Microtubule assembly in living cells after release from nocodazole block: The effects of metabolic inhibitors, taxol and PH

Brabander, M. De; Geuens, G.; Nuydens, Rony; Willebrords, R.; Mey, J. De

doi:10.1016/0309-1651(81)90206-x

Cited by 90 publications

(52 citation statements)

References 14 publications

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“…To ensure that our protocol could in fact detect a persistent bleached zone indicating stable microtubules, we photobleached meiotic midbodies obtained after artificial activation of injected oocytes. Microtubules of mitotic midbodies exhibit extraordinary stability to depolymerization by microtubule inhibitors such as nocodazole (30). Thus, as expected, microtubules of the oocyte midbody exhibited limited fluorescence recovery after photobleaching, averaging 22% ± 21% (mean ± SD) (data not shown).…”

Section: Resultsmentioning

confidence: 84%

Microtubules in the metaphase-arrested mouse oocyte turn over rapidly.

Gorbsky

Simerly

Schatten

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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After ovulation mammalian oocytes arrest in second meiotic metaphase. We asked whether the microtubules that comprise the meiotic spindle of mouse oocytes were stable or were undergoing rapid cycles of assembly and disassembly. Porcine brain tubulin, derivatized with biotin or x-rhodamine [5-(and -6)-carboxy-x-rhodamine], was microhijected into living oocytes. Biotinylated tubulin incorporated into the meiotic spindle to apparent equilibrium within 15 min. To assess quantitatively the rates of disassembly and assembly of the microtubules, small domains within the spindles of oocytes injected with x-rhodamine-tubulin were photobleached and their recovery was analyzed by digital imaging microscopy. Fluorescence recovery in the spindles was rapid and extensive, plateauing to an average of 83% at 4 min. The calculated half-time for turnover of the spindle microtubules was 77 sec. In contrast, fluorescence recovery of the spindle midbodies in telophase oocytes was much more limited, averaging '=22% at 4 min. These data indicate that most microtubules within the arrested metaphase spindle of the mouse oocyte undergo rapid cycles of assembly and disassembly. Microtubules of the telophase midbody are more stable.Recent studies of living cells microinjected with fluorescent tubulin (1-4) have eroded the notion that microtubules are static structures. For the bulk of the microtubule array in cultured cells in interphase, estimates of the half-time for disassembly and reassembly range from 5 to 15 min (3-5). Rapid length changes at the ends of microtubules, growth at 3.5-7.2 pum/min and disassembly at 4.3-18.5 Aum/min, have been directly observed in the thin lamellae of well-spread cells (6-8). Microtubule turnover is yet more rapid in the mitotic metaphase spindles of living mammalian cells and early echinoderm embryos, exhibiting half-times from 15 to 70 sec (1, 2, 9, 10). This rapid assembly and disassembly of microtubules may play an important role in the proper positioning and segregation of the chromosomes.In contrast to the generally inexorable transit of cells through mitosis, oocytes are often arrested at particular stages of meiosis that are characteristic for each species (11). In mammals such as mice, mature oocytes within the ovary are arrested at meiotic prophase. The oocyte, under the influence of luteinizing hormone, is released from the ovary and undergoes the first meiotic division with the production of the first polar body. The egg then enters another period of stasis in development, arresting at second meiotic metaphase as it moves into the oviduct. The egg may remain arrested in this state for several hours awaiting fertilization and final maturation. Recently it was shown that expression of the c-mos protooncogene is the "cytostatic factor" that arrests Xenopus oocytes in metaphase of meiosis II (12). The mosencoded protein was found to be proteolyzed upon fertilization of Xenopus eggs (13). Presumably, destruction of the mos protein allows progression through meiosis and initiation of the mi...

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Section: Resultsmentioning

confidence: 84%

Microtubules in the metaphase-arrested mouse oocyte turn over rapidly.

