“Wait anaphase” signals are not confined to the mitotic spindle

The spindle assembly checkpoint ensures the faithful inheritance of chromosomes by arresting mitotic progression in the presence of kinetochores that are not attached to spindle microtubules. This is achieved through inhibition of the anaphase-promoting complex/cyclosome by a kinetochore-derived “wait anaphase” signal known as the mitotic checkpoint complex. It remains unclear whether the localization and activity of these inhibitory complexes are restricted to the mitotic spindle compartment or are diffusible throughout the cytoplasm. Here we report that “wait anaphase” signals are indeed able to diffuse outside the confines of the mitotic spindle compartment. Using a cell fusion approach to generate multinucleate cells, we investigate the effects of checkpoint signals derived from one spindle compartment on a neighboring spindle compartment. We find that spindle compartments in close proximity wait for one another to align all chromosomes before entering anaphase synchronously. Synchrony is disrupted in cells with increased interspindle distances and cellular constrictions between spindle compartments. In addition, when mitotic cells are fused with interphase cells, “wait anaphase” signals are diluted, resulting in premature mitotic exit. Overall our studies reveal that anaphase inhibitors are diffusible and active outside the confines of the mitotic spindle from which they are derived.

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Intermediates in the assembly of mitotic checkpoint complexes and their role in the regulation of the anaphase-promoting complex

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1524551113 ◽

2016 ◽

Vol 113 (4) ◽

pp. 966-971 ◽

Cited By ~ 10

Author(s):

Sharon Kaisari ◽

Danielle Sitry-Shevah ◽

Shirly Miniowitz-Shemtov ◽

Avram Hershko

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Checkpoint Proteins ◽

Sister Chromatids ◽

Unknown Species ◽

Mitotic Checkpoint Complex

The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. Kinetochores that are not attached properly to the mitotic spindle produce an inhibitory signal that prevents progression into anaphase. The checkpoint system acts on the Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase, which targets for degradation inhibitors of anaphase initiation. APC/C is inhibited by the Mitotic Checkpoint Complex (MCC), which assembles when the checkpoint is activated. MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2, associated with the APC/C coactivator Cdc20. The intermediary processes in the assembly of MCC are not sufficiently understood. It is also not clear whether or not some subcomplexes of MCC inhibit the APC/C and whether Mad2 is required only for MCC assembly and not for its action on the APC/C. We used purified subcomplexes of mitotic checkpoint proteins to examine these problems. Our results do not support a model in which Mad2 catalytically generates a Mad2-free APC/C inhibitor. We also found that the release of Mad2 from MCC caused a marked (although not complete) decrease in inhibitory action, suggesting a role of Mad2 in MCC for APC/C inhibition. A previously unknown species of MCC, which consists of Mad2, BubR1, and two molecules of Cdc20, contributes to the inhibition of APC/C by the mitotic checkpoint system.

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The Mad2-Binding Protein p31comet as a potential target for human cancer therapy

Current Cancer Drug Targets ◽

10.2174/1568009621666210129095726 ◽

2021 ◽

Vol 21 ◽

Author(s):

Ana C. Henriques ◽

Patrícia M. A. Silva ◽

Bruno Sarmento ◽

Hassan Bousbaa

Keyword(s):

Cancer Therapy ◽

Spindle Assembly Checkpoint ◽

Human Cancer ◽

Binding Protein ◽

Mitotic Checkpoint ◽

Mitotic Exit ◽

Mitotic Progression ◽

Spindle Microtubules ◽

Surveillance Mechanism ◽

Mitotic Checkpoint Complex

: The spindle assembly checkpoint (SAC) is a surveillance mechanism that prevents mitotic exit at the metaphase-to-anaphase transition until all chromosomes have established correct bipolar attachment to spindle microtubules. Activation of SAC relies on the assembly of the mitotic checkpoint complex (MCC), which requires conformational change from inactive open Mad2 (O-Mad2) to the active closed Mad2 (C-Mad2) at unattached kinetochores. The Mad2-binding protein p31comet plays a key role in controlling timely mitotic exit by promoting SAC silencing, through preventing Mad2 activation and promoting MCC disassembly. Besides, increasing evidences highlight the p31comet potential as target for cancer therapy. Here, we provide an updated overview of the functional significance of p31comet in mitotic progression, and discuss the potential of deregulated expression of p31comet in cancer and in therapeutic strategies.

