| 1997 |
HEC/NDC80 protein localizes to nuclei of interphase cells and redistributes to centromeres during M phase; microinjection of anti-HEC antibodies during interphase severely disrupts subsequent mitosis, causing disordered sister chromatid alignment/separation and formation of micronuclei, demonstrating a direct role in chromosome segregation. |
Antibody microinjection, immunofluorescence, cell biology |
Molecular and cellular biology |
Medium |
9315664
|
| 2002 |
Hec1 is phosphorylated on serine 165 by Nek2 kinase both in vitro and in vivo during G2/M. Nek2 binds Hec1 specifically during G2/M. S165A or S201A mutations in yeast Hec1 fail to rescue lethality from Hec1 deletion, while S165E/S201E phosphomimetics partially rescue but increase segregation errors, establishing that Nek2-mediated Hec1 phosphorylation is essential for faithful chromosome segregation. |
In vitro kinase assay, co-immunoprecipitation, yeast complementation/rescue genetics, site-directed mutagenesis |
The Journal of biological chemistry |
High |
12386167
|
| 2002 |
Hec1 is required for recruitment of Mps1 kinase and Mad1/Mad2 complexes to kinetochores; depletion of Hec1 impairs chromosome congression and causes persistent spindle checkpoint activation; simultaneous depletion of Hec1 and Mad2 causes catastrophic mitotic exit. |
RNAi depletion, immunofluorescence, genetic epistasis (double depletion) |
Science |
High |
12351790
|
| 2003 |
The Xenopus Ndc80/Nuf2 complex physically interacts in a 190-kD complex at the outer kinetochore. Immunodepletion of the complex from Xenopus extracts abolishes kinetochore recruitment of Rod, Zw10, Dynactin, Mad1, Mad2, Bub1, and Bub3, demonstrating that the Ndc80 complex is required for functional kinetochore assembly. Function-blocking antibodies also abolish spindle checkpoint signaling. |
Co-immunoprecipitation, immunodepletion from Xenopus extracts, antibody injection, immunofluorescence |
Genes & development |
High |
12514103
|
| 2003 |
hSPC25 (a novel NDC80 complex subunit identified by immunoaffinity) interacts with HEC1 throughout the cell cycle and localizes to kinetochores. RNAi depletion of hSPC25 causes aberrant mitosis, multipolar spindles, cell death, and failure of MAD1 and HEC1 to localize to kinetochores, placing SPC25 as essential for NDC80 complex kinetochore assembly. |
Immunoaffinity purification, RNAi, immunofluorescence |
The Journal of biological chemistry |
Medium |
14699129
|
| 2003 |
Nuf2-Hec1 complex localizes to centrosomes during G1/S phases and moves to centromeres in G2; the complex is stably associated with centromeres during mitosis (as shown by FRAP). Conditional loss of Nuf2 or Hec1 in chicken DT40 cells causes prometaphase arrest; Mad2 localization is abolished while CENP-A/-C/-H and BubR1 remain, placing the Ndc80 complex upstream of Mad2 kinetochore recruitment. |
GFP live imaging, FRAP, conditional knockout in DT40 cells, immunofluorescence |
Journal of cell science |
High |
12829748
|
| 2003 |
Depletion of Nuf2 or Hec1 by RNAi in HeLa cells reduces both proteins at kinetochores, and causes Mad1 and Mad2 to become depleted from kinetochores in a microtubule-dependent manner during prolonged prometaphase, which is reversible upon spindle depolymerization. Nuf2 and Hec1 function to prevent microtubule-dependent stripping of Mad1/Mad2 from kinetochores lacking stable kinetochore-microtubule attachments. |
RNAi, immunofluorescence, spindle depolymerization rescue experiments |
Current biology : CB |
Medium |
14654001
|
| 2004 |
Hec1 and Nuf2 localize throughout the outer plate (not corona) of vertebrate kinetochores. They are required for formation/maintenance of the outer plate structure itself and for normal kinetochore microtubule attachment, as shown by quantitative EM and fluorescence microscopy after RNAi depletion. |
RNAi, immunofluorescence, electron microscopy, live cell imaging |
Molecular biology of the cell |
High |
15548592
|
| 2005 |
