scholarly journals Sequestration of CDH1 by MAD2L2 prevents premature APC/C activation prior to anaphase onset

2013 ◽  
Vol 203 (1) ◽  
pp. 87-100 ◽  
Author(s):  
Tamar Listovsky ◽  
Julian E. Sale
Keyword(s):  
Cyclin B1 ◽  
Human Cells ◽  
Anaphase Promoting Complex ◽  
Bistable Switch ◽  
Anaphase Onset ◽  
Subsequent Degradation ◽  
Sequential Activation

The switch from activation of the anaphase-promoting complex/cyclosome (APC/C) by CDC20 to CDH1 during anaphase is crucial for accurate mitosis. APC/CCDC20 ubiquitinates a limited set of substrates for subsequent degradation, including Cyclin B1 and Securin, whereas APC/CCDH1 has a broader specificity. This switch depends on dephosphorylation of CDH1 and the APC/C, and on the degradation of CDC20. Here we show, in human cells, that the APC/C inhibitor MAD2L2 also contributes to ensuring the sequential activation of the APC/C by CDC20 and CDH1. In prometaphase, MAD2L2 sequestered free CDH1 away from the APC/C. At the onset of anaphase, MAD2L2 was rapidly degraded by APC/CCDC20, releasing CDH1 to activate the dephosphorylated APC/C. Loss of MAD2L2 led to premature association of CDH1 with the APC/C, early destruction of APC/CCDH1 substrates, and accelerated mitosis with frequent mitotic aberrations. Thus, MAD2L2 helps to ensure a robustly bistable switch between APC/CCDC20 and APC/CCDH1 during the metaphase-to-anaphase transition, thereby contributing to mitotic fidelity.

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 Cdc20 hypomorphic mice fail to counteract de novo synthesis of cyclin B1 in mitosis

2010 ◽  
Vol 191 (2) ◽  
pp. 313-329 ◽  
Author(s):  
Liviu Malureanu ◽  
Karthik B. Jeganathan ◽  
Fang Jin ◽  
Darren J. Baker ◽  
Janine H. van Ree ◽  
...  
Keyword(s):  
De Novo ◽  
Cyclin B1 ◽  
Anaphase Promoting Complex ◽  
De Novo Synthesis ◽  
Cytoplasmic Polyadenylation ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Element Binding Protein ◽  
Sister Chromosome ◽  
Chromosome Separation

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome–microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element–binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset.

scholarly journals The RZZ complex requires the N-terminus of KNL1 to mediate optimal Mad1 kinetochore localization in human cells

Open Biology ◽  
2015 ◽  
Vol 5 (11) ◽  
pp. 150160 ◽  
Author(s):  
Gina V. Caldas ◽  
Tina R. Lynch ◽  
Ryan Anderson ◽  
Sana Afreen ◽  
Dileep Varma ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Protein S ◽  
Human Cells ◽  
Anaphase Promoting Complex ◽  
Anaphase Onset ◽  
N Terminus ◽  
Independent Mechanism ◽  
Direct Linkage ◽  
Surveillance Mechanism

The spindle assembly checkpoint is a surveillance mechanism that blocks anaphase onset until all chromosomes are properly attached to microtubules of the mitotic spindle. Checkpoint activity requires kinetochore localization of Mad1/Mad2 to inhibit activation of the anaphase promoting complex/cyclosome in the presence of unattached kinetochores. In budding yeast and Caenorhabditis elegans , Bub1, recruited to kinetochores through KNL1, recruits Mad1/Mad2 by direct linkage with Mad1. However, in human cells it is not yet established which kinetochore protein(s) function as the Mad1/Mad2 receptor. Both Bub1 and the RZZ complex have been implicated in Mad1/Mad2 kinetochore recruitment; however, their specific roles remain unclear. Here, we investigate the contributions of Bub1, RZZ and KNL1 to Mad1/Mad2 kinetochore recruitment. We find that the RZZ complex localizes to the N-terminus of KNL1, downstream of Bub1, to mediate robust Mad1/Mad2 kinetochore localization. Our data also point to the existence of a KNL1-, Bub1-independent mechanism for RZZ and Mad1/Mad2 kinetochore recruitment. Based on our results, we propose that in humans, the primary mediator for Mad1/Mad2 kinetochore localization is the RZZ complex.

scholarly journals Defining pathways of spindle checkpoint silencing: functional redundancy between Cdc20 ubiquitination and p31comet

