Aberrantly segregating centromeres activate the spindle assembly checkpoint in budding yeast.

The spindle assembly checkpoint is the mechanism or set of mechanisms that prevents cells with defects in chromosome alignment or spindle assembly from passing through mitosis. We have investigated the effects of mini-chromosomes on this checkpoint in budding yeast by performing pedigree analysis. This method allowed us to observe the frequency and duration of cell cycle delays in individual cells. Short, centromeric linear mini-chromosomes, which have a low fidelity of segregation, cause frequent delays in mitosis. Their circular counterparts and longer linear mini-chromosomes, which segregate more efficiently, show a much lower frequency of mitotic delays, but these delays occur much more frequently in divisions where the mini-chromosome segregates to only one of the two daughter cells. Using a conditional centromere to increase the copy number of a circular mini-chromosome greatly increases the frequency of delayed divisions. In all cases the division delays are completely abolished by the mad mutants that inactivate the spindle assembly checkpoint, demonstrating that the Mad gene products are required to detect the subtle defects in chromosome behavior that have been observed to arrest higher eukaryotic cells in mitosis.

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The equatorial position of the metaphase plate ensures symmetric cell divisions

eLife ◽

10.7554/elife.05124 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 12

Author(s):

Chia Huei Tan ◽

Ivana Gasic ◽

Sabina P Huber-Reggi ◽

Damian Dudka ◽

Marin Barisic ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Metaphase Plate ◽

Spindle Assembly ◽

Equatorial Position ◽

Asymmetric Cell Divisions ◽

Daughter Cells ◽

Cell Divisions ◽

Microtubule Stability ◽

Chromosome Alignment ◽

Metazoan Cell

Chromosome alignment in the middle of the bipolar spindle is a hallmark of metazoan cell divisions. When we offset the metaphase plate position by creating an asymmetric centriole distribution on each pole, we find that metaphase plates relocate to the middle of the spindle before anaphase. The spindle assembly checkpoint enables this centering mechanism by providing cells enough time to correct metaphase plate position. The checkpoint responds to unstable kinetochore–microtubule attachments resulting from an imbalance in microtubule stability between the two half-spindles in cells with an asymmetric centriole distribution. Inactivation of the checkpoint prior to metaphase plate centering leads to asymmetric cell divisions and daughter cells of unequal size; in contrast, if the checkpoint is inactivated after the metaphase plate has centered its position, symmetric cell divisions ensue. This indicates that the equatorial position of the metaphase plate is essential for symmetric cell divisions.

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The mitosis-regulating and protein-protein interaction activities of Astrin are controlled by Aurora-A-induced phosphorylation

AJP Cell Physiology ◽

10.1152/ajpcell.00164.2014 ◽

2014 ◽

Vol 307 (5) ◽

pp. C466-C478 ◽

Cited By ~ 10

Author(s):

Shao-Chih Chiu ◽

Jo-Mei Maureen Chen ◽

Tong-You Wade Wei ◽

Tai-Shan Cheng ◽

Ya-Hui Candice Wang ◽

...

Keyword(s):

Regulatory Networks ◽

Spindle Assembly Checkpoint ◽

Morphological Changes ◽

Spindle Assembly ◽

Aurora A ◽

Protein Protein Interaction ◽

Daughter Cells ◽

Sister Chromatids ◽

Complicated Process ◽

Spindle Stability

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser115. The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

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Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e10-08-0673 ◽

2011 ◽

Vol 22 (9) ◽

pp. 1473-1485 ◽

Cited By ~ 29

Author(s):

