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 10,214 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The spindle checkpoint maintains genome stability by inhibiting Cdc20-mediated activation of the anaphase promoting complex/cyclosome (APC/C) until all the chromosomes correctly align on the microtubule spindle apparatus via their kinetochores. BubR1, an essential component of this checkpoint, localises to kinetochores and its kinase activity is regulated by the kinesin-related motor protein Cenp-E. BubR1 also inhibits APC/C(Cdc20) in vitro, thus providing a molecular link between kinetochore-microtubule interactions and the proteolytic machinery that regulates mitotic progression. Several other protein kinases, including Bub1 and members of the Ipl1/aurora family, also regulate anaphase onset. However, in human somatic cells Bub1 and aurora B kinase activity do not appear to be essential for spindle checkpoint function. Specifically, when Bub1 is inhibited by RNA interference, or aurora kinase activity is inhibited with the small molecule ZM447439, cells arrest transiently in mitosis following exposure to spindle toxins that prevent microtubule polymerisation. Here, we show that mitotic arrest of Bub1-deficient cells is dependent on aurora kinase activity, and vice versa. We suggest therefore that the checkpoint is composed of two arms, one dependent on Bub1, the other on aurora B. Analysis of BubR1 complexes suggests that both of these arms converge on the mitotic checkpoint complex (MCC), which includes BubR1, Bub3, Mad2 and Cdc20. Although it is known that MCC components can bind and inhibit the APC/C, we show here for the first time that the binding of the MCC to the APC/C is dependent on an active checkpoint signal. Furthermore, we show that both Bub1 and aurora kinase activity are required to promote binding of the MCC to the APC/C. These observations provide a simple explanation of why BubR1 and Mad2 are essential for checkpoint function following spindle destruction, yet Bub1 and aurora B kinase activity are not. Taken together with other observations, we suggest that these two arms respond to different spindle cues: whereas the Bub1 arm monitors kinetochore-microtubule attachment, the aurora B arm monitors biorientation. This bifurcation in the signalling mechanism may help explain why many tumour cells mount a robust checkpoint response following spindle damage, despite exhibiting chromosome instability.
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PMID:Bub1 and aurora B cooperate to maintain BubR1-mediated inhibition of APC/CCdc20. 1604 81

The vertebrate kinetochore is a complex structure that specifies the attachments between the chromosomes and microtubules of the spindle and is thus essential for accurate chromosome segregation. Kinetochores are assembled on centromeric chromatin through complex pathways that are coordinated with the cell cycle. In the light of recent discoveries on how proteins assemble onto kinetochores and interact with each other, we review these findings in this article (which is part of the Chromosome Segregation and Aneuploidy series), and discuss their implications for the current mitotic checkpoint models - the template model and the two-step model. The template model proposes that Mad1-Mad2 at kinetochores acts as a template to change the conformation of another binding molecule of Mad2. This templated change in conformation is postulated as a mechanism for the amplification of the 'anaphase wait' signal. The two-step model proposes that the mitotic checkpoint complex (MCC) is the kinetochore-independent anaphase inhibitor, and the role of the unaligned kinetochore is to sensitize the anaphase-promoting complex/cyclosome (APC/C) to MCC-mediated inhibition.
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PMID:Kinetochore structure and function. 1621 39

To maintain the fidelity of chromosome inheritance, cells utilize a surveillance mechanism called the spindle checkpoint to sense improper attachment of sister chromatids to the mitotic spindle prior to chromosome segregation. The target of the spindle checkpoint is a ubiquitin ligase called the anaphase-promoting complex or cyclosome (APC/C). The spindle checkpoint protein Mad2 inhibits the activity of APC/C through direct binding to its activator Cdc20. Studies have shown that Mad2 has two distinct natively folded conformations and that the unusual two-state behavior of Mad2 plays a crucial role in checkpoint signaling. This article describes methods for the purification of the two Mad2 conformers and for the analysis of their activities in APC/C inhibition in Xenopus egg extracts.
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PMID:Purification and assay of Mad2: a two-state inhibitor of anaphase-promoting complex/cyclosome. 1627 33

Mad2 is a pivotal component of the spindle assembly checkpoint (SAC) which inhibits anaphase promoting complex/cyclo-some (APC/C) activity by sequestering Cdc20 thereby regulating the destruction of securin and cyclin B. During mitosis, spindle depolymerisation induces a robust Mad2-dependent arrest due to inhibition of securin and cyclin B destruction. In contrast to mitosis, the molecular details underpinning the meiosis I arrest experienced by mouse oocytes exposed to spindle depolymerisation remain incompletely characterised. Notably, the role of Mad2 and the fate of the anaphase-marker, securin, are unexplored. As shown previously, we find that spindle depolymerisation by nocodazole inhibits first polar body extrusion (PBE) and stabilises cyclin B and cyclin-dependent kinase 1 activity in mouse oocytes. Here we show that stabilisation of cyclin B in nocodazole can be sustained for several hours and is associated with stabilisation of securin. These effects are SAC-mediated as, in oocytes depleted of the majority of Mad2 by morpholino antisense, securin and cyclin B are destabilised and 15% of oocytes undergo PBE. This reflects premature APC/C activation as a mutant form of cyclin B lacking its APC/C degradation signal is stable in Mad2-depleted oocytes. Moreover, homologues do not disjoin during the prolonged meiosis I arrest (> 18 h) induced by nocodaozole indicating that a non-cleavage mechanism is insufficient on its own for resolution of arm cohesion in mammalian oocytes. In conclusion, when all kinetochores lack attachment and tension, mouse oocytes mount a robust Mad2-dependent meiosis I arrest which inhibits the destruction of securin and cyclin B.
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PMID:Mad2 is required for inhibiting securin and cyclin B degradation following spindle depolymerisation in meiosis I mouse oocytes. 1632 43