The recombinant human Ndc80 complex (Hec1:Nuf2:Spc24:Spc25 in 1:1:1:1 stoichiometry) forms a ~570 Å elongated rod with two stable subcomplexes—Hec1-Nuf2 and Spc24-Spc25—each forming a parallel heterodimeric coiled coil. The subcomplexes tetramerize via coiled-coil interactions. The Spc24/Spc25 end faces the centromere and Ndc80/Nuf2 end faces microtubules. |
Recombinant reconstitution, rotary-shadowing EM, limited proteolysis, antibody labeling, hydrodynamic analysis |
Proceedings of the National Academy of Sciences of the United States of America |
High |
15809444
|
| 2005 |
The recombinant human Ndc80 complex has hydrodynamic properties identical to endogenous HeLa complex and shows normal kinetochore localization upon injection into HeLa cells. Hec1-Nuf2 and Spc24-Spc25 form independently stable subcomplexes stabilized by parallel heterodimeric coiled coils. |
Recombinant expression, biochemical reconstitution, hydrodynamic analysis, microinjection/immunofluorescence |
The Journal of biological chemistry |
High |
15961401
|
| 2006 |
The most conserved N-terminal region of Hec1 folds into a calponin-homology (CH) domain, similar to the microtubule-binding domain of EB1. The Ndc80-Nuf2 heterodimer binds microtubules in vitro; the N-terminal segment of Ndc80 contributes to this interaction. |
Crystal structure, in vitro microtubule-binding assay |
Nature structural & molecular biology |
High |
17195848
|
| 2006 |
Hec1 directly interacts with Zwint-1, which is required for subsequent kinetochore recruitment of ZW10. Depletion of Hec1 impairs recruitment of both Zwint-1 and ZW10; depletion of Zwint-1 abolishes ZW10 but not Hec1 kinetochore localization, defining a Hec1→Zwint-1→ZW10 sequential recruitment hierarchy essential for spindle checkpoint and chromosome segregation. |
Co-immunoprecipitation, RNAi, immunofluorescence, epistasis |
Oncogene |
Medium |
16732327
|
| 2006 |
Hec1 N-terminal domain regulates kinetochore microtubule plus-end dynamics and attachment stability. Anti-Hec1 N-terminal antibody microinjection suppresses microtubule detachment and polymerization/depolymerization at kinetochores. The Hec1 N-terminus is phosphorylated by Aurora B kinase in vitro; nonphosphorylatable N-terminal Hec1 mutants exhibit increased merotelic attachments, centromere hyperstretching, and chromosome segregation errors. |
Antibody microinjection, in vitro kinase assay, site-directed mutagenesis, live cell imaging, fluorescence microscopy |
Cell |
High |
17129782
|
| 2008 |
Crystal structure of an engineered 'bonsai' Ndc80 complex reveals a microtubule-binding interface formed by a pair of tightly interacting calponin-homology (CH) domains in a novel arrangement. Interaction with microtubules is cooperative and predominantly electrostatic, involving positive charges in CH domains and Ndc80 N-terminal tail, and negative charges in tubulin C-terminal tails; this interaction is regulated by Aurora B kinase. |
X-ray crystallography, in vitro microtubule-binding assays, mutagenesis |
Cell |
High |
18455984
|
| 2008 |
Yeast Ndc80 complex EM shows a dramatic kink within the coiled-coil rod ~160 Å from the microtubule-binding globular head, at a position conserved across eukaryotes, suggesting this flexibility may contribute to kinetochore tension sensing. |
Electron microscopy (negative stain), rotary shadowing |
Journal of molecular biology |
Medium |
18793650
|
| 2008 |
The 80 amino acid disordered N-terminal tail of Hec1 is required for stable kinetochore-microtubule attachments. Deletion of the tail or mutation of nine positively charged residues within it abolishes stable attachment. The CH domain (but not the tail) is required for mitotic checkpoint function. |
RNAi rescue (gene silence and re-expression), deletion and point mutagenesis, live cell imaging |
Current biology : CB |
High |
19026543
|
| 2009 |
