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A Siz1/Siz2-mediated SUMOylation programme inactivates the shugoshin-associated phosphatase-kinase network to stabilize sister kinetochore biorientation

ABSTRACT: The accurate segregation of chromosomes during mitosis relies on the attachment of sister chromatids to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore-microtubule attachments remains unclear. Here we identify SUMOylation as a mechanism that promotes anaphase onset upon biorientation. SUMO ligases modify the tension-sensing pericentromere-localized chromatin protein, shugoshin, to stabilize bioriented sister kinetochore-microtubule attachments. In the absence of SUMOylation, Aurora B kinase removal from kinetochores is delayed. Shugoshin SUMOylation prevents its binding to protein phosphatase 2A (PP2A) and release of this interaction is important for stabilizing sister kinetochore biorientation. We propose that SUMOylation modulates the kinase-phosphatase network within pericentromeres to inactivate the error correction machinery, thereby allowing anaphase entry in response to biorientation.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Culture

SUBMITTER: Adele Marston

LAB HEAD: Adele Marston

PROVIDER: PXD019287 | Pride | 2021-03-25

REPOSITORIES: Pride

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SUMOylation regulates the metaphase-anaphase transition by stabilizing sister kinetochore biorientation

Project description:The accurate segregation of chromosomes during mitosis relies on the attachment of sister chromatids to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore-microtubule remains unclear. Here we identify SUMOylation as a mechanism that promotes anaphase onset upon biorientation. SUMO ligases modify the tension-sensing pericentromere-localized chromatin protein, shugoshin, to stabilize bioriented sister kinetochore-microtubule attachments and allow entry into anaphase. SUMOylation of shugoshin prevents binding to protein phosphatase 2A (PP2A), while enhancing chromosome passenger complex (CPC) interaction and removal from centromeres. Dissociation of the shugoshin-PP2A interaction is important for stabilizing sister kinetochore biorientation. Therefore, SUMOylation modulates protein interactions within pericentromeres to allow a signalling switch in response to biorientation.

2021-05-06 | PXD015565 | Pride

Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere

Project description:To protect against aneuploidy, chromosomes must attach to microtubules from opposite poles (“biorientation”) prior to their segregation during mitosis. Biorientation relies on the correction of erroneous attachments by the aurora B kinase, which destabilizes kinetochore-microtubule attachments that lack tension. Incorrect attachments are also avoided because sister kinetochores are intrinsically biased towards capture by microtubules from opposite poles. Here we show that shugoshin acts as a pericentromeric adaptor that plays dual roles in biorientation in budding yeast. Shugoshin maintains the aurora B kinase at kinetochores that lack tension, thereby engaging the error correction machinery. Shugoshin also recruits the chromosome-organising complex, condensin, to the pericentromere. Pericentromeric condensin biases sister kinetochores towards capture by microtubules from opposite poles. Overall, shugoshin integrates a bias to sister kinetochore capture with error correction to enable chromosome biorientation. Our findings uncover the molecular basis of the bias to sister kinetochore capture and expose shugoshin as a pericentromeric hub controlling chromosome biorientation.

2014-01-08 | GSE53856 | GEO

Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere

2014-01-08 | E-GEOD-53856 | biostudies-arrayexpress

Mps1 regulates kinetochore-microtubule attachment stability via the Ska complex to ensure error-free chromosome segregation

Project description:Spindle assembly checkpoint (SAC) regulators such as the Mps1 kinase not only delay anaphase onset, but also correct improper chromosome-spindle linkages that would otherwise lead to missegregation and aneuploidy. However, the substrates and mechanisms involved in this pathway of error correction remain poorly understood. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K-fibers) epistatically to Aurora B, the other major error-correcting kinase. Through chemical genetics and quantitative proteomics, we identify both known and novel sites of Mps1- regulated phosphorylation at the outer kinetochore. Modification of these substrates was sensitive to microtubule tension and counterbalanced by the PP2A-B56 phosphatase, a positive regulator of chromosome-spindle interactions. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the hinge region of the W-shaped Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation strongly destabilized K-fibers in vivo and inhibited the Ska complex’s conversion from lattice diffusion to end-coupled microtubule binding in vitro. Together these results provide new insights into how Mps1 modulates the microtubule-binding properties of the kinetochore to promote the selective stabilization of bipolar attachments and error-free chromosome segregation.

2018-10-24 | PXD006135 | Pride

A common molecular mechanism underlies the role of Mps1 in chromosome biorientation and the spindle assembly checkpoint

Project description:The conserved Mps1 kinase corrects improper kinetochore-microtubule attachments, thereby ensuring chromosome biorientation. Yet, its critical targets in this process remain elusive. Mps1 is also involved in the spindle assembly checkpoint (SAC), the surveillance mechanism halting chromosome segregation until biorientation is attained. Its role in SAC activation is antagonized by the PP1 phosphatase and involves phosphorylation of Knl1/Spc105, which recruits Bub1 to kinetochores to promote assembly of SAC effector complexes. A crucial question is whether error correction and SAC activation are part of a single device or separable pathways. Here we characterise a novel yeast mutant, mps1-3, defective in chromosome biorientation and SAC activation. Through an unbiased screen for suppressors, we found that mutations lowering PP1 levels at Spc105 or forced association of Bub1 with Spc105 reinstate both chromosome biorientation and SAC signalling in mps1-3 cells. Our data strongly argue that Mps1-dependent phosphorylation of the Knl1/Spc105 kinetochore scaffold is critical for Mps1 function in both chromosome biorientation and SAC activation, thus supporting the idea that a common sensory apparatus simultaneously elicits error correction and SAC signalling.