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Mode of interaction of TRIP13 AAA-ATPase with the Mad2-binding protein p31comet and with mitotic checkpoint complexes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1515358112 ◽

2015 ◽

Vol 112 (37) ◽

pp. 11536-11540 ◽

Cited By ~ 34

Author(s):

Shirly Miniowitz-Shemtov ◽

Ety Eytan ◽

Sharon Kaisari ◽

Danielle Sitry-Shevah ◽

Avram Hershko

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Thyroid Hormone Receptor ◽

Binding Protein ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Interacting Protein ◽

Aaa Atpase ◽

Oligomeric Form ◽

Mitotic Checkpoint Complex

The AAA-ATPase thyroid hormone receptor interacting protein 13 (TRIP13), jointly with the Mad2-binding protein p31comet, promotes the inactivation of the mitotic (spindle assembly) checkpoint by disassembling the mitotic checkpoint complex (MCC). This checkpoint system ensures the accuracy of chromosome segregation by delaying anaphase until correct bipolar attachment of chromatids to the mitotic spindle is achieved. MCC inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation securin, an inhibitor of anaphase initiation. MCC is composed of the checkpoint proteins Mad2, BubR1, and Bub3, in association with the APC/C activator Cdc20. The assembly of MCC in active checkpoint is initiated by the conversion of Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformation, which then binds tightly to Cdc20. Conversely, the disassembly of MCC that takes place when the checkpoint is turned off involves the conversion of C-Mad2 back to O-Mad2. Previously, we found that the latter process is mediated by TRIP13 together with p31comet, but the mode of their interaction remained unknown. Here, we report that the oligomeric form of TRIP13 binds both p31comet and MCC. Furthermore, p31comet and checkpoint complexes mutually promote the binding of each other to oligomeric TRIP13. We propose that p31comet bound to C-Mad2–containing checkpoint complex is the substrate for the ATPase and that the substrate-binding site of TRIP13 is composed of subsites specific for p31comet and C-Mad2–containing complex. The simultaneous occupancy of both subsites is required for high-affinity binding to TRIP13.

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Role of Polo-like kinase 1 in the regulation of the action of p31comet in the disassembly of mitotic checkpoint complexes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1902970116 ◽

2019 ◽

pp. 201902970 ◽

Cited By ~ 4

Author(s):

Sharon Kaisari ◽

Pnina Shomer ◽

Tamar Ziv ◽

Danielle Sitry-Shevah ◽

Shirly Miniowitz-Shemtov ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Hela Cells ◽

Mitotic Spindle ◽

Joint Action ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Futile Cycle ◽

Mitotic Checkpoint Complex ◽

Bi 2536

The Mad2-binding protein p31comet has important roles in the inactivation of the mitotic checkpoint system, which delays anaphase until chromosomes attach correctly to the mitotic spindle. The activation of the checkpoint promotes the assembly of a Mitotic Checkpoint Complex (MCC), which inhibits the action of the ubiquitin ligase APC/C (Anaphase-Promoting Complex/Cyclosome) to degrade inhibitors of anaphase initiation. The inactivation of the mitotic checkpoint requires the disassembly of MCC. p31comet promotes the disassembly of mitotic checkpoint complexes by liberating their Mad2 component in a joint action with the ATPase TRIP13. Here, we investigated the regulation of p31comet action. The release of Mad2 from checkpoint complexes in extracts from nocodazole-arrested HeLa cells was inhibited by Polo-like kinase 1 (Plk1), as suggested by the effects of selective inhibitors of Plk1. Purified Plk1 bound to p31comet and phosphorylated it, resulting in the suppression of its activity (with TRIP13) to disassemble checkpoint complexes. Plk1 phosphorylated p31comet on S102, as suggested by the prevention of the phosphorylation of this residue in checkpoint extracts by the selective Plk1 inhibitor BI-2536 and by the phosphorylation of S102 with purified Plk1. An S102A mutant of p31comet had a greatly decreased sensitivity to inhibition by Plk1 of its action to disassemble mitotic checkpoint complexes. We propose that the phosphorylation of p31comet by Plk1 prevents a futile cycle of MCC assembly and disassembly during the active mitotic checkpoint.

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Mitotic phosphorylation of tumor suppressor DAB2IP maintains spindle assembly checkpoint and chromosomal stability through activating PLK1-Mps1 signal pathway and stabilizing mitotic checkpoint complex

Oncogene ◽

10.1038/s41388-021-02106-8 ◽

2021 ◽

Author(s):

Lan Yu ◽

Yue Lang ◽

Ching-Cheng Hsu ◽

Wei-Min Chen ◽

Jui-Chung Chiang ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Independent Manner ◽

