纺锤体组装检控点

细胞有丝分裂过程中,纺锤体组装检控点监控着染色体在赤道板的队列和向纺锤体两极的分离,确保动粒-微管的黏附和有丝分裂器的完整,使所有的染色体都置于赤道板并双极定向后才进入后期,保证遗传物质均等地分配给两个子代细胞。纺锤体组装检控点缺陷将导致非整倍体的出现,并与一些肿瘤的发生密切相关。现就近年来纺锤体组装检控点蛋白以及纺锤体组装检控点功能缺陷与肿瘤的关系方面的研究进展作一简要综述。     

Bub1p Kinase Activates the Saccharomyces cerevisiae Spindle Assembly Checkpoint

Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function. In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase. We have identified a dominantly acting BUB1 allele that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles. High-level expression of BUB1-5 did not cause detectable spindle damage, yet it delayed yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation. Delayed cells possessed a G₂ DNA content and elevated Clb2p mitotic cyclin levels. Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein did not accumulate. Similar to cells overexpressing MPS1, the BUB1-5 delay was dependent upon the functions of the other checkpoint genes, including BUB2 and BUB3 and MAD1, MAD2, and MAD3. We found that the mitotic delay caused by BUB1-5 or MPS1 overexpression was interdependent upon the function of the other. This suggests that the Bub1p and Mps1p kinases act together at an early step in generating the spindle damage signal.     

Kinase Signaling in the Spindle Checkpoint

The spindle checkpoint is a cell cycle surveillance system that ensures the fidelity of chromosome segregation. In mitosis, it elicits the “wait anaphase” signal to inhibit the anaphase-promoting complex or cyclosome until all chromosomes achieve bipolar microtubule attachment and align at the metaphase plate. Because a single kinetochore unattached to microtubules activates the checkpoint, the wait anaphase signal is thought to be generated by this kinetochore and is then amplified and distributed throughout the cell to inhibit the anaphase-promoting complex/cyclosome. Several spindle checkpoint kinases participate in the generation and amplification of this signal. Recent studies have begun to reveal the activation mechanisms of these checkpoint kinases. Increasing evidence also indicates that the checkpoint kinases not only help to generate the wait anaphase signal but also actively correct kinetochore-microtubule attachment defects.     

A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells

The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole—the chromosomes decondensed and the nuclear envelope re-formed—whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.     

Reverse Engineering of the Spindle Assembly Checkpoint

The Spindle Assembly Checkpoint (SAC) is an intracellular mechanism that ensures proper chromosome segregation. By inhibiting Cdc20, a co-factor of the Anaphase Promoting Complex (APC), the checkpoint arrests the cell cycle until all chromosomes are properly attached to the mitotic spindle. Inhibition of Cdc20 is mediated by a conserved network of interacting proteins. The individual functions of these proteins are well characterized, but understanding of their integrated function is still rudimentary. We here describe our attempts to reverse-engineer the SAC network based on gene deletion phenotypes. We begun by formulating a general model of the SAC which enables us to predict the rate of chromosomal missegregation for any putative set of interactions between the SAC proteins. Next the missegregation rates of seven yeast strains are measured in response to the deletion of one or two checkpoint proteins. Finally, we searched for the set of interactions that correctly predicted the observed missegregation rates of all deletion mutants. Remarkably, although based on only seven phenotypes, the consistent network we obtained successfully reproduces many of the known properties of the SAC. Further insights provided by our analysis are discussed.     

Absence of the Spindle Assembly Checkpoint Restores Mitotic Fidelity upon Loss of Sister Chromatid Cohesion

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Loss of Kif18A Results in Spindle Assembly Checkpoint Activation at Microtubule-Attached Kinetochores

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Restaging the Spindle Assembly Checkpoint in Female Mammalian Meiosis I

In mammalian somatic cells, the spindle assembly checkpoint (SAC) is indispensable forensuring the fidelity of chromosome segregation by delaying cell-cycle progression in theface of even a single misaligned chromosome. In contrast, the role of the SAC inunperturbed mammalian oocytes is less well defined as progression through meiosis I isunaltered in mouse oocytes in the presence of one or a few misaligned chromosomes.Furthermore, attempts to disable the function of the SAC protein, Mad2, in mouseoocytes have produced conflicting results. To gain further insight into SAC functionduring female mammalian meiosis I, we recently utilised a morpholino-based antisenseapproach to deplete the majority of Mad2 in mouse oocytes. Our results define a clearrole for Mad2 in ensuring the proper timing of meiosis I events and ultimately, inensuring the fidelity of homologue disjunction. We discuss the implications of theseresults for the regulation of meiosis I in mammalian oocytes and for the genesis of humananeuploidy.     

