scholarly journals Inefficient degradation of cyclin B1 re-activates the spindle checkpoint right after sister chromatid disjunction

Cell Cycle ◽  
2014 ◽  
Vol 13 (15) ◽  
pp. 2370-2378 ◽  
Author(s):  
Linda Clijsters ◽  
Wouter van Zon ◽  
Bas ter Riet ◽  
Erik Voets ◽  
Michiel Boekhout ◽  
...  
Keyword(s):  
Sister Chromatid ◽  
Spindle Checkpoint ◽  
Cyclin B1

scholarly journals Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1

2002 ◽  
Vol 157 (7) ◽  
pp. 1125-1137 ◽  
Author(s):  
Anja Hagting ◽  
Nicole den Elzen ◽  
Hartmut C. Vodermaier ◽  
Irene C. Waizenegger ◽  
Jan-Michael Peters ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Spindle Checkpoint ◽  
Cyclin B1 ◽  
Mutant Form ◽  
Exact Time ◽  
Sister Chromatids ◽  
Sister Chromatid Separation

Progress through mitosis is controlled by the sequential destruction of key regulators including the mitotic cyclins and securin, an inhibitor of anaphase whose destruction is required for sister chromatid separation. Here we have used live cell imaging to determine the exact time when human securin is degraded in mitosis. We show that the timing of securin destruction is set by the spindle checkpoint; securin destruction begins at metaphase once the checkpoint is satisfied. Furthermore, reimposing the checkpoint rapidly inactivates securin destruction. Thus, securin and cyclin B1 destruction have very similar properties. Moreover, we find that both cyclin B1 and securin have to be degraded before sister chromatids can separate. A mutant form of securin that lacks its destruction box (D-box) is still degraded in mitosis, but now this is in anaphase. This destruction requires a KEN box in the NH2 terminus of securin and may indicate the time in mitosis when ubiquitination switches from APCCdc20 to APCCdh1. Lastly, a D-box mutant of securin that cannot be degraded in metaphase inhibits sister chromatid separation, generating a cut phenotype where one cell can inherit both copies of the genome. Thus, defects in securin destruction alter chromosome segregation and may be relevant to the development of aneuploidy in cancer.

scholarly journals The spindle checkpoint, APC/CCdc20, and APC/CCdh1 play distinct roles in connecting mitosis to S phase

2013 ◽  
Vol 201 (7) ◽  
pp. 1013-1026 ◽  
Author(s):  
Linda Clijsters ◽  
Janneke Ogink ◽  
Rob Wolthuis
Keyword(s):  
Spindle Checkpoint ◽  
Cyclin B1 ◽  
S Phase ◽  
Animal Cells ◽  
Proliferating Cells ◽  
Cell Cycle Entry ◽  
Sister Chromatids ◽  
Mitotic Cyclin ◽  
Bona Fide ◽  
Subsequent Activation

DNA replication depends on a preceding licensing event by Cdt1 and Cdc6. In animal cells, relicensing after S phase but before mitosis is prevented by the Cdt1 inhibitor geminin and mitotic cyclin activity. Here, we show that geminin, like cyclin B1 and securin, is a bona fide target of the spindle checkpoint and APC/CCdc20. Cyclin B1 and geminin are degraded simultaneously during metaphase, which directs Cdt1 accumulation on segregating sister chromatids. Subsequent activation of APC/CCdh1 leads to degradation of Cdc6 well before Cdt1 becomes unstable in a replication-coupled manner. In mitosis, the spindle checkpoint supports Cdt1 accumulation, which promotes S phase onset. We conclude that the spindle checkpoint, APC/CCdc20, and APC/CCdh1 act successively to ensure that the disappearance of licensing inhibitors coincides exactly with a peak of Cdt1 and Cdc6. Whereas cell cycle entry from quiescence requires Cdc6 resynthesis, our results indicate that proliferating cells use a window of time in mitosis, before Cdc6 is degraded, as an earlier opportunity to direct S phase.

scholarly journals The Role of Cdc55 in the Spindle Checkpoint Is through Regulation of Mitotic Exit in Saccharomyces cerevisiae

2006 ◽  
Vol 17 (2) ◽  
pp. 658-666 ◽  
Author(s):  
Christopher M. Yellman ◽  
Daniel J. Burke
Keyword(s):  
Sister Chromatid ◽  
Spindle Checkpoint ◽  
Sister Chromatid Cohesion ◽  
Mitotic Exit ◽  
Negative Regulator ◽  
Regulatory Subunit ◽  
Checkpoint Activation ◽  
Chromatid Cohesion ◽  
Phosphatase 2A