Chromosome Separation ◽

Surveillance Mechanism ◽

Mitotic Phosphorylation ◽

Mitotic Checkpoint Complex ◽

Mitotic Regulation

AbstractChromosomal instability (CIN) is a driving force for cancer development. The most common causes of CIN include the dysregulation of the spindle assembly checkpoint (SAC), which is a surveillance mechanism that prevents premature chromosome separation during mitosis by targeting anaphase-promoting complex/cyclosome (APC/C). DAB2IP is frequently silenced in advanced prostate cancer (PCa) and is associated with aggressive phenotypes of PCa. Our previous study showed that DAB2IP activates PLK1 and functions in mitotic regulation. Here, we report the novel mitotic phosphorylation of DAB2IP by Cdks, which mediates DAB2IP’s interaction with PLK1 and the activation of the PLK1-Mps1 pathway. DAB2IP interacts with Cdc20 in a phosphorylation-independent manner. However, the phosphorylation of DAB2IP inhibits the ubiquitylation of Cdc20 in response to SAC, and blocks the premature release of the APC/C-MCC. The PLK1-Mps1 pathway plays an important role in mitotic checkpoint complex (MCC) assembly. It is likely that DAB2IP acts as a scaffold to aid PLK1-Mps1 in targeting Cdc20. Depletion or loss of the Cdks-mediated phosphorylation of DAB2IP destabilizes the MCC, impairs the SAC, and increases chromosome missegregation and subsequent CIN, thus contributing to tumorigenesis. Collectively, these results demonstrate the mechanism of DAB2IP in SAC regulation and provide a rationale for targeting the SAC to cause lethal CIN against DAB2IP-deficient aggressive PCa, which exhibits a weak SAC.

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Spindle assembly checkpoint: the third decade

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2011.0072 ◽

2011 ◽

Vol 366 (1584) ◽

pp. 3595-3604 ◽

Cited By ~ 126

Author(s):

Andrea Musacchio

Keyword(s):

Cell Cycle Progression ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Checkpoint Control ◽

Checkpoint Response ◽

The Third ◽

Binding Interface ◽

Mitotic Checkpoint Complex

The spindle assembly checkpoint controls cell cycle progression during mitosis, synchronizing it with the attachment of chromosomes to spindle microtubules. After the discovery of the mitotic arrest deficient ( MAD ) and budding uninhibited by benzymidazole ( BUB ) genes as crucial checkpoint components in 1991, the second decade of checkpoint studies (2001–2010) witnessed crucial advances in the elucidation of the mechanism through which the checkpoint effector, the mitotic checkpoint complex, targets the anaphase-promoting complex (APC/C) to prevent progression into anaphase. Concomitantly, the discovery that the Ndc80 complex and other components of the microtubule-binding interface of kinetochores are essential for the checkpoint response finally asserted that kinetochores are crucial for the checkpoint response. Nevertheless, the relationship between kinetochores and checkpoint control remains poorly understood. Crucial advances in this area in the third decade of checkpoint studies (2011–2020) are likely to be brought about by the characterization of the mechanism of kinetochore recruitment, activation and inactivation of checkpoint proteins, which remains elusive for the majority of checkpoint components. Here, we take a molecular view on the main challenges hampering this task.

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Bub1 is not required for the checkpoint response to unattached kinetochores in diploid human cells

10.1101/278820 ◽

2018 ◽

Cited By ~ 4

Author(s):

Cerys E. Currie ◽

Mar Mora-Santos ◽

Chris Smith ◽

Andrew D. McAinsh ◽

Jonathan B.A. Millar

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Mitotic Checkpoint ◽

Human Cells ◽

Anaphase Promoting Complex ◽

Pathway Model ◽

Checkpoint Response ◽

Conflicting Evidence ◽

Dual Pathway ◽

Mitotic Checkpoint Complex

AbstractError-free chromosome segregation during mitosis depends on a functional spindle assembly checkpoint (SAC). The SAC is a multi-component signaling system that is recruited to incorrectly attached kinetochores to catalyze the formation of a soluble inhibitor, known as the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex [1]. We have previously proposed that two separable pathways, composed of KNL1-Bub3-Bub1 (KBB) and Rod-Zwilch-Zw10 (RZZ), recruit Mad1-Mad2 complexes to human kinetochores to activate the SAC [2]. We refer to this as the dual pathway model. Although Bub1 is absolutely required for MCC formation in yeast (which lack RZZ), there is conflicting evidence as to whether this is also the case in human cells based on siRNA studies [2–5]. Here we report, using genome editing, that Bub1 is not strictly required for the SAC response to unattached kinetochores in human diploid hTERT-RPE1 cells, consistent with the dual pathway model.

