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  • 1
    Electronic Resource
    Electronic Resource
    Identity and polymerization-stimulatory activity of the nontubulin proteins associated with microtubules (1977)
    s.l. : American Chemical Society
    Biochemistry 16 (1977), S. 2598-2605 
    Details
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1021/bi00631a004
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  • 2
    Electronic Resource
    Electronic Resource
    Direct observation of microtubule dynamics in living cells (1988)
    [s.l.] : Nature Publishing Group
    Nature 332 (1988), S. 724-726 
    Details
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] To observe microtubule dynamics, single microtubules must be detected with adequate clarity. To confirm that we could detect single fluorescent microtubules, in vitro preparations were observed by both light and electron microscopy. Figure 1 a shows microtubules assembled from a mixture of 5- and ...
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1038/332724a0
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  • 3
    Electronic Resource
    Electronic Resource
    Self-centring activity of cytoplasm (1997)
    [s.l.] : Nature Publishing Group
    Nature 386 (1997), S. 170-173 
    Details
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] Nascent fragments of cultured black tetra melanophores, which had been induced to aggregate, initially formed the aggregate at the cut edge. Remarkably, after 2-3 min delay, the aggregate relocated to the centre (Fig. la) with kinetics approximated by a single declining exponential (f1/2 = 150 ...
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1038/386170a0
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  • 4
    Electronic Resource
    Electronic Resource
    The attachment of kinetochores to the pro-metaphase spindle in PtK1 cells (1981)
    Springer
    Chromosoma 82 (1981), S. 693-716 
    Details
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract When late prophase PtK1 cells are chilled to 6 ° C the nuclear envelope (NE) breaks down as in normal cells but the spindle is inhibited from forming. When these cells are subsequently warmed to 18 ° C the spindle slowly forms and pro-metaphase congression ensues. Using this approach we have been able to experimentally eliminate the influence of asynchronous NE breakdown on the formation and development of the spindle, and also to slow down (and thus increase the temporal separation of) the subsequent events which occur during the initial stages of spindle formation. Correlative light and high voltage electron microscopic studies on these cells, fixed after various times of recovery, reveal the following results: 1) the centrosomes generate microtubules (MTs) well before MTs are seen to be associated with the kinetochores; 2) as in untreated PtK1 cells (Roos, 1973a, 1976) the order in which chromosomes attach to the forming spindle is influenced by their proximity to a centrosome-kinetochores closest to a centrosome appear stretched towards the centrosome at a time during recovery when other kinetochores, more distal to the centrosome appear unstretched and unoriented; 3) as in untreated cells (Heneen, 1970; Roos, 1976) the predominant behavior during recovery is for a chromosome to initially mono-orient and associate with the near centrosome and only later to develop a bipolar association; and 4) MTs associated with early pro-metaphase kinetochores are almost always oriented towards a centrosome. — From our results we conclude that the proximity effect and the tendency of pro-metaphase chromosomes in PtK1 to initially mono-orient and associate with the near centrosome cannot be ascribed, as suggested by Roos (1976), to influences arising during the asynchronous breakdown of the NE. Rather, our data clearly demonstrate that a kinetochore-centrosome interaction occurs during spindle formation which cannot be attributed to transient influences. The proximity effect and the predominant tendency of PtK1 pro-metaphase chromosomes to mono-orient to the near pole are taken to signify the existance of a centrosomal influence on the attachment and orientation of chromosomes. Two possible mechanisms for this influence, both involving a structural interaction between the centrosome and the kinetochore, are outlined.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00285776
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  • 5
    Electronic Resource
    Electronic Resource
    Structure of kinetochore fibers: Microtubule continuity and inter-microtubule bridges (1981)
    Springer
    Chromosoma 83 (1981), S. 523-540 
    Details
