scholarly journals Loading of the 3F3/2 Antigen onto Kinetochores Is Dependent on the Ordered Assembly of the Spindle Checkpoint Proteins

2006 ◽  
Vol 17 (10) ◽  
pp. 4390-4399 ◽  
Author(s):  
Oi Kwan Wong ◽  
Guowei Fang
Keyword(s):  
Chromosome Segregation ◽  
Spindle Checkpoint ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Assembly Pathway ◽  
Checkpoint Pathway ◽  
Unknown Protein ◽  
Ordered Assembly

Accurate chromosome segregation is controlled by the spindle checkpoint, which responds to the lack of microtubule–kinetochore attachment or of tension across sister kinetochores through phosphorylation of kinetochore proteins by the Mps1, Bub1, BubR1, Aurora B, and Plk1/Plx1 kinases. The presence of the 3F3/2 phosphoepitope on kinetochores, generated by Plk1/Plx1-mediated phosphorylation of an unknown protein, correlates with the activation of the tension-sensitive checkpoint pathway. Using immunodepletion approach and a rephosphorylation assay in Xenopus extracts, we report here that not only the formation of the 3F3/2 phosphoepitope is dependent on the checkpoint activation but also the loading of the 3F3/2 substrate to kinetochores requires the prior assembly of Mps1, Bub1 and BubR1 onto kinetochores. Interestingly, generation of the 3F3/2 epitope in checkpoint extracts requires the kinase activities of Mps1 and Bub1 but not that of BubR1. Furthermore, we demonstrate that checkpoint proteins in Xenopusextracts are assembled onto kinetochores in a highly ordered pathway consisting of three steps. Mps1 and Bub1 are loaded first, and BubR1 and Plx1 second, followed by Mad1 and Mad2. The characterization of this ordered assembly pathway provides a framework for the biochemical mechanism of the checkpoint signaling and will aid in the eventual identification of the 3F3/2 substrate.

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

2019 ◽  
Vol 218 (4) ◽  
pp. 1108-1117 ◽  
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.

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 Regulation of Kinetochore Recruitment of Two Essential Mitotic Spindle Checkpoint Proteins by Mps1 Phosphorylation

2009 ◽  
Vol 20 (1) ◽  
pp. 10-20 ◽  
Author(s):  
Quanbin Xu ◽  
Songcheng Zhu ◽  
Wei Wang ◽  
Xiaojuan Zhang ◽  
William Old ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mitotic Spindle ◽  
Kinase Activity ◽  
Spindle Checkpoint ◽  
Signaling Cascade ◽  
Checkpoint Activation ◽  
Mitotic Spindle Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Anaphase Onset

Mps1 is a protein kinase that plays essential roles in spindle checkpoint signaling. Unattached kinetochores or lack of tension triggers recruitment of several key spindle checkpoint proteins to the kinetochore, which delays anaphase onset until proper attachment or tension is reestablished. Mps1 acts upstream in the spindle checkpoint signaling cascade, and kinetochore targeting of Mps1 is required for subsequent recruitment of Mad1 and Mad2 to the kinetochore. The mechanisms that govern recruitment of Mps1 or other checkpoint proteins to the kinetochore upon spindle checkpoint activation are incompletely understood. Here, we demonstrate that phosphorylation of Mps1 at T12 and S15 is required for Mps1 recruitment to the kinetochore. Mps1 kinetochore recruitment requires its kinase activity and autophosphorylation at T12 and S15. Mutation of T12 and S15 severely impairs its kinetochore association and markedly reduces recruitment of Mad2 to the kinetochore. Our studies underscore the importance of Mps1 autophosphorylation in kinetochore targeting and spindle checkpoint signaling.

scholarly journals Distinct Chromosome Segregation Roles for Spindle Checkpoint Proteins

2002 ◽  
Vol 13 (9) ◽  
pp. 3029-3041 ◽  
Author(s):  
Cheryl D. Warren ◽  
D. Michelle Brady ◽  
Raymond C. Johnston ◽  
Joseph S. Hanna ◽  
Kevin G. Hardwick ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chromosome Segregation ◽  
Chromosome Instability ◽  
Spindle Checkpoint ◽  
Adjacent Segment ◽  
Effect Analysis ◽  
Checkpoint Proteins ◽  
Chromosome Transmission ◽  
Acid Fragment ◽  
Molecular Functions

