Kinetochore-microtubule Attachments Research Articles

A communication hub for phosphoregulation of kinetochore-microtubule attachment

The Mps1 and Aurora B kinases regulate and monitor kinetochore attachment to spindle microtubules during cell division, ultimately ensuring accurate chromosome segregation. In yeast, the critical spindle attachment components are the Ndc80 and Dam1 complexes (Ndc80c and DASH/Dam1c, respectively). Ndc80c is a 600-Å-long heterotetramer that binds microtubules through a globular "head" at one end and centromere-proximal kinetochore components through a globular knob at the other end. Dam1c is a heterodecamer that forms a ring of 16-17 protomers around the shaft of the single kinetochore microtubule in point-centromere yeast. The ring coordinates the approximately eight Ndc80c rods per kinetochore. In published work, we showed that a site on the globular "head" of Ndc80c, including residues from both Ndc80 and Nuf2, binds a bipartite segment in the long C-terminal extension of Dam1. Results reported here show, both by invitro binding experiments and by crystal structure determination, that the same site binds a conserved segment in the long N-terminal extension of Mps1. It also binds, less tightly, a conserved segment in the N-terminal extension of Ipl1 (yeast Aurora B). Together with results from experiments in yeast cells and from biochemical assays reported in two accompanying papers, the structures and graded affinities identify a communication hub for ensuring uniform bipolar attachment and for signaling anaphase onset.

Microtubule end-on attachment maturation regulates Mps1 association with its kinetochore receptor

Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents premature anaphase onset with incomplete attachments. However, how microtubule attachment and checkpoint signaling are coordinated remains unclear. The conserved kinase Mps1 initiates SAC signaling by localizing transiently to kinetochores in prometaphase and is released upon bi-orientation. Using biochemistry, structure predictions, and cellular assays, we shed light on this dynamic behavior in Saccharomyces cerevisiae. A conserved N-terminal segment of Mps1 binds the neck region of Ndc80:Nuf2, the main microtubule receptor of kinetochores. Mutational disruption of this interface, located at the backside of the paired CH domains and opposite the microtubule-binding site, prevents Mps1 localization, eliminates SAC signaling, and impairs growth. The same interface of Ndc80:Nuf2 binds the microtubule-associated Dam1 complex. We demonstrate that the error correction kinase Ipl1/Aurora B controls the competition between Dam1 and Mps1 for the same binding site. Thus, binding of the Dam1 complex to Ndc80:Nuf2 may release Mps1 from the kinetochore to promote anaphase onset.

Abstract SY22-01: A strategy to intervene mitotic kinases to induce mitotic catastrophe in cancer cells

Abstract Chromosomal instability (CIN) is an outstanding feature of cancer, which relates to intratumor heterogeneity, therapeutic resistance, and poor prognosis. While cancer cells can proliferate with CIN, experimentally induced aneuploidy is known to suppress cell growth, leading to the idea that extreme ploidy changes are detrimental to cancer cells. One of the major etiologies of CIN is abnormal kinetochore microtubule attachments, followed by chromosome missegregation. To avoid this, cells have evolved a robust system including two molecular networks for stabilizing and destabilizing microtubule attachments. Two mitotic kinases are involved in this system which have opposite but balanced mode of actions. We previously found that a wide range of cancer cells are defective for destabilization and results in an elevated rate of missegregation. In this study, we aimed to manipulate this kinase balance and found that weighing the balance to stabilizers can cause catastrophic chromosome segregation in mitosis, and such effect could be induced predominantly in cancer cells. These findings prompted us to undertake a screen for small molecules targeting the balanced activity of mitotic kinases. Citation Format: Motoko Takahashi, Minji Jo, Nana Kamakura, Chang Liu, Utako Kato, Toru Hirota. A strategy to intervene mitotic kinases to induce mitotic catastrophe in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY22-01.

Kinesin KIFC3 is essential for microtubule stability and cytokinesis in oocyte meiosis

KIFC3 is a member of Kinesin-14 family motor proteins, which play a variety of roles such as centrosome cohesion, cytokinesis, vesicles transportation and cell proliferation in mitosis. Here, we investigated the functional roles of KIFC3 in meiosis. Our findings demonstrated that KIFC3 exhibited expression and localization at centromeres during metaphase I, followed by translocation to the midbody at telophase I throughout mouse oocyte meiosis. Disruption of KIFC3 activity resulted in defective polar body extrusion. We observed aberrant meiotic spindles and misaligned chromosomes, accompanied by the loss of kinetochore-microtubule attachment, which might be due to the failed recruitment of BubR1/Bub3. Coimmunoprecipitation data revealed that KIFC3 plays a crucial role in maintaining the acetylated tubulin level mediated by Sirt2, thereby influencing microtubule stability. Additionally, our findings demonstrated an interaction between KIFC3 and PRC1 in regulating midbody formation during telophase I, which is involved in cytokinesis regulation. Collectively, these results underscore the essential contribution of KIFC3 to spindle assembly and cytokinesis during mouse oocyte meiosis.

Kinesin-7 CENP-E in tumorigenesis: Chromosome instability, spindle assembly checkpoint, and applications.

