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Spindle Assembly (spindle + assembly)

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Life Sciences100%

Terms modified by Spindle Assembly

  • spindle assembly checkpoint
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    Selected Abstracts

    TOGp regulates microtubule assembly and density during mitosis and contributes to chromosome directional instability

    CYTOSKELETON, Issue 8 2009
    Lynne Cassimeris
    Abstract TOGp, a member of the XMAP215 MAP family, is required for bipolar mitotic spindle assembly. To understand how TOGp contributes to spindle assembly, we examined microtubule dynamics after depleting TOGp by siRNA. Fluorescence recovery after photobleaching of GFP-tubulin demonstrated that spindle microtubule turnover is slowed two-fold in the absence of TOGp. Consistent with photobleaching results, microtubule regrowth after washout of the microtubule depolymerizing drug nocodazole was slower at the centrosomes and in the vicinity of mitotic chromatin in cells depleted of TOGp. The slower microtubule turnover is likely due to either nucleation or the transitions of dynamic instability because TOGp depletion did not effect the rate of plus end growth, measured by tracking EB1-GFP at microtubule ends. In contrast, microtubule regrowth after nocodazole washout was unaffected by prior depletion of TACC3, a centrosomal protein that interacts with TOGp. Kinetochore fibers in both untreated and TOGp-depleted cells were stable to incubation at 4°C or lysis in buffer containing calcium indicating that stable kinetochore-microtubule attachments are formed in the absence of TOGp. Depletion of TOGp, but not TACC3, reduced kinetochore oscillations during prometaphase/metaphase. Defects in oscillations are not due simply to multipolarity or loss of centrosome focus in the TOGp-depleted cells, since kinetochore oscillations appear normal in cells treated with the proteosome inhibitor MG132, which also results in multipolar spindles and centrosome fragmentation. We hypothesize that TOGp is required for chromosome motility as a downstream consequence of reduced microtubule dynamics and/or density. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source]

    Live-cell analysis of mitotic spindle formation in taxol-treated cells

    CYTOSKELETON, Issue 8 2008
    Jessica E. Hornick
    Abstract Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-, tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible,but before NEB is complete,results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source]

    Arabidopsis thaliana protein, ATK1, is a minus-end directed kinesin that exhibits non-processive movement

    CYTOSKELETON, Issue 3 2002
    Adam I. Marcus
    Abstract The microtubule cytoskeleton forms the scaffolding of the meiotic spindle. Kinesins, which bind to microtubules and generate force via ATP hydrolysis, are also thought to play a critical role in spindle assembly, maintenance, and function. The A. thaliana protein, ATK1 (formerly known as KATA), is a member of the kinesin family based on sequence similarity and is implicated in spindle assembly and/or maintenance. Thus, we want to determine if ATK1 behaves as a kinesin in vitro, and if so, determine the directionality of the motor activity and processivity character (the relationship between molecular "steps" and microtubule association). The results show that ATK1 supports microtubule movement in an ATP-dependent manner and has a minus-end directed polarity. Furthermore, ATK1 exhibits non-processive movement along the microtubule and likely requires at least four ATK1 motors bound to the microtubule to support movement. Based on these results and previous data, we conclude that ATK1 is a non-processive, minus-end directed kinesin that likely plays a role in generating forces in the spindle during meiosis. Cell Motil. Cytoskeleton 52:144,150, 2002. © 2002 Wiley-Liss, Inc. [source]

    Mutagenesis of ,-tubulin cysteine residues in Saccharomyces cerevisiae: Mutation of cysteine 354 results in cold-stable microtubules

    CYTOSKELETON, Issue 2 2001
    Mohan L. Gupta Jr.
    Abstract Cysteine residues play important roles in the control of tubulin function. To determine which of the six cysteine residues in ,-tubulin are critical to tubulin function, we mutated the cysteines in Saccharomyces cerevisiae ,-tubulin individually to alanine and serine residues. Of the twelve mutations, only three produced significant effects: C12S, C354A, and C354S. The C12S mutation was lethal in the haploid, but the C12A mutation had no observable phenotype. Based on interactive views of the electron crystallographic structure of tubulin, we suggest that substitution of serine for cysteine at this position has a destabilizing effect on the interaction of tubulin with the exchangeable GTP. The two C354 mutations, although not lethal, produced dramatic effects on microtubules and cellular processes that require microtubules. The C354 mutant cells had decreased growth rates, a slowed mitosis, increased resistance to benomyl, and impaired nuclear migration and spindle assembly. The C354A mutation produced a more severe phenotype than the C354S mutation: the haploid cells had chromosome segregation defects, only 50% of cells in a culture were viable, and a significant percentage of the cells were misshapened. Cytoplasmic microtubules in the C354S and C354A cells were longer than in the control strain and spindle structures appeared shorter and thicker. Both cytoplasmic and spindle microtubules in the two C354 mutants were extremely stable to cold temperature. After 24 h at 4°C, the microtubules were still present and, in fact, very long and thick tubulin polymers had formed. Evidence exists to indicate that the C354 residue in mammalian tubulin is near the colchicine binding site and the electron crystal structure of tubulin places the residue at the interface between the ,- and ,-subunits. The sulfhydryl group is situated in a polar environment, which may explain why the alanine mutation is more severe than the serine mutation. When the C12S and the two C354 mutations were made in a diploid strain, the mutated tubulin was incorporated into microtubules and the resulting heterozygotes had phenotypes that were intermediate between those of the mutated haploids and the wild-type strains. The results suggest that the C12 and C354 residues play important roles in the structure and function of tubulin. Cell Motil. Cytoskeleton 49:67,77, 2001. © 2001 Wiley-Liss, Inc. [source]

