Stu2 uses a 15-nm parallel coiled coil for kinetochore localization and concomitant regulation of the mitotic spindle

Haase, Karen Plevock; Fox, Jaime C.; Byrnes, Amy E.; Adikes, Rebecca C.; Speed, Sarah K.; Haase, Julian; Friedman, Brandon; Cook, Diana M.; Bloom, Kerry; Rusan, Nasser M.; Slep, Kevin C.

doi:10.1091/mbc.e17-01-0057

Cited by 6 publications

(9 citation statements)

References 40 publications

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“…How so many proteins compete for a small region of Stu2 raises interesting questions about cellular regulation that will need to be resolved in future studies. We also found that Stu2's coiled-coil domain is also required for its kinetochore association, consistent with recent work in cells (Haase et al, 2018;Humphrey et al, 2018).…”

Section: Stu2 Utilizes Multiple Domains To Achieve Kinetochore Associsupporting

confidence: 91%

“…We identified two domains in Stu2 required for its kinetochore association, the extreme Cterminus and the coiled-coil domain, consistent with prior work (Haase et al, 2018;Humphrey et al, 2018). We propose that Stu2's C-terminal tail domain (residues 855-888) directly interacts with Ndc80c, and that two copies of Stu2's C-terminal domain are required to "sandwich" its binding motif on Ndc80c.…”

Section: Stu2 Utilizes Multiple Domains To Achieve Kinetochore Associsupporting

confidence: 86%

“…Given that XMAP215 family members, including human ch-TOG, display a conserved association with the kinetochore's Ndc80c (Aravamudhan et al, 2014;Haase et al, 2018;Hsu and Toda, 2011;Humphrey et al, 2018;Miller et al, 2016;Tang et al, 2013), it will be important to investigate whether intrinsic tension-selectivity is a wide-spread feature of kinetochore-microtubule interactions, whether mutations B) Exponentially growing stu2-AID cultures expressing an ectopic copy of STU2 (STU2 WT , SBY13901; stu2 TOG1∆ , SBY13904; stu2 TOG2∆ , SBY13907; stu2 SK-rich∆ , SBY13913; stu2 cc∆ , SBY13916; or stu2 C-term∆(855-888) , SBY14269) that also contained Dsn1-6His-3Flag were treated with auxin 30 min prior to harvesting. Protein lysates were subsequently prepared and kinetochore particles were purified by α-Flag immunoprecipitation (IP) and analyzed by immunoblotting.…”

Section: Discussionmentioning

confidence: 99%

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A Stu2-mediated intrinsic tension-sensing pathway promotes chromosome biorientation in vivo

Miller

Evans

Zelter

et al. 2018

Preprint

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Accurate segregation of chromosomes to daughter cells is a critical aspect of cell division. It requires the kinetochores on duplicated chromosomes to biorient, attaching to microtubules from opposite poles of the cell. Bioriented attachments come under tension, while incorrect attachments lack tension and must be destabilized. A well-studied error correction pathway is mediated by the Aurora B kinase, which destabilizes low tension-bearing attachments. We recently discovered that in vitro, kinetochores display an additional intrinsic tension-sensing pathway that utilizes Stu2. This pathway's contribution to error correction in cells, however, was unknown. Here, we identify a Stu2 mutant that abolishes its kinetochore function and show that it causes error correction defects in vivo. We also show that this intrinsic tensionsensing pathway functions in concert with the Aurora B-mediated pathway. Together, our work indicates that cells employ at least two pathways to ensure biorientation and the accuracy of chromosome segregation.

