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Trisha N. Davis - One of the best experts on this subject based on the ideXlab platform.
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tight bending of the Ndc80 Complex provides intrinsic regulation of its binding to microtubules
eLife, 2019Co-Authors: Emily Anne Scarborough, Trisha N. Davis, Charles L. AsburyAbstract:Regulation of the outer kinetochore Complex Ndc80 is essential to ensure correct kinetochore-microtubule attachments during mitosis. Here, we present a novel mechanism of regulation that is intrinsic to its structure; tight bending of the Ndc80 Complex inhibits its microtubule binding. Using single molecule Forster resonance energy transfer (FRET), we show that the Saccharomyces cerevisiae Ndc80 Complex can fluctuate between straight and bent forms, and that binding of the Complex to microtubules selects for straightened forms. The loop region of the Complex enables its bent conformation, as deletion of the loop promotes straightening. In addition, the kinetochore Complex MIND enhances microtubule binding by opposing the tightly bent, auto-inhibited conformation of the Ndc80 Complex. We suggest that prior to its assembly at the kinetochore, the Ndc80 Complex interchanges between bent (auto-inhibited) and open conformations. Once assembled, its association with MIND stabilizes the Ndc80 Complex in a straightened form for higher affinity microtubule binding.
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human ska Complex and Ndc80 Complex interact to form a load bearing assembly that strengthens kinetochore microtubule attachments
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Luke A Helgeson, Alex Zelter, Michael Riffle, Charles L. Asbury, Michael J Maccoss, Trisha N. DavisAbstract:Accurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska Complex and Ndc80 Complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore–microtubule attachments in vivo. While purified Ska Complex tracks with disassembling microtubule tips, it remains unclear whether the Ska Complex–microtubule interaction is sufficiently strong to make a significant contribution to kinetochore–microtubule coupling. Alternatively, Ska Complex might affect kinetochore coupling indirectly, through recruitment of phosphoregulatory factors. Using optical tweezers, we show that the Ska Complex itself bears load on microtubule tips, strengthens Ndc80 Complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Cross-linking mass spectrometry suggests the Ska Complex directly binds Ndc80 Complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 Complex subunit. Deletion of the Ska Complex microtubule-binding domain or the Ska3 C terminus prevents Ska Complex from strengthening Ndc80 Complex-based attachments. Together, our results indicate that the Ska Complex can directly strengthen the kinetochore–microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 Complex and the microtubule.
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The human Ska Complex and Ndc80 Complex interact to form a load-bearing assembly that strengthens kinetochore-microtubule attachments
2018Co-Authors: Luke A Helgeson, Alex Zelter, Michael Riffle, Charles L. Asbury, Michael J Maccoss, Trisha N. DavisAbstract:Accurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska Complex and Ndc80 Complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore-microtubule attachments in vivo. While purified Ska Complex tracks with disassembling microtubule tips, it remains unclear whether the Ska Complex-microtubule interaction is sufficiently strong to make a significant contribution to kinetochore-microtubule coupling. Alternatively, Ska Complex might affect kinetochore coupling indirectly, through recruitment of phospho-regulatory factors. Using optical tweezers, we show that the Ska Complex itself bears load on microtubule tips, strengthens Ndc80 Complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Crosslinking mass spectrometry suggests the Ska Complex directly binds Ndc80 Complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 Complex subunit. Deletion of the Ska Complex microtubule-binding domain or the Ska3 C-terminus prevents Ska Complex from strengthening Ndc80 Complex-based attachments. Together our results indicate that the Ska Complex can directly strengthen the kinetochore microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 Complex and the microtubule.
