scholarly journals Adaptation to the spindle checkpoint is regulated by the interplay between Cdc28/Clbs and PP2ACdc55

2013 ◽  
Vol 202 (5) ◽  
pp. 765-778 ◽  
Author(s):  
Claudio Vernieri ◽  
Elena Chiroli ◽  
Valentina Francia ◽  
Fridolin Gross ◽  
Andrea Ciliberto
Keyword(s):  
Chromosome Segregation ◽  
Protein Phosphatase ◽  
Feedback Loop ◽  
Spindle Checkpoint ◽  
Place Cells ◽  
Double Negative ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Mitotic Kinase ◽  
Mitotic Cyclin

The spindle checkpoint arrests cells in metaphase until all chromosomes are properly attached to the chromosome segregation machinery. Thereafter, the anaphase promoting complex (APC/C) is activated and chromosome segregation can take place. Cells remain arrested in mitosis for hours in response to checkpoint activation, but not indefinitely. Eventually, they adapt to the checkpoint and proceed along the cell cycle. In yeast, adaptation requires the phosphorylation of APC/C. Here, we show that the protein phosphatase PP2ACdc55 dephosphorylates APC/C, thereby counteracting the activity of the mitotic kinase Cdc28. We also observe that the key regulator of Cdc28, the mitotic cyclin Clb2, increases before cells adapt and is then abruptly degraded at adaptation. Adaptation is highly asynchronous and takes place over a range of several hours. Our data suggest the presence of a double negative loop between PP2ACdc55 and APC/CCdc20 (i.e., a positive feedback loop) that controls APC/CCdc20 activity. The circuit could guarantee sustained APC/CCdc20 activity after Clb2 starts to be degraded.

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

2015 ◽  
Vol 209 (4) ◽  
pp. 519-527 ◽  
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.

scholarly journals Spindle checkpoint activation at meiosis I advances anaphase II onset via meiosis-specific APC/C regulation

2008 ◽  
Vol 182 (2) ◽  
pp. 277-288 ◽  
Author(s):  
Ayumu Yamamoto ◽  
Kenji Kitamura ◽  
Daisuke Hihara ◽  
Yukinobu Hirose ◽  
Satoshi Katsuyama ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Related Factor ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Anaphase Onset ◽  
Yeast Meiosis ◽  
Meiosis I

During mitosis, the spindle assembly checkpoint (SAC) inhibits the Cdc20-activated anaphase-promoting complex/cyclosome (APC/CCdc20), which promotes protein degradation, and delays anaphase onset to ensure accurate chromosome segregation. However, the SAC function in meiotic anaphase regulation is poorly understood. Here, we examined the SAC function in fission yeast meiosis. As in mitosis, a SAC factor, Mad2, delayed anaphase onset via Slp1 (fission yeast Cdc20) when chromosomes attach to the spindle improperly. However, when the SAC delayed anaphase I, the interval between meiosis I and II shortened. Furthermore, anaphase onset was advanced and the SAC effect was reduced at meiosis II. The advancement of anaphase onset depended on a meiosis-specific, Cdc20-related factor, Fzr1/Mfr1, which contributed to anaphase cyclin decline and anaphase onset and was inefficiently inhibited by the SAC. Our findings show that impacts of SAC activation are not confined to a single division at meiosis due to meiosis-specific APC/C regulation, which has probably been evolved for execution of two meiotic divisions.

scholarly journals Structure of an intermediate conformer of the spindle checkpoint protein Mad2

2015 ◽  
Vol 112 (36) ◽  
pp. 11252-11257 ◽  
Author(s):  
Mayuko Hara ◽  
Engin Özkan ◽  
Hongbin Sun ◽  
Hongtao Yu ◽  
Xuelian Luo
Keyword(s):  
Chromosome Segregation ◽  
Structural Transition ◽  
Spindle Checkpoint ◽  
Structural Elements ◽  
Anaphase Promoting Complex ◽  
Core Complex ◽  
Checkpoint Protein ◽  
Closed Conformation ◽  
Allosteric Communication ◽  
Insight Into

The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint protein mitotic arrest deficient 2 (Mad2) is an unusual protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1–Mad2 core complex. In mitosis, kinetochore-bound Mad1–C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. Here, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2. Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2–binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2–C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2.

scholarly journals Two Complexes of Spindle Checkpoint Proteins Containing Cdc20 and Mad2 Assemble during Mitosis Independently of the Kinetochore in Saccharomyces cerevisiae

