Mad2-independent Spindle Assembly Checkpoint Activation and Controlled Metaphase–Anaphase Transition inDrosophilaS2 Cells

The spindle assembly checkpoint is essential to maintain genomic stability during cell division. We analyzed the role of the putative Drosophila Mad2 homologue in the spindle assembly checkpoint and mitotic progression. Depletion of Mad2 by RNAi from S2 cells shows that it is essential to prevent mitotic exit after spindle damage, demonstrating its conserved role. Mad2-depleted cells also show accelerated transit through prometaphase and premature sister chromatid separation, fail to form metaphases, and exit mitosis soon after nuclear envelope breakdown with extensive chromatin bridges that result in severe aneuploidy. Interestingly, preventing Mad2-depleted cells from exiting mitosis by a checkpoint-independent arrest allows congression of normally condensed chromosomes. More importantly, a transient mitotic arrest is sufficient for Mad2-depleted cells to exit mitosis with normal patterns of chromosome segregation, suggesting that all the associated phenotypes result from a highly accelerated exit from mitosis. Surprisingly, if Mad2-depleted cells are blocked transiently in mitosis and then released into a media containing a microtubule poison, they arrest with high levels of kinetochore-associated BubR1, properly localized cohesin complex and fail to exit mitosis revealing normal spindle assembly checkpoint activity. This behavior is specific for Mad2 because BubR1-depleted cells fail to arrest in mitosis under these experimental conditions. Taken together our results strongly suggest that Mad2 is exclusively required to delay progression through early stages of prometaphase so that cells have time to fully engage the spindle assembly checkpoint, allowing a controlled metaphase–anaphase transition and normal patterns of chromosome segregation.

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Interactions between N-Terminal Modules in MPS1 Enable Spindle Checkpoint Silencing

10.1101/438903 ◽

2018 ◽

Author(s):

Spyridon T. Pachis ◽

Yoshitaka Hiruma ◽

Anastassis Perrakis ◽

Geert J.P.L. Kops

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Intramolecular Interactions ◽

Mitotic Progression ◽

Microtubule Attachment ◽

Anaphase Onset ◽

Regulatory Input ◽

Tpr Domain

ABSTRACTFaithful chromosome segregation relies on the ability of the spindle assembly checkpoint (SAC) to delay anaphase onset until all chromosomes are attached to the mitotic spindle via their kinetochores. MPS1 kinase is recruited to unattached kinetochores to initiate SAC signaling, and is removed from kinetochores once stable microtubule attachments have been formed to allow normal mitotic progression. Here we show that a helical fragment within the kinetochore-targeting NTE module of MPS1 is required for interactions with kinetochores, and also forms intramolecular interactions with its adjacent TPR domain. Bypassing this NTE-TPR interaction results in high MPS1 levels at kinetochores due to loss of regulatory input into MPS1 localization, ineffecient MPS1 delocalization from kinetochores upon microtubule attachment, and SAC silencing defects. These results show that SAC responsiveness to attachments relies on regulated intramolecular interactions in MPS1 and highlight the sensitivity of mitosis to perturbations in the dynamics of the MSP1-NDC80-C interactions.

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Aurora B Tension Sensing Mechanisms in the Kinetochore Ensure Accurate Chromosome Segregation

International Journal of Molecular Sciences ◽

10.3390/ijms22168818 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8818

Author(s):

Shelby L. McVey ◽

Jenna K. Cosby ◽

Natalie J. Nannas

Keyword(s):

Chromosome Segregation ◽

Birth Defects ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Aurora B ◽

Sister Chromatids ◽

Congenital Birth Defects ◽

Biochemical Signals ◽

Segregation Of Chromosomes

The accurate segregation of chromosomes is essential for the survival of organisms and cells. Mistakes can lead to aneuploidy, tumorigenesis and congenital birth defects. The spindle assembly checkpoint ensures that chromosomes properly align on the spindle, with sister chromatids attached to microtubules from opposite poles. Here, we review how tension is used to identify and selectively destabilize incorrect attachments, and thus serves as a trigger of the spindle assembly checkpoint to ensure fidelity in chromosome segregation. Tension is generated on properly attached chromosomes as sister chromatids are pulled in opposing directions but resisted by centromeric cohesin. We discuss the role of the Aurora B kinase in tension-sensing and explore the current models for translating mechanical force into Aurora B-mediated biochemical signals that regulate correction of chromosome attachments to the spindle.