Mps1 kinase interacts physically with the N-terminal domain of Ndc80 (residues 1-257) and phosphorylates it in vitro; Mps1 facilitates Ndc80 phosphorylation in vivo. Phosphorylation-deficient (14×Ala) mutants show compromised spindle assembly checkpoint signaling; phosphomimetic (14×Asp) mutants cause constitutive SAC activation with bipolar kinetochore-microtubule attachment, revealing that Mps1-mediated Ndc80 phosphorylation is important for SAC activation at kinetochores. |
Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis, cell biology/genetic rescue in yeast |
The EMBO journal |
High |
19300438
|
| 2009 |
Hec1 colocalizes with Hice1 (a centrosomal microtubule-binding protein) at spindle poles during mitosis. The C-terminal region of Hec1 directly binds Hice1 coiled-coil domain 1. Hec1 or Hice1 siRNA reduces centrosomal microtubule nucleation; anti-Hec1/Hice1 antibodies impair microtubule aster formation from purified mitotic centrosomes in vitro, demonstrating Hec1 contributes to centrosomal microtubule growth. |
Co-immunoprecipitation, siRNA, in vitro centrosome aster assay, immunofluorescence |
Molecular biology of the cell |
Medium |
19776357
|
| 2009 |
Drosophila Dgt6 (augmin subunit) coprecipitates with Ndc80 and Nuf2, and is required for kinetochore-driven k-fiber formation; RNAi of Ndc80/Hec1 reduced but did not abolish k-fiber regrowth. |
Co-immunoprecipitation, RNAi, microtubule regrowth assay |
Current biology : CB |
Medium |
19836241
|
| 2010 |
Cryo-EM reconstruction of human Ndc80 complex on microtubules at sub-nanometer resolution reveals the complex binds microtubules with a tubulin-monomer repeat, recognizing both α- and β-tubulin at intra- and inter-dimer interfaces. Ndc80 complexes self-associate along protofilaments via the amino-terminal tail of NDC80, which is the site of Aurora B phosphoregulation, suggesting oligomerization regulates load-bearing attachment stability. |
Cryo-electron microscopy, crystal structure docking, image reconstruction |
Nature |
High |
20944740
|
| 2010 |
The budding yeast Dam1 complex acts as a processivity factor for the Ndc80 complex, enhancing its ability to form load-bearing attachments to and track with dynamic microtubule tips in vitro. Phosphorylation of the Dam1 complex by yeast Aurora B kinase (Ipl1) abolishes the Ndc80-Dam1 interaction, providing a mechanism for Aurora B to reset aberrant kinetochore-microtubule attachments. |
In vitro reconstitution with purified proteins, single-molecule microtubule tracking, kinase assays |
The Journal of cell biology |
High |
20479468
|
| 2010 |
CENP-U is a novel interacting partner of Hec1 (identified by co-immunoprecipitation). CENP-U cooperates with Hec1 in microtubule binding in vitro. CENP-U is a substrate of Aurora B; phosphorylation of CENP-U reduces kinetochore-microtubule interaction contributing to Aurora B error-correction function. |
Co-immunoprecipitation, in vitro microtubule binding, kinase assay, shRNA knockdown |
The Journal of biological chemistry |
Medium |
21056971
|
| 2011 |
Multiple serine residues in the Hec1 N-terminus are phosphorylated by Aurora B in an Aurora-B-dependent manner during mitosis. Phosphorylation is high in early mitosis and decreases as chromosomes bi-orient. Once dephosphorylated, Hec1 is not highly rephosphorylated after loss of attachment/tension, suggesting Hec1 phosphorylation drives destabilization in early mitosis while dephosphorylation maintains stable attachments in late mitosis. |
Phospho-specific antibodies, kinase inhibitor experiments, phosphorylation site mutagenesis, immunofluorescence quantification |
Journal of cell science |
High |
21266467
|
| 2011 |
The CH domain of Hec1, the CH domain of Nuf2, and the Hec1 tail each contribute distinctly to kinetochore-microtubule attachment: Hec1 CH domain mutations cause the most severe attachment defects; Hec1 tail mutations cause intermediate defects; Nuf2 CH domain mutations generate stable attachments but fail to produce wild-type interkinetochore tension and delay anaphase entry. |
RNAi rescue with domain-specific mutants, live cell imaging, kinetochore-microtubule attachment assay |
Molecular biology of the cell |
High |
21270439
|
| 2011 |