2005 ◽  
Vol 4 (5) ◽  
pp. 867-878 ◽  
Author(s):  
Atasi Poddar ◽  
P. Todd Stukenberg ◽  
Daniel J. Burke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Budding Yeast ◽  
Spindle Checkpoint ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
In Cells

ABSTRACT Favored models of spindle checkpoint signaling propose that two inhibitory complexes (Mad2-Cdc20 and Mad2-Mad3-Bub3-Cdc20) must be assembled at kinetochores in order to inhibit mitosis. We have directly tested this model in the budding yeast Saccharomyces cerevisiae. The proteins Mad2, Mad3, Bub3, Cdc20, and Cdc27 in yeast were quantified, and there are sufficient amounts to form stoichiometric inhibitors of Cdc20 and the anaphase-promoting complex. Mad2 is present in two separate complexes in cells arrested in mitosis with nocodazole. There is a small amount of Mad2-Mad3-Bub3-Cdc20 and a much larger amount of a complex that contains Mad2-Cdc20. We use conditional mutants to show that both Mad2 and Mad3 are essential for establishment and maintenance of the spindle checkpoint. Both spindle checkpoint complexes containing Mad2 form in mitosis, not in response to checkpoint activation. The kinetochore is not required to form either complex. We propose that the conversion of Mad1-Mad2 to Cdc20-Mad2, a key step in generating inhibitory checkpoint complexes, is limited to mitosis by the availability of Cdc20 and is kinetochore independent.

scholarly journals Delineating the contribution of Spc105-bound PP1 to spindle checkpoint silencing and kinetochore microtubule attachment regulation

2019 ◽  
Vol 218 (12) ◽  
pp. 3926-3942 ◽  
Author(s):  
Babhrubahan Roy ◽  
Vikash Verma ◽  
Janice Sim ◽  
Adrienne Fontan ◽  
Ajit P. Joglekar
Keyword(s):  
Cell Division ◽  
Error Correction ◽  
Chromosome Segregation ◽  
Protein Phosphatase ◽  
Spindle Assembly Checkpoint ◽  
Spindle Checkpoint ◽  
Spindle Assembly ◽  
Protein Phosphatase 1 ◽  
Microtubule Attachment ◽  
Error Correction Mechanism

Accurate chromosome segregation during cell division requires the spindle assembly checkpoint (SAC), which detects unattached kinetochores, and an error correction mechanism that destabilizes incorrect kinetochore–microtubule attachments. While the SAC and error correction are both regulated by protein phosphatase 1 (PP1), which silences the SAC and stabilizes kinetochore–microtubule attachments, how these distinct PP1 functions are coordinated remains unclear. Here, we investigate the contribution of PP1, docked on its conserved kinetochore receptor Spc105/Knl1, to SAC silencing and attachment regulation. We find that Spc105-bound PP1 is critical for SAC silencing but dispensable for error correction; in fact, reduced PP1 docking on Spc105 improved chromosome segregation and viability of mutant/stressed states. We additionally show that artificially recruiting PP1 to Spc105/Knl1 before, but not after, chromosome biorientation interfered with error correction. These observations lead us to propose that recruitment of PP1 to Spc105/Knl1 is carefully regulated to ensure that chromosome biorientation precedes SAC silencing, thereby ensuring accurate chromosome segregation.

scholarly journals Regulation of the Cyclin B Degradation System by an Inhibitor of Mitotic Proteolysis

1998 ◽  
Vol 9 (7) ◽  
pp. 1817-1831 ◽  
Author(s):  
Elisabeth Vorlaufer ◽  
Jan-Michael Peters
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Cyclin B ◽  
Protein Phosphatase 1 ◽  
Anaphase Promoting Complex ◽  
Egg Extracts ◽  
Mitotic Cyclin ◽  
Phosphatase 2A ◽  
Cytostatic Factor ◽  
Xenopus Egg Extracts

