Kinetochore life histories reveal an Aurora-B-dependent error correction mechanism in anaphase.
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ABSTRACT:
SUBMITTER: Sen O
PROVIDER: S-EPMC8629432 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:To establish chromosome biorientation, aberrant kinetochore-microtubule interaction must be resolved (error correction) by Aurora B kinase. 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 unclear how kinetochore-microtubule interactions are exchanged during error correction. Here, we reconstituted the budding yeast kinetochore-microtubule interface in vitro by attaching the Ndc80 complexes 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. Similar reconstitutions with purified kinetochore particles were used for comparison. We suggest 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.
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Project description:Kinetochores are superprotein complexes that orchestrate chromosome segregation via a dynamic interaction with spindle microtubules. A physical connection between CENP-C and the Mis12-Ndc80-Knl1 (KMN) protein network is an important pathway that is used to assemble kinetochores on CENP-A nucleosomes. Multiple outer kinetochore components are phosphorylated by Aurora B kinase to activate the spindle assembly checkpoint (SAC) and to ensure accurate chromosome segregation. However, it is unknown whether Aurora B can phosphorylate inner kinetochore components to facilitate proper mitotic chromosome segregation. Here, we reported the structure of the fission yeast Schizosaccharomyces pombe Mis12-Nnf1 complex and showed that N-terminal residues 26-50 in Cnp3 (the CENP-C homolog of S. pombe) are responsible for interacting with the Mis12 complex. Interestingly, Thr28 of Cnp3 is a substrate of Ark1 (the Aurora B homolog of S. pombe), and phosphorylation impairs the interaction between the Cnp3 and Mis12 complex. The expression of a phosphorylation-mimicking Cnp3 mutant results in defective chromosome segregation due to improper kinetochore assembly. These results establish a previously uncharacterized regulatory mechanism involved in CENP-C-Mis12-facilitated kinetochore attachment error correction to ensure accurate chromosome segregation during mitosis.
| S-EPMC5740674 | biostudies-literature
Project description:Kinetochores assemble on chromosomes in mitosis to allow microtubules to attach and bring about accurate chromosome segregation. The kinases Cyclin B-Cdk1 and Aurora B are crucial for the formation of stable kinetochores. However, the activity of these two kinases appears to decline dramatically at centromeres during anaphase onset, precisely when microtubule attachments are required to move chromosomes toward opposite poles of the dividing cell. We find that, although Aurora B leaves centromeres at anaphase, a gradient of Aurora B activity centered on the central spindle is still able to phosphorylate kinetochore substrates such as Dsn1 to modulate kinetochore stability in anaphase and to regulate kinetochore disassembly as cells enter telophase. We provide a model to explain how Aurora B co-operates with Cyclin B-Cdk1 to maintain kinetochore function in anaphase.
| S-EPMC8595645 | biostudies-literature
Project description:Robust kinetochore-microtubule (kMT) attachment is critical for accurate chromosome segregation. G2/M-specific depletion of human Cdt1 that localizes to kinetochores in an Ndc80 complex-dependent manner leads to abnormal kMT attachments and mitotic arrest. This indicates an independent mitotic role for Cdt1 in addition to its prototypic function in DNA replication origin licensing. Here, we show that Cdt1 directly binds to microtubules (MTs). Endogenous or transiently expressed Cdt1 localizes to both mitotic spindle MTs and kinetochores. Deletion mapping of Cdt1 revealed that the regions comprising the middle and C-terminal winged-helix domains but lacking the N-terminal unstructured region were required for efficient MT binding. Mitotic kinase Aurora B interacts with and phosphorylates Cdt1. Aurora B-phosphomimetic Cdt1 exhibited attenuated MT binding, and its cellular expression induced defective kMT attachments with a concomitant delay in mitotic progression. Thus we provide mechanistic insight into how Cdt1 affects overall kMT stability in an Aurora B kinase phosphorylation-dependent manner; which is envisioned to augment the MT-binding of the Ndc80 complex.