The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete. In this study, we find that known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae. Loss of Bub1 or Bub3 protein elicits the largest effect. Analysis of Bub1p reveals the presence of two molecular functions. An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation. A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism. Both molecular functions depend on association with Bub3p. A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay. An adjacent segment is required for Mad1p binding, detected by deletion analysis and coimmunoprecipitation. Finally, overexpression of wild-type BUB1 or MAD3 genes leads to chromosome instability. Analysis of this activity indicates that the Bub3p-binding domain of Bub1p contributes to this phenotype through disruption of checkpoint activity as well as through introduction of kinetochore or spindle damage.

scholarly journals The ESCRT protein Chmp4c regulates mitotic spindle checkpoint signaling

2018 ◽  
Vol 217 (3) ◽  
pp. 861-876 ◽  
Author(s):  
Eleni Petsalaki ◽  
Maria Dandoulaki ◽  
George Zachos
Keyword(s):  
Mitotic Spindle ◽  
Spindle Checkpoint ◽  
Metaphase Plate ◽  
Membrane Remodeling ◽  
Mitotic Progression ◽  
Mitotic Spindle Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Endosomal Sorting ◽  
Anaphase Onset

The mitotic spindle checkpoint delays anaphase onset in the presence of unattached kinetochores, and efficient checkpoint signaling requires kinetochore localization of the Rod–ZW10–Zwilch (RZZ) complex. In the present study, we show that human Chmp4c, a protein involved in membrane remodeling, localizes to kinetochores in prometaphase but is reduced in chromosomes aligned at the metaphase plate. Chmp4c promotes stable kinetochore–microtubule attachments and is required for proper mitotic progression, faithful chromosome alignment, and segregation. Depletion of Chmp4c diminishes localization of RZZ and Mad1-Mad2 checkpoint proteins to prometaphase kinetochores and impairs mitotic arrest when microtubules are depolymerized by nocodazole. Furthermore, Chmp4c binds to ZW10 through a small C-terminal region, and constitutive Chmp4c kinetochore targeting causes a ZW10-dependent checkpoint metaphase arrest. In addition, Chmp4c spindle functions do not require endosomal sorting complex required for transport–dependent membrane remodeling. These results show that Chmp4c regulates the mitotic spindle checkpoint by promoting localization of the RZZ complex to unattached kinetochores.

scholarly journals Systematic Analysis in Caenorhabditis elegans Reveals that the Spindle Checkpoint Is Composed of Two Largely Independent Branches

2009 ◽  
Vol 20 (4) ◽  
pp. 1252-1267 ◽  
Author(s):  
Anthony Essex ◽  
Alexander Dammermann ◽  
Lindsay Lewellyn ◽  
Karen Oegema ◽  
Arshad Desai
Keyword(s):  
Caenorhabditis Elegans ◽  
Spindle Checkpoint ◽  
Checkpoint Activation ◽  
Systematic Analysis ◽  
Checkpoint Signaling ◽  
Epistasis Analysis ◽  
Kinetochore Assembly ◽  
Anaphase Onset ◽  
Monopolar Spindle ◽  
Spindle Microtubules

Kinetochores use the spindle checkpoint to delay anaphase onset until all chromosomes have formed bipolar attachments to spindle microtubules. Here, we use controlled monopolar spindle formation to systematically define the requirements for spindle checkpoint signaling in the Caenorhabditis elegans embryo. The results, when interpreted in light of kinetochore assembly epistasis analysis, indicate that checkpoint activation is coordinately directed by the NDC-80 complex, the Rod/Zwilch/Zw10 complex, and BUB-1—three components independently targeted to the outer kinetochore by the scaffold protein KNL-1. These components orchestrate the integration of a core Mad1MDF-1/Mad2MDF-2-based signal, with a largely independent Mad3SAN-1/BUB-3 pathway. Evidence for independence comes from the fact that subtly elevating Mad2MDF-2 levels bypasses the requirement for BUB-3 and Mad3SAN-1 in kinetochore-dependent checkpoint activation. Mad3SAN-1 does not accumulate at unattached kinetochores and BUB-3 kinetochore localization is independent of Mad2MDF-2. We discuss the rationale for a bipartite checkpoint mechanism in which a core Mad1MDF-1/Mad2MDF-2 signal generated at kinetochores is integrated with a separate cytoplasmic Mad3SAN-1/BUB-3–based pathway.

scholarly journals Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Ivana Primorac ◽  
John R Weir ◽  
Elena Chiroli ◽  
Fridolin Gross ◽  
Ingrid Hoffmann ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Binding Mode ◽  
Signaling Networks ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Downstream Signaling ◽  
Signaling Components ◽  
Insight Into