Cdc55, a B-type regulatory subunit of protein phosphatase 2A, has been implicated in mitotic spindle checkpoint activity and maintenance of sister chromatid cohesion during metaphase. The spindle checkpoint is composed of two independent pathways, one leading to inhibition of the metaphase-to-anaphase transition by checkpoint proteins, including Mad2, and the other to inhibition of mitotic exit by Bub2. We show that Cdc55 is a negative regulator of mitotic exit. A cdc55 mutant, like a bub2 mutant, prematurely releases Cdc14 phosphatase from the nucleolus during spindle checkpoint activation, and premature exit from mitosis indirectly leads to loss of sister chromatid cohesion and inviability in nocodazole. The role of Cdc55 is separable from Bub2 and inhibits release of Cdc14 through a mechanism independent of the known negative regulators of mitotic exit. Epistasis experiments indicate Cdc55 acts either downstream or independent of the mitotic exit network kinase Cdc15. Interestingly, the B-type cyclin Clb2 is partially stable during premature activation of mitotic exit in a cdc55 mutant, indicating mitotic exit is incomplete.

Molecular Mechanisms of Spindle Checkpoint-Apoptosis Linkage and Karyotypic Stability in Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3361-3361
Author(s):  
Charlie Mantel ◽  
Sara Rhorabough ◽  
Ying Guo ◽  
Man-Ryul Lee ◽  
Myung-Kwan Han ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Spindle Checkpoint ◽  
Cyclin B1 ◽  
Polyploid Cell ◽  
Mitotic Slippage

Abstract Ex-vivo expansion of human HSC prior to bone marrow transplantation is still an unrealized goal that could greatly extend the usefulness of this mainstay strategy for treating numerous human hematologic diseases. The safety of this process for potential use in humans depends in large part on the maintenance of karyotypic stability of HSC during expansion, a lack of which could contribute to serious, even fatal, complications such as cancer, and could also contribute to engraftment failure. The spindle checkpoint and its linkage to apoptosis initiation is one of the most important cellular processes that helps maintain chromosomal stability in rapidly proliferating cell populations by removing aneuploid and karyotypically abnormal cells via activation of cell death programs. Detailed understanding of the molecular regulation of this vital cell cycle checkpoint is important to maximize safety of in-vitro HSC expansion techniques. It is widely accepted that mammalian cells enter the next G1-phase with 4N DNA after slippage from prolonged drug-induced mitotic block caused by activation of the transient spindle checkpoint that it is from this state that polyploid/aneuploid cells initiate apoptosis. However, definitive biochemical evidence for G1 is scarce or unconvincing; in part because of methods of protein extraction required for immunoblot analysis that cannot take into account the cell cycle heterogeneity of cell cultures. We used single-cell-intracellular-flow-cytometric analysis to define important factors determining cell fate after mitotic slippage. Results from human and mouse embryonic stem cells that reenter polyploid cell cycles are compared to human somatic hematopoietic cells that die after MS. We now report for the first time that phosphorylation status of pRb, p53, CDK1, and cyclin B1 levels are important for cell fate/apoptosis decision in mitotic-slippage cells, which occurs in a unique, intervening, non-G1, tetraploid subphase. Hyperphosphorylated Rb was extremely abundant in mitotic-slippage cells, a cell signaling event usually associated with early G1-S phase transition. P53 was phosphorylated at sites known to be associated with apoptosis regulation. Cyclin A and B1 were undetectable in mitotic slippage cells; yet, CDK1 was phosphorylated at sites typically associated with its activation. Evidence is also presented raising the possibility of cyclin B1-independent CDK1 activity in mitotic-slippage cells. These findings challenge the current models of spindle checkpoint-apoptosis linkages. Our new model could have important implications for methods to maintain karyotypic stability during ex-vivo HSC expansion.

scholarly journals Securin and not CDK1/cyclin B1 regulates sister chromatid disjunction during meiosis II in mouse eggs

2008 ◽  
Vol 321 (2) ◽  
pp. 379-386 ◽  
Author(s):  
Ibtissem Nabti ◽  
Alexandra Reis ◽  
Mark Levasseur ◽  
Olaf Stemmann ◽  
Keith T. Jones
Keyword(s):  
Sister Chromatid ◽  
Cyclin B1 ◽  
Mouse Eggs

scholarly journals Sil Phosphorylation in a Pin1 Binding Domain Affects the Duration of the Spindle Checkpoint

