scholarly journals Chk2 prevents mitotic exit when the majority of kinetochores are unattached

2014 ◽  
Vol 205 (3) ◽  
pp. 339-356 ◽  
Author(s):  
Eleni Petsalaki ◽  
George Zachos
Keyword(s):  
Spindle Checkpoint ◽  
Mitotic Exit ◽  
Aurora B ◽  
Anaphase Onset ◽  
Chromosome Alignment ◽  
Essential Function ◽  
In Cells

The spindle checkpoint delays exit from mitosis in cells with spindle defects. In this paper, we show that Chk2 is required to delay anaphase onset when microtubules are completely depolymerized but not in the presence of relatively few unattached kinetochores. Mitotic exit in Chk2-deficient cells correlates with reduced levels of Mps1 protein and increased Cdk1–tyrosine 15 inhibitory phosphorylation. Chk2 localizes to kinetochores and is also required for Aurora B–serine 331 phosphorylation in nocodazole or unperturbed early prometaphase. Serine 331 phosphorylation contributed to prometaphase accumulation in nocodazole after partial Mps1 inhibition and was required for spindle checkpoint establishment at the beginning of mitosis. In addition, expression of a phosphomimetic S331E mutant Aurora B rescued chromosome alignment or segregation in Chk2-deficient cells. We propose that Chk2 stabilizes Mps1 and phosphorylates Aurora B–serine 331 to prevent mitotic exit when most kinetochores are unattached. These results highlight mechanisms of an essential function of Chk2 in mitosis.

scholarly journals Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores

2003 ◽  
Vol 161 (2) ◽  
pp. 267-280 ◽  
Author(s):  
Claire Ditchfield ◽  
Victoria L. Johnson ◽  
Anthony Tighe ◽  
Rebecca Ellston ◽  
Carolyn Haworth ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Spindle Checkpoint ◽  
Aurora Kinase ◽  
Multiple Roles ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Checkpoint Proteins ◽  
Anaphase Onset ◽  
Chromosome Alignment

The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

scholarly journals The Ki-67 and RepoMan mitotic phosphatases assemble via an identical, yet novel mechanism

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Ganesan Senthil Kumar ◽  
Ezgi Gokhan ◽  
Sofie De Munter ◽  
Mathieu Bollen ◽  
Paola Vagnarelli ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Mitotic Chromosome ◽  
Cancer Therapeutics ◽  
Mitotic Exit ◽  
Protein Phosphatase 1 ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Ki 67 ◽  
Anaphase Onset

Ki-67 and RepoMan have key roles during mitotic exit. Previously, we showed that Ki-67 organizes the mitotic chromosome periphery and recruits protein phosphatase 1 (PP1) to chromatin at anaphase onset, in a similar manner as RepoMan (<xref ref-type="bibr" rid="bib2">Booth et al., 2014</xref>). Here we show how Ki-67 and RepoMan form mitotic exit phosphatases by recruiting PP1, how they distinguish between distinct PP1 isoforms and how the assembly of these two holoenzymes are dynamically regulated by Aurora B kinase during mitosis. Unexpectedly, our data also reveal that Ki-67 and RepoMan bind PP1 using an identical, yet novel mechanism, interacting with a PP1 pocket that is engaged only by these two PP1 regulators. These findings not only show how two distinct mitotic exit phosphatases are recruited to their substrates, but also provide immediate opportunities for the design of novel cancer therapeutics that selectively target the Ki-67:PP1 and RepoMan:PP1 holoenzymes.

scholarly journals The spindle and kinetochore–associated (Ska) complex enhances binding of the anaphase-promoting complex/cyclosome (APC/C) to chromosomes and promotes mitotic exit

2014 ◽  
Vol 25 (5) ◽  
pp. 594-605 ◽  
Author(s):  
Sushama Sivakumar ◽  
John R. Daum ◽  
Aaron R. Tipton ◽  
Susannah Rankin ◽  
Gary J. Gorbsky
Keyword(s):  
Cyclin B1 ◽  
Mitotic Exit ◽  
Anaphase Promoting Complex ◽  
Metaphase Arrest ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Chromosome Alignment ◽  
Partial Degradation ◽  
Interfering Rna ◽  
Transient Alignment