The initiation of anaphase and exit from mitosis depend on the anaphase-promoting complex (APC), which mediates the ubiquitin-dependent proteolysis of anaphase-inhibiting proteins and mitotic cyclins. We have analyzed whether protein phosphatases are required for mitotic APC activation. In Xenopus egg extracts APC activation occurs normally in the presence of protein phosphatase 1 inhibitors, suggesting that the anaphase defects caused by protein phosphatase 1 mutation in several organisms are not due to a failure to activate the APC. Contrary to this, the initiation of mitotic cyclin B proteolysis is prevented by inhibitors of protein phosphatase 2A such as okadaic acid. Okadaic acid induces an activity that inhibits cyclin B ubiquitination. We refer to this activity as inhibitor of mitotic proteolysis because it also prevents the degradation of other APC substrates. A similar activity exists in extracts of Xenopus eggs that are arrested at the second meiotic metaphase by the cytostatic factor activity of the protein kinase mos. In Xenopus eggs, the initiation of anaphase II may therefore be prevented by an inhibitor of APC-dependent ubiquitination.

scholarly journals Cyclin-Specific Control of Ribosomal DNA Segregation

2008 ◽  
Vol 28 (17) ◽  
pp. 5328-5336 ◽  
Author(s):  
Matt Sullivan ◽  
Liam Holt ◽  
David O. Morgan
Keyword(s):  
Protein Kinase ◽  
Chromosome Segregation ◽  
Ribosomal Dna ◽  
S Phase ◽  
Anaphase Promoting Complex ◽  
Sister Chromatids ◽  
Early Anaphase ◽  
Mitotic Cyclin ◽  
Mitosis And Meiosis ◽  
Specific Control

ABSTRACT Following chromosome duplication in S phase of the cell cycle, the sister chromatids are linked by cohesin. At the onset of anaphase, separase cleaves cohesin and thereby initiates sister chromatid separation. Separase activation results from the destruction of its inhibitor, securin, which is triggered by a ubiquitin ligase called the anaphase-promoting complex (APC). Here, we show in budding yeast that securin destruction and, thus, separase activation are not sufficient for the efficient segregation of the repetitive ribosomal DNA (rDNA). We find that rDNA segregation also requires the APC-mediated destruction of the S-phase cyclin Clb5, an activator of the protein kinase Cdk1. Mutations that prevent Clb5 destruction are lethal and cause defects in rDNA segregation and DNA synthesis. These defects are distinct from the mitotic-exit defects caused by stabilization of the mitotic cyclin Clb2, emphasizing the importance of cyclin specificity in the regulation of late-mitotic events. Efficient rDNA segregation, both in mitosis and meiosis, also requires APC-dependent destruction of Dbf4, an activator of the protein kinase Cdc7. We speculate that the dephosphorylation of Clb5-specific Cdk1 substrates and Dbf4-Cdc7 substrates drives the resolution of rDNA in early anaphase. The coincident destruction of securin, Clb5, and Dbf4 coordinates bulk chromosome segregation with segregation of rDNA.

scholarly journals A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Zhejian Ji ◽  
Haishan Gao ◽  
Luying Jia ◽  
Bing Li ◽  
Hongtao Yu
Keyword(s):  
Spindle Checkpoint ◽  
Mitotic Checkpoint ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Phosphorylation Cascade ◽  
Mitotic Checkpoint Complex

The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when phosphorylated by Mps1, recruits checkpoint complexes Bub1–Bub3 and BubR1–Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1–Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/CCdc20) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1–Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment.

scholarly journals Ubc1 turnover contributes to the spindle assembly checkpoint in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Heather E Arsenault ◽  
Julie M Ghizzoni ◽  
Cassandra M Leech ◽  
Anne R Diers ◽  
Stephane Gesta ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Spindle Checkpoint ◽  
Spindle Assembly ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Cycle Arrest ◽  
Microtubule Poisons

Abstract The spindle assembly checkpoint protects the integrity of the genome by ensuring that chromosomes are properly attached to the mitotic spindle before they are segregated during anaphase. Activation of the spindle checkpoint results in inhibition of the Anaphase Promoting Complex (APC), an E3 ubiquitin ligase that triggers the metaphase-anaphase transition. Here we show that levels of Ubc1, an E2 enzyme that functions in complex with the APC, modulate the response to spindle checkpoint activation in Saccharomyces cerevisiae. Overexpression of Ubc1 increased resistance to microtubule poisons, whereas Ubc1 shut-off sensitized cells. We also found that Ubc1 levels are regulated by the spindle checkpoint. Checkpoint activation or direct APC inhibition led to a decrease in Ubc1 levels, charging and half-life. Additionally, stabilization of Ubc1 prevented its downregulation by the spindle checkpoint and increased resistance to checkpoint-activating drugs. These results suggest that downregulation of Ubc1 in response to spindle checkpoint signaling is necessary for a robust cell cycle arrest.

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