2005 ◽  
Vol 25 (15) ◽  
pp. 6660-6672 ◽  
Author(s):  
Stefano Campaner ◽  
Philipp Kaldis ◽  
Shai Izraeli ◽  
Ilan R. Kirsch
Keyword(s):  
Kinase Activity ◽  
Phosphorylation Site ◽  
Spindle Checkpoint ◽  
Cyclin B1 ◽  
Early Gene ◽  
Microtubule Inhibitors ◽  
Cycle Dependent ◽  
Mitotic Phosphorylation ◽  
Cdc2 Activity ◽  
In Cells

ABSTRACT SIL is an immediate-early gene that is essential for embryonic development and is implicated in T-cell leukemia-associated translocations. We now show that the Sil protein is hyperphosphorylated during mitosis or in cells blocked at prometaphase by microtubule inhibitors. Cell cycle-dependent phosphorylation of Sil is required for its interaction with Pin1, a regulator of mitosis. Point mutation of the seven (S/T)P sites between amino acids 567 and 760 reduces mitotic phosphorylation of Sil, Pin1 binding, and spindle checkpoint duration. When a phosphorylation site mutant Sil is stably expressed, the duration of the spindle checkpoint is shortened in cells challenged with taxol or nocodazole, and the cells revert to a G2-like state. This event is associated with the downregulation of the kinase activity of the Cdc2/cyclin B1 complex and the dephosphorylation of the threonine 161 on the Cdc2 subunit. Sil downregulation by plasmid-mediated RNA interference limited the ability of cells to activate the spindle checkpoint and correlated with a reduction of Cdc2/cyclin B1 activity and phosphorylation on T161 on the Cdc2 subunit. These data suggest that a critical region of Sil is required to mediate the presentation of Cdc2 activity during spindle checkpoint arrest.

scholarly journals The APC/C recruits cyclin B1–Cdk1–Cks in prometaphase before D box recognition to control mitotic exit

2010 ◽  
Vol 190 (4) ◽  
pp. 587-602 ◽  
Author(s):  
Wouter van Zon ◽  
Janneke Ogink ◽  
Bas ter Riet ◽  
René H. Medema ◽  
Hein te Riele ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Spindle Checkpoint ◽  
Cyclin A ◽  
Cyclin B1 ◽  
Mitotic Exit ◽  
Dependent Manner ◽  
Anaphase Promoting Complex ◽  
Checkpoint Control ◽  
N Terminus ◽  
Mitotic Phosphorylation

The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) is activated at prometaphase by mitotic phosphorylation and binding of its activator, Cdc20. This initiates cyclin A degradation, whereas cyclin B1 is stabilized by the spindle checkpoint. Upon checkpoint release, the RXXL destruction box (D box) was proposed to direct cyclin B1 to core APC/C or Cdc20. In this study, we report that endogenous cyclin B1–Cdk1 is recruited to checkpoint-inhibited, phosphorylated APC/C in prometaphase independently of Cdc20 or the cyclin B1 D box. Like cyclin A, cyclin B1 binds the APC/C by the Cdk cofactor Cks and the APC3 subunit. Prior binding to APC/CCdc20 makes cyclin B1 a better APC/C substrate in metaphase, driving mitotic exit and cytokinesis. We conclude that in prometaphase, the phosphorylated APC/C can recruit both cyclin A and cyclin B1 in a Cks-dependent manner. This suggests that the spindle checkpoint blocks D box recognition of APC/C-bound cyclin B1, whereas distinctive complexes between the N terminus of cyclin A and Cdc20 evade checkpoint control.

scholarly journals Zwilch, a New Component of the ZW10/ROD Complex Required for Kinetochore Functions

2003 ◽  
Vol 14 (4) ◽  
pp. 1379-1391 ◽  
Author(s):  
Byron C. Williams ◽  
ZeXiao Li ◽  
Songtao Liu ◽  
Erika V. Williams ◽  
Garmay Leung ◽  
...  
Keyword(s):  
Hela Cell ◽  
Chromosome Segregation ◽  
Mass Spectroscopy ◽  
Sister Chromatid ◽  
Spindle Checkpoint ◽  
Cytoplasmic Dynein ◽  
Immunoaffinity Chromatography ◽  
Cell Extracts ◽  
Evolutionarily Conserved ◽  
Additional Protein