    Modulation of Alp4 function in Schizosaccharomyces pombe induces novel phenotypes that imply distinct functions for nuclear and cytoplasmic ,-tubulin complexes

    GENES TO CELLS, Issue 4 2006
    Hirohisa Masuda
    The ,-tubulin complex acts as a nucleation unit for microtubule assembly. It remains unknown, however, how spatial and temporal regulation of the complex activity affects microtubule-mediated cellular processes. Alp4 is one of the essential components of the S. pombe,-tubulin complex. We show here that overproduction of a carboxy-terminal form of Alp4 (Alp4C) and its derivatives tagged to a nuclear localization signal or to a nuclear export signal affect localization of ,-tubulin complexes and induces novel phenotypes that reflect distinct functions of nuclear and cytoplasmic ,-tubulin complexes. Nuclear Alp4C induces a Wee1-dependent G2 delay, reduces the levels of the ,-tubulin complex at the spindle pole body, and results in defects in mitotic progression including spindle assembly, cytoplasmic microtubule disassembly, and chromosome segregation. In contrast, cytoplasmic Alp4C induces oscillatory nuclear movement and affects levels of cell polarity markers, Bud6 and Tip1, at the cell ends. These results demonstrate that regulation of nuclear ,-tubulin complex activity is essential for cell cycle progression through the G2/M boundary and M phase, whereas regulation of cytoplasmic ,-tubulin complex activity is important for nuclear positioning and cell polarity control during interphase. [source]

    Potential roles of the nucleotide exchange factor ECT2 and Cdc42 GTPase in spindle assembly in Xenopus egg cell-free extracts,

    JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2003
    Takashi Tatsumoto
    Abstract The ECT2 protooncogene encodes a guanine nucleotide exchange factor for the Rho family of small GTPases. ECT2 contains motifs of cell cycle regulators at its N-terminal domain. We previously showed that ECT2 plays a critical role in cytokinesis. Here, we report a potential role of XECT2, the Xenopus homologue of the human ECT2, in spindle assembly in cell-free Xenopus egg extracts. Cloned XECT2 cDNA encodes a 100 kDa protein closely related to human ECT2. XECT2 is specifically phosphorylated in M phase extracts. Affinity-purified anti-XECT2 antibody strongly inhibited mitosis in Xenopus cell-free extracts. Instead of bipolar spindles, where chromosomes are aligned at the metaphase plane in control extracts, the addition of anti-XECT2 resulted in the appearance of abnormal spindles including monopolar and multipolar spindles as well as bipolar spindles with misaligned chromosomes. In these in vitro synthesized spindle structures, XECT2 was found to tightly associate with mitotic spindles. The N-terminal half of XECT2 lacking the catalytic domain also strongly inhibited spindle assembly in vitro, resulting in the formation of mitotic spindles with a low density. Among the representative Rho GTPases, a dominant-negative form of Cdc42 strongly inhibited spindle assembly in vitro. These results suggest that the Rho family GTPase Cdc42 and its exchange factor XECT2 are critical regulators of spindle assembly in Xenopus egg extracts. Published 2003 Wiley-Liss, Inc., [source]

    The Ras pathway and spindle assembly collide?

    BIOESSAYS, Issue 4 2001
    Marisa Segal
    Although alterations in Ras signalling are found in about 30% of human cancers, the transforming activity of oncogenic Ras is not fully understood. In a recent paper, a putative Ras1 effector in S. pombe, named Scd1, was reported to localize to mitotic apindies. Scd1 physically associates with Moe1, a factor that may contribute to the inherent inatability of microtubules (MTs) and appears to be needed for proper apindle function. Altered MT dynamics within the spindle are likely to affect spindle assembly and chromosome capture, processes that need to be delicately controlled if cells are to guard against genome instability adn transformation. BloEssays 23: 307-310,2001.©2001 John Willey & Sons, Inc. [source]