scholarly journals The Dam1 complex confers microtubule plus end–tracking activity to the Ndc80 kinetochore complex

2010 ◽  
Vol 189 (4) ◽  
pp. 641-649 ◽  
Author(s):  
Fabienne Lampert ◽  
Peter Hornung ◽  
Stefan Westermann
Keyword(s):  
Fluorescence Microscopy ◽  
Chromosome Segregation ◽  
Molecular Basis ◽  
Total Internal Reflection ◽  
Functional Relationship ◽  
Budding Yeast ◽  
Ndc80 Complex ◽  
Dam1 Complex ◽  
Necessary And Sufficient ◽  
Dynamic Microtubule

Kinetochores must remain associated with microtubule ends, as they undergo rapid transitions between growth and shrinkage. The molecular basis for this essential activity that ensures correct chromosome segregation is unclear. In this study, we have used reconstitution of dynamic microtubules and total internal reflection fluorescence microscopy to define the functional relationship between two important budding yeast kinetochore complexes. We find that the Dam1 complex is an autonomous plus end–tracking complex. The Ndc80 complex, despite being structurally related to the general tip tracker EB1, fails to recognize growing ends efficiently. Dam1 oligomers are necessary and sufficient to recruit Ndc80 to dynamic microtubule ends, where both complexes remain continuously associated. The interaction occurs specifically in the presence of microtubules and is subject to regulation by Ipl1 phosphorylation. These findings can explain how the force harvested by Dam1 is transmitted to the rest of the kinetochore via the Ndc80 complex.

scholarly journals Meiotic regulation of the Ndc80 complex composition and function

2021 ◽  
Author(s):  
Jingxun Chen ◽  
Elçin Ünal
Keyword(s):  
Chromosome Segregation ◽  
Budding Yeast ◽  
Complex Composition ◽  
The Past ◽  
Ndc80 Complex ◽  
Health And Disease ◽  
Yeast Meiosis ◽  
And Function ◽  
Protein Synthesis And Degradation ◽  
Synthesis And Degradation

AbstractThis review describes the current models for how the subunit abundance of the Ndc80 complex, a key kinetochore component, is regulated in budding yeast and metazoan meiosis. The past decades of kinetochore research have established the Ndc80 complex to be a key microtubule interactor and a central hub for regulating chromosome segregation. Recent studies further demonstrate that Ndc80 is the limiting kinetochore subunit that dictates the timing of kinetochore activation in budding yeast meiosis. Here, we discuss the molecular circuits that regulate Ndc80 protein synthesis and degradation in budding yeast meiosis and compare the findings with those from metazoans. We envision the regulatory principles discovered in budding yeast to be conserved in metazoans, thereby providing guidance into future investigations on kinetochore regulation in human health and disease.

scholarly journals Structural view of the yeast Dam1 complex, a ring-shaped molecular coupler for the dynamic microtubule end

2020 ◽  
Vol 64 (2) ◽  
pp. 359-370
Author(s):  
Shaowen Wu ◽  
Ekaterina L. Grishchuk
Keyword(s):  
Self Assembly ◽  
Eukaryotic Cell ◽  
Chromosome Loss ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ndc80 Complex ◽  
Unified View ◽  
Dam1 Complex ◽  
Ring Architecture ◽  
Chromosome Motion ◽  
Dynamic Microtubule

Abstract In a dividing eukaryotic cell, proper chromosome segregation requires the dynamic yet persistent attachment of kinetochores to spindle microtubules. In the budding yeast Saccharomyces cerevisiae, this function is especially crucial because each kinetochore is attached to a single microtubule; consequently, loss of attachment could lead to unrecoverable chromosome loss. The highly specialized heterodecameric Dam1 protein complex achieves this coupling by assembling into a microtubule-encircling ring that glides near the end of the dynamic microtubule to mediate chromosome motion. In recent years, we have learned a great deal about the structural properties of the Dam1 heterodecamer, its mechanism of self-assembly into rings, and its tethering to the kinetochore by the elongated Ndc80 complex. The most remarkable progress has resulted from defining the fine structures of helical bundles within Dam1 heterodecamer. In this review, we critically analyze structural observations collected by diverse approaches with the goal of obtaining a unified view of Dam1 ring architecture. A considerable consistency between different studies supports a coherent model of the circular core of the Dam1 ring. However, there are persistent uncertainties about the composition of ring protrusions and flexible extensions, as well as their roles in mediating ring core assembly and interactions with the Ndc80 complex and microtubule.