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Role of ubiquitylation of components of mitotic checkpoint complex in their dissociation from anaphase-promoting complex/cyclosome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1720312115 ◽

2018 ◽

Vol 115 (8) ◽

pp. 1777-1782 ◽

Cited By ~ 4

Author(s):

Danielle Sitry-Shevah ◽

Sharon Kaisari ◽

Adar Teichner ◽

Shirly Miniowitz-Shemtov ◽

Avram Hershko

Keyword(s):

Chromosome Segregation ◽

Ubiquitin Ligase ◽

Mitotic Spindle ◽

Mitotic Checkpoint ◽

Terminal Region ◽

Anaphase Promoting Complex ◽

Lysine Residues ◽

Mitotic Checkpoint Complex

The mitotic checkpoint system ensures the fidelity of chromosome segregation in mitosis by preventing premature initiation of anaphase until correct bipolar attachment of chromosomes to the mitotic spindle is reached. It promotes the assembly of a mitotic checkpoint complex (MCC), composed of BubR1, Bub3, Cdc20, and Mad2, which inhibits the activity of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. When the checkpoint is satisfied, anaphase is initiated by the disassembly of MCC. Previous studies indicated that the dissociation of APC/C-bound MCC requires ubiquitylation and suggested that the target of ubiquitylation is the Cdc20 component of MCC. However, it remained unknown how ubiquitylation causes the release of MCC from APC/C and its disassembly and whether ubiquitylation of additional proteins is involved in this process. We find that ubiquitylation causes the dissociation of BubR1 from Cdc20 in MCC and suggest that this may lead to the release of MCC components from APC/C. BubR1 in MCC is ubiquitylated by APC/C, although to a lesser degree than Cdc20. The extent of BubR1 ubiquitylation was markedly increased in recombinant MCC that contained a lysine-less mutant of Cdc20. Mutation of lysine residues to arginines in the N-terminal region of BubR1 partially inhibited its ubiquitylation and slowed down the release of MCC from APC/C, provided that Cdc20 ubiquitylation was also blocked. It is suggested that ubiquitylation of both Cdc20 and BubR1 may be involved in their dissociation from each other and in the release of MCC components from APC/C.

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Aneuploid abortion correlates positively with MAD1 overexpression and miR-125b down-regulation

Molecular Cytogenetics ◽

10.1186/s13039-021-00538-1 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Juan Zhao ◽

Hui Li ◽

Guangxin Chen ◽

Lijun Du ◽

Peiyan Xu ◽

...

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Mitotic Checkpoint ◽

Early Embryo ◽

Vital Role ◽

Control Group ◽

Embryo Abortion ◽

Protein Levels ◽

Mitotic Checkpoint Complex

Abstract Background Aneuploidy is the most frequent cause of early-embryo abortion. Any defect in chromosome segregation would fail to satisfy the spindle assembly checkpoint (SAC) during mitosis, halting metaphase and causing aneuploidy. The mitotic checkpoint complex (MCC), comprising MAD1, MAD2, Cdc20, BUBR1 and BUB3, plays a vital role in SAC activation. Studies have confirmed that overexpression of MAD2 and BUBR1 can facilitate correct chromosome segregation and embryo stability. Research also proves that miR-125b negatively regulates MAD1 expression by binding to its 3′UTR. However, miR-125b, Mad1 and Bub3 gene expression in aneuploid embryos of spontaneous abortion has not been reported to date. Methods In this study, embryonic villi from miscarried pregnancies were collected and divided into two groups (aneuploidy and euploidy) based on High-throughput ligation-dependent probe amplification (HLPA) and Fluorescence in situ hybridization (FISH) analyses. RNA levels of miR-125b, MAD1 and BUB3 were detected by Quantitative real-time PCR (qRT-PCR); protein levels of MAD1 and BUB3 were analysed by Western blotting. Results statistical analysis (p < 0.05) showed that miR-125b and BUB3 were significantly down-regulated in the aneuploidy group compared to the control group and that MAD1 was significantly up-regulated. Additionally, the MAD1 protein level was significantly higher in aneuploidy abortion villus, but BUB3 protein was only mildly increased. Correlation analysis revealed that expression of MAD1 correlated negatively with miR-125b. Conclusion These results suggest that aneuploid abortion correlates positively with MAD1 overexpression, which might be caused by insufficient levels of miR-125b. Taken together, our findings first confirmed the negative regulatory mode between MAD1 and miR-125b, providing a basis for further mechanism researches in aneuploid abortion.

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Mechanism of APC/CCDC20 activation by mitotic phosphorylation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1604929113 ◽

2016 ◽

Vol 113 (19) ◽

pp. E2570-E2578 ◽

Cited By ~ 69

Author(s):

Renping Qiao ◽

Florian Weissmann ◽

Masaya Yamaguchi ◽

Nicholas G. Brown ◽

Ryan VanderLinden ◽

...

Keyword(s):

Chromosome Segregation ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Loop Region ◽

Evolutionarily Conserved ◽

Terminal Loop ◽

Different Types ◽

Mitotic Phosphorylation ◽

Mitotic Checkpoint Complex

Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/CCDC20 activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/CCDC20 activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/CCDC20 activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis.

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