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  • calcium  (2)
  • video microscopy  (2)
  • 1
    Electronic Resource
    Electronic Resource
    Microtubule-associated motility in cytoplasmic extracts of sea urchin eggs (1993)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 24 (1993), S. 167-178 
    Details
    ISSN: 0886-1544
    Keywords: cytoplasmic dynein ; kinesin ; bundling ; crosslinking ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have developed a method for producing sea urchin egg cytoplasmic extracts which support substantial microtubule-associated motility, particularly minus end-directed motility characteristic of cytoplasmic dynein. Particles translocated along microtubules and axonemes predominantly in the minus end direction; microtubules and axonemes glided across the coverslip surface only in the plus end direction (as expected for a minus-end directed motor bound to the coverslip surface); and microtubules crosslinked into bundles in an antiparallel orientation. Velocities of particle and microtubule translocation were in the range of 0.5-1.8 μm/sec. Vanadate at 10 μM inhibited all gliding of the microtubules and axonemes, yet bidirectional particle transport persisted. Vanadate at concentrations of 25 μM and higher inhibited nearly all microtubule-based motility in the preparation and produced parallel bundling of the microtubules. Motility was slowed but not stopped in the presence of 5 mM AMP-PNP.Usually when a particle bound to a microtubule wall, it moved to the microtubule minus end. These particles often remained attached to the minus end. When a microtubule plus end in the shortening phase of dynamic instability reached a stationary particle on the microtubule, sometimes normal minus enddirected motility was activated, or at other times the particle remained attached to the shortening plus end. © 1993 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970240304
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  • 2
    Electronic Resource
    Electronic Resource
    Buffer conditions and non-tubulin factors critically affect the microtubule dynamic instability of sea urchin egg tubulin (1992)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 1-14 
    Details
    ISSN: 0886-1544
    Keywords: Pipes ; Hepes ; calcium ; VE-DIC microscopy ; cytoplasmic extracts ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The dynamic instability of individual microtubules (Mts) in cytoplasmic extracts or assembled from highly purified sea urchin egg tubulin was examined using video-enhanced, differential-interference contrast (VE-DIC) light microscopy. Extract Mts (endogenous tubulin = 12.1 μM) displayed only plus-ended growth. The elongation velocity was 7.8 μm/min for an average duration of 1.3 min before switching (catastrophe) to rapid shortening, which occurred at 13.0 μm/ min for an average duration of 0.5 min before switching (rescue) back to the elongation phase. These parameters are typical of interphase Mt dynamic instability. Surprisingly, Mts assembled from purified urchin egg tubulin in standard buffers were less dynamic that those reported for purified brain tubulin or Mts in the extract. Buffer parameters were changed in an attempt to mimic the extract Mt results. The pH buffer itself, Hepes or Pipes, drastically altered Mt dynamics but could not achieve high elongation velocity with high catastrophe frequencies. Calcium at 1 μM had negligible effects, while increasing pH from 6.9 to 7.2 stimulated elongation velocity. Finally, Mt dynamics of purified egg tubulin (11.9 μM) were assayed in ultrafiltiates (MW cut-off 〈30 kD) of the cytoplasmic extracts. Mts elongated slowly at 1.2 μm/min for 26 min before a catastrophe and rapid shortening at 11.8 μm/min. Rescue was less frequent than unfiltered extracts, minus-ended growth was observed, and self-assembly occurred at slightly higher tubulin concentrations. Therefore, the egg extracts and cytoplasm must contain non-buffer factors which stimulate elongation velocity by 6.5-fold without self-assembly, increase catastrophe frequency by 20-fold, and block minus-ended growth.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970210102
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  • 3
    Electronic Resource
    Electronic Resource
    Taxol stabilization of mitotic spindle microtubules: Analysis using calcium induced depolymerization (1984)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 4 (1984), S. 155-167 
    Details
    ISSN: 0886-1544
    Keywords: taxol ; microtubules ; mitosis ; mitotic spindle ; calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Taxol stabilizes or promotes the assembly of microtubules. In this report we characterize the rate, extent, and reversibility of taxol stabilization of calciumlabile microtubules in isolated mitotic spindles, principally from embryos of the sand dollar Echinarachnius parma. The intense depolymerizing action of 100 μM Ca2+ was used to assess the extent of stabilization by taxol. Changes in spindle microtubule assembly were evaluated and recorded by measuring changes in spindle birefringent retardation (BR). Membrane-free mitotic spindles, isolated with a calcium-chelating, nonionic detergent buffer, were stored in an EGTA-gylcerol storage buffer to prevent microtubule depolymerization. When perfused with an EGTA-buffer without glycerol, microtubules in these isolated spindles depolymerized gradually over 60-120 min; but in isolated spindles perfused with buffer that contained 100 μM Ca2+, BR decreased by 90% within 2-5 sec. In contrast, spindles that were pretreated for 3 min with 1 μM taxol, or for about 30 sec with 10 μM taxol, lost less than 10% of their initial BR when perfused with buffer containing 100 μM Ca2+. The rate and extent of microtubule stabilization by taxol depended on both the concentration and the duration of exposure to taxol. Taxol stabilization was reversible. After a 15 min preincubation with 1 μM or 10 μM taxol then washout, stability of spindle BR to 100 μM Ca2+ decreased exponentially with a time constant of 30-60 min. Thus taxol dissociates from spindle microtubules at significant rates; taxol-stabilized microtubules are not “fixed.”
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970040302
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  • 4
    Electronic Resource
    Electronic Resource
    Microtubule dynamics in the chromosomal spindle fiber: Analysis by fluorescence and high-resolution polarization microscopy (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 185-196 
    Details
    ISSN: 0886-1544
    Keywords: mitosis ; kinetochore ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24°C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24°C.In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into “rods” composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100123
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