Zuzana Storchová ◽

Justin S. Becker ◽

Nicolas Talarek ◽

Sandra Kögelsberger ◽

David Pellman

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Budding Yeast ◽

Spindle Pole ◽

Growth Defect ◽

Aurora B ◽

Mitotic Kinase ◽

Sister Chromatids ◽

Chromosome Alignment ◽

Chromosomal Passenger

The conserved mitotic kinase Bub1 performs multiple functions that are only partially characterized. Besides its role in the spindle assembly checkpoint and chromosome alignment, Bub1 is crucial for the kinetochore recruitment of multiple proteins, among them Sgo1. Both Bub1 and Sgo1 are dispensable for growth of haploid and diploid budding yeast, but they become essential in cells with higher ploidy. We find that overexpression of SGO1 partially corrects the chromosome segregation defect of bub1Δ haploid cells and restores viability to bub1Δ tetraploid cells. Using an unbiased high-copy suppressor screen, we identified two members of the chromosomal passenger complex (CPC), BIR1 (survivin) and SLI15 (INCENP, inner centromere protein), as suppressors of the growth defect of both bub1Δ and sgo1Δ tetraploids, suggesting that these mutants die due to defects in chromosome biorientation. Overexpression of BIR1 or SLI15 also complements the benomyl sensitivity of haploid bub1Δ and sgo1Δ cells. Mutants lacking SGO1 fail to biorient sister chromatids attached to the same spindle pole (syntelic attachment) after nocodazole treatment. Moreover, the sgo1Δ cells accumulate syntelic attachments in unperturbed mitoses, a defect that is partially corrected by BIR1 or SLI15 overexpression. We show that in budding yeast neither Bub1 nor Sgo1 is required for CPC localization or affects Aurora B activity. Instead we identify Sgo1 as a possible partner of Mps1, a mitotic kinase suggested to have an Aurora B–independent function in establishment of biorientation. We found that Sgo1 overexpression rescues defects caused by metaphase inactivation of Mps1 and that Mps1 is required for Sgo1 localization to the kinetochore. We propose that Bub1, Sgo1, and Mps1 facilitate chromosome biorientation independently of the Aurora B–mediated pathway at the budding yeast kinetochore and that both pathways are required for the efficient turnover of syntelic attachments.

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PP2A-B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes spindle assembly checkpoint silencing

The Journal of Cell Biology ◽

10.1083/jcb.201406109 ◽

2014 ◽

Vol 206 (7) ◽

pp. 833-842 ◽

Cited By ~ 96

Author(s):

Antonio Espert ◽

Pelin Uluocak ◽

Ricardo Nunes Bastos ◽

Davinderpreet Mangat ◽

Philipp Graab ◽

...

Keyword(s):

Chromosome Segregation ◽

Mammalian Cells ◽

Feedback Loop ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Eukaryotic Cells ◽

Aurora B ◽

Safeguard Mechanism

The spindle assembly checkpoint (SAC) monitors correct attachment of chromosomes to microtubules, an important safeguard mechanism ensuring faithful chromosome segregation in eukaryotic cells. How the SAC signal is turned off once all the chromosomes have successfully attached to the spindle remains an unresolved question. Mps1 phosphorylation of Knl1 results in recruitment of the SAC proteins Bub1, Bub3, and BubR1 to the kinetochore and production of the wait-anaphase signal. SAC silencing is therefore expected to involve a phosphatase opposing Mps1. Here we demonstrate in vivo and in vitro that BubR1-associated PP2A-B56 is a key phosphatase for the removal of the Mps1-mediated Knl1 phosphorylations necessary for Bub1/BubR1 recruitment in mammalian cells. SAC silencing is thus promoted by a negative feedback loop involving the Mps1-dependent recruitment of a phosphatase opposing Mps1. Our findings extend the previously reported role for BubR1-associated PP2A-B56 in opposing Aurora B and suggest that BubR1-bound PP2A-B56 integrates kinetochore surveillance and silencing of the SAC.

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Cdk1 and Plk1 mediate a CLASP2 phospho-switch that stabilizes kinetochore–microtubule attachments

The Journal of Cell Biology ◽

10.1083/jcb.201203091 ◽

2012 ◽

Vol 199 (2) ◽

pp. 285-301 ◽

Cited By ~ 60

Author(s):

Ana R.R. Maia ◽

Zaira Garcia ◽

Lilian Kabeche ◽

Marin Barisic ◽

Stefano Maffini ◽

...

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Regulatory Mechanism ◽

Stable Condition ◽

Spindle Assembly ◽

Chromosome Alignment ◽

Chromosome Movements

Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)–microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT–MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs was enhanced by CLASP2 phosphorylation on S1234. This was specifically required to stabilize KT–MT attachments important for chromosome alignment and to coordinate KT and non-KT MT dynamics necessary to maintain spindle bipolarity. CLASP2 C-terminal phosphorylation by Plk1 was also required for chromosome alignment and timely satisfaction of the SAC. We propose that Cdk1 and Plk1 mediate a fine CLASP2 “phospho-switch” that temporally regulates KT–MT attachment stability.

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Roles of the 2 microns gene products in stable maintenance of the 2 microns plasmid of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.7.10.3566 ◽

1987 ◽

Vol 7 (10) ◽

pp. 3566-3573 ◽

Cited By ~ 66

Author(s):

A E Reynolds ◽

A W Murray ◽

J W Szostak

Keyword(s):

Saccharomyces Cerevisiae ◽

Copy Number ◽

Gene Products ◽

Site Specific ◽

Cell Cycles ◽

Daughter Cells ◽

Repeat Sequences ◽

Copy Numbers ◽

Mother And Daughter ◽

Stable Maintenance

We have examined the replication and segregation of the Saccharomyces cerevisiae 2 microns circle. The amplification of the plasmid at low copy numbers requires site-specific recombination between the 2 microns inverted repeat sequences catalyzed by the plasmid-encoded FLP gene. No other 2 microns gene products are required. The overexpression of FLP in a strain carrying endogenous 2 microns leads to uncontrolled plasmid replication, longer cell cycles, and cell death. Two different assays show that the level of Flp activity decreases with increasing 2 microns copy number. This regulation requires the products of the REP1 and REP2 genes. These gene products also act together to ensure that 2 microns molecules are randomly segregated between mother and daughter cells at cell division.

