scholarly journals Bod1, a novel kinetochore protein required for chromosome biorientation

2007 ◽  
Vol 179 (2) ◽  
pp. 187-197 ◽  
Author(s):  
Iain M. Porter ◽  
Sarah E. McClelland ◽  
Guennadi A. Khoudoli ◽  
Christopher J. Hunter ◽  
Jens S. Andersen ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Hela Cells ◽  
Aurora B ◽  
Macromolecular Complex ◽  
Mitotic Spindles ◽  
Kinetochore Protein ◽  
Spindle Poles ◽  
Interfering Rna ◽  
Rna Depletion

We have combined the proteomic analysis of Xenopus laevis in vitro–assembled chromosomes with RNA interference and live cell imaging in HeLa cells to identify novel factors required for proper chromosome segregation. The first of these is Bod1, a protein conserved throughout metazoans that associates with a large macromolecular complex and localizes with kinetochores and spindle poles during mitosis. Small interfering RNA depletion of Bod1 in HeLa cells produces elongated mitotic spindles with severe biorientation defects. Bod1-depleted cells form syntelic attachments that can oscillate and generate enough force to separate sister kinetochores, suggesting that microtubule–kinetochore interactions were intact. Releasing Bod1-depleted cells from a monastrol block increases the frequency of syntelic attachments and the number of cells displaying biorientation defects. Bod1 depletion does not affect the activity or localization of Aurora B but does cause mislocalization of the microtubule depolymerase mitotic centromere- associated kinesin and prevents its efficient phosphorylation by Aurora B. Therefore, Bod1 is a novel kinetochore protein that is required for the detection or resolution of syntelic attachments in mitotic spindles.

scholarly journals RMD-1, a novel microtubule-associated protein, functions in chromosome segregation in Caenorhabditis elegans

2007 ◽  
Vol 179 (6) ◽  
pp. 1149-1162 ◽  
Author(s):  
Kumiko Oishi ◽  
Hideyuki Okano ◽  
Hitoshi Sawa
Keyword(s):  
Caenorhabditis Elegans ◽  
Chromosome Segregation ◽  
Growth Defect ◽  
Aurora B ◽  
C Elegans ◽  
Spindle Poles ◽  
Protein Functions ◽  
Microtubule Growth ◽  
Mild Defect

For proper chromosome segregation, the sister kinetochores must attach to microtubules extending from the opposite spindle poles. Any errors in microtubule attachment can induce aneuploidy. In this study, we identify a novel conserved Caenorhabditis elegans microtubule-associated protein, regulator of microtubule dynamics 1 (RMD-1), that localizes to spindle microtubules and spindle poles. Depletion of RMD-1 induces severe defects in chromosome segregation, probably through merotelic attachments between microtubules and chromosomes. Although rmd-1 embryos also have a mild defect in microtubule growth, we find that mutants of the microtubule growth regulator XMAP215/ZYG-9 show much weaker segregation defects. This suggests that the microtubule growth defect in rmd-1 embryos does not cause abnormal chromosome segregation. We also see that RMD-1 interacts with aurora B in vitro. Our results suggest that RMD-1 functions in chromosome segregation in C. elegans embryos, possibly through the aurora B–mediated pathway. Human homologues of RMD-1 could also bind microtubules, which would suggest a function for these proteins in chromosome segregation during mitosis in other organisms as well.

scholarly journals chTOG is a conserved mitotic error correction factor

2020 ◽  
Author(s):  
Jacob A. Herman ◽  
Matthew P. Miller ◽  
Sue Biggins
Keyword(s):  
Protein Kinase ◽  
Error Correction ◽  
Chromosome Segregation ◽  
Correction Factor ◽  
Budding Yeast ◽  
Aurora B ◽  
Spindle Poles ◽  
Low Tension ◽  
Intrinsic Error