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the Ndc80 Complex bridges two dam1 Complex rings
eLife, 2017Co-Authors: Jae Ook Kim, Alex Zelter, Michael Riffle, Neil T Umbreit, Charles L. Asbury, Michael J Maccoss, Athena Bollozos, Richard J Johnson, Trisha N. DavisAbstract:Strong kinetochore-microtubule attachments are essential for faithful segregation of sister chromatids during mitosis. The Dam1 and Ndc80 Complexes are the main microtubule binding components of the Saccharomyces cerevisiae kinetochore. Cooperation between these two Complexes enhances kinetochore-microtubule coupling and is regulated by Aurora B kinase. We show that the Ndc80 Complex can simultaneously bind and bridge across two Dam1 Complex rings through a tripartite interaction, each component of which is regulated by Aurora B kinase. Mutations in any one of the Ndc80p interaction regions abrogates the Ndc80 Complex's ability to bind two Dam1 rings in vitro, and results in kinetochore biorientation and microtubule attachment defects in vivo. We also show that an extra-long Ndc80 Complex, engineered to space the two Dam1 rings further apart, does not support growth. Taken together, our work suggests that each kinetochore in vivo contains two Dam1 rings and that proper spacing between the rings is vital.
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regulation of outer kinetochore Ndc80 Complex based microtubule attachments by the central kinetochore mis12 mind Complex
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Emily M Kudalkar, Alex Zelter, Michael Riffle, Richard S Johnson, Neil T Umbreit, Charles L. Asbury, Michael J Maccoss, Daniel R Gestaut, Emily Anne Scarborough, Trisha N. DavisAbstract:Multiple protein subComplexes of the kinetochore cooperate as a cohesive molecular unit that forms load-bearing microtubule attachments that drive mitotic chromosome movements. There is intriguing evidence suggesting that central kinetochore components influence kinetochore–microtubule attachment, but the mechanism remains unclear. Here, we find that the conserved Mis12/MIND (Mtw1, Nsl1, Nnf1, Dsn1) and Ndc80 (Ndc80, Nuf2, Spc24, Spc25) Complexes are connected by an extensive network of contacts, each essential for viability in cells, and collectively able to withstand substantial tensile load. Using a single-molecule approach, we demonstrate that an individual MIND Complex enhances the microtubule-binding affinity of a single Ndc80 Complex by fourfold. MIND itself does not bind microtubules. Instead, MIND binds Ndc80 Complex far from the microtubule-binding domain and confers increased microtubule interaction of the Complex. In addition, MIND activation is redundant with the effects of a mutation in Ndc80 that might alter its ability to adopt a folded conformation. Together, our results suggest a previously unidentified mechanism for regulating microtubule binding of an outer kinetochore component by a central kinetochore Complex.
Iain M. Cheeseman - One of the best experts on this subject based on the ideXlab platform.
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astrin skap Complex reconstitution reveals its kinetochore interaction with microtubule bound Ndc80
eLife, 2017Co-Authors: David M Kern, Elizabeth M Wilsonkubalek, Julie K Monda, Iain M. CheesemanAbstract: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.
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astrin skap Complex reconstitution reveals its kinetochore interaction with microtubule bound Ndc80
bioRxiv, 2017Co-Authors: David M Kern, Elizabeth M Wilsonkubalek, Iain M. CheesemanAbstract: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, 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.
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distinct organization and regulation of the outer kinetochore kmn network downstream of cenp c and cenp t
Current Biology, 2015Co-Authors: Florencia Rago, Karen E Gascoigne, Iain M. CheesemanAbstract:Summary The kinetochore provides a vital connection between chromosomes and spindle microtubules [1, 2]. Defining the molecular architecture of the core kinetochore components is critical for understanding the mechanisms by which the kinetochore directs chromosome segregation. The KNL1/Mis12 Complex/Ndc80 Complex (KMN) network acts as the primary microtubule-binding interface at kinetochores [3] and provides a platform to recruit regulatory proteins [4]. Recent work found that the inner kinetochore components CENP-C and CENP-T act in parallel to recruit the KMN network to kinetochores [5–8]. However, due to the presence of these dual pathways, it has not been possible to distinguish differences in the nature of kinetochore assembly downstream of CENP-C or CENP-T. Here, we separated these pathways by targeting CENP-C and CENP-T independently to an ectopic chromosomal locus in human cells. Our work reveals that the organization of the KMN network components downstream of CENP-C and CENP-T is distinct. CENP-C recruits the Ndc80 Complex through its interactions with KNL1 and the Mis12 Complex. In contrast, CENP-T directly interacts with Ndc80, which in turn promotes KNL1/Mis12 Complex recruitment through a separate region on CENP-T, resulting in functional relationships for KMN network localization that are inverted relative to the CENP-C pathway. We also find that distinct regulatory paradigms control the assembly of these pathways, with Aurora B kinase promoting KMN network recruitment to CENP-C and cyclin-dependent kinase (CDK) regulating KMN network recruitment to CENP-T. This work reveals unexpected Complexity for the architecture and regulation of the core components of the kinetochore-microtubule interface.