Hec1 Ser165 is phosphorylated at kinetochores predominantly by Nek2, and the pS165 mark localizes to kinetochores of misaligned chromosomes. S165A mutant shows normal metaphase but accelerated metaphase-to-anaphase transition with defective Mad1/Mad2 kinetochore localization. S165E (phosphomimetic) causes defective chromosome alignment and severe mitotic arrest. PP1 phosphatase dephosphorylates pS165 during SAC silencing. |
Phospho-specific antibodies, RNAi rescue with point mutants, immunofluorescence, kinase/phosphatase inhibition |
Molecular biology of the cell |
High |
21832156
|
| 2011 |
The Ndc80 complex uses a tripartite microtubule-attachment mechanism in human cells: the positively charged Hec1 N-terminal tail, the Hec1 CH domain, and the Nuf2 CH domain each contribute independently to microtubule binding. Point mutations in the Hec1 CH domain abolish chromosome alignment and stable microtubule attachments without affecting kinetochore architecture or checkpoint protein recruitment. Cooperative binding in vitro is driven by positive charge on the tail. |
RNAi rescue with point mutants, in vitro microtubule binding, immunofluorescence |
Molecular biology of the cell |
High |
21325630
|
| 2012 |
Human Ndc80 complex directly stabilizes tips of disassembling microtubules and promotes rescue in vitro in a reconstituted system. Aurora B phosphomimetic mutations of the Ndc80 N-terminal domain are defective at promoting microtubule rescue even when robustly coupled to disassembling tips, identifying a role for Aurora B in controlling microtubule dynamics through Ndc80 phosphorylation beyond regulating attachment stability. |
In vitro reconstitution, single-molecule tracking, microtubule dynamics assay, phosphomimetic mutagenesis |
Proceedings of the National Academy of Sciences of the United States of America |
High |
22908300
|
| 2012 |
Cryo-EM and biochemical analyses reveal that the Ndc80 tail has two segments that become ordered at the microtubule surface: one contacts tubulin, the second contacts an adjacent Ndc80 head. Both interfaces are disrupted by Aurora B phosphorylation, revealing multimodal microtubule binding through cooperative interactions. |
Cryo-EM, biochemical MT binding assays, mutagenesis |
Nature structural & molecular biology |
High |
23085714
|
| 2012 |
Structural elements of the Ndc80 complex required for Ndc80-Dam1 interaction were identified. A new ndc80 allele selectively impaired in Dam1 binding shows growth and chromosome segregation defects in vivo; combining with N-terminal truncation is lethal, demonstrating partially redundant essential roles. CH domain mutations in Ndc80 abrogate kinetochore function and cannot be rescued by Dam1. |
In vitro binding assay, mutagenesis, yeast genetics/epistasis, chromosome segregation assay |
The Journal of cell biology |
High |
23277429
|
| 2013 |
Hec1 interacts with Mps1 and specifies its kinetochore localization via its CH domain and N-terminal 80 amino acids. Aurora B phosphorylation of Hec1 weakens microtubule interaction but promotes Hec1 binding to Mps1. Phosphomimetic Hec1 induces SAC hyperactivation, defining an Aurora B-Hec1-Mps1 signaling axis that couples kinetochore-microtubule attachment status to SAC activation. |
Co-immunoprecipitation, in vitro binding, kinase assay, mutagenesis, immunofluorescence |
The Journal of biological chemistry |
Medium |
24187132
|
| 2013 |
In mouse oocytes, Hec1 depletion compromises the G2-M transition due to impaired Cdk1 activation. Hec1 protects cyclin B2 from APC(Cdh1)-mediated destruction; this protection is important through early prometaphase spindle assembly. By late M phase, APC(Cdc20) triggers cyclin B2 destruction despite Hec1 stability, revealing a non-kinetochore role for Hec1 in M phase entry. |
siRNA depletion in mouse oocytes, immunoblotting, live imaging, epistasis with APC components |
Developmental cell |
Medium |
23541922
|
| 2013 |