AbstractAccurate chromosome segregation requires kinetochores on duplicated chromatids to biorient by attaching to dynamic microtubules from opposite spindle poles, which exerts forces to bring kinetochores under tension. However, kinetochores initially bind to MTs indiscriminately, resulting in errors that must be corrected. While the Aurora B protein kinase destabilizes low-tension attachments by phosphorylating kinetochores, low-tension attachments are intrinsically less stable than those under higher tension in vitro independent of Aurora activity. Intrinsic tensionsensitive behavior requires the microtubule regulator Stu2 (budding yeast Dis1/XMAP215 ortholog), which we demonstrate here is likely a conserved function for the TOG protein family. The human TOG protein, chTOG, localizes to kinetochores independent of microtubules by interacting with Hec1. We identify a chTOG mutant that regulates microtubule dynamics but accumulates erroneous kinetochore-microtubule attachments that Aurora B fails to destabilize. Thus, TOG proteins confer a unique, intrinsic error correction activity to kinetochores that ensures accurate chromosome segregation.

scholarly journals Aurora B switches relative strength between kinetochore–microtubule attachment modes to promote error correction

2018 ◽  
Author(s):  
Harinath Doodhi ◽  
Taciana Kasciukovic ◽  
Lesley Clayton ◽  
Tomoyuki U. Tanaka
Keyword(s):  
Error Correction ◽  
Chromosome Segregation ◽  
Relative Strength ◽  
Lateral Side ◽  
Aurora B ◽  
Spindle Poles ◽  
Microtubule Attachment ◽  
Attachment Strength ◽  
Dam1 Complex

AbstractFor proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles; this is called bi-orientation. To establish bi-orientation prior to chromosome segregation, any aberrant kinetochore–microtubule interaction must be resolved (error correction) by Aurora B kinase that phosphorylates outer kinetochore components. Aurora B differentially regulates kinetochore attachment to the microtubule plus end and its lateral side (end-on and lateral attachment, respectively). However, it is still not fully understood how kinetochore–microtubule interactions are exchanged during error correction. Here we reconstituted the kinetochore–microtubule interface of budding yeast in vitro by attaching the Ndc80 complexes (Ndc80C) to nanobeads. These Ndc80C–nanobeads recapitulated in vitro the lateral and end-on attachments of authentic kinetochores, on dynamic microtubules loaded with the Dam1 complex. This in vitro assay enabled the direct comparison of lateral and end-on attachment strength and showed that Dam1 phosphorylation by Aurora B makes the end-on attachment weaker than the lateral attachment. We suggest that the Dam1 phosphorylation weakens interaction with the Ndc80 complex, disrupts the end-on attachment and promotes the exchange to a new lateral attachment, leading to error correction. Our study reveals a fundamental mechanism of error correction for establishment of bi-orientation.

scholarly journals Tension on kinetochore substrates is insufficient to prevent Aurora-triggered detachment

2018 ◽  
Author(s):  
Anna K. de Regt ◽  
Charles L. Asbury ◽  
Sue Biggins
Keyword(s):  
Chromosome Segregation ◽  
Direct Evidence ◽  
Underlying Mechanism ◽  
Aurora B ◽  
Trial And Error ◽  
Spindle Poles ◽  
Pulling Forces ◽  
Low Tension

Introduction / AbstractChromosome segregation requires large macromolecular structures called kinetochores to attach dynamic microtubules from opposite spindle poles1,2. Attachments are made iteratively, through a trial-and-error process, and proper attachments come under tension from the pulling forces of microtubules3,4. However, if sister kinetochores bind microtubules from the same pole1,2, these defective attachments lack tension and must be destabilized to give another chance for proper attachments to form. This vital error correction process requires Aurora B kinase, which phosphorylates kinetochores lacking tension to reduce their affinity for microtubules5-11. An unresolved question is how Aurora B distinguishes the level of tension on kinetochores. There are conflicting reports on the underlying mechanism12-16, owing in part to the difficulties of manipulating kinetochore tension in vivo and distinguishing kinase from opposing phosphatase activity. To address these issues, we have reconstituted Aurora B-triggered kinetochore detachment in an in vitro optical trapping-based flow assay. Here, we test an outstanding model by determining whether kinetochore tension is sufficient to prevent kinase-triggered detachments. Strikingly, Aurora B detaches kinetochores from microtubules under both high and low tension, providing direct evidence that the kinase does not distinguish correct versus incorrect attachments by recognizing tension-dependent changes in the conformation of its kinetochore substrates.

