Mad2 and BubR1 Function in a Single Checkpoint Pathway that Responds to a Loss of Tension

The spindle checkpoint monitors microtubule attachment and tension at kinetochores to ensure proper chromosome segregation. Previously, PtK1 cells in hypothermic conditions (23°C) were shown to have a pronounced mitotic delay, despite having normal numbers of kinetochore microtubules. At 23°C, we found that PtK1 cells remained in metaphase for an average of 101 min, compared with 21 min for cells at 37°C. The metaphase delay at 23°C was abrogated by injection of Mad2 inhibitors, showing that Mad2 and the spindle checkpoint were responsible for the prolonged metaphase. Live cell imaging showed that kinetochore Mad2 became undetectable soon after chromosome congression. Measurements of the stretch between sister kinetochores at metaphase found a 24% decrease in tension at 23°C, and metaphase kinetochores at 23°C exhibited higher levels of 3F3/2, Bub1, and BubR1 compared with 37°C. Microinjection of anti-BubR1 antibody abolished the metaphase delay at 23°C, indicating that the higher kinetochore levels of BubR1 may contribute to the delay. Disrupting both Mad2 and BubR1 function induced anaphase with the same timing as single inhibitions, suggesting that these checkpoint genes function in the same pathway. We conclude that reduced tension at kinetochores with a full complement of kinetochore microtubules induces a checkpoint dependent metaphase delay associated with elevated amounts of kinetochore 3F3/2, Bub1, and BubR1 labeling.

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

The Journal of Cell Biology ◽

10.1083/jcb.200111001 ◽

2002 ◽

Vol 157 (7) ◽

pp. 1125-1137 ◽

Cited By ~ 198

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.

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Microinjection of mitotic cells with the 3F3/2 anti-phosphoepitope antibody delays the onset of anaphase.

The Journal of Cell Biology ◽

10.1083/jcb.129.5.1195 ◽

1995 ◽

Vol 129 (5) ◽

pp. 1195-1204 ◽

Cited By ~ 77

Author(s):

M S Campbell ◽

G J Gorbsky

Keyword(s):

Monoclonal Antibody ◽

Chromosome Segregation ◽

Mammalian Cells ◽

Metaphase Plate ◽

Chromosome Movement ◽

Normal Cells ◽

Microtubule Attachment ◽

Checkpoint Pathway ◽

Chromosome Congression ◽

Asymmetric Expression

The transition from metaphase to anaphase is regulated by a checkpoint system that prevents chromosome segregation in anaphase until all the chromosomes have aligned at the metaphase plate. We provide evidence indicating that a kinetochore phosphoepitope plays a role in this checkpoint pathway. The 3F3/2 monoclonal antibody recognizes a kinetochore phosphoepitope in mammalian cells that is expressed on chromosomes before their congression to the metaphase plate. Once chromosomes are aligned, expression is lost and cells enter anaphase shortly thereafter. When microinjected into prophase cells, the 3F3/2 antibody caused a concentration-dependent delay in the onset of anaphase. Injected antibody inhibited the normal dephosphorylation of the 3F3/2 phosphoepitope at kinetochores. Microinjection of the antibody eliminated the asymmetric expression of the phosphoepitope normally seen on sister kinetochores of chromosomes during their movement to the metaphase plate. Chromosome movement to the metaphase plate appeared unaffected in cells injected with the antibody suggesting that asymmetric expression of the phosphoepitope on sister kinetochores is not required for chromosome congression to the metaphase plate. In antibody-injected cells, the epitope remained expressed at kinetochores throughout the prolonged metaphase, but had disappeared by the onset of anaphase. When normal cells in metaphase, lacking the epitope at kinetochores, were treated with agents that perturb microtubules, the 3F3/2 phosphoepitope quickly reappeared at kinetochores. Immunoelectron microscopy revealed that the 3F3/2 epitope is concentrated in the middle electronlucent layer of the trilaminar kinetochore structure. We propose that the 3F3/2 kinetochore phosphoepitope is involved in detecting stable kinetochore-microtubule attachment or is a signaling component of the checkpoint pathway regulating the metaphase to anaphase transition.

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Yeast Kinetochores Do Not Stabilize Stu2p-dependent Spindle Microtubule Dynamics

Molecular Biology of the Cell ◽

10.1091/mbc.e03-03-0180 ◽

2003 ◽

Vol 14 (10) ◽

pp. 4181-4195 ◽

Cited By ~ 61

Author(s):

Chad G. Pearson ◽

Paul S. Maddox ◽

Ted R. Zarzar ◽

E.D. Salmon ◽

Kerry Bloom

Keyword(s):