Dependency of the Spindle Assembly Checkpoint on Cdk1 Renders the Anaphase Transition Irreversible

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APOLLON Protein Promotes Early Mitotic CYCLIN A Degradation Independent of the Spindle Assembly Checkpoint

In the mammalian cell cycle, both CYCLIN A and CYCLIN B are required for entry into mitosis, and their elimination is also essential to complete the process. During mitosis, CYCLIN A and CYCLIN B are ubiquitylated by the anaphase-promoting complex/cyclosome (APC/C) and then subjected to proteasomal degradation. However, CYCLIN A, but not CYCLIN B, begins to be degraded in the prometaphase when APC/C is inactivated by the spindle assembly checkpoint (SAC). Here, we show that APOLLON (also known as BRUCE or BIRC6) plays a role in SAC-independent degradation of CYCLIN A in early mitosis. APPOLON interacts with CYCLIN A that is not associated with cyclin-dependent kinases. APPOLON also interacts with APC/C, and it facilitates CYCLIN A ubiquitylation. In APPOLON-deficient cells, mitotic degradation of CYCLIN A is delayed, and the total, but not the cyclin-dependent kinase-bound, CYCLIN A level was increased. We propose APPOLON to be a novel regulator of mitotic CYCLIN A degradation independent of SAC.     

Role of the Mad2 Dimerization Interface in the Spindle Assembly Checkpoint Independent of Kinetochores

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Highlights? Mad2 overexpression activates the SAC independently from Mad1 in budding yeast ? Mad2 dimerization surface is required for the SAC independently from Mad1 ? Mad2 dimerization downstream of kinetochores is negligible for the SAC ? Mad2 dimerization surface interacts with Mad3 and is required for MCC stability     

TRIP13 Functions in the Establishment of the Spindle Assembly Checkpoint by Replenishing O-MAD2

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Oxidative Stress Overrides the Spindle Checkpoint

Aneuploidy, an abnormal chromosome set, can ensue from failure of the spindle checkpoint, the safeguard mechanism that halts anaphase onset until mitotic spindle assembly. Inefficiency of cells to maintain the normal chromosome set across cell generations has been linked to tumorigenesis and senescence. Here we show that oxidative stress overrides the spindle checkpoint mechanism. Oxidant challenge of checkpoint-arrested cells led to proteolysis of the anaphase inhibitor securin and mitotic cyclins. This appeared consequent to loss of cyclin B-cdk1 activity caused by oxidant-induced reversal of cdk1 inhibitory phosphorylation. These observations may provide a link between aneuploidy occurrence and oxidative stress.     

Phosphorylation of Microtubule-binding Protein Hec1 by Mitotic Kinase Aurora B Specifies Spindle Checkpoint Kinase Mps1 Signaling at the Kinetochore

The spindle assembly checkpoint (SAC) is a quality control device to ensure accurate chromosome attachment to spindle microtubule for equal segregation of sister chromatid. Aurora B is essential for SAC function by sensing chromosome bi-orientation via spatial regulation of kinetochore substrates. However, it has remained elusive as to how Aurora B couples kinetochore-microtubule attachment to SAC signaling. Here, we show that Hec1 interacts with Mps1 and specifies its kinetochore localization via its calponin homology (CH) domain and N-terminal 80 amino acids. Interestingly, phosphorylation of the Hec1 by Aurora B weakens its interaction with microtubules but promotes Hec1 binding to Mps1. Significantly, the temporal regulation of Hec1 phosphorylation orchestrates kinetochore-microtubule attachment and Mps1 loading to the kinetochore. Persistent expression of phosphomimetic Hec1 mutant induces a hyperactivation of SAC, suggesting that phosphorylation-elicited Hec1 conformational change is used as a switch to orchestrate SAC activation to concurrent destabilization of aberrant kinetochore attachment. Taken together, these results define a novel role for Aurora B-Hec1-Mps1 signaling axis in governing accurate chromosome segregation in mitosis.