The internal loop of fission yeast Ndc80 binds Alp7/TACC-Alp14/TOG complex. An ndc80 internal-loop point mutant (ndc80-NH12) assembles the Ndc80 complex normally but has impaired Alp7/TACC-Alp14/TOG kinetochore localization and end-on attachment. Forced targeting of Alp7-Alp14 to the kinetochore rescues ndc80-NH12 phenotypes. The loop also binds Dis1/TOG independently. |
Yeast genetics, co-immunoprecipitation, mutagenesis, forced-targeting rescue experiments, chromosome segregation assays |
Molecular biology of the cell |
High |
23427262
|
| 2015 |
Multiple Aurora B phosphorylation events on the Hec1 unstructured tail are additively integrated (not cooperatively) to gradually tune NDC80-microtubule binding affinity in vitro and in silico. Conformational plasticity of the tail enables it to function as a phosphorylation-controlled rheostat. Cooperativity between NDC80 complexes is weak and unaffected by phosphorylation. |
In vitro quantitative microtubule binding assays, molecular dynamics simulations, phosphomimetic mutagenesis |
Molecular biology of the cell |
High |
25808492
|
| 2015 |
CENP-T and CENP-C independently recruit Ndc80 complexes to human kinetochores. Quantification reveals ~244 Ndc80 complexes per kinetochore (~14 per kinetochore microtubule); ~151 are part of the KMN network. Each CENP-T molecule recruits ~2 Ndc80 complexes; ~40% of CENP-C recruits only a KMN network. |
Fluorescence copy number quantification, selective protein depletion, kinetochore reconstitution experiments |
Nature communications |
High |
26345214
|
| 2015 |
The central kinetochore Mis12/MIND complex binds the Ndc80 complex through an extensive network of contacts and enhances the microtubule-binding affinity of a single Ndc80 complex by fourfold in single-molecule assays. MIND itself does not bind microtubules but acts from a distal position; activation is redundant with a mutation that might prevent Ndc80 from adopting a folded conformation. |
Single-molecule reconstitution, microtubule-binding assay, mutagenesis, yeast viability assays |
Proceedings of the National Academy of Sciences of the United States of America |
High |
26430240
|
| 2017 |
Aurora A kinase phosphorylates Hec1 at serine 69 (a previously uncharacterized site in the Hec1 tail) and regulates kinetochore-microtubule dynamics of metaphase chromosomes. Aurora A associates with INCENP during mitosis, and INCENP can drive accumulation of Aurora A to the centromere region, revealing both Aurora A and B contribute to Hec1-mediated kinetochore-microtubule attachment dynamics. |
In vitro kinase assay, phospho-specific antibody, phosphomimetic/non-phosphorylatable mutagenesis rescue, co-immunoprecipitation, live cell imaging |
The Journal of cell biology |
High |
29187526
|
| 2017 |
Cdk1 phosphorylates Ska3 to promote its direct binding to the Ndc80 complex during mitosis, and this interaction is required for kinetochore localization of the Ska complex. Ska3 phosphomutants support chromosome alignment but delay anaphase onset. |
In vitro kinase assay, co-immunoprecipitation, cell biology (mutagenesis rescue), immunofluorescence |
Current biology : CB |
High |
28479321
|
| 2017 |
Electron microscopic tomography reveals that Ndc80 recruits the Ska complex so that the V-shaped Ska dimer interacts along microtubule protofilaments. A mutant Ndc80 tail that is deficient in Ska recruitment also fails to cluster along protofilaments, while retaining normal microtubule-binding affinity, identifying the Ndc80 tail as the site of Ska recruitment and suggesting clustering is required for Ska orientation. |
Electron microscopic tomography, in vitro binding assays, mutagenesis |
Developmental cell |
High |
28535377
|
| 2017 |
Removing the Ndc80 tail in C. elegans embryo has no effect on kinetochore-microtubule attachments in vivo, despite compromising in vitro binding. However, preventing Aurora B phosphorylation of the tail causes prematurely stable attachments requiring the Ska complex, indicating that Ndc80-tail dephosphorylation promotes attachment stabilization via the Ska complex rather than solely through direct electrostatic interactions. |