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resonance assignments of the microtubule binding domain of the c elegans spindle and kinetochore associated protein 1
Biomolecular Nmr Assignments, 2014Co-Authors: Jens C Schmidt, Andras Boeszoermenyi, Monika Oberer, Iain M. Cheeseman, Gerhard Wagner, Haribabu ArthanariAbstract:During mitosis, kinetochores coordinate the attachment of centromeric DNA to the dynamic plus ends of microtubules, which is hypothesized to pull sister chromatids toward opposing poles of the mitotic spindle. The outer kinetochore Ndc80 Complex acts synergistically with the Ska (spindle and kinetochore-associated) Complex to harness the energy of depolymerizing microtubules and power chromosome movement. The Ska Complex is a hexamer consisting of two copies of the proteins Ska1, Ska2 and Ska3, respectively. The C-terminal domain of the spindle and kinetochore-associated protein 1 (Ska1) is the microtubule-binding domain of the Ska Complex. We solved the solution structure of the C. elegans microtubule-binding domain (MTBD) of the protein Ska1 using NMR spectroscopy. Here, we report the resonance assignments of the MTBD of C. elegans Ska1.
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cenp t provides a structural platform for outer kinetochore assembly
The EMBO Journal, 2013Co-Authors: Tatsuya Nishino, Tetsuya Hori, Iain M. Cheeseman, Florencia Rago, Kentaro Tomii, Tatsuo FukagawaAbstract:The kinetochore forms a dynamic interface with microtubules from the mitotic spindle during mitosis. The Ndc80 Complex acts as the key microtubule-binding Complex at kinetochores. However, it is unclear how the Ndc80 Complex associates with the inner kinetochore proteins that assemble upon centromeric chromatin. Here, based on a high-resolution structural analysis, we demonstrate that the N-terminal region of vertebrate CENP-T interacts with the ‘RWD' domain in the Spc24/25 portion of the Ndc80 Complex. Phosphorylation of CENP-T strengthens a cryptic hydrophobic interaction between CENP-T and Spc25 resulting in a phospho-regulated interaction that occurs without direct recognition of the phosphorylated residue. The Ndc80 Complex interacts with both CENP-T and the Mis12 Complex, but we find that these interactions are mutually exclusive, supporting a model in which two distinct pathways target the Ndc80 Complex to kinetochores. Our results provide a model for how the multiple protein Complexes at kinetochores associate in a phospho-regulated manner.
Eva Nogales - One of the best experts on this subject based on the ideXlab platform.
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molecular requirements for the formation of a kinetochore microtubule interface by dam1 and Ndc80 Complexes
Journal of Cell Biology, 2013Co-Authors: Fabienne Lampert, Christine Mieck, Gregory M Alushin, Eva Nogales, Stefan WestermannAbstract:Kinetochores are large protein Complexes that link sister chromatids to the spindle and transduce microtubule dynamics into chromosome movement. In budding yeast, the kinetochore-microtubule interface is formed by the plus end-associated Dam1 Complex and the kinetochore-resident Ndc80 Complex, but how they work in combination and whether a physical association between them is critical for chromosome segregation is poorly understood. Here, we define structural elements required for the Ndc80-Dam1 interaction and probe their function in vivo. A novel Ndc80 allele, selectively impaired in Dam1 binding, displayed growth and chromosome segregation defects. Its combination with an N-terminal truncation resulted in lethality, demonstrating essential but partially redundant roles for the Ndc80 N-tail and Ndc80-Dam1 interface. In contrast, mutations in the calponin homology domain of Ndc80 abrogated kinetochore function and were not compensated by the presence of Dam1. Our experiments shed light on how microtubule couplers cooperate and impose important constraints on structural models for outer kinetochore assembly.