| S-EPMC6168275 | biostudies-literature
Project description:Although assembly of acentrosomal meiotic spindles has been extensively studied, little is known about the segregation of chromosomes on these spindles. Here, we show in Caenorhabditis elegans oocytes that the kinetochore protein, KNL-1, directs assembly of meiotic kinetochores that orient chromosomes. However, in contrast to mitosis, chromosome separation during meiotic anaphase is kinetochore-independent. Before anaphase, meiotic kinetochores and spindle poles disassemble along with the microtubules on the poleward side of chromosomes. During anaphase, microtubules then form between the separating chromosomes. Functional analysis implicated a set of proteins that localize to a ring-shaped domain between kinetochores during pre-anaphase spindle assembly and anaphase separation. These proteins are localized by the chromosomal passenger complex, which regulates the loss of meiotic chromosome cohesion. Thus, meiotic segregation in C. elegans is a two-stage process, where kinetochores orient chromosomes, but are then dispensable for their separation. We suggest that separation is controlled by a meiosis-specific chromosomal domain to coordinate cohesin removal and chromosome segregation.
| S-EPMC3052858 | biostudies-literature
Project description:For proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles (bi-orientation). To establish bi-orientation, aberrant kinetochore-microtubule attachments are disrupted (error correction) by aurora B kinase (Ipl1 in budding yeast). Paradoxically, during this disruption, new attachments are still formed efficiently to enable fresh attempts at bi-orientation. How this is possible remains an enigma. Here we show that kinetochore attachment to the microtubule lattice (lateral attachment) is impervious to aurora B regulation, but attachment to the microtubule plus end (end-on attachment) is disrupted by this kinase. Thus, a new lateral attachment is formed without interference, then converted to end-on attachment and released if incorrect. This process continues until bi-orientation is established and stabilized by tension across sister kinetochores. We reveal how aurora B specifically promotes disruption of the end-on attachment through phospho-regulation of kinetochore components Dam1 and Ndc80. Our results reveal fundamental mechanisms for promoting error correction for bi-orientation.
| S-EPMC4380510 | biostudies-literature
Project description:Mutations in the STAG2 gene are present in ∼20% of tumors from different tissues of origin. STAG2 encodes a subunit of the cohesin complex, and tumors with loss-of-function mutations are usually aneuploid and display elevated frequencies of lagging chromosomes during anaphase. Lagging chromosomes are a hallmark of chromosomal instability (CIN) arising from persistent errors in kinetochore-microtubule (kMT) attachment. To determine whether the loss of STAG2 increases the rate of formation of kMT attachment errors or decreases the rate of their correction, we examined mitosis in STAG2-deficient cells. STAG2 depletion does not impair bipolar spindle formation or delay mitotic progression. Instead, loss of STAG2 permits excessive centromere stretch along with hyperstabilization of kMT attachments. STAG2-deficient cells display mislocalization of Bub1 kinase, Bub3 and the chromosome passenger complex. Importantly, strategically destabilizing kMT attachments in tumor cells harboring STAG2 mutations by overexpression of the microtubule-destabilizing enzymes MCAK (also known as KIF2C) and Kif2B decreased the rate of lagging chromosomes and reduced the rate of chromosome missegregation. These data demonstrate that STAG2 promotes the correction of kMT attachment errors to ensure faithful chromosome segregation during mitosis.
| S-EPMC4179491 | biostudies-literature
Project description:Chromosome biorientation, where sister kinetochores attach to microtubules (MTs) from opposing spindle poles, is the configuration that best ensures equal partitioning of the genome during cell division. Erroneous kinetochore-MT attachments are commonplace but are often corrected prior to anaphase. Error correction, thought to be mediated primarily by the centromere-enriched Aurora B kinase (ABK), typically occurs near spindle poles; however, the relevance of this locale is unclear. Furthermore, polar ejection forces (PEFs), highest near poles, can stabilize improper attachments by pushing mal-oriented chromosome arms away from spindle poles. Hence, there is a conundrum: erroneous kinetochore-MT attachments are weakened where PEFs are most likely to strengthen them. Here, we report that Aurora A kinase (AAK) opposes the stabilizing effect of PEFs. AAK activity contributes to phosphorylation of kinetochore substrates near poles and its inhibition results in chromosome misalignment and an increased incidence of erroneous kinetochore-MT attachments. Furthermore, AAK directly phosphorylates a site in the N-terminal tail of Ndc80/Hec1 that has been implicated in reducing the affinity of the Ndc80 complex for MTs when phosphorylated. We propose that an AAK activity gradient contributes to correcting mal-oriented kinetochore-MT attachments in the vicinity of spindle poles.