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Bub3 promotes Cdc20-dependent activation of the APC/C in S. cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.201412036 ◽

2015 ◽

Vol 209 (4) ◽

pp. 519-527 ◽

Cited By ~ 13

Author(s):

Yang Yang ◽

Dai Tsuchiya ◽

Soni Lacefield

Keyword(s):

Chromosome Segregation ◽

Unknown Function ◽

Budding Yeast ◽

Spindle Checkpoint ◽

Anaphase Promoting Complex ◽

Mitotic Progression ◽

Checkpoint Activation ◽

Anaphase Onset ◽

Impaired Binding

The spindle checkpoint ensures accurate chromosome segregation by sending a signal from an unattached kinetochore to inhibit anaphase onset. Numerous studies have described the role of Bub3 in checkpoint activation, but less is known about its functions apart from the spindle checkpoint. In this paper, we demonstrate that Bub3 has an unexpected role promoting metaphase progression in budding yeast. Loss of Bub3 resulted in a metaphase delay that was not a consequence of aneuploidy or the activation of a checkpoint. Instead, bub3Δ cells had impaired binding of the anaphase-promoting complex/cyclosome (APC/C) with its activator Cdc20, and the delay could be rescued by Cdc20 overexpression. Kinetochore localization of Bub3 was required for normal mitotic progression, and Bub3 and Cdc20 colocalized at the kinetochore. Although Bub1 binds Bub3 at the kinetochore, bub1Δ cells did not have compromised APC/C and Cdc20 binding. The results demonstrate that Bub3 has a previously unknown function at the kinetochore in activating APC/C-Cdc20 for normal mitotic progression.

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ULK1 phosphorylates Mad1 to regulate spindle assembly checkpoint

Nucleic Acids Research ◽

10.1093/nar/gkz602 ◽

2019 ◽

Vol 47 (15) ◽

pp. 8096-8110 ◽

Cited By ~ 7

Author(s):

Fengjie Yuan ◽

Ximin Jin ◽

Dan Li ◽

Yuanshuai Song ◽

Nan Zhang ◽

...

Keyword(s):

Chromosome Segregation ◽

Cell Cycle Progression ◽

Spindle Assembly Checkpoint ◽

Chromosome Instability ◽

Spindle Assembly ◽

Cancer Cell Growth ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Significant Impairment

Abstract The spindle assembly checkpoint (SAC) ensures the fidelity of chromosome segregation during mitosis. Here, we show that ULK1, a serine/threonine kinase that plays a key role in initiation of autophagy, also has an important function in the activation of SAC. ULK1 phosphorylates the SAC protein Mad1 at Ser546 to recruit Mad1 to kinetochores. Furthermore, Rod/ZW10/Zwilch (RZZ) complex may serve as a receptor for phos-Ser546-Mad1 at kinetochore, since phosphorylation of Mad1 by ULK1 strengthens the interaction between Mad1 and RZZ complex. In addition, deletion of ULK1 increases chromosome instability and cytotoxicity of paclitaxel, resulting in significant impairment of cancer cell growth. These findings highlight the role of ULK1 as a protein kinase controlling the fidelity of chromosome segregation and cell-cycle progression.

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Mammalian SWI/SNF chromatin remodeler is essential for reductional meiosis in males

10.1101/2020.04.28.066647 ◽

2020 ◽

Author(s):

Debashish U. Menon ◽

Terry Magnuson

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Male Meiosis ◽

Chromatin Modifications ◽

Histone H2a ◽

Centromeric Chromatin ◽

Chromosome Passenger Complex ◽

Metaphase I

AbstractBRG1, a catalytic subunit of the mammalian SWI/SNF nucleosome remodeler is essential for male meiosis1. In addition to BRG1, multiple subunits (~10-14) some of which are mutually exclusive, constitute biochemically distinct SWI/SNF subcomplexes, whose functions in gametogenesis remain unknown. Here, we identify a role for the PBAF (Polybromo - Brg1 Associated Factor) complex in the regulation of meiotic cell division. The germ cell-specific depletion of PBAF specific subunit, ARID2 resulted in a metaphase-I arrest. Arid2cKO metaphase-I spermatocytes displayed defects in chromosome organization and spindle assembly. Additionally, mutant centromeres were devoid of Polo-like kinase1 (PLK1), a known regulator of the spindle assembly checkpoint (SAC)2. The loss of PLK1 coincided with an abnormal chromosome-wide expansion of centromeric chromatin modifications such as Histone H3 threonine3 phosphorylation (H3T3P) and Histone H2A threonine120 phosphorylation (H2AT120P) that are critical for chromosome segregation3,4. Consistent with the known role of these histone modifications in chromosome passenger complex (CPC) recruitment, Arid2cKO metaphase-I chromosomes display defects in CPC association. We propose that ARID2 facilitates metaphase-I exit by regulating spindle assembly and centromeric chromatin.