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multimodal microtubule binding by the Ndc80 kinetochore Complex
Nature Structural & Molecular Biology, 2012Co-Authors: Gregory M Alushin, Eva Nogales, Todd P Stukenberg, John G Tooley, Vivek Musinipally, Daniel R MatsonAbstract:The Ndc80 Complex is a key site of kinetochore-microtubule attachment during cell division. The human Complex engages microtubules with a globular 'head' formed by tandem calponin-homology domains and an 80-amino-acid unstructured 'tail' that contains sites of phosphoregulation by the Aurora B kinase. Using biochemical, cell biological and electron microscopy analyses, we dissected the roles of the tail in binding of microtubules and mediation of cooperative interactions between Ndc80 Complexes. Two segments of the tail that contain Aurora B phosphorylation sites become ordered at interfaces; one with tubulin and the second with an adjacent Ndc80 head on the microtubule surface, forming interactions that are disrupted by phosphorylation. We propose a model in which Ndc80's interaction with either growing or shrinking microtubule ends can be tuned by the phosphorylation state of its tail.
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the Ndc80 kinetochore Complex forms oligomeric arrays along microtubules
Nature, 2010Co-Authors: Gregory M Alushin, Eva Nogales, Andrea Musacchio, Sebastiano Pasqualato, Vincent H Ramey, David A Ball, Nikolaus GrigorieffAbstract:The Ndc80 Complex is a key site of regulated kinetochore-microtubule attachment (a process required for cell division), but the molecular mechanism underlying its function remains unknown. Here we present a subnanometre-resolution cryo-electron microscopy reconstruction of the human Ndc80 Complex bound to microtubules, sufficient for precise docking of crystal structures of the component proteins. We find that the Ndc80 Complex binds the microtubule with a tubulin monomer repeat, recognizing α- and β-tubulin at both intra- and inter-tubulin dimer interfaces in a manner that is sensitive to tubulin conformation. Furthermore, Ndc80 Complexes self-associate along protofilaments through interactions mediated by the amino-terminal tail of the Ndc80 protein, which is the site of phospho-regulation by Aurora B kinase. The Complex's mode of interaction with the microtubule and its oligomerization suggest a mechanism by which Aurora B could regulate the stability of load-bearing kinetochore-microtubule attachments.
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architecture and flexibility of the yeast Ndc80 kinetochore Complex
Journal of Molecular Biology, 2008Co-Authors: Hongwei Wang, Eva Nogales, Stefan Westermann, Claudio Ciferri, Sydney Long, David G Drubin, Georjana BarnesAbstract:Kinetochores mediate microtubule-chromosome attachment and ensure accurate segregation of sister chromatids. The highly conserved Ndc80 kinetochore Complex makes direct contacts with the microtubule and is essential for spindle checkpoint signaling. It contains a long coiled-coil region with globular domains at each end involved in kinetochore localization and microtubule binding, respectively. We have directly visualized the architecture of the yeast Ndc80 Complex and found a dramatic kink within the 560-A coiled-coil rod located about 160 A from the larger globular head. Comparison of our electron microscopy images to the structure of the human Ndc80 Complex allowed us to position the kink proximal to the microtubule-binding end and to define the conformational range of the Complex. The position of the kink coincides with a coiled-coil breaking region conserved across eukaryotes. We hypothesize that the kink in Ndc80 is essential for correct kinetochore geometry and could be part of a tension-sensing mechanism at the kinetochore.