The spindle and kinetochore–associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA–mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore–microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit.

scholarly journals Kinetochore-localized BUB-1/BUB-3 complex promotes anaphase onset in C. elegans

2015 ◽  
Vol 209 (4) ◽  
pp. 507-517 ◽  
Author(s):  
Taekyung Kim ◽  
Mark W. Moyle ◽  
Pablo Lara-Gonzalez ◽  
Christian De Groot ◽  
Karen Oegema ◽  
...  
Keyword(s):  
Spindle Checkpoint ◽  
Kinase Domain ◽  
Mitotic Chromosomes ◽  
Checkpoint Signaling ◽  
C Elegans ◽  
Anaphase Onset ◽  
Checkpoint Pathway ◽  
Chromosome Alignment ◽  
Kinetochore Region

The conserved Bub1/Bub3 complex is recruited to the kinetochore region of mitotic chromosomes, where it initiates spindle checkpoint signaling and promotes chromosome alignment. Here we show that, in contrast to the expectation for a checkpoint pathway component, the BUB-1/BUB-3 complex promotes timely anaphase onset in Caenorhabditis elegans embryos. This activity of BUB-1/BUB-3 was independent of spindle checkpoint signaling but required kinetochore localization. BUB-1/BUB-3 inhibition equivalently delayed separase activation and other events occurring during mitotic exit. The anaphase promotion function required BUB-1’s kinase domain, but not its kinase activity, and this function was independent of the role of BUB-1/BUB-3 in chromosome alignment. These results reveal an unexpected role for the BUB-1/BUB-3 complex in promoting anaphase onset that is distinct from its well-studied functions in checkpoint signaling and chromosome alignment, and suggest a new mechanism contributing to the coordination of the metaphase-to-anaphase transition.

scholarly journals Haspin inhibitors reveal centromeric functions of Aurora B in chromosome segregation

2012 ◽  
Vol 199 (2) ◽  
pp. 251-268 ◽  
Author(s):  
Fangwei Wang ◽  
Natalia P. Ulyanova ◽  
John R. Daum ◽  
Debasis Patnaik ◽  
Anna V. Kateneva ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Metaphase Chromosome ◽  
Histone H3 ◽  
Spindle Checkpoint ◽  
Aurora B ◽  
Docking Site ◽  
Chromosome Movement ◽  
Checkpoint Signaling ◽  
Chromosome Alignment ◽  
Spindle Midzone

Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), providing a docking site for the Aurora B complex at centromeres. Aurora B functions to correct improper kinetochore–microtubule attachments and alert the spindle checkpoint to the presence of misaligned chromosomes. We show that Haspin inhibitors decreased H3T3ph, resulting in loss of centromeric Aurora B and reduced phosphorylation of centromere and kinetochore Aurora B substrates. Consequently, metaphase chromosome alignment and spindle checkpoint signaling were compromised. These effects were phenocopied by microinjection of anti-H3T3ph antibodies. Retargeting Aurora B to centromeres partially restored checkpoint signaling and Aurora B–dependent phosphorylation at centromeres and kinetochores, bypassing the need for Haspin activity. Haspin inhibitors did not obviously affect phosphorylation of histone H3 at serine-10 (H3S10ph) by Aurora B on chromosome arms but, in Aurora B reactivation assays, recovery of H3S10ph was delayed. Haspin inhibitors did not block Aurora B localization to the spindle midzone in anaphase or Aurora B function in cytokinesis. Thus, Haspin inhibitors reveal centromeric roles of Aurora B in chromosome movement and spindle checkpoint signaling.

scholarly journals Delayed Chromosome Alignment to the Spindle Equator Increases the Rate of Chromosome Missegregation in Cancer Cell Lines

Biomolecules ◽  
2018 ◽  
Vol 9 (1) ◽  
pp. 10 ◽  
Author(s):  
Kinue Kuniyasu ◽  
Kenji Iemura ◽  
Kozo Tanaka
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Chromosome Missegregation ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
In Cells