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Recent Progress on the Localization of the Spindle Assembly Checkpoint Machinery to Kinetochores

Cells ◽

10.3390/cells8030278 ◽

2019 ◽

Vol 8 (3) ◽

pp. 278 ◽

Cited By ~ 14

Author(s):

Zhen Dou ◽

Diogjena Prifti ◽

Ping Gui ◽

Xing Liu ◽

Sabine Elowe ◽

...

Keyword(s):

Chromosome Segregation ◽

Genome Stability ◽

Cell Cycle Progression ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Progression ◽

Cycle Progression ◽

Daughter Cells ◽

Microtubule Attachment ◽

Surveillance Mechanism

Faithful chromosome segregation during mitosis is crucial for maintaining genome stability. The spindle assembly checkpoint (SAC) is a surveillance mechanism that ensures accurate mitotic progression. Defective SAC signaling leads to premature sister chromatid separation and aneuploid daughter cells. Mechanistically, the SAC couples the kinetochore microtubule attachment status to the cell cycle progression machinery. In the presence of abnormal kinetochore microtubule attachments, the SAC prevents the metaphase-to-anaphase transition through a complex kinase-phosphatase signaling cascade which results in the correct balance of SAC components recruited to the kinetochore. The correct kinetochore localization of SAC proteins is a prerequisite for robust SAC signaling and, hence, accurate chromosome segregation. Here, we review recent progresses on the kinetochore recruitment of core SAC factors.

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BubR1 alterations that reinforce mitotic surveillance act against aneuploidy and cancer

eLife ◽

10.7554/elife.16620 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 6

Author(s):

Robbyn L Weaver ◽

Jazeel F Limzerwala ◽

Ryan M Naylor ◽

Karthik B Jeganathan ◽

Darren J Baker ◽

...

Keyword(s):

Transgenic Mice ◽

Error Correction ◽

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Binding Domain ◽

Amino Terminal ◽

Binding Domains

BubR1 is a key component of the spindle assembly checkpoint (SAC). Mutations that reduce BubR1 abundance cause aneuploidization and tumorigenesis in humans and mice, whereas BubR1 overexpression protects against these. However, how supranormal BubR1 expression exerts these beneficial physiological impacts is poorly understood. Here, we used Bub1b mutant transgenic mice to explore the role of the amino-terminal (BubR1N) and internal (BubR1I) Cdc20-binding domains of BubR1 in preventing aneuploidy and safeguarding against cancer. BubR1N was necessary, but not sufficient to protect against aneuploidy and cancer. In contrast, BubR1 lacking the internal Cdc20-binding domain provided protection against both, which coincided with improved microtubule-kinetochore attachment error correction and SAC activity. Maximal SAC reinforcement occurred when both the Phe- and D-box of BubR1I were disrupted. Thus, while under- or overexpression of most mitotic regulators impairs chromosome segregation fidelity, certain manipulations of BubR1 can positively impact this process and therefore be therapeutically exploited.

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FANCA Controls Mitotic Phosphosignaling Networks To Ensure Genome Stability During Cell Division

Blood ◽

10.1182/blood.v122.21.801.801 ◽

2013 ◽

Vol 122 (21) ◽

pp. 801-801

Author(s):

Rikki Enzor ◽

Zahi Abdul Sater ◽

Donna Cerabona ◽

Zejin Sun ◽

Su-jung Park ◽

...

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Chromosome Segregation ◽

Fanconi Anemia ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Signaling Network ◽