Stephen C. Harrison - One of the best experts on this subject based on the ideXlab platform.
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structural basis of stu2 recruitment to yeast kinetochores
eLife, 2021Co-Authors: Jacob A Zahm, Stephen C. Harrison, Michael G Stewart, Joseph S Carrier, Matthew P MillerAbstract:Chromosome segregation during cell division requires engagement of kinetochores of sister chromatids with microtubules emanating from opposite poles. As the corresponding microtubules shorten, these 'bioriented' sister kinetochores experience tension-dependent stabilization of microtubule attachments. The yeast XMAP215 family member and microtubule polymerase, Stu2, associates with kinetochores and contributes to tension-dependent stabilization in vitro. We show here that a C-terminal segment of Stu2 binds the four-way junction of the Ndc80 Complex (Ndc80c) and that residues conserved both in yeast Stu2 orthologs and in their metazoan counterparts make specific contacts with Ndc80 and Spc24. Mutations that perturb this interaction prevent association of Stu2 with kinetochores, impair cell viability, produce biorientation defects, and delay cell cycle progression. Ectopic tethering of the mutant Stu2 species to the Ndc80c junction restores wild-type function in vivo. These findings show that the role of Stu2 in tension-sensing depends on its association with kinetochores by binding with Ndc80c.
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structure of the dash dam1 Complex shows its role at the yeast kinetochore microtubule interface
Science, 2018Co-Authors: Simon Jenni, Stephen C. HarrisonAbstract:Kinetochores connect mitotic-spindle microtubules with chromosomes, allowing microtubule depolymerization to pull chromosomes apart during anaphase while resisting detachment as the microtubule shortens. The heterodecameric DASH/Dam1 Complex (DASH/Dam1c), an essential component of yeast kinetochores, assembles into a microtubule-encircling ring. The ring associates with rodlike Ndc80 Complexes to organize the kinetochore-microtubule interface. We report the cryo–electron microscopy structure (at ~4.5-angstrom resolution) of a DASH/Dam1c ring and a molecular model of its ordered components, validated by evolutionary direct-coupling analysis. Integrating this structure with that of the Ndc80 Complex and with published interaction data yields a molecular picture of kinetochore-microtubule attachment, including how flexible, C-terminal extensions of DASH/Dam1c subunits project and contact widely separated sites on the Ndc80 Complex rod and how phosphorylation at previously identified sites might regulate kinetochore assembly.
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conserved tetramer junction in the kinetochore Ndc80 Complex
Cell Reports, 2016Co-Authors: Roberto Valverde, Jessica R Ingram, Stephen C. HarrisonAbstract:Summary The heterotetrameric Ndc80 Complex establishes connectivity along the principal longitudinal axis of a kinetochore. Its two heterodimeric subComplexes, each with a globular end and a coiled-coil shaft, connect end-to-end to create a ∼600 A long rod spanning the gap from centromere-proximal structures to spindle microtubules. Neither subComplex has a known function on its own, but the heterotetrameric organization and the characteristics of the junction are conserved from yeast to man. We have determined crystal structures of two shortened ("dwarf") Ndc80 Complexes that contain the full tetramer junction and both globular ends. The junction connects two α-helical coiled coils through regions of four-chain and three-chain overlap. The Complexity of its structure depends on interactions among conserved amino-acid residues, suggesting a binding site for additional cellular factor(s) not yet identified.