2011 ◽  
Vol 22 (22) ◽  
pp. 4227-4235 ◽  
Author(s):  
Luying Jia ◽  
Bing Li ◽  
Ross T. Warrington ◽  
Xing Hao ◽  
Shixuan Wang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Spindle Checkpoint ◽  
Functional Redundancy ◽  
Mitotic Checkpoint ◽  
Human Cells ◽  
Unresolved Issue ◽  
Anaphase Promoting Complex ◽  
Anaphase Onset ◽  
Mitotic Checkpoint Complex

The spindle checkpoint senses unattached or improperly attached kinetochores during mitosis, inhibits the anaphase-promoting complex or cyclosome (APC/C), and delays anaphase onset to prevent aneuploidy. The mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20 is a critical APC/C-inhibitory checkpoint complex in human cells. At the metaphase–anaphase transition, the spindle checkpoint turns off, and MCC disassembles to allow anaphase onset. The molecular mechanisms of checkpoint inactivation are poorly understood. A major unresolved issue is the role of Cdc20 autoubiquitination in this process. Although Cdc20 autoubiquitination can promote Mad2 dissociation from Cdc20, a nonubiquitinatable Cdc20 mutant still dissociates from Mad2 during checkpoint inactivation. Here, we show that depletion of p31comet delays Mad2 dissociation from Cdc20 mutants that cannot undergo autoubiquitination. Thus both p31comet and ubiquitination of Cdc20 are critical mechanisms of checkpoint inactivation. They act redundantly to promote Mad2 dissociation from Cdc20.

Cyclin A and Nek2A: APC/C–Cdc20 substrates invisible to the mitotic spindle checkpoint

2010 ◽  
Vol 38 (1) ◽  
pp. 72-77 ◽  
Author(s):  
Wouter van Zon ◽  
Rob M.F. Wolthuis
Keyword(s):  
Nuclear Envelope ◽  
Spindle Checkpoint ◽  
Cyclin A ◽  
Cyclin B1 ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
Spindle Microtubules

Active cyclin B1–Cdk1 (cyclin-dependent kinase 1) keeps cells in mitosis, allowing time for spindle microtubules to capture the chromosomes and for incorrect chromosome-spindle attachments to be repaired. Meanwhile, securin, an inhibitor of separase, secures cohesion between sister chromatids, preventing anaphase onset. The spindle checkpoint is a signalling pathway emerging from improperly attached chromosomes that inhibits Cdc20, the mitotic activator of the APC/C (anaphase-promoting complex/cyclosome) ubiquitin ligase. Blocking Cdc20 stabilizes cyclin B1 and securin to delay mitotic exit and anaphase until all chromosomes reach bipolar spindle attachments. Cells entering mitosis in the absence of a functional spindle checkpoint degrade cyclin B1 and securin right after nuclear-envelope breakdown, in prometaphase. Interestingly, two APC/C substrates, cyclin A and Nek2A, are normally degraded at nuclear-envelope breakdown, even when the spindle checkpoint is active. This indicates that the APC/C is activated early in mitosis, whereas cyclin B1 and securin are protected as long as the spindle checkpoint inhibits Cdc20. Remarkably, destruction of cyclin A and Nek2A also depends on Cdc20. The paradox of Cdc20 being both active and inhibited in prometaphase could be explained if cyclin A and Nek2A are either exceptionally efficient Cdc20 substrates, or if they are equipped with ‘stealth’ mechanisms to effectively escape detection by the spindle checkpoint. In the present paper, we discuss recently emerging models for spindle-checkpoint-independent APC/C–Cdc20 activity, which might even have implications for cancer therapy.

scholarly journals The ubiquitin ligase CRL2ZYG11 targets cyclin B1 for degradation in a conserved pathway that facilitates mitotic slippage

2016 ◽  
Vol 215 (2) ◽  
pp. 151-166 ◽  
Author(s):  
Riju S. Balachandran ◽  
Cassandra S. Heighington ◽  
Natalia G. Starostina ◽  
James W. Anderson ◽  
David L. Owen ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Spindle Assembly Checkpoint ◽  
Cyclin B1 ◽  
Human Cells ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Animal Cells ◽  
C Elegans ◽  
Chemotherapy Drugs ◽  
Mitotic Slippage