Chromosome Segregation ◽

Live Cell Imaging ◽

Cell Imaging ◽

Regulatory Mechanism ◽

Metaphase Plate ◽

Microtubule Dynamics ◽

Spindle Microtubule ◽

Microtubule Binding ◽

Microtubule Stabilization ◽

Kinetochore Function

The interaction of kinetochores with dynamic microtubules during mitosis is essential for proper centromere motility, congression to the metaphase plate, and subsequent anaphase chromosome segregation. Budding yeast has been critical in the discovery of proteins necessary for this interaction. However, the molecular mechanism for microtubule–kinetochore interactions remains poorly understood. Using live cell imaging and mutations affecting microtubule binding proteins and kinetochore function, we identify a regulatory mechanism for spindle microtubule dynamics involving Stu2p and the core kinetochore component, Ndc10p. Depleting cells of the microtubule binding protein Stu2p reduces kinetochore microtubule dynamics. Centromeres remain under tension but lack motility. Thus, normal microtubule dynamics are not required to maintain tension at the centromere. Loss of the kinetochore (ndc10-1, ndc10-2, and ctf13-30) does not drastically affect spindle microtubule turnover, indicating that Stu2p, not the kinetochore, is the foremost governor of microtubule dynamics. Disruption of kinetochore function with ndc10-1 does not affect the decrease in microtubule turnover in stu2 mutants, suggesting that the kinetochore is not required for microtubule stabilization. Remarkably, a partial kinetochore defect (ndc10-2) suppresses the decreased spindle microtubule turnover in the absence of Stu2p. These results indicate that Stu2p and Ndc10p differentially function in controlling kinetochore microtubule dynamics necessary for centromere movements.

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MAD1-dependent recruitment of CDK1-CCNB1 to kinetochores promotes spindle checkpoint signaling

The Journal of Cell Biology ◽

10.1083/jcb.201808015 ◽

2019 ◽

Vol 218 (4) ◽

pp. 1108-1117 ◽

Cited By ~ 32

Author(s):

Tatiana Alfonso-Pérez ◽

Daniel Hayward ◽

James Holder ◽

Ulrike Gruneberg ◽

Francis A. Barr

Keyword(s):

Feedback Loop ◽

Spindle Checkpoint ◽

Mitotic Exit ◽

Checkpoint Signaling ◽

Checkpoint Proteins ◽

Mitotic Entry ◽

Upstream Regulator ◽

Microtubule Attachment ◽

Integral Component ◽

Checkpoint Pathway

Cyclin B–dependent kinase (CDK1-CCNB1) promotes entry into mitosis. Additionally, it inhibits mitotic exit by activating the spindle checkpoint. This latter role is mediated through phosphorylation of the checkpoint kinase MPS1 and other spindle checkpoint proteins. We find that CDK1-CCNB1 localizes to unattached kinetochores and like MPS1 is lost from these structures upon microtubule attachment. This suggests that CDK1-CCNB1 is an integral component and not only an upstream regulator of the spindle checkpoint pathway. Complementary proteomic and cell biological analysis demonstrate that the spindle checkpoint protein MAD1 is one of the major components of CCNB1 complexes, and that CCNB1 is recruited to unattached kinetochores in an MPS1-dependent fashion through interaction with the first 100 amino acids of MAD1. This MPS1 and MAD1-dependent pool of CDK1-CCNB1 creates a positive feedback loop necessary for timely recruitment of MPS1 to kinetochores during mitotic entry and for sustained spindle checkpoint arrest. CDK1-CCNB1 is therefore an integral component of the spindle checkpoint, ensuring the fidelity of mitosis.

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

The Journal of Cell Biology ◽

10.1083/jcb.201810172 ◽

2019 ◽

Vol 218 (12) ◽

pp. 3926-3942 ◽

Cited By ~ 7

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.

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Kinetochore Targeting of Fission Yeast Mad and Bub Proteins Is Essential for Spindle Checkpoint Function but Not for All Chromosome Segregation Roles of Bub1p

Molecular and Cellular Biology ◽

10.1128/mcb.24.22.9786-9801.2004 ◽

2004 ◽

Vol 24 (22) ◽

pp. 9786-9801 ◽

Cited By ~ 59

Author(s):

Vincent Vanoosthuyse ◽

Rebekka Valsdottir ◽

Jean-Paul Javerzat ◽

Kevin G. Hardwick

Keyword(s):

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Chromosome Segregation ◽

Chromatin Immunoprecipitation ◽

Spindle Checkpoint ◽

Central Domain ◽

Checkpoint Response ◽

Terminal Domain ◽

Microtubule Attachment ◽

Lines Of Evidence

ABSTRACT Several lines of evidence suggest that kinetochores are organizing centers for the spindle checkpoint response and the synthesis of a “wait anaphase” signal in cases of incomplete or improper kinetochore-microtubule attachment. Here we characterize Schizosaccharomyces pombe Bub3p and study the recruitment of spindle checkpoint components to kinetochores. We demonstrate by chromatin immunoprecipitation that they all interact with the central domain of centromeres, consistent with their role in monitoring kinetochore-microtubule interactions. Bub1p and Bub3p are dependent upon one another, but independent of the Mad proteins, for their kinetochore localization. We demonstrate a clear role for the highly conserved N-terminal domain of Bub1p in the robust targeting of Bub1p, Bub3p, and Mad3p to kinetochores and show that this is crucial for an efficient checkpoint response. Surprisingly, neither this domain nor kinetochore localization is required for other functions of Bub1p in chromosome segregation.