The Zeste-White 10 (ZW10) and Rough Deal (ROD) proteins are part of a complex necessary for accurate chromosome segregation. This complex recruits cytoplasmic dynein to the kinetochore and participates in the spindle checkpoint. We used immunoaffinity chromatography and mass spectroscopy to identify theDrosophila proteins in this complex. We found that the complex contains an additional protein we name Zwilch. Zwilch localizes to kinetochores and kinetochore microtubules in a manner identical to ZW10 and ROD. We have also isolated azwilch mutant, which exhibits the same mitotic phenotypes associated with zw10 and rodmutations: lagging chromosomes at anaphase and precocious sister chromatid separation upon activation of the spindle checkpoint. Zwilch's role within the context of this complex is evolutionarily conserved. The human Zwilch protein (hZwilch) coimmunoprecipitates with hZW10 and hROD from HeLa cell extracts and localizes to the kinetochores at prometaphase. Finally, we discuss immunoaffinity chromatography results that suggest the existence of a weak interaction between the ZW10/ROD/Zwilch complex and the kinesin-like kinetochore component CENP-meta.

Spindle Checkpoint Insufficiency and Unscheduled APC-Dependent Proteolysis in Acute Myeloid Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2082-2082
Author(s):  
Dominik Schnerch ◽  
Julia Felthaus ◽  
Monika Engelhardt ◽  
Ralph M. Waesch
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Spindle Assembly Checkpoint ◽  
Spindle Checkpoint ◽  
Cyclin B1 ◽  
Leukemic Cells ◽  
Competent Cell ◽  
Cell Cycle Regulators ◽  
Acute Myeloid

Abstract Genetic instability including aneuploidy is frequent in most cancers. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism responsible for accurate chromosome segregation. Unattached chromosomes or lack of spindle tension are sensed by the SAC. The activated SAC inhibits the ubiquitin-ligase anaphase-promoting complex (APC), which prevents the proteolysis of cell cycle regulators in order to delay progression through mitosis and allow cells to recover from defective mitotic spindle attachment. Spindle checkpoint malfunction proved to favor the generation of aneuploidy. In our recent work we investigated the roles of essential SAC proteins in acute myeloid leukemia (AML). We found the SAC-protein Bub1 to be posttranscriptionally downregulated in all investigated AML cell lines. As a consequence, after exposure to the microtubule disrupting agent nocodazole we observed a defective mitotic delay mechanism in comparison to SAC-competent cell lines and increased apoptosis consistent with the effects of Bub1 downregulation by RNA interference. At the molecular level we found a dramatic decline in mitotic regulator levels such as cyclin B1 and securin despite lasting spindle disruption. Additional data showed that the levels of these regulator proteins can be efficiently restored by exposure to the proteasome inhibitor MG-132 indicating that APC-dependent proteolysis is directly involved in SAC insufficiency. Thus, continuous activation of the APC triggers degradation of essential regulator proteins even in leukemic cells faced to mitotic stress such as complete spindle disruption. Such defects can lead to establishment of aneuploidy in vivo. Our findings emphasize a role of SAC insufficiency and unscheduled proteolysis in rise and progression of AML with complex karyotype.

scholarly journals Control of localization of a spindle checkpoint protein, Mad2, in fission yeast

2002 ◽  
Vol 115 (8) ◽  
pp. 1603-1610 ◽  
Author(s):  
Amy E. Ikui ◽  
Kanji Furuya ◽  
Mitsuhiro Yanagida ◽  
Tomohiro Matsumoto
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Nuclear Membrane ◽  
Sister Chromatid ◽  
Nuclear Periphery ◽  
Spindle Checkpoint ◽  
Chromatin Domain ◽  
Checkpoint Protein ◽  
Higher Eukaryotes ◽  
Sister Chromatid Separation

To ensure accurate chromosome segregation, the spindle checkpoint delays the onset of sister chromatid separation when the spindle is not attached to a kinetochore. Mad2, a component of the checkpoint, targets fission yeast Slp1/budding yeast Cdc20/human p55CDC and prevents it from promoting proteolysis, which is a prerequisite to sister chromatid separation. The protein is localized to unattached kinetochores in higher eukaryotes, and it is thought to be required for activation of the checkpoint as well. In this study, Mad2 and its target Slp1 were visualized in a tractable organism,fission yeast Schizosaccharomyces pombe. When cells were arrested at a prometaphase-like stage, the Mad2-Slp1 complex was stable and the two proteins were colocalized to unattached kinetochores. When the spindle attachment was completed, the complex was no longer detectable and only Mad2 was found associated to the spindle. These results would suggest that unattached kinetochores provide sites for assembly of the Mad2-Slp1 complex. During interphase, Mad2 was localized to the nuclear periphery as well as to the chromatin domain. This localization was abolished in a yeast strain lacking Mad1, a protein that physically interacts with Mad2. Mad1 may anchor Mad2 to the nuclear membrane and regulate its entry into the nucleus.

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