CENP-A Is Required for Accurate Chromosome Segregation and Sustained Kinetochore Association of BubR1

ABSTRACT CENP-A is an evolutionarily conserved, centromere-specific variant of histone H3 that is thought to play a central role in directing kinetochore assembly and in centromere function. Here, we have analyzed the consequences of disrupting the CENP-A gene in the chicken DT40 cell line. In CENP-A-depleted cells, kinetochore protein assembly is impaired, as indicated by mislocalization of the inner kinetochore proteins CENP-I, CENP-H, and CENP-C as well as the outer components Nuf2/Hec1, Mad2, and CENP-E. However, BubR1 and the inner centromere protein INCENP are efficiently recruited to kinetochores. Following CENP-A depletion, chromosomes are deficient in proper congression on the mitotic spindle and there is a transient delay in prometaphase. CENP-A-depleted cells further proceed through anaphase and cytokinesis with unequal chromosome segregation, suggesting that some kinetochore function remains following substantial depletion of CENP-A. We furthermore demonstrate that CENP-A-depleted cells exhibit a specific defect in maintaining kinetochore localization of the checkpoint protein BubR1 under conditions of checkpoint activation. Our data thus point to a specific role for CENP-A in assembly of kinetochores competent in the maintenance of mitotic checkpoint signaling.

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The Constitutive Centromere Component CENP-50 Is Required for Recovery from Spindle Damage

Molecular and Cellular Biology ◽

10.1128/mcb.25.23.10315-10328.2005 ◽

2005 ◽

Vol 25 (23) ◽

pp. 10315-10328 ◽

Cited By ~ 54

Author(s):

Yukinori Minoshima ◽

Tetsuya Hori ◽

Masahiro Okada ◽

Hiroshi Kimura ◽

Tokuko Haraguchi ◽

...

Keyword(s):

Cell Cycle ◽

Growth Rate ◽

Mitotic Checkpoint ◽

Loss Of Function ◽

Wild Type ◽

Centromere Function ◽

Dt40 Cells ◽

Precise Role ◽

Chicken Dt40 Cell

ABSTRACT We identified CENP-50 as a novel kinetochore component. We found that CENP-50 is a constitutive component of the centromere that colocalizes with CENP-A and CENP-H throughout the cell cycle in vertebrate cells. To determine the precise role of CENP-50, we examined its role in centromere function by generating a loss-of-function mutant in the chicken DT40 cell line. The CENP-50 knockout was not lethal; however, the growth rate of cells with this mutation was slower than that of wild-type cells. We observed that the time for CENP-50-deficient cells to complete mitosis was longer than that for wild-type cells. Centromeric localization of CENP-50 was abolished in both CENP-H- and CENP-I-deficient cells. Coimmunoprecipitation experiments revealed that CENP-50 interacted with the CENP-H/CENP-I complex in chicken DT40 cells. We also observed severe mitotic defects in CENP-50-deficient cells with apparent premature sister chromatid separation when the mitotic checkpoint was activated, indicating that CENP-50 is required for recovery from spindle damage.

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Molecular requirements for the formation of a kinetochore–microtubule interface by Dam1 and Ndc80 complexes

The Journal of Cell Biology ◽

10.1083/jcb.201210091 ◽

2012 ◽

Vol 200 (1) ◽

pp. 21-30 ◽

Cited By ~ 49

Author(s):

Fabienne Lampert ◽

Christine Mieck ◽

Gregory M. Alushin ◽

Eva Nogales ◽

Stefan Westermann

Keyword(s):

Chromosome Segregation ◽

Protein Complexes ◽

Structural Elements ◽

Large Protein ◽

Kinetochore Assembly ◽

Sister Chromatids ◽

Dam1 Complex ◽

Shed Light ◽

Kinetochore Function

Kinetochores are large protein complexes that link sister chromatids to the spindle and transduce microtubule dynamics into chromosome movement. In budding yeast, the kinetochore–microtubule interface is formed by the plus end–associated Dam1 complex and the kinetochore-resident Ndc80 complex, but how they work in combination and whether a physical association between them is critical for chromosome segregation is poorly understood. Here, we define structural elements required for the Ndc80–Dam1 interaction and probe their function in vivo. A novel ndc80 allele, selectively impaired in Dam1 binding, displayed growth and chromosome segregation defects. Its combination with an N-terminal truncation resulted in lethality, demonstrating essential but partially redundant roles for the Ndc80 N-tail and Ndc80–Dam1 interface. In contrast, mutations in the calponin homology domain of Ndc80 abrogated kinetochore function and were not compensated by the presence of Dam1. Our experiments shed light on how microtubule couplers cooperate and impose important constraints on structural models for outer kinetochore assembly.

