with attached ribbons in the presence of GTP, rings in the presence of GDP, and protofilament spirals in the presence of vinblastine. Undigested tubulin does not assemble under these conditions. Rings and Vinca-induced spiral structures are assembled from undigested tubulin only when microtubule-associated proteins, high Mg2+ concentrations, or polycations are present. Thus, cleavage with subtilisin affects assembly in a manner similar to the addition of these agents. It appears that binding of positively charged substances may act by neutralizing the charge on the highly acidic C-terminal regions of the α- and β-subunits, while cleavage with subtilisin produces the same effect by removing these peptides. Undigested and subtilisin-digested tubulin form sheets of protofilaments in the presence of Zn2+, which indicates that the binding sites for the 2-3 Zn2+ ions necessary to induce sheet formation do not reside in the C-terminal regions of the monomers.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 8 (1987), S. 143-154 
    ISSN: 0886-1544
    Keywords: amphibian egg ; Nile blue stain ; microtubules ; subcortial rotation ; cytoplasmic movement ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The amphibian egg undergoes a 30° rotation of its subcortical contents relative to its surface during the first cell cycle, a displacement of 350 μm in 50 min. This is directly visualized by following the movement of an array of Nile blue (a subcortical stain) spots applied to the egg periphery (Vincent, Oster, and Gerhart: Dev Bio 113:484-500, '86). We have investigated the mechanochemical basis of this unusual cell motility. Subcortical rotation depends on microtubule integrity during its entire course and is insensitive to inhibitors of microfilament assembly. It does not depend on newly synthesized proteins for its operation or timing, and it does not involve calcium-dependent processes. Finally, we show that vegetal fragments of the egg can complete rotation on their own, indicating that mechanochemical components can operate locally in this hemisphere.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 237-245 
    ISSN: 0886-1544
    Keywords: video microscopy ; digital image processing ; fluorescence photobleaching ; microtubule dynamics ; living cell dynamics ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The ability to tag biological molecules fluorescently and to detect their distribution in living cells has promoted the study of cytoplasmic organization in general and microtubule dynamics in particular. The techniques that we have selected and developed allowed the determination of spatial and temporal changes of the microtubule network in living fibroblasts at the level of individual microtubules. We have employed two general approaches for determining pattern changes: direct video microscopy and photobleaching and subsequent observation. Direct observation of fluorescent microtubules by high-definition video microscopy provided good spatial resolution at several time points, but was limited to the less congested and thinner periphery of the cell. This approach was made possible by a relatively bright, photostable reporter, xrhodamine-tubulin, and showed that microtubules underwent rounds of assembly and disassembly from their ends. Bleaching and subsequent observation of lysed cells improved the signal to noise ratio by extracting soluble chromophore and permitted observations in congested areas, but was limited to a single time interval. This approach demonstrated that microtubule domains were replaced one by one and that turnover was most rapid at the cell periphery. Antibodies specific for nonbleached chromophore can be used to enhance the signal to noise ratio further or to extend spatial resolution by the use of immunoelectron microscopy. Direct video microscopy and photo-bleaching are two approaches to the study of dynamics that have complementary strengths and wide application to the biology of living cells.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 11 (1985), S. 191-221 
    ISSN: 0148-7280
    Keywords: ovulation ; follicle cell ; granulose cell ; viviparity ; mammal arthropod ; lower vertebrate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Additional Material: 17 Ill.
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  • 8
    ISSN: 0148-7280
    Keywords: rhesus monkey ; Macaca mulatta spermatozoa ; capacitation ; zona-free ; hamster egg penetration ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A major problem in development of nonhuman primate in vitro fertilization is the selection of donor males and repeated collection of consistent sperm samples. In practice, collection of a viable semen sample is highly dependent on operator technique and the type of animal restraint. We report an updated method for semen collection from the rhesus monkey (Macaca mulatta), use of TES-Tris (TEST) Yolk Buffer (TYB) for prolonged sperm storage and improved results of hamster ovum penetration assay. Semen was obtained from adult males restrained with 2.0 mg/kg IM ketamine hydrochloride prior to direct penile stimulation (Grass SD-9, frequency 150, delay 9, duration 7, volts 12-18, repeat mode, twin pulse). Liquified semen was washed and centrifuged twice at 100 × g for 5 min in BWW, Ham's F-10 and TALP and allowed to swim-up 60 min at 37° in 5% CO2 and air. Alternatively, semen was mixed 1:1 with TYB, refrigerated 20 h at 4°C, centrifuged at 100 × g for 5 min, and the pellet resuspended in 1.0 ml of TALP or BWW prior to use. Hamster ova penetration was achieved with capacitated macaque sperm. Penetration was significantly improved (P 〈 .001) with preincubation in TYB followed by resuspension in TALP (79%).
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    BioEssays 2 (1985), S. 32-34 
    ISSN: 0265-9247
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: During development some cells are migratory whilst others are stationary. However, the same cell may change its behaviour depending upon its environment. Recent evidence has implicated the extracellular matrix protein fibronectin in the regulation of migratory behaviour. As the structure of this molecule becomes elucidated, it is also becoming possible to interpret this regulation in precise molecular terms.
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  • 10
    ISSN: 0265-9247
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
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