Coupling Unbiased Mutagenesis to High-throughput DNA Sequencing Uncovers Functional Domains in the Ndc80 Kinetochore Protein of <i>Saccharomyces cerevisiae</i>

Tien, Jerry F.; Fong, Kimberly K.; Umbreit, Neil T.; Payen, Célia; Zelter, Alex; Asbury, Charles L.; Dunham, Maitreya J.; Davis, Trisha N.

doi:10.1534/genetics.113.152728

Cited by 21 publications

(36 citation statements)

References 54 publications

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“…In total, our approach revealed 277 unique crosslinks and 85 unique loop-links within the Ndc80 complex with $95% confidence ( Figure S2, C and D, Table S4, and Table S5). The cross-links observed are in excellent agreement with the current structural model of the Ndc80 complex and confirm the location of the tetramerization domain as previously proposed (Wei et al 2005;Maiolica et al 2007;Ciferri et al 2008;Tien et al 2013). The N-terminal tail of Ndc80 cross-linked to multiple regions of the complex (Figure S2,C and D).…”

Section: The S Cerevisiae Ndc80 Complex Can Adopt a Tightly Folded Csupporting

confidence: 89%

“…We recently performed an insertional mutagenesis screen to uncover essential regions of NDC80 in S. cerevisiae (Tien et al 2013). The functions of four of these essential regions, identified as clusters of lethal insertions, have not yet been identified.…”

Section: Isolation Of the Ndc80-121 Temperature-sensitive Allelementioning

confidence: 99%

“…However, it remains to be determined if these conformations arise from the flexibility of the complex at the loop observed in vitro. Furthermore, no previous study has examined whether the bending flexibility of the Ndc80 complex is important in vivo.Our recent results from a mutagenesis screen suggest that several small regions of the coiled coil are essential for cell viability, including one region near the predicted loop (Tien et al 2013). Here, biochemical characterization of the Ndc80 complex was combined with genetic analysis of a novel ndc80 mutant to reveal that the loop region acts as a hinge in vivo, enabling the complex to adopt a tightly folded conformation.…”

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confidence: 99%

“…Our recent results from a mutagenesis screen suggest that several small regions of the coiled coil are essential for cell viability, including one region near the predicted loop (Tien et al 2013). Here, biochemical characterization of the Ndc80 complex was combined with genetic analysis of a novel ndc80 mutant to reveal that the loop region acts as a hinge in vivo, enabling the complex to adopt a tightly folded conformation.…”

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confidence: 99%

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Kinetochore Biorientation in Saccharomyces cerevisiae Requires a Tightly Folded Conformation of the Ndc80 Complex

et al. 2014

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Accurate transmission of genetic material relies on the coupling of chromosomes to spindle microtubules by kinetochores. These linkages are regulated by the conserved Aurora B/Ipl1 kinase to ensure that sister chromatids are properly attached to spindle microtubules. Kinetochore-microtubule attachments require the essential Ndc80 complex, which contains two globular ends linked by large coiled-coil domains. In this study, we isolated a novel ndc80 mutant in Saccharomyces cerevisiae that contains mutations in the coiled-coil domain. This ndc80 mutant accumulates erroneous kinetochore-microtubule attachments, resulting in misalignment of kinetochores on the mitotic spindle. Genetic analyses with suppressors of the ndc80 mutant and in vitro cross-linking experiments suggest that the kinetochore misalignment in vivo stems from a defect in the ability of the Ndc80 complex to stably fold at a hinge in the coiled coil. Previous studies proposed that the Ndc80 complex can exist in multiple conformations: elongated during metaphase and bent during anaphase. However, the distinct functions of individual conformations in vivo are unknown. Here, our analysis revealed a tightly folded conformation of the Ndc80 complex that is likely required early in mitosis. This conformation is mediated by a direct, intracomplex interaction and involves a greater degree of folding than the bent form of the complex at anaphase. Furthermore, our results suggest that this conformation is functionally important in vivo for efficient error correction by Aurora B/Ipl1 and, consequently, to ensure proper kinetochore alignment early in mitosis. KINETOCHORES mediate the linkage between chromosomes and spindle microtubules during mitosis. This attachment is highly regulated to promote the fidelity of chromosome segregation. At metaphase, replicated chromosomes are attached to microtubules emanating from opposite poles, in a "bioriented" alignment. This ensures that each daughter cell receives a full complement of genetic material after chromosome segregation. Errors can occur in the form of "syntelic" attachments, when both sister kinetochores attach to microtubules emanating from the same pole. These erroneous kinetochore-microtubule attachments are detected and detached by the conserved Aurora B/Ipl1 kinase Tanaka et al. 2002;Pinsky et al. 2006). In the current prevailing model, Aurora B/Ipl1 corrects syntelic attachments by destabilizing linkages that are under low levels of tension (Nicklas and Koch 1969;Tanaka et al. 2002;Liu et al. 2009;Cane et al. 2013). The Aurora B/Ipl1 error detection system is coupled to the spindle checkpoint, a separate surveillance system that delays anaphase until all kinetochores are attached to spindle microtubules (reviewed in Hauf 2013). Together, these systems ensure that the physical separation of replicated chromatids does not occur until all kinetochore-microtubule attachments are bioriented.The attachment of spindle microtubules to kinetochores requires the conserved Ndc80 complex ( Figure 1A) rod-sh...