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Live Cell Imaging of Chromosome Segregation During Mitosis

Journal of Visualized Experiments ◽

10.3791/57389 ◽

2018 ◽

Author(s):

Prajakta Varadkar ◽

Kazuyo Takeda ◽

Brenton McCright

Keyword(s):

Chromosome Segregation ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell

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The RZZ complex integrates spindle checkpoint maintenance with dynamic expansion of unattached kinetochores

10.1101/297580 ◽

2018 ◽

Cited By ~ 2

Author(s):

Jose-Antonio Rodriguez-Rodriguez ◽

Kara L. McKinley ◽

Vitali Sikirzhytski ◽

Jennifer Corona ◽

John Maciejowski ◽

...

Keyword(s):

Live Cell Imaging ◽

Gene Editing ◽

Structural Changes ◽

Spindle Assembly Checkpoint ◽

Cell Imaging ◽

Mitotic Checkpoint ◽

Nuclear Envelope Breakdown ◽

Chromosome Congression ◽

Mitotic Checkpoint Complex

SummaryThe Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls mitosis through assembly of a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1, 2]. Mad1-Mad2 first catalyzes MCC assembly at interphase nuclear pores [3], then migrates to kinetochores at nuclear envelope breakdown (NEBD) and resumes MCC assembly until bipolar spindle attachment is complete [1, 2]. There is significant debate about the factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes [4-9]. Through gene editing and live-cell imaging, we found that the human Rod-Zw10-Zwilch (RZZ) complex is dispensable for cell viability and initial recruitment of Mad1-Mad2 to kinetochores at NEBD, but then becomes necessary to tether Mad1-Mad2 at kinetochores and sustain SAC arrest in cells challenged with spindle poisons. We also show that RZZ forms the mesh-like fibrous corona, a structural expansion of the outer kinetochore important for timely chromosome congression [10-13] once Mps1 phosphorylates the N-terminus of Rod. Artificially tethering Mad1-Mad2 to kinetochores enabled long-term mitotic arrest in the absence of RZZ. Conversely, blocking early RZZ-independent recruitment of Mad1-Mad2 eliminated the transient SAC response in RZZ-null cells. We conclude that RZZ drives structural changes in the outer kinetochore that facilitate chromosome bi-orientation and chronic SAC transduction, a key determinant of cytotoxicity during anti-mitotic drug therapy [14-16].

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Mitotic Centromere–associated Kinesin Is Important for Anaphase Chromosome Segregation

The Journal of Cell Biology ◽

10.1083/jcb.142.3.787 ◽

1998 ◽

Vol 142 (3) ◽

pp. 787-801 ◽

Cited By ~ 201

Author(s):

Todd Maney ◽

Andrew W. Hunter ◽

Mike Wagenbach ◽

Linda Wordeman

Keyword(s):

Chromosome Segregation ◽

Live Cell Imaging ◽

Cell Imaging ◽

Single Gene ◽

Live Cell ◽

Spindle Microtubule ◽

Eventual Loss ◽

Sister Centromere

Mitotic centromere–associated kinesin (MCAK) is recruited to the centromere at prophase and remains centromere associated until after telophase. MCAK is a homodimer that is encoded by a single gene and has no associated subunits. A motorless version of MCAK that binds centromeres but not microtubules disrupts chromosome segregation during anaphase. Antisense-induced depletion of MCAK results in the same defect. MCAK overexpression induces centromere-independent bundling and eventual loss of spindle microtubule polymer suggesting that centromere-associated bundling and/or depolymerization activity is required for anaphase. Live cell imaging indicates that MCAK may be required to coordinate the onset of sister centromere separation.

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CENP-F stabilizes kinetochore-microtubule attachments and limits dynein stripping of corona cargoes

The Journal of Cell Biology ◽

10.1083/jcb.201905018 ◽

2020 ◽

Vol 219 (5) ◽

Cited By ~ 3

Author(s):

Philip Auckland ◽

Emanuele Roscioli ◽

Helena Louise Elvidge Coker ◽

Andrew D. McAinsh

Keyword(s):

Chromosome Segregation ◽

Direct Interaction ◽

Antagonistic Activity ◽

Early Event ◽

Microtubule Binding ◽

Binding Domains ◽

Microtubule Attachment ◽

Dynein Motor ◽

Chromosome Congression ◽

Kinetochore Function

Accurate chromosome segregation demands efficient capture of microtubules by kinetochores and their conversion to stable bioriented attachments that can congress and then segregate chromosomes. An early event is the shedding of the outermost fibrous corona layer of the kinetochore following microtubule attachment. Centromere protein F (CENP-F) is part of the corona, contains two microtubule-binding domains, and physically associates with dynein motor regulators. Here, we have combined CRISPR gene editing and engineered separation-of-function mutants to define how CENP-F contributes to kinetochore function. We show that the two microtubule-binding domains make distinct contributions to attachment stability and force transduction but are dispensable for chromosome congression. We further identify a specialized domain that functions to limit the dynein-mediated stripping of corona cargoes through a direct interaction with Nde1. This antagonistic activity is crucial for maintaining the required corona composition and ensuring efficient kinetochore biorientation.

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