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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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Systematic Analysis in Caenorhabditis elegans Reveals that the Spindle Checkpoint Is Composed of Two Largely Independent Branches

Molecular Biology of the Cell ◽

10.1091/mbc.e08-10-1047 ◽

2009 ◽

Vol 20 (4) ◽

pp. 1252-1267 ◽

Cited By ~ 59

Author(s):

Anthony Essex ◽

Alexander Dammermann ◽

Lindsay Lewellyn ◽

Karen Oegema ◽

Arshad Desai

Keyword(s):

Caenorhabditis Elegans ◽

Spindle Checkpoint ◽

Checkpoint Activation ◽

Systematic Analysis ◽

Checkpoint Signaling ◽

Epistasis Analysis ◽

Kinetochore Assembly ◽

Anaphase Onset ◽

Monopolar Spindle ◽

Spindle Microtubules

Kinetochores use the spindle checkpoint to delay anaphase onset until all chromosomes have formed bipolar attachments to spindle microtubules. Here, we use controlled monopolar spindle formation to systematically define the requirements for spindle checkpoint signaling in the Caenorhabditis elegans embryo. The results, when interpreted in light of kinetochore assembly epistasis analysis, indicate that checkpoint activation is coordinately directed by the NDC-80 complex, the Rod/Zwilch/Zw10 complex, and BUB-1—three components independently targeted to the outer kinetochore by the scaffold protein KNL-1. These components orchestrate the integration of a core Mad1MDF-1/Mad2MDF-2-based signal, with a largely independent Mad3SAN-1/BUB-3 pathway. Evidence for independence comes from the fact that subtly elevating Mad2MDF-2 levels bypasses the requirement for BUB-3 and Mad3SAN-1 in kinetochore-dependent checkpoint activation. Mad3SAN-1 does not accumulate at unattached kinetochores and BUB-3 kinetochore localization is independent of Mad2MDF-2. We discuss the rationale for a bipartite checkpoint mechanism in which a core Mad1MDF-1/Mad2MDF-2 signal generated at kinetochores is integrated with a separate cytoplasmic Mad3SAN-1/BUB-3–based pathway.

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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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Relief of the Dma1-mediated checkpoint requires Dma1 autoubiquitination and dynamic localization

Molecular Biology of the Cell ◽

10.1091/mbc.e18-04-0261 ◽

2018 ◽

Vol 29 (18) ◽

pp. 2176-2189 ◽

Cited By ~ 1

Author(s):

Christine M. Jones ◽

Jun-Song Chen ◽

Alyssa E. Johnson ◽

Zachary C. Elmore ◽

Sierra N. Cullati ◽

...

Keyword(s):

Cell Division ◽

Chromosome Segregation ◽

Ubiquitin Ligase ◽

Spindle Pole Body ◽

Mitotic Checkpoint ◽

Spindle Pole ◽

Checkpoint Activation ◽

Pole Body ◽

Septation Initiation Network ◽

Anaphase B

Chromosome segregation and cell division are coupled to prevent aneuploidy and cell death. In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) promotes cytokinesis, but upon mitotic checkpoint activation, the SIN is actively inhibited to prevent cytokinesis from occurring before chromosomes have safely segregated. SIN inhibition during the mitotic checkpoint is mediated by the E3 ubiquitin ligase Dma1. Dma1 binds to the CK1-phosphorylated SIN scaffold protein Sid4 at the spindle pole body (SPB), and ubiquitinates it. Sid4 ubiquitination antagonizes the SPB localization of the Pololike kinase Plo1, the major SIN activator, so that SIN signaling is delayed. How this checkpoint is silenced once spindle defects are resolved has not been clear. Here we establish that Dma1 transiently leaves SPBs during anaphase B due to extensive autoubiquitination. The SIN is required for Dma1 to return to SPBs later in anaphase. Blocking Dma1 removal from SPBs by permanently tethering it to Sid4 prevents SIN activation and cytokinesis. Therefore, controlling Dma1’s SPB dynamics in anaphase is an essential step in S. pombe cell division and the silencing of the Dma1-dependent mitotic checkpoint.

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CENP-E–dependent BubR1 autophosphorylation enhances chromosome alignment and the mitotic checkpoint

The Journal of Cell Biology ◽

10.1083/jcb.201202152 ◽

2012 ◽

Vol 198 (2) ◽

pp. 205-217 ◽

Cited By ~ 47

Author(s):

Yige Guo ◽

Christine Kim ◽

Sana Ahmad ◽

Jiayin Zhang ◽

Yinghui Mao

Keyword(s):