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Section: The S Cerevisiae Ndc80 Complex Can Adopt a Tightly Folded Csupporting

confidence: 89%

Section: Isolation Of the Ndc80-121 Temperature-sensitive Allelementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Kinetochore Biorientation in Saccharomyces cerevisiae Requires a Tightly Folded Conformation of the Ndc80 Complex

et al. 2014

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“…Ndc80p helps organize and stabilize kinetochore-microtubule interaction in order to facilitate proper chromosome segregation [Wei et al, 2011]. Ndc80p forms a "dumbbell-like" heterotetramer with Nuf2p, Spc24p, and Spc25p to form the Ndc80 complex [Cheeseman et al, 2006;Tien et al, 2013]. The Ndc80 complex also helps localize spindle assembly checkpoint proteins to the kinetochore [Gillett et al, 2004;Maiato et al, 2004].…”

Section: Ndc80pmentioning

confidence: 99%

Emerging roles of sumoylation in the regulation of actin, microtubules, intermediate filaments, and septins

et al. 2015

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Sumoylation is a powerful regulatory system that controls many of the critical processes in the cell, including DNA repair, transcriptional regulation, nuclear transport, and DNA replication. Recently, new functions for SUMO have begun to emerge. SUMO is covalently attached to components of each of the four major cytoskeletal networks, including microtubule‐associated proteins, septins, and intermediate filaments, in addition to nuclear actin and actin‐regulatory proteins. However, knowledge of the mechanisms by which this signal transduction system controls the cytoskeleton is still in its infancy. One story that is beginning to unfold is that SUMO may regulate the microtubule motor protein dynein by modification of its adaptor Lis1. In other instances, cytoskeletal elements can both bind to SUMO non‐covalently and also be conjugated by it. The molecular mechanisms for many of these new functions are not yet clear, but are under active investigation. One emerging model links the function of MAP sumoylation to protein degradation through SUMO‐targeted ubiquitin ligases, also known as STUbL enzymes. Other possible functions for cytoskeletal sumoylation are also discussed. © 2015 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc.

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MAPping the Ndc80 loop in cancer: A possible link between Ndc80/Hec1 overproduction and cancer formation

Tang

Toda

2015

BioEssays

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Mis-regulation (e.g. overproduction) of the human Ndc80/Hec1 outer kinetochore protein has been associated with aneuploidy and tumourigenesis, but the genetic basis and underlying mechanisms of this phenomenon remain poorly understood. Recent studies have identified the ubiquitous Ndc80 internal loop as a protein-protein interaction platform. Binding partners include the Ska complex, the replication licensing factor Cdt1, the Dam1 complex, TACC-TOG microtubule-associated proteins (MAPs) and kinesin motors. We review the field and propose that the overproduction of Ndc80 may unfavourably absorb these interactors through the internal loop domain and lead to a change in the equilibrium of MAPs and motors in the cells. This sequestration will disrupt microtubule dynamics and the proper segregation of chromosomes in mitosis, leading to aneuploid formation. Further investigation of Ndc80 internal loop-MAPs interactions will bring new insights into their roles in kinetochore-microtubule attachment and tumourigenesis.

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Coupling Unbiased Mutagenesis to High-throughput DNA Sequencing Uncovers Functional Domains in the Ndc80 Kinetochore Protein of Saccharomyces cerevisiae

Cited by 21 publications

References 54 publications

Kinetochore Biorientation in Saccharomyces cerevisiae Requires a Tightly Folded Conformation of the Ndc80 Complex

Kinetochore Biorientation in Saccharomyces cerevisiae Requires a Tightly Folded Conformation of the Ndc80 Complex

Emerging roles of sumoylation in the regulation of actin, microtubules, intermediate filaments, and septins

MAPping the Ndc80 loop in cancer: A possible link between Ndc80/Hec1 overproduction and cancer formation

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