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit ubiquitin-ligase whose major functions in the cell cycle are the initiation of sister chromatid separation and the inactivation of cyclin-dependent kinases. This complex is also essential for meiosis, a specialized form of the cell cycle characterized by two consecutive rounds of chromosome segregation. To ensure a proper meiotic cell cycle, the activity of APC/C needs to be tightly controlled. It is now evident that inhibitors of APC/C play pivotal roles to avert its untimely activation. During prophase I, this ubiquitin-ligase must be kept inactive to prevent precocious sister chromatid separation. Studies in yeast showed that this inhibition is mediated by a specific subunit of the complex. Accurate chromosome segregation in meiosis I depends on spindle checkpoint proteins such as Mad2 which delay APC/C activation in response to an erroneous spindle attachment of chromosomes. Additional APC/C antagonists are known to block complete cyclin destruction between meiosis I and II, thereby ensuring that cyclin dependent kinases remain active and that DNA replication does not occur. Inhibitors of APC/C also mediate the cytostatic factor induced metaphase II arrest of oocytes. This review highlights the current knowledge about the role and relevance of these diverse regulators of the meiotic APC/C.
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PMID:Preventing fatal destruction: inhibitors of the anaphase-promoting complex in meiosis. 1647 60

Cdc20 (cell division cycle 20) and Cdh1 are the activating subunits of APC (anaphase-promoting complex), an E3-ubiquitin ligase that drives cells into anaphase by inducing degradation of cyclin B and the anaphase inhibitor securin. To prevent chromosome missegregation due to early degradation of cyclin B and securin, mitotic checkpoint protein complexes consisting of BubR1, Bub3 and Mad2 bind to and inhibit APC(Cdc20) until all chromosomes are properly attached to the mitotic spindle and aligned in the metaphase plate. The nuclear transport factors Rae1 and Nup98, which convert into mitotic checkpoint proteins in M-phase, further prevent chromosome missegregation by assembling into a complex with APC(Cdh1) and delaying APC(Cdh1)-mediated ubiquitination of securin. Disruption of Mad2, BubR1, Bub3 or Rae1 in mice results in substantial aneuploidy in somatic tissues, but whether these genes are equally important for accurate chromosome segregation during meiosis has not yet been established. To address this issue, we generated cohorts of male mice in which Mad2, BubR1, Bub3, Rae1 and Nup98 were disrupted either individually or in combination. We tested the fertility of these mice and performed chromosome counts on secondary spermatocytes. We found that male fertility and accurate chromosome segregation during spermatogenesis are highly dependent on BubR1, but not Mad2, Bub3, Rae1 and Nup98. Our results suggest that the mechanisms ensuring accurate chromosome segregation differ between mitotic and meiotic cells.
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PMID:Differential mitotic checkpoint protein requirements in somatic and germ cells. 1685 67

The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. It affects the activity of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets inhibitors of anaphase initiation for degradation. The mechanisms by which this system regulates APC/C remain obscure. Some models propose that the system promotes sequestration of the APC/C activator Cdc20 by binding to the checkpoint proteins Mad2 and BubR1. A different model suggests that a mitotic checkpoint complex (MCC) composed of BubR1, Bub3, Cdc20, and Mad2 inhibits APC/C in mitotic checkpoint [Sudakin V, Chan GKT, Yen TJ (2001) J Cell Biol 154:925-936]. We examined this problem by using extracts from nocodazole-arrested cells that reproduce some downstream events of the mitotic checkpoint system, such as lag kinetics of the degradation of APC/C substrate. Incubation of extracts with adenosine-5'-(gamma-thio)triphosphate (ATP[gammaS]) stabilized the checkpoint-arrested state, apparently by stable thiophosphorylation of some proteins. By immunoprecipitation of APC/C from stably checkpoint-arrested extracts, followed by elution with increased salt concentration, we isolated inhibitory factors associated with APC/C. A part of the inhibitory material consists of Cdc20 associated with BubR1 and Mad2, and is thus similar to MCC. Contrary to the original MCC hypothesis, we find that MCC disassembles upon exit from the mitotic checkpoint. Thus, the requirement of the mitotic checkpoint system for the binding of Mad2 and BubR1 to Cdc20 may be for the assembly of the inhibitory complex rather than for Cdc20 sequestration.
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PMID:Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint. 1736 Mar 35