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structure of the mind Complex defines a regulatory focus for yeast kinetochore assembly
Cell, 2016Co-Authors: Yoana N Dimitrova, Simon Jenni, Stephen C. Harrison, Roberto Valverde, Yadana KhinAbstract:Kinetochores connect centromeric nucleosomes with mitotic-spindle microtubules through conserved, cross-interacting protein subassemblies. In budding yeast, the heterotetrameric MIND Complex (Mtw1, Nnf1, Nsl1, Dsn1), ortholog of the metazoan Mis12 Complex, joins the centromere-proximal components, Mif2 and COMA, with the principal microtubule-binding component, the Ndc80 Complex (Ndc80C). We report the crystal structure of Kluyveromyces lactis MIND and examine its partner interactions, to understand the connection from a centromeric nucleosome to a much larger microtubule. MIND resembles an elongated, asymmetric Y; two globular heads project from a coiled-coil shaft. An N-terminal extension of Dsn1 from one head regulates interactions of the other head, blocking binding of Mif2 and COMA. Dsn1 phosphorylation by Ipl1/Aurora B relieves this autoinhibition, enabling MIND to join an assembling kinetochore. A C-terminal extension of Dsn1 recruits Ndc80C to the opposite end of the shaft. The structure and properties of MIND show how it integrates phospho-regulatory inputs for kinetochore assembly and disassembly.
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the Ndc80 hec1 Complex is a contact point for kinetochore microtubule attachment
Nature Structural & Molecular Biology, 2007Co-Authors: Ronnie Wei, Jawdat Albassam, Stephen C. HarrisonAbstract:Kinetochores are multicomponent assemblies that connect chromosomal centromeres to mitotic-spindle microtubules. The Ndc80 Complex is an essential core element of kinetochores, conserved from yeast to humans. It is a rod-like assembly of four proteins- Ndc80p (HEC1 in humans), Nuf2p, Spc24p and Spc25p. We describe here the crystal structure of the most conserved region of HEC1, which lies at one end of the rod and near the N terminus of the polypeptide chain. It folds into a calponin-homology domain, resembling the microtubule-binding domain of the plus-end-associated protein EB1. We show that an Ndc80p-Nuf2p heterodimer binds microtubules in vitro. The less conserved, N-terminal segment of Ndc80p contributes to the interaction and may be a crucial regulatory element. We propose that the Ndc80 Complex forms a direct link between kinetochore core components and spindle microtubules.
Charles L. Asbury - One of the best experts on this subject based on the ideXlab platform.
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tight bending of the Ndc80 Complex provides intrinsic regulation of its binding to microtubules
eLife, 2019Co-Authors: Emily Anne Scarborough, Trisha N. Davis, Charles L. AsburyAbstract:Regulation of the outer kinetochore Complex Ndc80 is essential to ensure correct kinetochore-microtubule attachments during mitosis. Here, we present a novel mechanism of regulation that is intrinsic to its structure; tight bending of the Ndc80 Complex inhibits its microtubule binding. Using single molecule Forster resonance energy transfer (FRET), we show that the Saccharomyces cerevisiae Ndc80 Complex can fluctuate between straight and bent forms, and that binding of the Complex to microtubules selects for straightened forms. The loop region of the Complex enables its bent conformation, as deletion of the loop promotes straightening. In addition, the kinetochore Complex MIND enhances microtubule binding by opposing the tightly bent, auto-inhibited conformation of the Ndc80 Complex. We suggest that prior to its assembly at the kinetochore, the Ndc80 Complex interchanges between bent (auto-inhibited) and open conformations. Once assembled, its association with MIND stabilizes the Ndc80 Complex in a straightened form for higher affinity microtubule binding.
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the kinetoplastid kinetochore protein kkt4 is an unconventional microtubule tip coupling protein
Journal of Cell Biology, 2018Co-Authors: Aida Llauro, Hanako Hayashi, Megan E Bailey, Alexander C Wilson, Patryk Ludzia, Charles L. Asbury, Bungo AkiyoshiAbstract:: Kinetochores are multiprotein machines that drive chromosome segregation by maintaining persistent, load-bearing linkages between chromosomes and dynamic microtubule tips. Kinetochores in commonly studied eukaryotes bind microtubules through widely conserved components like the Ndc80 Complex. However, in evolutionarily divergent kinetoplastid species such as Trypanosoma brucei, which causes sleeping sickness, the kinetochores assemble from a unique set of proteins lacking homology to any known microtubule-binding domains. Here, we show that the T. brucei kinetochore protein KKT4 binds directly to microtubules and maintains load-bearing attachments to both growing and shortening microtubule tips. The protein localizes both to kinetochores and to spindle microtubules in vivo, and its depletion causes defects in chromosome segregation. We define a microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Thus, despite its lack of significant similarity to other known microtubule-binding proteins, KKT4 has key functions required for driving chromosome segregation. We propose that it represents a primary element of the kinetochore-microtubule interface in kinetoplastids.