Auxin-inducible degron/gene editing in C. elegans, live imaging, epistasis with Ska complex |
Developmental cell |
High |
28535376
|
| 2017 |
Hec1 tail phosphorylation tunes friction (coupling) along polymerizing microtubules but does not compromise the kinetochore's ability to grip depolymerizing microtubules. This differential regulation was determined by laser ablation to decouple sister kinetochores and probe Hec1 function at each microtubule end type separately. |
Laser ablation, live cell imaging, phosphomimetic/non-phosphorylatable Hec1 mutants |
Current biology : CB |
Medium |
28552353
|
| 2017 |
The Ndc80 complex bridges two Dam1 complex rings simultaneously through a tripartite interaction, each component regulated by Aurora B kinase. Mutations in any one Ndc80-Dam1 interaction region abolish dual-ring bridging in vitro and cause biorientation/microtubule attachment defects in vivo. Proper spacing between the two Dam1 rings is vital, as an extra-long Ndc80 complex does not support growth. |
In vitro binding assays, electron microscopy, yeast genetics, mutagenesis, chromosome segregation assay |
eLife |
High |
28191870
|
| 2018 |
Reconstitution of a 26-subunit human kinetochore shows that the N-terminal basic tail of the NDC80 complex binds microtubules and cooperates with CENP-Q (which also has a basic microtubule-binding tail) in microtubule binding; the NDC80 tail can functionally replace the CENP-Q tail, revealing unexpected functional similarities. |
Recombinant reconstitution of 26-subunit particle, microtubule-binding assay, mutagenesis, cell biology |
Molecular cell |
High |
30174292
|
| 2018 |
NDC80 binding to microtubules in living human cells is modulated in a chromosome-autonomous fashion over prometaphase/metaphase, predominantly regulated by centromere tension. This tension-dependency requires Hec1 N-terminal tail phosphorylation and proper Aurora B localization, as measured by FLIM-FRET in living cells. |
FLIM-FRET in live cells, phosphomimetic mutagenesis, Aurora B inhibition |
eLife |
High |
30044223
|
| 2018 |
TIP60 acetyltransferase acetylates Hec1 at Lys-53 and Lys-59. This acetylation is reversed by SIRT1 deacetylase. TIP60-mediated acetylation weakens Aurora B-mediated phosphorylation of Hec1 at Ser-55 and Ser-62, regulating NDC80-microtubule dynamics and chromosome segregation fidelity, defining a TIP60/Aurora B/SIRT1 signaling hierarchy for Hec1 regulation. |
Pulldown assays, site-directed mutagenesis, in vitro acetylation assay, immunofluorescence, cell biology |
The Journal of biological chemistry |
Medium |
30409912
|
| 2019 |
The Ska-Ndc80 interaction is phosphorylation-dependent and does not require microtubules, applied force, the Ndc80-loop, or the Ndc80-tail. The Ndc80-tail is essential for microtubule end-tracking (not Ska recruitment). Both the Ndc80-tail and Ndc80-bound Ska stabilize microtubule ends in a stalled conformation. |
In vitro binding assays, microtubule end-tracking reconstitution, force-coupling experiments, mutagenesis |
eLife |
High |
31804178
|
| 2020 |
Aurora B-dependent phosphorylation of Ndc80 N-terminus (including a 27-residue sequence and phosphorylation sites) is both necessary and sufficient for APCAma1-mediated, proteasome-dependent Ndc80 degradation during meiotic prophase in budding yeast, in a microtubule-independent manner. Defects in this regulated turnover predispose meiotic cells to chromosome mis-segregation. |
Yeast genetics, mutagenesis (deletion/phosphomimetic), proteasome inhibition, cell biology |
Genes & development |
High |
31919192
|
| 2020 |
The Hec1 tail domain is dispensable for kinetochore-microtubule attachment formation and Ska complex recruitment in human cells. Instead, the Ska complex is recruited via the coiled-coil region of Ndc80. Hec1 tail phosphorylation regulates attachment stability independently of the Ska complex. The Hec1 tail is required for force generation at kinetochores. |
RNAi rescue with tail-deletion mutants, in vitro binding assays, immunofluorescence, laser-trapping force measurements |