Regulation of macromolecular interactions by phosphorylation is crucial in signaling networks. In the spindle assembly checkpoint (SAC), which enables errorless chromosome segregation, phosphorylation promotes recruitment of SAC proteins to tensionless kinetochores. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on the kinetochore subunit Spc105/Knl1. The phosphorylated MELT motifs (MELTP) then promote recruitment of downstream signaling components. How MELTP motifs are recognized is unclear. In this study, we report that Bub3, a 7-bladed β-propeller, is the MELTP reader. It contains an exceptionally well-conserved interface that docks the MELTP sequence on the side of the β-propeller in a previously unknown binding mode. Mutations targeting the Bub3 interface prevent kinetochore recruitment of the SAC kinase Bub1. Crucially, they also cause a checkpoint defect, showing that recognition of phosphorylated targets by Bub3 is required for checkpoint signaling. Our data provide the first detailed mechanistic insight into how phosphorylation promotes recruitment of checkpoint proteins to kinetochores.

scholarly journals Kinetochore-localized BUB-1/BUB-3 complex promotes anaphase onset in C. elegans

2015 ◽  
Vol 209 (4) ◽  
pp. 507-517 ◽  
Author(s):  
Taekyung Kim ◽  
Mark W. Moyle ◽  
Pablo Lara-Gonzalez ◽  
Christian De Groot ◽  
Karen Oegema ◽  
...  
Keyword(s):  
Spindle Checkpoint ◽  
Kinase Domain ◽  
Mitotic Chromosomes ◽  
Checkpoint Signaling ◽  
C Elegans ◽  
Anaphase Onset ◽  
Checkpoint Pathway ◽  
Chromosome Alignment ◽  
Kinetochore Region

The conserved Bub1/Bub3 complex is recruited to the kinetochore region of mitotic chromosomes, where it initiates spindle checkpoint signaling and promotes chromosome alignment. Here we show that, in contrast to the expectation for a checkpoint pathway component, the BUB-1/BUB-3 complex promotes timely anaphase onset in Caenorhabditis elegans embryos. This activity of BUB-1/BUB-3 was independent of spindle checkpoint signaling but required kinetochore localization. BUB-1/BUB-3 inhibition equivalently delayed separase activation and other events occurring during mitotic exit. The anaphase promotion function required BUB-1’s kinase domain, but not its kinase activity, and this function was independent of the role of BUB-1/BUB-3 in chromosome alignment. These results reveal an unexpected role for the BUB-1/BUB-3 complex in promoting anaphase onset that is distinct from its well-studied functions in checkpoint signaling and chromosome alignment, and suggest a new mechanism contributing to the coordination of the metaphase-to-anaphase transition.

scholarly journals In vivo, regulated reconstitution of spindle checkpoint arrest and silencing through chemical-induced dimerisation

2018 ◽  
Author(s):  
Priya Amin ◽  
Sadhbh Soper Ní Chafraidh ◽  
Ioanna Leontiou ◽  
Kevin G. Hardwick
Keyword(s):  
Protein Interactions ◽  
Protein Structures ◽  
Spindle Checkpoint ◽  
Specific Protein ◽  
Genetic Dissection ◽  
Protein Protein Interactions ◽  
Checkpoint Activation ◽  
Checkpoint Proteins ◽  
Reductionist Approach

AbstractChemical-induced dimerisation (CID) uses small molecules to control specific protein-protein interactions. Here, we employ CID dependent on the plant hormone abscisic acid (ABA) to reconstitute spindle checkpoint signalling in fission yeast. The spindle checkpoint signal usually originates at unattached or inappropriately attached kinetochores. These are complex, multi-protein structures with several important functions. To bypass kinetochore complexity, we take a reductionist approach to study checkpoint signalling. We generate a synthetic checkpoint arrest ectopically by inducing hetero-dimerisation of the checkpoint proteins Mph1Mps1 and Spc7KNL1. These proteins are engineered such that they can’t localise to kinetochores, and only form a complex in the presence of ABA. Using this novel assay we are able to checkpoint arrest a synchronous population of cells within 30 minutes of ABA addition. This assay allows for detailed genetic dissection of checkpoint activation and importantly it also provides a valuable tool for studying checkpoint silencing. To analyse silencing of the checkpoint and the ensuing mitotic exit, we simply wash-out the ABA from arrested cells. We show here that silencing is critically-dependent on PP1Dis2 recruitment to Mph1Mps1-Spc7KNL1 signalling platforms.

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