During mitosis, the genome duplicated during S-phase is synchronously and accurately segregated to the two daughter cells. The spindle checkpoint prevents premature sister-chromatid separation and mitotic exit. The anaphase-promoting complex/cyclosome (APC/C) is a key target of the spindle checkpoint. Upon checkpoint activation, the mitotic checkpoint complex (MCC) containing Mad2, Bub3, Mad3/BubR1 and Cdc20 inhibits APC/C. Two independent studies in budding yeast have now shed light on the mechanism by which MCC inhibits APC/C. These studies indicate that Mad3 binds to the mitotic activator of APC/C Cdc20 using peptide motifs commonly found in APC/C substrates and thus competes with APC/C substrates for APC/CCdc20 binding. In addition, Mad3 binding to APC/CCdc20 induces Cdc20 ubiquitination by APC/C, leading to the dissociation of MCC. Meanwhile, two other studies have shown that a deubiquitinating enzyme is required for the spindle checkpoint whereas APC/C-dependent ubiquitination is needed for checkpoint inactivation. Collectively, these studies suggest a dynamic model for APC/CCdc20 regulation by MCC in which APC/C- and Mad3-dependent ubiquitination of Cdc20 constitutes a self-regulated switch that rapidly inactivates the spindle checkpoint upon correct chromosome attachment.
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PMID:Running on a treadmill: dynamic inhibition of APC/C by the spindle checkpoint. 1765 Mar 7

KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) inhibited cancer cell growth in vitro and in vivo. Flow cytometry studies showed that KRIBB3 caused cell cycle arrest at the G(2)/M phase and subsequent apoptosis. This was confirmed as accumulation of Cyclin B1 and cleavage of poly(ADP-ribose) polymerase (PARP) were detected. While transient inhibition by KRIBB3 led to reversible mitotic arrest, prolonged exposure to KRIBB3-induced apoptosis. Co-immunoprecipitation experiments showed that KRIBB3 initially induced association of inhibitory Mad2 with p55CDC (mammalian homologue of CDC20), an activator of APC/C (anaphase-promoting complex/cyclosome), suggesting that the mitotic spindle checkpoint was activated by KRIBB3. However, the level of this inhibitory complex of Mad2 with p55CDC was gradually decreased 24 h after KRIBB3 treatment, and was hardly detectable after 48 h, indicating some slipping of the mitotic checkpoint. Consistent with these observations, KRIBB3 activated the mitotic spindle checkpoint by disrupting the microtubule cytoskeleton. KRIBB3 was proven to be a tubulin inhibitor using in vitro polymerization assays and in vivo indirect immunofluorescence staining. The temporal pattern of Bax activation by KRIBB3 was similar to PARP cleavage, suggesting that Bax is a mediator of KRIBB3-dependent apoptosis. Furthermore, when KRIBB3 was administered intraperitoneally into nude mice at 50 mg/kg or 100 mg/kg, it inhibited 49.5 or 70.3% of tumor growth, respectively. These results suggest that KRIBB3 is a good drug candidate for cancer therapy.
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PMID:KRIBB3, a novel microtubule inhibitor, induces mitotic arrest and apoptosis in human cancer cells. 1791 94

The spindle assembly checkpoint (SAC) monitors attachment to microtubules and tension on chromosomes in mitosis and meiosis. It represents a surveillance mechanism that halts cells in M-phase in the presence of unattached chromosomes, associated with accumulation of checkpoint components, in particular, Mad2, at the kinetochores. A complex between the anaphase promoting factor/cylosome (APC/C), its accessory protein Cdc20 and proteins of the SAC renders APC/C inactive, usually until all chromosomes are properly assembled at the spindle equator (chromosome congression) and under tension from spindle fibres. Upon release from the SAC the APC/C can target proteins like cyclin B and securin for degradation by the proteasome. Securin degradation causes activation of separase proteolytic enzyme, and in mitosis cleavage of cohesin proteins at the centromeres and arms of sister chromatids. In meiosis I only the cohesin proteins at the sister chromatid arms are cleaved. This requires meiosis specific components and tight regulation by kinase and phosphatase activities. There is no S-phase between meiotic divisions. Second meiosis resembles mitosis. Mammalian oocytes arrest constitutively at metaphase II in presence of aligned chromosomes, which is due to the activity of the cytostatic factor (CSF). The SAC has been identified in spermatogenesis and oogenesis, but gender-differences may contribute to sex-specific differential responses to aneugens. The age-related reduction in expression of components of the SAC in mammalian oocytes may act synergistically with spindle and other cell organelles' dysfunction, and a partial loss of cohesion between sister chromatids to predispose oocytes to errors in chromosome segregation. This might affect dose-response to aneugens. In view of the tendency to have children at advanced maternal ages it appears relevant to pursue studies on consequences of ageing on the susceptibility of human oocytes to the induction of meiotic error by aneugens and establish models to assess risks to human health by environmental exposures.
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PMID:Spindle formation, chromosome segregation and the spindle checkpoint in mammalian oocytes and susceptibility to meiotic error. 1809 27


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