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is known to target the degradation of cyclin B1, which is crucial for mitotic progression in animal cells. In this study, we show that the ubiquitin ligase CRL2ZYG-11 redundantly targets the degradation of cyclin B1 in Caenorhabditis elegans and human cells. In C. elegans, both CRL2ZYG-11 and APC/C are required for proper progression through meiotic divisions. In human cells, inactivation of CRL2ZYG11A/B has minimal effects on mitotic progression when APC/C is active. However, when APC/C is inactivated or cyclin B1 is overexpressed, CRL2ZYG11A/B-mediated degradation of cyclin B1 is required for normal progression through metaphase. Mitotic cells arrested by the spindle assembly checkpoint, which inactivates APC/C, often exit mitosis in a process termed “mitotic slippage,” which generates tetraploid cells and limits the effectiveness of antimitotic chemotherapy drugs. We show that ZYG11A/B subunit knockdown, or broad cullin–RING ubiquitin ligase inactivation with the small molecule MLN4924, inhibits mitotic slippage in human cells, suggesting the potential for antimitotic combination therapy.

scholarly journals Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis

2006 ◽  
Vol 174 (6) ◽  
pp. 791-801 ◽  
Author(s):  
Suzanne Madgwick ◽  
David V. Hansen ◽  
Mark Levasseur ◽  
Peter K. Jackson ◽  
Keith T. Jones
Keyword(s):  
Fluorescent Protein ◽  
Polar Body ◽  
Cyclin B1 ◽  
Anaphase Promoting Complex ◽  
Morpholino Knockdown ◽  
Short Time ◽  
Mitotic Inhibitor ◽  
Antisense Morpholino ◽  
Meiosis I

During interkinesis, a metaphase II (MetII) spindle is built immediately after the completion of meiosis I. Oocytes then remain MetII arrested until fertilization. In mouse, we find that early mitotic inhibitor 2 (Emi2), which is an anaphase-promoting complex inhibitor, is involved in both the establishment and the maintenance of MetII arrest. In MetII oocytes, Emi2 needs to be degraded for oocytes to exit meiosis, and such degradation, as visualized by fluorescent protein tagging, occurred tens of minutes ahead of cyclin B1. Emi2 antisense morpholino knockdown during oocyte maturation did not affect polar body (PB) extrusion. However, in interkinesis the central spindle microtubules from meiosis I persisted for a short time, and a MetII spindle failed to assemble. The chromatin in the oocyte quickly decondensed and a nucleus formed. All of these effects were caused by the essential role of Emi2 in stabilizing cyclin B1 after the first PB extrusion because in Emi2 knockdown oocytes a MetII spindle was recovered by Emi2 rescue or by expression of nondegradable cyclin B1 after meiosis I.

scholarly journals Loss of Cdc20 Causes a Securin-Dependent Metaphase Arrest in Two-Cell Mouse Embryos

2007 ◽  
Vol 27 (9) ◽  
pp. 3481-3488 ◽  
Author(s):  
Min Li ◽  
J. Philippe York ◽  
Pumin Zhang
Keyword(s):  
Ubiquitin Ligase ◽  
Cyclin B1 ◽  
Mitotic Exit ◽  
Adapter Proteins ◽  
Anaphase Promoting Complex ◽  
Metaphase Arrest ◽  
Cell Stage ◽  
Gene Trapping ◽  
Protein Substrates

ABSTRACT The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase mediating targeted proteolysis through ubiquitination of protein substrates to control the progression of mitosis. The APC/C recognizes its substrates through two adapter proteins, Cdc20 and Cdh1, which contain similar C-terminal domains composed of seven WD-40 repeats believed to be involved in interacting with their substrates. During the transition from metaphase to anaphase, APC/C-Cdc20 mediates the ubiquitination of securin and cyclin B1, allowing the activation of separase and the onset of anaphase and mitotic exit. APC/C-Cdc20 and APC/C-Cdh1 have overlapping substrates. It is unclear whether they are redundant for mitosis. Using a gene-trapping approach, we have obtained mice which lack Cdc20 function. These mice show failed embryogenesis. The embryos were arrested in metaphase at the two-cell stage with high levels of cyclin B1, indicating an essential role of Cdc20 in mitosis that is not redundant with that of Cdh1. Interestingly, Cdc20 and securin double mutant embryos could not maintain the metaphase arrest, suggesting a role of securin in preventing mitotic exit.

scholarly journals The RAD51 recombinase protects mitotic chromatin in human cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Isabel E. Wassing ◽  
Emily Graham ◽  
Xanita Saayman ◽  
Lucia Rampazzo ◽  
Christine Ralf ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Genome Stability ◽  
Spindle Assembly Checkpoint ◽  
De Novo ◽  
Genomic Stability ◽  
Human Cells ◽  
Genome Integrity ◽  
Chromosomal Replication ◽  
Anaphase Onset ◽  
Mitotic Chromatin

AbstractThe RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.

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