2020-02-05 | GSE144741 | GEO

Small changes in phospho-occupancy at the kinetochore-microtubule interface drive mitotic fidelity

Project description:Kinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e. all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded ~50% phospho-occupancy, and the cumulative phospho-occupancy changed by only ~20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained, in part, by ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. The low inherent phospho-occupancy promotes microtubule attachment to kinetochores while the high sensitivity of kinetochore-microtubule attachments to small changes in phospho-occupancy drives error correction and ensures high mitotic fidelity.

2022-06-27 | MSV000089738 | MassIVE

The proteomic landscape of centromeric chromatin reveals an essential role for the Ctf19 complex in meiotic kinetochore assembly.

Project description:Faithful gamete formation during meiosis requires that kinetochores take on new functions that impact homolog pairing/recombination and their attachments to microtubules. We used a proteomics pipeline to determine the global composition of centromeric chromatin and kinetochores at distinct meiotic stages. Our datasets uncover an unanticipated role for the Ctf19CCAN inner kinetochore sub-complex in gametogenesis. We show that components of the Ctf19 complex that are dispensable for mitotic growth become critical for assembly of a functional kinetochore upon meiotic entry. Consequently, in the absence of these factors, chromosomes fail to attach to meiotic spindles and produce inviable gametes due to gross segregation errors. Our evidence suggests that functional kinetochore assembly is driven by Ctf19CCAN complex-dependent Ipl1AURORA B positioning at the inner kinetochore. Thus, our global view of kinetochore composition in meiosis reveals the extent of kinetochore remodelling during gametogenesis and uncovers a kinetochore assembly pathway that becomes critical in meiosis.

2021-02-23 | PXD019754 | Pride

CX-MS of Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore

Project description:Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the S. cerevisiae outer kinetochore, assembling into 3 MDa-sized microtubule-embracing rings, but how ring assembly is specifically initiated at kinetochores remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi-orientation. We show that Dam1c and the general microtubule plus-end-associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c-Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c-Bim1 binding by relieving an intramolecular inhibition of the Dam1 C-terminus. In addition, Bim1 recruits Bik1/CLIP-170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end-on attachments are formed during the process of attachment error correction.

2021-06-28 | PXD026935 | Pride

Mapping of cohesion factor association sites in S. cerevisiae - Meiosis I chromosome segregation is established through regulation of microtubule-kinetochore interactions.

Project description:During meiosis, a single round of DNA replication is followed by two consecutive rounds of nuclear divisions called meiosis I and meiosis II. In meiosis I, homologous chromosomes segregate, while sister chromatids remain together. Determining how this unusual chromosome segregation behavior is established is central to understanding germ cell development. Here we show that preventing microtubule-kinetochore interactions during premeiotic S phase and prophase I is essential for establishing the meiosis I chromosome segregation pattern. Premature interactions of kinetochores with microtubules transform meiosis I into a mitosis-like division by disrupting two key meiosis I events: coorientation of sister kinetochores and protection of centromeric cohesin removal from chromosomes. Furthermore we find that restricting outer kinetochore assembly contributes to preventing premature engagement of microtubules with kinetochores. We propose that inhibition of microtubule-kinetochore interactions during premeiotic S phase and prophase I is central to establishing the unique meiosis I chromosome segregation pattern. The association of the cohesion factors Rec8, Pds5, and Sgo1 were measured by ChIP-chip analysis in wild-type and CUP-CLB3 strains.

2012-10-05 | E-GEOD-41339 | biostudies-arrayexpress

Kinetochore protein Cnn1 promotes sister chromatid bi-orientation by modulating intraKMN affinities in a biphasic, phospho-regulated manner.

Project description:Kinetochores are large proteinaceous structures that assemble on centromeres to align sister chromatids to the mitotic spindle and orchestrate their segregation to the daughter cells. At the core of kinetochores lies a network of proteins known as KMN, whose microtubule-binding activity is regulated by the Ipl1/Aurora B and Mps1 kinases. Here, we have characterized a novel structural yeast kinetochore protein named Cnn1 and show that it regulates KMN-spindle activity by modulating the affinity between the KMN subcomplexes at their basis. Cnn1 supports KMN from S phase to anaphase, and is controlled by the Cdc28, Mps1 and Ipl1 kinases. Cnn1 has evolved to associate the KMN complexes into a coherent network activity to ensure efficient

2012-05-06 | GSE31567 | GEO

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