Gorbsky

Simerly

Schatten

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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“…To determine whether Mad2 binds to the kinetochore outer domain, we took advantage of the morphological changes that occur for unattached kinetochores when depleted of spindle microtubules for several hours by inhibition of microtubule assembly with nocodazole (DeBrabender et al, 1981;Thrower et al, 1996). We found that a 4-h treatment of PtK1 cells in 20 M nocodazole caused Mad2 along with CENP-E, cytoplasmic dynein, and Bub1R, but not the inner core CREST antigens, to assemble into an expanded crescent, or C-shape ( Figure 1).…”

Section: Mad2 Like Cenp-e Cytoplasmic Dynein Bubr1 Bub3 and 3f3/mentioning

confidence: 99%

Microtubule-dependent Changes in Assembly of Microtubule Motor Proteins and Mitotic Spindle Checkpoint Proteins at PtK1 Kinetochores

Hoffman

Pearson

Yen

et al. 2001

MBoC

332

351

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The ability of kinetochores to recruit microtubules, generate force, and activate the mitotic spindle checkpoint may all depend on microtubule-and/or tension-dependent changes in kinetochore assembly. With the use of quantitative digital imaging and immunofluorescence microscopy of PtK1 tissue cells, we find that the outer domain of the kinetochore, but not the CREST-stained inner core, exhibits three microtubule-dependent assembly states, not directly dependent on tension. First, prometaphase kinetochores with few or no kinetochore microtubules have abundant punctate or oblate fluorescence morphology when stained for outer domain motor proteins CENP-E and cytoplasmic dynein and checkpoint proteins BubR1 and Mad2. Second, microtubule depolymerization induces expansion of the kinetochore outer domain into crescent and ring morphologies around the centromere. This expansion may enhance recruitment of kinetochore microtubules, and occurs with more than a 20-to 100-fold increase in dynein and relatively little change in CENP-E, BubR1, and Mad2 in comparison to prometaphase kinetochores. Crescents disappear and dynein decreases substantially upon microtubule reassembly. Third, when kinetochores acquire their full metaphase complement of kinetochore microtubules, levels of CENP-E, dynein, and BubR1 decrease by three-to sixfold in comparison to unattached prometaphase kinetochores, but remain detectable. In contrast, Mad2 decreases by 100-fold and becomes undetectable, consistent with Mad2 being a key factor for the "wait-anaphase" signal produced by unattached kinetochores. Like previously found for Mad2, the average amounts of CENP-E, dynein, or BubR1 at metaphase kinetochores did not change with the loss of tension induced by taxol stabilization of microtubules.

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“…According to their working mechanisms, chemodrugs can be divided into several categories; one includes microtubule inhibitors such as paclitaxel (Taxol) (16), vincristine (17), and JIMB01 (18). These microtubule-disrupting agents are thought to arrest cells in mitosis by triggering mitotic checkpoint activation, resulting cells arrested in mitotic phase without entering anaphase (19).…”

Section: Introductionmentioning

confidence: 99%

Inhibition of synuclein-γ expression increases the sensitivity of breast cancer cells to paclitaxel treatment

Zhou

Inaba²,

Li³

2006

Int J Oncol

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Abstract. Anti-microtubule drugs that cause mitotic arrest and subsequent apoptosis of cancer cells are frequently used to treat breast cancer patients with advanced or metastatic diseases. However, patient response rates to this class of chemotherapeutic agents vary significantly. Identification of cellular and genetic factors that are associated with the sensitivity to anti-microtubule drug treatment would have great clinical implications. Our previous studies have demonstrated that the neuronal protein, synuclein-Á (SNCG), plays oncogenic roles in breast carcinogenesis and is abnormally expressed at high levels in advanced and metastatic breast carcinomas but not expressed in normal or benign breast tissues. In this study, we show that responses of 12 breast cancer cell lines to paclitaxel-induced mitotic arrest and cytotoxicity highly correlate with SNCG expression status. SNCG-positive cells exhibit a significant higher resistance to paclitaxel-induced mitotic arrest than SNCG-negative cells (p<0.01). Moreover, we demonstrate that down-regulation of SNCG expression directly increased the effectiveness of anti-microtubule druginduced cytotoxicity in breast cancer cells without altering cell responses to doxorubicin. These new findings suggest that SNCG expression in breast carcinomas is probably a causal factor contributing to the poor patient response to paclitaxel treatment.

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Microtubule assembly in living cells after release from nocodazole block: The effects of metabolic inhibitors, taxol and PH

Cited by 90 publications

References 14 publications

Microtubules in the metaphase-arrested mouse oocyte turn over rapidly.

Microtubules in the metaphase-arrested mouse oocyte turn over rapidly.

Microtubule-dependent Changes in Assembly of Microtubule Motor Proteins and Mitotic Spindle Checkpoint Proteins at PtK1 Kinetochores

Inhibition of synuclein-γ expression increases the sensitivity of breast cancer cells to paclitaxel treatment

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