scholarly journals chTOG is a conserved mitotic error correction factor

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jacob A Herman ◽  
Matthew P Miller ◽  
Sue Biggins
Keyword(s):  
Protein Kinase ◽  
Error Correction ◽  
Chromosome Segregation ◽  
Correction Factor ◽  
Aurora B ◽  
Spindle Poles ◽  
Low Tension ◽  
Intrinsic Error ◽  
Sensitive Behavior

Accurate chromosome segregation requires kinetochores on duplicated chromatids to biorient by attaching to dynamic microtubules from opposite spindle poles, which exerts forces to bring kinetochores under tension. However, kinetochores initially bind to microtubules indiscriminately, resulting in errors that must be corrected. While the Aurora B protein kinase destabilizes low-tension attachments by phosphorylating kinetochores, low-tension attachments are intrinsically less stable than those under higher tension in vitro independent of Aurora activity. Intrinsic tension-sensitive behavior requires the microtubule regulator Stu2 (budding yeast Dis1/XMAP215 ortholog), which we demonstrate here is likely a conserved function for the TOG protein family. The human TOG protein, chTOG, localizes to kinetochores independent of microtubules by interacting with Hec1. We identify a chTOG mutant that regulates microtubule dynamics but accumulates erroneous kinetochore-microtubule attachments that are not destabilized by Aurora B. Thus, TOG proteins confer a unique, intrinsic error correction activity to kinetochores that ensures accurate chromosome segregation.

scholarly journals Interaction between Poly(ADP-ribose) and NuMA Contributes to Mitotic Spindle Pole Assembly

2009 ◽  
Vol 20 (21) ◽  
pp. 4575-4585 ◽  
Author(s):  
Paul Chang ◽  
Margaret Coughlin ◽  
Timothy J. Mitchison
Keyword(s):  
Binding Proteins ◽  
Magnetic Beads ◽  
Spindle Pole ◽  
Covalent Modification ◽  
Mitotic Spindles ◽  
Spindle Poles ◽  
Tankyrase 1 ◽  
Mitotic Spindle Pole

Poly(ADP-ribose) (pADPr), made by PARP-5a/tankyrase-1, localizes to the poles of mitotic spindles and is required for bipolar spindle assembly, but its molecular function in the spindle is poorly understood. To investigate this, we localized pADPr at spindle poles by immuno-EM. We then developed a concentrated mitotic lysate system from HeLa cells to probe spindle pole assembly in vitro. Microtubule asters assembled in response to centrosomes and Ran-GTP in this system. Magnetic beads coated with pADPr, extended from PARP-5a, also triggered aster assembly, suggesting a functional role of the pADPr in spindle pole assembly. We found that PARP-5a is much more active in mitosis than interphase. We used mitotic PARP-5a, self-modified with pADPr chains, to capture mitosis-specific pADPr-binding proteins. Candidate binding proteins included the spindle pole protein NuMA previously shown to bind to PARP-5a directly. The rod domain of NuMA, expressed in bacteria, bound directly to pADPr. We propose that pADPr provides a dynamic cross-linking function at spindle poles by extending from covalent modification sites on PARP-5a and NuMA and binding noncovalently to NuMA and that this function helps promote assembly of exactly two poles.

scholarly journals Central-spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

2019 ◽  
Vol 30 (19) ◽  
pp. 2503-2514 ◽  
Author(s):  
Che-Hang Yu ◽  
Stefanie Redemann ◽  
Hai-Yin Wu ◽  
Robert Kiewisz ◽  
Tae Yeon Yoo ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Tomographic Reconstruction ◽  
Mitotic Spindles ◽  
Strongly Coupled ◽  
Central Spindle ◽  
Spindle Poles ◽  
Meiotic Spindles ◽  
Spindle Microtubules ◽  
Anaphase B ◽  
Chromosome Motion

Spindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, that is, on the region between chromosomes and poles. In comparison, microtubules in the central-spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central-spindle microtubules during chromosome segregation in human mitotic spindles and Caenorhabditis elegans mitotic and female meiotic spindles. We found that these central-spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move toward spindle poles. In these systems, damaging central-spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central-spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central-spindle microtubules during chromosome segregation in diverse spindles and suggest that central-spindle microtubules and chromosomes are strongly coupled in anaphase.