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract To understand how microtubules interact in forming the mitotic apparatus and orienting and moving chromosomes, the precise arrangement of microtubules in kinetochore fibers in Chinese hamster ovary cells was examined. Individual microtubules were traced, using high voltage electron microscopy of serial 0.25 μm sections, from the kinetochore toward the pole. Microtubule arrangement in kinetochore fibers in untreated mitotic cells and in cells recovering from Colcemid arrest were similar in two respects: the number of microtubules per kinetochore (mean 14 and 12, respectively) and the nearest neighbor intermicrotubule distance (mean∼90 nm). In Colcemid recovered cells, over 90% of the microtubules in kinetochore fibers were attached to the kinetochore (i.e. kinetochore microtubules) and extended most or all of the distance to the pole. Few free microtubules were present in the kinetochore fibers; most non-kinetochore microtubles terminated in the pole. Since kinetochores in this Colcemid-recovered system have been demonstrated to nucleate microtubules (Witt et al., 1980), it seems likely that most if not all of these kinetochore microtubules originated at the kinetochore. Some of the reconstructed kinetochore fibers were attached to chromosomes with bipolar orientation, suggesting that kinetochore microtubules need not interact with many polar microtubules for orientation to occur. In Colcemid recovered cells lysed to reduce cytoplasmic background, microtubules in kinetochore fibers were preferentially preserved. The parallel and near-hexagonal order typical of microtubules in kinetochore fibers was maintained, as was the number of kinetochore microtubules (mean, 13). The intermicrotubule distance was slightly reduced in lysed cells (mean, 60 nm). Crossbridges about 5 nm wide and 30–40 nm long were visible in kinetochore fibers of lysed cells. Such crossbridges probably contribute to the stabilization and parallel order of microtubules in kinetochore fibers, and may have a functional role as well.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00328277
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  • 6
    Electronic Resource
    Electronic Resource
    Origin of kinetochore microtubules in Chinese hamster ovary cells (1980)
    Springer
    Chromosoma 81 (1980), S. 483-505 
    Details
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We have attempted to determine whether chromosomal microtubules arise by kinetochore nucleation or by attachment of pre-existing microtubules. The appearance of new microtubules was investigated in vivo on kinetochores to which microtubules had not previously been attached. The mitotic apparatus of Chinese hamster ovary cells was reconstructed in three dimensions from 0.25 μm thick serial sections, and the location of chromosomes, kinetochore outer disks, centrioles, virus-like particles and microtubules determined. Central to the interpretation of these data is a synchronization scheme in which cells entered Colcemid arrest without forming mitotic microtubules. Cells were synchronized by the excess thymidine method and exposed to 0.3 μg/ml Colcemid for 8 h. Electron microscopic examination showed that this Colcemid concentration eliminated all microtubules. Mitotic cells were collected by shaking off, and cell counts showed that over 95% of the cells were in interphase when treatment began and thus were arrested without the kinetochores having been previously attached to microtubules. Cells were then incubated in fresh medium and fixed for high voltage electron microscopy at intervals during recovery. — In early stages of recovery, short microtubules were observed near and in contact with kinetochores and surrounding centrioles. Microtubules were associated with kinetochores facing away from centrosomes and far from any centrosomal microtubules, and thus were not of centrosomal origin. At a later stage of recovery, long parallel bundles of microtubules, terminating in the kinetochore outer disk, extended from kinetochores both toward and away from centrosomes. Because microtubules had never been attached to kinetochores, the possibility that kinetochore microtubles were initiated by microtubule stubs resistant to Colcemid was eliminated. Therefore we conclude that mammalian kinetochores can initiate microtubules in vivo, thus serving as microtubule organizing centers for the mitotic spindle, and that formation of kinetochore-microtubule bundles is not dependent on centrosomal activity.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00368158
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  • 7
    Electronic Resource
    Electronic Resource
    Decoration of microtubules by fluorescently labeled microtubule-associated protein 2 (MAP2) does not interfere with their spatial organization and progress through mitosis in living fibroblasts (1986)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 6 (1986), S. 570-579 
    Details
    ISSN: 0886-1544
    Keywords: microinjection ; mitosis ; microtubule-associated proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubule-associated protein 2 (MAP2) derivatized with iodoacetamidotetramethylrhodamine or with iodoacetamidofluorescein binds to microtubules after injection into living interphase cells [Scherson et al, 1984]. The binding of derivatized MAP2 stabilized microtubules in vitro; it was therefore important to check if the binding of MAP2 in vivo perturbed the dynamics and organization of the microtubule network. We have addressed these questions by studying the effect of the injection of derivatized MAP2 on mitosis in PtK 1 cells and on the recovery of the microtubule network from low temperature incubation in interphase cells. We found that the presence of derivatized MAP2 did not change the duration of any mitotic