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human ska Complex and Ndc80 Complex interact to form a load bearing assembly that strengthens kinetochore microtubule attachments
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Luke A Helgeson, Alex Zelter, Michael Riffle, Charles L. Asbury, Michael J Maccoss, Trisha N. DavisAbstract:Accurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska Complex and Ndc80 Complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore–microtubule attachments in vivo. While purified Ska Complex tracks with disassembling microtubule tips, it remains unclear whether the Ska Complex–microtubule interaction is sufficiently strong to make a significant contribution to kinetochore–microtubule coupling. Alternatively, Ska Complex might affect kinetochore coupling indirectly, through recruitment of phosphoregulatory factors. Using optical tweezers, we show that the Ska Complex itself bears load on microtubule tips, strengthens Ndc80 Complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Cross-linking mass spectrometry suggests the Ska Complex directly binds Ndc80 Complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 Complex subunit. Deletion of the Ska Complex microtubule-binding domain or the Ska3 C terminus prevents Ska Complex from strengthening Ndc80 Complex-based attachments. Together, our results indicate that the Ska Complex can directly strengthen the kinetochore–microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 Complex and the microtubule.
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The human Ska Complex and Ndc80 Complex interact to form a load-bearing assembly that strengthens kinetochore-microtubule attachments
2018Co-Authors: Luke A Helgeson, Alex Zelter, Michael Riffle, Charles L. Asbury, Michael J Maccoss, Trisha N. DavisAbstract:Accurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska Complex and Ndc80 Complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore-microtubule attachments in vivo. While purified Ska Complex tracks with disassembling microtubule tips, it remains unclear whether the Ska Complex-microtubule interaction is sufficiently strong to make a significant contribution to kinetochore-microtubule coupling. Alternatively, Ska Complex might affect kinetochore coupling indirectly, through recruitment of phospho-regulatory factors. Using optical tweezers, we show that the Ska Complex itself bears load on microtubule tips, strengthens Ndc80 Complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Crosslinking mass spectrometry suggests the Ska Complex directly binds Ndc80 Complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 Complex subunit. Deletion of the Ska Complex microtubule-binding domain or the Ska3 C-terminus prevents Ska Complex from strengthening Ndc80 Complex-based attachments. Together our results indicate that the Ska Complex can directly strengthen the kinetochore microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 Complex and the microtubule.
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the Ndc80 Complex bridges two dam1 Complex rings
eLife, 2017Co-Authors: Jae Ook Kim, Alex Zelter, Michael Riffle, Neil T Umbreit, Charles L. Asbury, Michael J Maccoss, Athena Bollozos, Richard J Johnson, Trisha N. DavisAbstract:Strong kinetochore-microtubule attachments are essential for faithful segregation of sister chromatids during mitosis. The Dam1 and Ndc80 Complexes are the main microtubule binding components of the Saccharomyces cerevisiae kinetochore. Cooperation between these two Complexes enhances kinetochore-microtubule coupling and is regulated by Aurora B kinase. We show that the Ndc80 Complex can simultaneously bind and bridge across two Dam1 Complex rings through a tripartite interaction, each component of which is regulated by Aurora B kinase. Mutations in any one of the Ndc80p interaction regions abrogates the Ndc80 Complex's ability to bind two Dam1 rings in vitro, and results in kinetochore biorientation and microtubule attachment defects in vivo. We also show that an extra-long Ndc80 Complex, engineered to space the two Dam1 rings further apart, does not support growth. Taken together, our work suggests that each kinetochore in vivo contains two Dam1 rings and that proper spacing between the rings is vital.