Mitotic Apparatus ◽

Risk Of Cancer

Abstract Fanconi anemia (FA) is a heterogenous genome instability syndrome with a high risk of cancer. The FA proteins are essential for interphase DNA damage repair. However, it is incompletely understood why FA-deficient cells also develop gross aneuploidy and multinucleation, which are symptoms of error-prone chromosome segregation. Emerging evidence indicates that the FA signaling network functions as a guardian of the genome throughout the cell cycle, including chromosome segregation during mitosis. However, the mechanistic aspects of the critical role of the FA signaling in mitosis remain poorly understood. We have recently shown that the FA signaling network localizes to the mitotic apparatus to control the spindle assembly checkpoint and centrosome maintenance (J Clin Invest 2013, in press). The spindle assembly checkpoint (SAC) is a complex tumor suppressor signaling network that prevents premature separation of sister chromatids by delaying the metaphase-to-anaphase transition until all the kinetochores are properly attached to the mitotic spindle. Since weakened SAC promotes stochastic chromosome segregation, mutagenesis and cancer, these findings shed new light on the role of FA signaling in maintenance of genomic stability. We found the subcellular localization of FA proteins to the mitotic apparatus is spatiotemporally regulated as cells divide. Our new data revealed the pathways connecting the FANCA protein with canonical mitotic phosphosignaling networks. We have employed unbiased kinome-wide phospho-mass spectrometry to compare the landscape of abnormalities of mitotic signaling pathways in primary FANCA-/- patient cells and gene-corrected isogenic cells. These experiments led us to identify and quantify a wide range of phosphorylation abnormalities of multiple FANCA-dependent centrosome-, kinetochore- and chromosome-associated regulators of mitosis. Our data illuminated the role for FA signaling in three critical stages of cell division: (1) the spindle assembly checkpoint, (2) anaphase and (3) cytokinesis. Thus, we employed live phase-contrast imaging of primary FANCA-/- patient cells in comparison to gene-corrected cells to separately quantify aberrations in (1) chromosome congression and metaphase-anaphase transition (SAC malfunction), (2) execution of anaphase and (3) completion of cytokinesis. Our findings further our understanding of human cell cycle control and provide new insights into the origins of genomic instability in Fanconi anemia by establishing mechanistic connection between the FANCA protein and key mitotic signaling networks. The identification of cell division pathways regulated by FANCA has implications for future targeted drug development in Fanconi anemia and FA-deficient malignancies in the general population. Disclosures: No relevant conflicts of interest to declare.

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Accumulation of Mad2–Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.200602109 ◽

2006 ◽

Vol 174 (1) ◽

pp. 39-51 ◽

Cited By ~ 37

Author(s):

Luigi Nezi ◽

Giulia Rancati ◽

Anna De Antoni ◽

Sebastiano Pasqualato ◽

Simonetta Piatti ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Sister Chromatid ◽

Hela Cells ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Progression ◽

Checkpoint Activation ◽

Mutant Proteins ◽

Checkpoint Protein

The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a mad2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution.

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Dual Regulation of Mad2 Localization on Kinetochores by Bub1 and Dam1/DASH that Ensure Proper Spindle Interaction

Molecular Biology of the Cell ◽

10.1091/mbc.e08-03-0298 ◽

2008 ◽

Vol 19 (9) ◽

pp. 3885-3897 ◽

Cited By ~ 11

Author(s):

Shigeaki Saitoh ◽

Yasuyo Kobayashi ◽

Yuki Ogiyama ◽

Kohta Takahashi

Keyword(s):

Fission Yeast ◽

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

The State ◽

Abnormal Chromosome ◽

Mitotic Progression ◽

Checkpoint Activation ◽

Molecular Sensors ◽

Checkpoint Proteins ◽

Spindle Attachment

The spindle assembly checkpoint monitors the state of spindle–kinetochore interaction to prevent premature onset of anaphase. Although checkpoint proteins, such as Mad2, are localized on kinetochores that do not interact properly with the spindle, it remains unknown how the checkpoint proteins recognize abnormalities in spindle–kinetochore interaction. Here, we report that Mad2 localization on kinetochores in fission yeast is regulated by two partially overlapping but distinct pathways: the Dam1/DASH and the Bub1 pathways. We show that Mad2 is localized on “unattached” as well as “tensionless” kinetochores. Our observations suggest that Bub1 is required for Mad2 to detect tensionless kinetochores, whereas Dam1/DASH is crucial for Mad2 to detect unattached kinetochores. In cells lacking both Bub1 and Dam1/DASH, Mad2 localization on kinetochores is diminished, and mitotic progression appears to be accelerated despite the frequent occurrence of abnormal chromosome segregation. Furthermore, we found that Dam1/DASH is required for promotion of spindle association with unattached kinetochores. In contrast, there is accumulating evidence that Bub1 is involved in resolution of erroneous spindle attachment on tensionless kinetochores. These pathways may act as molecular sensors determining the state of spindle association on each kinetochore, enabling proper regulation of the checkpoint activation as well as promotion/resolution of spindle attachment.

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