stage and that the injected cell normally completed mitosis. We subsequently showed that the injected MAP2 bound to the microtubules within 5 minutes after injection and remained bound throughout the course of mitosis. The reorganization of the microtubule network upon cooling and rewarming was studied in the cytoplasm of human foreskin fibroblasts (356 cells). During the recovery, the distribution of the fluorescent MAP2 in living cells was identical with the microtubule pattern visualized by immunofluorescence in lysed and fixed cells.In these experiments, the fluorescent MAP2 bound to microtubules can be considered as a nonperturbing reporter of the microtubule network. This result is discussed in terms of the role of MAPs in the dynamics and organization of microtubules in living cells.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970060605
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  • 8
    Electronic Resource
    Electronic Resource
    FRAP analysis of the stability of the microtubule population along the neurites of chick sensory neurons (1993)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 25 (1993), S. 59-72 
    Details
    ISSN: 0886-1544
    Keywords: microtubule dynamics ; photobleaching ; neurite elongation ; microtubule stability ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In order to study microtubule turnover in elongating neurites, chick embryo sensory neurons were microinjected with x-rhodamine tubulin, and after 6-12 hours, short segments along chosen neurites were photobleached at multiple sites. Previous studies [Lim et al., 1989; 1990] indicated that recovery of fluorescence (FRAP) in neurites occurs by the dynamic turnover of stationary microtubules. In all cases, distal bleached zones recovered fluorescence faster than bleached zones more proximally located along the same neurites. Bleached zones at growth cones completely recovered in 30-40 minutes, while bleached zones located more proximally usually recovered in 50-120 minutes. In the most proximal regions of long neurites, recovery of fluorescence was often incomplete, indicating that a significant fraction of the microtubules in these regions were very stable. These studies indicate that there are differences in microtubule stability along the lenght of growing neurites. These differences may arise from the combined effects of (1) modifications that stabilize and lengthen microtubules in maturing neurites and (2) the dynamic instability of the distally oriented microtubule plus ends. © 1993 Wiley-Liss, Inc.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970250108
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  • 9
    Electronic Resource
    Electronic Resource
    Independence of centriole formation and initiation of DNA synthesis in Chinese hamster ovary cells (1986)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 6 (1986), S. 355-362 
    Details
    ISSN: 0886-1544
    Keywords: centriole ; DNA synthesis ; cell cycle ; Chinese hamster ovary cells ; taxol ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The relationship between centriole formation and DNA synthesis was investigated by examining the effect of taxol on the centriole cycle and the initiation of DNA synthesis in synchronized cells. The centriole cycle was monitored by electron microscopy of whole-mount preparations [Kuriyama and Borisy, J. Cell Biol., 1981, 91:814-821]. A short daughter centriole appeared in perpendicular orientation to each parent during late G1 or early S and elongated slowly during S to G2. Addition of 5-20 μg/ml taxol to a synchronous population of cells in S phase did not inhibit centriole elongation; rather, elongation was accelerated. In contrast, when taxol was added to M phase or early G1 cells, centriole duplication was completely inhibited. The taxol block was reversible since nucleation and elongation of centrioles resumed as soon as the drug was removed. Cells exposed to taxol progressed through the cell cycle and became blocked in mitosis, as indicated by an increase in the mitotic index, but eventually the mitotic arrest was overcome, resulting in formation of multinucleated cells. A peak in mitotic index was seen in the following generation, indicating that chromosomes duplicated in the presence of taxol. Incorporation of 3H-thymidine followed by autoradiography confirmed that DNA synthesis was initiated in the presence of taxol even though formation of daughter centrioles was inhibited. It seems, therefore, that centriole duplication is not a prerequisite for entry into S phase. Since DNA synthesis has already been demonstrated not to be necessary for centriole duplication, these two events, normally coordinated in time, appear to be independent of each other.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970060402
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  • 10
    Electronic Resource
    Electronic Resource
    Detection of single fluorescent microtubules and methods for determining their dynamics in living cells (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 237-245 
    Details
    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.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100128
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