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A stochastic model for error correction of kinetochore-microtubule attachments and its coupling to the spindle assembly checkpoint

10.1101/541573 ◽

2019 ◽

Author(s):

Anand Banerjee ◽

Neil Adames ◽

Jean Peccoud ◽

John J. Tyson

Keyword(s):

Stochastic Model ◽

Error Correction ◽

Spindle Assembly Checkpoint ◽

Budding Yeast ◽

Analytical Formula ◽

Spindle Assembly ◽

Experimental Information ◽

Checkpoint Signaling ◽

Balance Point ◽

Checkpoint Signal

AbstractTo divide replicated chromosomes equally between daughter cells kinetochores must attach to microtubules emanating from opposite poles of the mitotic spindle. Two mechanisms, namely, error correction and ‘spindle assembly checkpoint’ work together to facilitate this process. The error correction mechanism recognizes and detaches erroneous kinetochore-microtubule attachments, and the spindle assembly checkpoint delays the onset of anaphase until all the kinetochores are properly attached. Kinases and phosphatases at the kinetochore play a key role in proper functioning of these two mechanisms. Here we present a stochastic model to study how the opposing activities of kinases and phosphatases at the kinetochore affect error correction of kinetochore-microtubule attachments and checkpoint signaling in budding yeast, Saccharomyces cerevisiae. We show that error correction and biorientation of chromosomes occurs efficiently when the ratio between kinase activity of Ipl1 and the activity of an opposing phosphatase is a constant (balance point), and derive an approximate analytical formula that defines the balance point. Analysis of the coupling of the spindle assembly checkpoint signal to error correction shows that its strength remains high when the Ipl1 activity is equal to (or larger than) the value specified by the balance point, and the activity of another kinase, Mps1, is much larger (approximately 30 times larger) than its opposing phosphatase (PP1). We also find that the geometrical orientation of sister chromatids does not significantly improve the probability of their reaching biorientation, which depends entirely on Ipl1-dependent microtubule detachment.Author summaryThe kinetochore, the master regulator of chromosome segregation, integrates signals from different chromosome attachment states to generate an appropriate response, like the destabilization of erroneous kinetochore-microtubule attachments, stabilization of correct attachments, maintenance of the spindle assembly checkpoint signal until all kinetochores are properly attached, and finally silencing of checkpoint when biorientation is achieved. At a molecular level the job is carried out by kinases and phosphatases. The complexity of the interactions between these kinases and phosphatases makes intuitive analysis of the control network impossible, and a systems-level model is needed to put experimental information together and to generate testable hypotheses. Here we present such a model for the process of error correction and its coupling to the spindle assembly checkpoint in budding yeast. Using the model, we characterize the balance between kinase and phosphatase activities required for removing erroneous attachments and then establishing correct stable attachments between kinetochore and microtubule. We also analyze how the balance affects the strength of the spindle assembly checkpoint signal.

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The copy-number and varied strengths of MELT motifs in Spc105 balance the strength and responsiveness the Spindle Assembly Checkpoint

10.1101/2020.01.07.897876 ◽

2020 ◽

Cited By ~ 1

Author(s):

Babhrubahan Roy ◽

Simon JY Han ◽

Adrienne N. Fontan ◽

Ajit P. Joglekar

Keyword(s):

Chromosome Segregation ◽

Copy Number ◽

Genome Stability ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Evolutionary Trend ◽

Binding Affinities ◽

Chromosome Missegregation ◽

Anaphase Onset ◽

Maintain Genome Stability

SummaryThe Spindle Assembly Checkpoint (SAC) maintains genome stability while enabling timely anaphase onset. To maintain genome stability, the SAC must be strong so that it delays cell division even if one chromosome is unattached, but for timely anaphase onset, it must be responsive to silencing mechanisms. How it meets these potentially antagonistic requirements is unclear. Here we show that the balance between SAC strength and responsiveness is determined by the number of ‘MELT’ motifs in the kinetochore protein Spc105/KNL1 and their Bub3-Bub1 binding affinities. Spc105/KNL1 must contain many strong MELT motifs to prevent chromosome missegregation, but not too many, because this delays SAC silencing and anaphase onset. We demonstrate this by constructing a Spc105 variant that trades SAC responsiveness for significantly improved chromosome segregation accuracy. We propose that the necessity of balancing SAC strength with responsiveness drives the evolutionary trend of MELT motif number amplification and degeneration of their functionally optimal amino acid sequence.

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