For appropriate chromosome segregation, kinetochores on sister chromatids have to attach to microtubules from opposite spindle poles (bi-orientation). Chromosome alignment at the spindle equator, referred to as congression, can occur through the attachment of kinetochores to the lateral surface of spindle microtubules, facilitating bi-orientation establishment. However, the contribution of this phenomenon to mitotic fidelity has not been clarified yet. Here, we addressed whether delayed chromosome alignment to the spindle equator increases the rate of chromosome missegregation. Cancer cell lines depleted of Kid, a chromokinesin involved in chromosome congression, showed chromosome alignment with a slight delay, and increased frequency of lagging chromosomes. Delayed chromosome alignment concomitant with an increased rate of lagging chromosomes was also seen in cells depleted of kinesin family member 4A (KIF4A), another chromokinesin. Cells that underwent chromosome missegregation took relatively longer time to align chromosomes in both control and Kid/KIF4A-depleted cells. Tracking of late-aligning chromosomes showed that they exhibit a higher rate of lagging chromosomes. Intriguingly, the metaphase of cells that underwent chromosome missegregation was shortened, and delaying anaphase onset ameliorated the increased chromosome missegregation. These data suggest that late-aligning chromosomes do not have sufficient time to establish bi-orientation, leading to chromosome missegregation. Our data imply that delayed chromosome alignment is not only a consequence, but also a cause of defective bi-orientation establishment, which can lead to chromosomal instability in cells without severe mitotic defects.

scholarly journals Cytoplasmic dynein participates in meiotic checkpoint inactivation in mouse oocytes by transporting cytoplasmic mitotic arrest-deficient (Mad) proteins from kinetochores to spindle poles

Reproduction ◽  
2007 ◽  
Vol 133 (4) ◽  
pp. 685-695 ◽  
Author(s):  
Dong Zhang ◽  
Shen Yin ◽  
Man-Xi Jiang ◽  
Wei Ma ◽  
Yi Hou ◽  
...  
Keyword(s):  
Spindle Checkpoint ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Mitotic Arrest ◽  
Checkpoint Proteins ◽  
Meiotic Progression ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Oocyte Meiosis ◽  
And Function

The present study was designed to investigate the localization and function of cytoplasmic dynein (dynein) during mouse oocyte meiosis and its relationship with two major spindle checkpoint proteins, mitotic arrest-deficient (Mad) 1 and Mad2. Oocytes at various stages during the first meiosis were fixed and immunostained for dynein, Mad1, Mad2, kinetochores, microtubules, and chromosomes. Some oocytes were treated with nocodazole before examination. Anti-dynein antibody was injected into the oocytes at germinal vesicle (GV) stage before the examination of its effects on meiotic progression or Mad1 and Mad2 localization. Results showed that dynein was present in the oocytes at various stages from GV to metaphase II and the locations of Mad1 and Mad2 were associated with dynein’s movement. Both Mad1 and Mad2 had two existing states: one existed in the cytoplasm (cytoplasmic Mad1 or cytoplasmic Mad2), which did not bind to kinetochores, while the other bound to kinetochores (kinetochore Mad1 or kinetochore Mad2). The equilibrium between the two states varied during meiosis and/or in response to the changes of the connection between microtubules and kinetochores. Cytoplasmic Mad1 and Mad2 recruited to chromosomes when the connection between microtubules and chromosomes was destroyed. Inhibition of dynein interferes with cytoplasmic Mad1 and Mad2 transportation from chromosomes to spindle poles, thus inhibits checkpoint silence and delays anaphase onset. These results indicate that dynein may play a role in spindle checkpoint inactivation.

scholarly journals Differential requirement for Bub1 and Bub3 in regulation of meiotic versus mitotic chromosome segregation

2020 ◽  
Vol 219 (4) ◽  
Author(s):  
Gisela Cairo ◽  
Anne M. MacKenzie ◽  
Soni Lacefield
Keyword(s):  
Chromosome Segregation ◽  
Protein Phosphatase ◽  
Mitotic Chromosome ◽  
Budding Yeast ◽  
Meiotic Chromosome ◽  
Protein Phosphatase 1 ◽  
Aurora B ◽  
Chromosome Missegregation ◽  
Anaphase Onset ◽  
Spindle Microtubules

Accurate chromosome segregation depends on the proper attachment of kinetochores to spindle microtubules before anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore–microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1–Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome–spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1–Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.

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