scholarly journals A small-molecule inhibitor of Haspin alters the kinetochore functions of Aurora B

2012 ◽  
Vol 199 (2) ◽  
pp. 269-284 ◽  
Author(s):  
Anna De Antoni ◽  
Stefano Maffini ◽  
Stefan Knapp ◽  
Andrea Musacchio ◽  
Stefano Santaguida
Keyword(s):  
Phosphatase Activity ◽  
Small Molecule ◽  
Hela Cells ◽  
Spindle Assembly Checkpoint ◽  
Histone H3 ◽  
Aurora B ◽  
Molecule Inhibitor ◽  
Chromosome Passenger Complex ◽  
Kinase A

By phosphorylating Thr3 of histone H3, Haspin promotes centromeric recruitment of the chromosome passenger complex (CPC) during mitosis. Aurora B kinase, a CPC subunit, sustains chromosome bi-orientation and the spindle assembly checkpoint (SAC). Here, we characterize the small molecule 5-iodotubercidin (5-ITu) as a potent Haspin inhibitor. In vitro, 5-ITu potently inhibited Haspin but not Aurora B. Consistently, 5-ITu counteracted the centromeric localization of the CPC without affecting the bulk of Aurora B activity in HeLa cells. Mislocalization of Aurora B correlated with dephosphorylation of CENP-A and Hec1 and SAC override at high nocodazole concentrations. 5-ITu also impaired kinetochore recruitment of Bub1 and BubR1 kinases, and this effect was reversed by concomitant inhibition of phosphatase activity. Forcing localization of Aurora B to centromeres in 5-ITu also restored Bub1 and BubR1 localization but failed to rescue the SAC override. This result suggests that a target of 5-ITu, possibly Haspin itself, may further contribute to SAC signaling downstream of Aurora B.

scholarly journals Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

2003 ◽  
Vol 162 (5) ◽  
pp. 757-764 ◽  
Author(s):  
Yasuhiko Terada ◽  
Yumi Uetake ◽  
Ryoko Kuriyama
Keyword(s):  
Mammalian Cells ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Mitotic Spindles ◽  
Microtubule Nucleation ◽  
Microtubule Organization ◽  
Centrosomal Protein ◽  
Spindle Poles

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of γ-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with γ-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring γ-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

scholarly journals Centromere Targeting of the Chromosomal Passenger Complex Requires a Ternary Subcomplex of Borealin, Survivin, and the N-Terminal Domain of INCENP

2006 ◽  
Vol 17 (6) ◽  
pp. 2547-2558 ◽  
Author(s):  
Ulf R. Klein ◽  
Erich A. Nigg ◽  
Ulrike Gruneberg
Keyword(s):  
Functional Module ◽  
Aurora B ◽  
Serine Threonine Kinase ◽  
Chromosomal Passenger Complex ◽  
Chromosomal Passenger ◽  
Core Components ◽  
Interfering Rna ◽  
Centromere Assembly

The chromosomal passenger complex (CPC), consisting of the serine/threonine kinase Aurora B, the inner centromere protein INCENP, Survivin, and Borealin/DasraB, has essential functions at the centromere in ensuring correct chromosome alignment and segregation. Despite observations that small interfering RNA-mediated knockdown of any one member of the CPC abolishes localization of the other subunits, it remains unclear how the complex is targeted to the centromere. We have now identified a ternary subcomplex of the CPC comprising Survivin, Borealin, and the N-terminal 58 amino acids of INCENP in vitro and in vivo. This subcomplex was found to be essential and sufficient for targeting to the centromere. Notably, Aurora B kinase, the enzymatic core of the CPC, was not required for centromere localization of the subcomplex. We demonstrate that CPC targeting to the centromere does not depend on CENP-A and hMis12, two core components for kinetochore/centromere assembly, and provide evidence that the CPC may be directed to centromeric DNA directly via the Borealin subunit. Our findings thus establish a functional module within the CPC that assembles on the N terminus of INCENP and controls centromere recruitment.

Sign in / Sign up

Export Citation Format

Share Document