scholarly journals Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

2018 ◽  
Author(s):  
Tae Yeon Yoo ◽  
Jeong-Mo Choi ◽  
William Conway ◽  
Che-Hang Yu ◽  
Rohit V. Pappu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Molecular Basis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Aurora B ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Ndc80 Complex

AbstractProper kinetochore-microtubule attachments, mediated by the NDC80 complex, are required for error-free chromosome segregation. Erroneous attachments are corrected by the tension dependence of kinetochore-microtubule interactions. Here, we present a method, based on fluorescence lifetime imaging microscopy and Förster resonance energy transfer, to quantitatively measure the fraction of NDC80 complexes bound to microtubules at individual kinetochores in living human cells. We found that NDC80 binding is modulated in a chromosome autonomous fashion over prometaphase and metaphase, and is predominantly regulated by centromere tension. We show that this tension dependency requires phosphorylation of the N-terminal tail of Hec1, a component of the NDC80 complex, and the proper localization of Aurora B kinase, which modulates NDC80 binding. Our results lead to a mathematical model of the molecular basis of tension-dependent NDC80 binding to kinetochore microtubules in vivo.

scholarly journals Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
David M Kern ◽  
Julie K Monda ◽  
Kuan-Chung Su ◽  
Elizabeth M Wilson-Kubalek ◽  
Iain M Cheeseman
Keyword(s):  
Chromosome Segregation ◽  
Human Cells ◽  
Cross Linking ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Anaphase Onset ◽  
Ndc80 Complex ◽  
Outstanding Question ◽  
Dynamic Microtubule

Chromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attachments are modulated to ensure that bi-oriented attachments are selectively stabilized and maintained. The Astrin-SKAP complex localizes preferentially to properly bi-oriented sister kinetochores, representing the final outer kinetochore component recruited prior to anaphase onset. Here, we reconstitute the 4-subunit Astrin-SKAP complex, including a novel MYCBP subunit. Our work demonstrates that the Astrin-SKAP complex contains separable kinetochore localization and microtubule binding domains. In addition, through cross-linking analysis in human cells and biochemical reconstitution, we show that the Astrin-SKAP complex binds synergistically to microtubules with the Ndc80 complex to form an integrated interface. We propose a model in which the Astrin-SKAP complex acts together with the Ndc80 complex to stabilize correctly formed kinetochore-microtubule interactions.

scholarly journals Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80

2017 ◽  
Author(s):  
David M. Kern ◽  
Elizabeth M. Wilson-Kubalek ◽  
Iain M. Cheeseman
Keyword(s):  
Chromosome Segregation ◽  
Human Cells ◽  
Cross Linking ◽  
Core Component ◽  
The Core ◽  
Binding Domains ◽  
Anaphase Onset ◽  
Ndc80 Complex ◽  
Outstanding Question ◽  
Dynamic Microtubule

AbstractChromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attachments are modulated to ensure that bi-oriented attachments are selectively stabilized and maintained. The Astrin-SKAP complex localizes preferentially to properly bi-oriented sister kinetochores, representing the final outer kinetochore component recruited prior to anaphase onset. Here, we reconstitute the 4-subunit Astrin-SKAP complex, including a novel MYCBP subunit. Our work demonstrates that the Astrin-SKAP complex contains separable kinetochore localization and microtubule binding domains. In addition, through cross-linking analysis in human cells and biochemical reconstitution, we show that the Astrin-SKAP complex binds synergistically to microtubules with the Ndc80 complex, the core component of the kinetochore-microtubule interface, to form an integrated interface. We propose a model in which the Astrin-SKAP complex acts together with the Ndc80 complex to stabilize correctly formed kinetochore-microtubule interactions.

scholarly journals Structural comparison of the Caenorhabditis elegans and human Ndc80 complexes bound to microtubules reveals distinct binding behavior

2016 ◽  
Vol 27 (8) ◽  
pp. 1197-1203 ◽  
Author(s):  
Elizabeth M. Wilson-Kubalek ◽  
Iain M. Cheeseman ◽  
Ronald A. Milligan
Keyword(s):  
Caenorhabditis Elegans ◽  
Molecular Basis ◽  
Structural Comparison ◽  
Direct Role ◽  
C Elegans ◽  
Binding Behavior ◽  
Cryo Electron Microscopy ◽  
Ndc80 Complex ◽  
Structural Differences ◽  
Dynamic Microtubule