| S-EPMC4509859 | biostudies-literature
Project description:BackgroundOverexpression of Aurora-A (AURKA) is a feature of breast cancer and associates with adverse prognosis. The selective Aurora-A inhibitor alisertib (MLN8237) has recently demonstrated promising antitumor responses as a single agent in various cancer types but its phase III clinical trial was reported as a failure since MLN8237 did not show an apparent effect in prolonging the survival of patients. Thus, identification of potential targets that could enhance the activity of MLN8237 would provide a rationale for drug combination to achieve better therapeutic outcome.MethodsHere, we conducted a systematic synthetic lethality CRISPR/Cas9 screening of 507 kinases using MLN8237 in breast cancer cells and identified a number of targetable kinases that displayed synthetic lethality interactions with MLN8237. Then, we performed competitive growth assays, colony formation assays, cell viability assays, apoptosis assays, and xenograft murine model to evaluate the synergistic therapeutic effects of Haspin (GSG2) depletion or inhibition with MLN8237. For mechanistic studies, immunofluorescence was used to detect the state of microtubules and the localization of Aurora-B and mitotic centromere-associated kinesin (MCAK).ResultsAmong the hits, we observed that Haspin depletion or inhibition marginally inhibited breast cancer cell growth but could substantially enhance the killing effects of MLN8237. Mechanistic studies showed that co-treatment with Aurora-A and Haspin inhibitors abolished the recruitment of Aurora-B and mitotic centromere-associated kinesin (MCAK) to centromeres which were associated with excessive microtubule depolymerization, kinetochore-microtubule (KT-MT) attachment failure, and severe mitotic catastrophe. We further showed that the combination of MLN8237 and the Haspin inhibitor CHR-6494 synergistically reduced breast cancer cell viability and significantly inhibited both in vitro and in vivo tumor growth.ConclusionsThese findings establish Haspin as a synthetic lethal target and demonstrate CHR-6494 as a potential combinational drug for promoting the therapeutic effects of MLN8237 on breast cancer.
| S-EPMC7896750 | biostudies-literature
Project description:The kinetochore complex is a conserved machinery that connects chromosomes to spindle microtubules. During meiosis, the kinetochore is restructured to accommodate a specialized chromosome segregation pattern. In budding yeast, meiotic kinetochore remodeling is mediated by the temporal changes in the abundance of a single subunit called Ndc80. We previously described the regulatory events that control the timely synthesis of Ndc80. Here, we report that Ndc80 turnover is also tightly regulated in meiosis: Ndc80 degradation is active in meiotic prophase, but not in metaphase I. Ndc80 degradation depends on the ubiquitin ligase APCAma1 and is mediated by the proteasome. Importantly, Aurora B-dependent Ndc80 phosphorylation, a mark that has been previously implicated in correcting erroneous microtubule-kinetochore attachments, is essential for Ndc80 degradation in a microtubule-independent manner. The N terminus of Ndc80, including a 27-residue sequence and Aurora B phosphorylation sites, is both necessary and sufficient for kinetochore protein degradation. Finally, defects in Ndc80 turnover predispose meiotic cells to chromosome mis-segregation. Our study elucidates the mechanism by which meiotic cells modulate their kinetochore composition through regulated Ndc80 degradation, and demonstrates that Aurora B-dependent regulation of kinetochores extends beyond altering microtubule attachments.
| S-EPMC7000919 | biostudies-literature