Mitotic Checkpoint ◽

The State ◽

Chromosome Loss ◽

Spindle Microtubule ◽

Single Chromosome ◽

Checkpoint Signaling ◽

Mitotic Kinase ◽

Chromosome Alignment ◽

Kinetochore Function ◽

Microtubule Capture

How the state of spindle microtubule capture at the kinetochore is translated into mitotic checkpoint signaling remains largely unknown. In this paper, we demonstrate that the kinetochore-associated mitotic kinase BubR1 phosphorylates itself in human cells and that this autophosphorylation is dependent on its binding partner, the kinetochore motor CENP-E. This CENP-E–dependent BubR1 autophosphorylation at unattached kinetochores is important for a full-strength mitotic checkpoint to prevent single chromosome loss. Replacing endogenous BubR1 with a nonphosphorylatable BubR1 mutant, as well as depletion of CENP-E, the BubR1 kinase activator, results in metaphase chromosome misalignment and a decrease of Aurora B–mediated Ndc80 phosphorylation at kinetochores. Furthermore, expressing a phosphomimetic BubR1 mutant substantially reduces the incidence of polar chromosomes in CENP-E–depleted cells. Thus, the state of CENP-E–dependent BubR1 autophosphorylation in response to spindle microtubule capture by CENP-E is important for kinetochore function in achieving accurate chromosome segregation.

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Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)–related protein Sli15 during chromosome segregation

The Journal of Cell Biology ◽

10.1083/jcb.200105029 ◽

2001 ◽

Vol 155 (5) ◽

pp. 763-774 ◽

Cited By ~ 121

Author(s):

Jung-seog Kang ◽

Iain M. Cheeseman ◽

George Kallstrom ◽

Soundarapandian Velmurugan ◽

Georjana Barnes ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Segregation ◽

Mitotic Spindle ◽

Kinase Activity ◽

Related Protein ◽

Centromeric Dna ◽

Centromere Protein ◽

Kinetochore Function

We have shown previously that Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. We show here that Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1–Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome–spindle interactions or in the movement of kinetochores along microtubules.

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TRF1 Depletion Reveals Mutual Regulation Between Telomeres, Kinetochores, and Inner Centromeres in Mouse Oocytes

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.749116 ◽

2021 ◽

Vol 9 ◽

Author(s):

Hyuk-Joon Jeon ◽

Jeong Su Oh

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Telomere Shortening ◽

Mouse Oocytes ◽

Kinetochore Assembly ◽

Telomere Protection ◽

Telocentric Chromosomes ◽

Close Proximity ◽

Mutual Regulation ◽

Kinetochore Function

In eukaryotic chromosomes, the centromere and telomere are two specialized structures that are essential for chromosome stability and segregation. Although centromeres and telomeres often are located in close proximity to form telocentric chromosomes in mice, it remained unclear whether these two structures influence each other. Here we show that TRF1 is required for inner centromere and kinetochore assembly in addition to its role in telomere protection in mouse oocytes. TRF1 depletion caused premature chromosome segregation by abrogating the spindle assembly checkpoint (SAC) and impairing kinetochore-microtubule (kMT) attachment, which increased the incidence of aneuploidy. Notably, TRF1 depletion disturbed the localization of Survivin and Ndc80/Hec1 at inner centromeres and kinetochores, respectively. Moreover, SMC3 and SMC4 levels significantly decreased after TRF1 depletion, suggesting that TRF1 is involved in chromosome cohesion and condensation. Importantly, inhibition of inner centromere or kinetochore function led to a significant decrease in TRF1 level and telomere shortening. Therefore, our results suggest that telomere integrity is required to preserve inner centromere and kinetochore architectures, and vice versa, suggesting mutual regulation between telomeres and centromeres.

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Relief of the Dma1-mediated checkpoint requires Dma1 autoubiquitination and dynamic localization

10.1101/322214 ◽

2018 ◽

Author(s):

Christine M. Jones ◽

Jun-Song Chen ◽

Alyssa E. Johnson ◽

Zachary C. Elmore ◽

Sierra N. Cullati ◽

...

Keyword(s):

Cell Death ◽

Cell Division ◽

Chromosome Segregation ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Mitotic Checkpoint ◽

Checkpoint Activation ◽

Essential Step ◽

Septation Initiation Network ◽

Anaphase B

AbstractChromosome segregation and cell division are coupled to prevent aneuploidy and cell death. In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) promotes cytokinesis, but upon mitotic checkpoint activation, the SIN is actively inhibited to prevent cytokinesis from occurring before chromosomes have safely segregated. SIN inhibition during the mitotic checkpoint is mediated by the E3 ubiquitin ligase Dma1. Dma1 binds to the CK1-phosphorylated SIN scaffold protein, Sid4, at the SPB, and ubiquitinates it. Sid4 ubiquitination antagonizes the SPB localization of the Polo-like kinase Plo1, the major SIN activator, so that SIN signaling is delayed. How this checkpoint is silenced once spindle defects are resolved has not been clear. Here we establish that Dma1 transiently leaves SPBs during anaphase B due to extensive auto-ubiquitination. The SIN is required for Dma1 to return to SPBs later in anaphase. Blocking Dma1 removal from SPBs by permanently tethering it to Sid4 prevents SIN activation and cytokinesis. Therefore, controlling Dma1’s SPB dynamics in anaphase is an essential step in S. pombe cell division and the silencing of the Dma1-dependent mitotic checkpoint.

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