During cell division, kinetochores must remain tethered to the plus ends of dynamic microtubule polymers. However, the molecular basis for robust kinetochore–microtubule interactions remains poorly understood. The conserved four-subunit Ndc80 complex plays an essential and direct role in generating dynamic kinetochore–microtubule attachments. Here we compare the binding of the Caenorhabditis elegans and human Ndc80 complexes to microtubules at high resolution using cryo–electron microscopy reconstructions. Despite the conserved roles of the Ndc80 complex in diverse organisms, we find that the attachment mode of these complexes for microtubules is distinct. The human Ndc80 complex binds every tubulin monomer along the microtubule protofilament, whereas the C. elegans Ndc80 complex binds more tightly to β-tubulin. In addition, the C. elegans Ndc80 complex tilts more toward the adjacent protofilament. These structural differences in the Ndc80 complex between different species may play significant roles in the nature of kinetochore–microtubule interactions.

scholarly journals An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast

2018 ◽  
Author(s):  
Jackie Lang ◽  
Adrienne Barber ◽  
Sue Biggins
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
De Novo ◽  
Budding Yeast ◽  
Centromeric Dna ◽  
Kinetochore Assembly ◽  
Ndc80 Complex ◽  
Spindle Microtubules ◽  
Underlying Mechanisms ◽  
Mitotic Phosphorylation

ABSTRACTChromosome segregation depends on the kinetochore, the machine that establishes force-bearing attachments between DNA and spindle microtubules. Kinetochores are formed every cell cycle via a highly regulated process that requires coordinated assembly of multiple subcomplexes on specialized chromatin. To elucidate the underlying mechanisms, we developed an assay to assemble kinetochores de novo using centromeric DNA and budding yeast extracts. Assembly is enhanced by mitotic phosphorylation of the Dsn1 kinetochore protein and generates kinetochores capable of binding microtubules. We used this assay to investigate why kinetochores recruit the microtubule-binding Ndc80 complex via two receptors: the Mis12 complex and CENP-T. Although the CENP-T pathway is non-essential in yeast, we demonstrate that it becomes essential for viability and Ndc80c recruitment when the Mis12 pathway is crippled by defects in Dsn1 phosphorylation. Assembling kinetochores de novo in yeast extracts provides a powerful and genetically tractable method to elucidate critical regulatory events in the future.

scholarly journals Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Tae Yeon Yoo ◽  
Jeong-Mo Choi ◽  
William Conway ◽  
Che-Hang Yu ◽  
Rohit V Pappu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Molecular Basis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Aurora B ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Ndc80 Complex

Proper kinetochore-microtubule attachments, mediated by the NDC80 complex, are required for error-free chromosome segregation. Erroneous attachments are corrected by the tension dependence of kinetochore-microtubule interactions. Here, we present a method, based on fluorescence lifetime imaging microscopy and Förster resonance energy transfer, to quantitatively measure the fraction of NDC80 complexes bound to microtubules at individual kinetochores in living human cells. We found that NDC80 binding is modulated in a chromosome autonomous fashion over prometaphase and metaphase, and is predominantly regulated by centromere tension. We show that this tension dependency requires phosphorylation of the N-terminal tail of Hec1, a component of the NDC80 complex, and the proper localization of Aurora B kinase, which modulates NDC80 binding. Our results lead to a mathematical model of the molecular basis of tension-dependent NDC80 binding to kinetochore microtubules in vivo.

scholarly journals An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Jackie Lang ◽  
Adrienne Barber ◽  
Sue Biggins
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
De Novo ◽  
Budding Yeast ◽  
Centromeric Dna ◽  
Kinetochore Assembly ◽  
Ndc80 Complex ◽  
Spindle Microtubules ◽  
Underlying Mechanisms ◽  
Mitotic Phosphorylation

Chromosome segregation depends on the kinetochore, the machine that establishes force-bearing attachments between DNA and spindle microtubules. Kinetochores are formed every cell cycle via a highly regulated process that requires coordinated assembly of multiple subcomplexes on specialized chromatin. To elucidate the underlying mechanisms, we developed an assay to assemble kinetochores de novo using centromeric DNA and budding yeast extracts. Assembly is enhanced by mitotic phosphorylation of the Dsn1 kinetochore protein and generates kinetochores capable of binding microtubules. We used this assay to investigate why kinetochores recruit the microtubule-binding Ndc80 complex via two receptors: the Mis12 complex and CENP-T. Although the CENP-T pathway is non-essential in yeast, we demonstrate that it becomes essential for viability and Ndc80c recruitment when the Mis12 pathway is crippled by defects in Dsn1 phosphorylation. Assembling kinetochores de novo in yeast extracts provides a powerful and genetically tractable method to elucidate critical regulatory events in the future.

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