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Section: Stu2 Utilizes Multiple Domains To Achieve Kinetochore Associsupporting

confidence: 91%

Section: Stu2 Utilizes Multiple Domains To Achieve Kinetochore Associsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A Stu2-mediated intrinsic tension-sensing pathway promotes chromosome biorientation in vivo

Miller

Evans

Zelter

et al. 2018

Preprint

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“…This domain coincides with the domain responsible for Stu2p dimerization (Wolyniak et al, ). This domain of Stu2p also is required for its recruitment to the kinetochore and SPB (Haase et al, ). As SUMO is known to regulate the dimerization of several of its targets (Bossis et al, ; Rojas‐Fernandez et al, ), we are currently working to determine whether SUMO regulates the dimerization of Stu2p.…”

Section: Discussionmentioning

confidence: 99%

“…In addition to its role in microtubule polymerization and nucleation, it is responsible for microtubule anchorage at MTOCs (Podolski et al, ; Usui, Maekawa, Pereira, & Schiebel, ; Wang & Huffaker, ). Stu2p also functions at the kinetochore, helping to attach MTs to the outer kinetochore plaque (Haase et al, ; Miller et al, ; Pearson, Maddox, Zarzar, Salmon, & Bloom, ; Suzuki et al, ). The kinetochore contains several sumoylated proteins, including its sumoylated partner, Ndc80p (Aravamudhan et al, ; Montpetit et al, ).…”

Section: Discussionmentioning

confidence: 99%

The TOG protein Stu2/XMAP215 interacts covalently and noncovalently with SUMO

et al. 2018

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Stu2p is the yeast member of the XMAP215/Dis1/ch-TOG family of microtubule-associated proteins that promote microtubule polymerization. However, the factors that regulate its activity are not clearly understood. Here we report that Stu2p in the budding yeast Saccharomyces cerevisiae interacts with SUMO by covalent and noncovalent mechanisms. Stu2p interacted by two-hybrid analysis with the yeast SUMO Smt3p, its E2 Ubc9p, and the E3 Nfi1p. A region of Stu2p containing the dimerization domain was both necessary and sufficient for interaction with SUMO and Ubc9p. Stu2p was found to be sumoylated both in vitro and in vivo. Stu2p copurified with SUMO in a pull-down assay and vice versa. Stu2p also bound to a nonconjugatable form of SUMO, suggesting that Stu2p can interact noncovalently with SUMO. In addition, Stu2p interacted with the STUbL enzyme Ris1p. Stu2p also copurified with ubiquitin in a pull-down assay, suggesting that it can be modified by both SUMO and ubiquitin. Tubulin, a major binding partner of Stu2p, also interacted noncovalently with SUMO. By two-hybrid analysis, the beta-tubulin Tub2p interacted with SUMO independently of the microtubule stressor, benomyl. Together, these findings raise the possibility that the microtubule polymerization activities mediated by Stu2p are regulated through sumoylation pathways.

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Structure of the native γ-tubulin ring complex capping spindle microtubules

Dendooven,

Yatskevich,

Burt

et al. 2024

Nat Struct Mol Biol

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Microtubule (MT) filaments, composed of α/β-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They are nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, the molecular mechanism of nucleation remains elusive. Here we used cryo-electron tomography to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, γTuRC presents a ring of γ-tubulin subunits to seed nucleation of exclusively 13-protofilament MTs, adopting an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-electron tomography reconstruction revealed that a coiled-coil protein staples the first row of α/β-tubulin of the MT to alternating positions along the γ-tubulin ring of γTuRC. This positioning of α/β-tubulin onto γTuRC suggests a role for the coiled-coil protein in augmenting γTuRC-mediated MT nucleation. Based on our results, we describe a molecular model for budding yeast γTuRC activation and MT nucleation.

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Stu2 uses a 15-nm parallel coiled coil for kinetochore localization and concomitant regulation of the mitotic spindle

Abstract: The yeast microtubule polymerase Stu2’s C-terminal domain is a 15-nm parallel, homodimeric coiled coil with two spatially distinct conserved regions. Determinants in these conserved regions optimally position Stu2 on the mitotic spindle to drive proper spindle structure and dynamics.

Cited by 6 publications

References 40 publications

A Stu2-mediated intrinsic tension-sensing pathway promotes chromosome biorientation in vivo

A Stu2-mediated intrinsic tension-sensing pathway promotes chromosome biorientation in vivo

The TOG protein Stu2/XMAP215 interacts covalently and noncovalently with SUMO

Structure of the native γ-tubulin ring complex capping spindle microtubules

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