Molecular biology of the cell |
High |
32401635
|
| 2021 |
Kinetochore-bound Mps1 (yeast) phosphorylates Ndc80 to weaken kinetochore-microtubule attachments. This phosphorylation contributes to error correction independently of Aurora B; phospho-deficient Ndc80 mutants show genetic interactions and segregation defects combined with other error correction pathway mutants. Mps1 phosphorylation of Ndc80 is stimulated at kinetochores lacking tension. |
In vitro kinetochore reconstitution, kinase assay, mutagenesis, genetic epistasis in yeast |
The Journal of cell biology |
High |
34647959
|
| 2021 |
Aurora A phosphorylates Hec1 at Ser-55 during metaphase on the spindle, dependent on chromosome oscillation amplitude. Hec1-S55 and S69 phosphorylation by Aurora A is required for efficient chromosome oscillation and for eliminating erroneous kinetochore-microtubule attachments, establishing a positive-feedback relationship between chromosome oscillation and Aurora A-dependent error correction. |
Phospho-specific antibodies, Aurora A inhibition, mutagenesis rescue, live-cell imaging of oscillation amplitude |
The Journal of cell biology |
Medium |
33988677
|
| 2022 |
Two copies of Ndc80 complex on CENP-T (one via Mis12C, one via direct binding) are needed for proper kinetochore-microtubule interactions in chicken DT40 cells. Artificial direct tethering of two Ndc80 copies to CENP-T can substitute for the Mis12C-Ndc80 interaction, demonstrating it is the number and geometry of Ndc80 copies—not Mis12C per se—that is critical. |
Genetic engineering (DT40 conditional mutants), artificial tethering, chromosome segregation assay |
Nature communications |
High |
35165266
|
| 2023 |
Cryo-EM structure of budding yeast outer kinetochore Ndc80 and Dam1 ring complexes assembled onto microtubules reveals multiple Ndc80-Dam1 ring interfaces and a 'staple' within Dam1 that aids ring assembly. Aurora B error-correction phosphorylation sites are located at Ndc80-Dam1 ring interfaces and the Dam1 staple, mechanistically explaining how kinetochore-microtubule attachments are destabilized for error correction. Force-rupture assays confirm that disruption of these interfaces impairs attachment. |
Cryo-EM structure determination, site-directed mutagenesis, force-rupture assays, yeast viability |
Science |
High |
38060647
|
| 2023 |
The Ndc80 loop folds into a rigid α-helical 'switchback' structure (by AlphaFold2 prediction validated by crystallography). The loop promotes direct Ndc80-Ndc80 interactions on microtubules; loop mutations impair these interactions, prevent force-resistant kinetochore-microtubule attachments, and cause prolonged mitotic arrest that cannot be rescued by phospho-deficient Hec1 tail mutations or by Ska complex recruitment. |
AlphaFold2 structure prediction, X-ray crystallography, in vitro microtubule binding, cell biology mutagenesis rescue |
The EMBO journal |
High |
37203876
|
| 2023 |
Crystal structures of the Ndc80 loop (as a stiff α-helical switchback) and of the Ndc80:Nuf2 globular head-Dam1 interaction surface were solved. Conserved stretches of the Dam1 C-terminus bind Ndc80c; phosphorylation of Dam1 Ser-257, Ser-265, and Ser-292 by Ipl1/Aurora B releases this contact, explaining how error correction phosphorylation destabilizes attachments. |
X-ray crystallography, AlphaFold2 modeling, in vitro binding/mutagenesis |
Open biology |
High |
36883282
|
| 2024 |
A conserved interaction domain in Nuf2's CH domain (two segments forming a binding site) recruits Mps1 to the yeast Ndc80 complex. This site also binds the Dam1 complex, suggesting Mps1 recruitment is subject to competitive regulation. Mutations disrupting this 'interaction hub' exhibit SAC dysfunction and severe chromosome segregation errors; restoring Mps1-Ndc80 complex association rescues these defects. |
Mutational analysis, co-immunoprecipitation, yeast genetics, chromosome segregation assays |
Current biology : CB |
Medium |
38776906
|