Kinesin motors are a large family of molecular motors that walk along microtubules to fulfill many roles in intracellular transport, microtubule organization, and chromosome alignment. Kinesin-7 CENP-E (Centromere protein E) is a chromosome scaffold-associated protein that is located in the corona layer of centromeres, which participates in kinetochore-microtubule attachment, chromosome alignment, and spindle assembly checkpoint. Over the past 3decades, CENP-E has attracted great interest as a promising new mitotic target for cancer therapy and drug development. In this review, we describe expression patterns of CENP-E in multiple tumors and highlight the functions of CENP-E in cancer cell proliferation. We summarize recent advances in structural domains, roles, and functions of CENP-E in cell division. Notably, we describe the dual functions of CENP-E in inhibiting and promoting tumorigenesis. We summarize the mechanisms by which CENP-E affects tumorigenesis through chromosome instability and spindle assembly checkpoints. Finally, we overview and summarize the CENP-E-specific inhibitors, mechanisms of drug resistances and their applications.

The Arabidopsis BUB1/MAD3 family protein BMF3 requires BUB3.3 to recruit CDC20 to kinetochores in spindle assembly checkpoint signaling

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during cell division by monitoring kinetochore-microtubule attachment. Plants produce both sequence-conserved and diverged SAC components, and it has been largely unknown how SAC activation leads to the assembly of these proteins at unattached kinetochores to prevent cells from entering anaphase. In Arabidopsis thaliana, the noncanonical BUB3.3 protein was detected at kinetochores throughout mitosis, unlike MAD1 and the plant-specific BUB1/MAD3 family protein BMF3 that associated with unattached chromosomes only. When BUB3.3 was lost by a genetic mutation, mitotic cells often entered anaphase with misaligned chromosomes and presented lagging chromosomes after they were challenged by low doses of the microtubule depolymerizing agent oryzalin, resulting in the formation of micronuclei. Surprisingly, BUB3.3 was not required for the kinetochore localization of other SAC proteins or vice versa. Instead, BUB3.3 specifically bound to BMF3 through two internal repeat motifs that were not required for BMF3 kinetochore localization. This interaction enabled BMF3 to recruit CDC20, a downstream SAC target, to unattached kinetochores. Taken together, our findings demonstrate that plant SAC utilizes unconventional protein interactions for arresting mitosis, with BUB3.3 directing BMF3's role in CDC20 recruitment, rather than the recruitment of BUB1/MAD3 proteins observed in fungi and animals. This distinct mechanism highlights how plants adapted divergent versions of conserved cell cycle machinery to achieve specialized SAC control.

KNL1 and NDC80 represent new universal markers for the detection of functional centromeres in plants.

Centromere is the chromosomal site of kinetochore assembly and microtubule attachment for chromosome segregation. Given its importance, markers that allow specific labeling of centromeric chromatin throughout the cell cycle and across all chromosome types are sought for facilitating various centromere studies. Antibodies against the N-terminal region of CENH3 are commonly used for this purpose, since CENH3 is the near-universal marker of functional centromeres. However, because the N-terminal region of CENH3 is highly variable among plant species, antibodies directed against this region usually function only in a small group of closely related species. As a more versatile alternative, we present here antibodies targeted to the conserved domains of two outer kinetochore proteins, KNL1 and NDC80. Sequence comparison of these domains across more than 350 plant species revealed a high degree of conservation, particularly within a six amino acid motif, FFGPVS in KNL1, suggesting that both antibodies would function in a wide range of plant species. This assumption was confirmed by immunolabeling experiments in angiosperm (monocot and dicot) and gymnosperm species, including those with mono-, holo-, and meta-polycentric chromosomes. In addition to centromere labeling on condensed chromosomes during cell division, both antibodies detected the corresponding regions in the interphase nuclei of most species tested. These results demonstrated that KNL1 and NDC80 are better suited for immunolabeling centromeres than CENH3, because antibodies against these proteins offer incomparably greater versatility across different plant species which is particularly convenient for studying the organization and function of the centromere in non-model species.

PLK1 phosphorylation of ZW10 guides accurate chromosome segregation in mitosis.

Stable transmission of genetic information during cell division requires faithful chromosome segregation. Mounting evidence has demonstrated that PLK1 dynamics at kinetochores control correct kinetochore-microtubule attachments and subsequent silencing of the spindle checkpoint. However, the mechanisms underlying PLK1-mediated silencing of the spindle checkpoint remain elusive. Here, we identified a regulatory mechanism by which PLK1-elicited ZW10 phosphorylation regulates spindle checkpoint silencing in mitosis. ZW10 is a cognate substrate of PLK1, and the phosphorylation of ZW10 at Ser12 enables dynamic ZW10-Zwint1 interactions. Inhibition of ZW10 phosphorylation resulted in misaligned chromosomes, while persistent expression of phospho-mimicking ZW10 mutant caused premature anaphase, in which sister chromatids entangled as cells entered anaphase. These findings reveal the previously uncharacterized PLK1-ZW10 interaction through which dynamic phosphorylation of ZW10 fine-tunes accurate chromosome segregation in mitosis.

Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling

Accurate mitosis is coordinated by the spindle assembly checkpoint (SAC) through the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C). As an essential regulator, Cdc20 promotes mitotic exit through activating APC/C and monitors kinetochore-microtubule attachment through activating SAC. Cdc20 requires multiple interactions with APC/C and MCC subunits to elicit these functions. Functionally assessing these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNA interference (RNAi). Here we generated Cdc20 RNAi-sensitive cell lines which display a penetrant metaphase arrest by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron, was found critical for SAC by promoting MCC formation and its interaction with APC/C. These data reveal additional regulation within the SAC and establish a novel method to interrogate Cdc20.

Prothioconazole exposure disrupts oocyte maturation and fertilization by inducing mitochondrial dysfunction and apoptosis in mice

Prothioconazole (PTC), a novel broad-spectrum triazole fungicide, has attracted widespread concern due to its wide use and toxicological effects on non-target organisms. However, little is known about the impact of PTC on oocyte quality and female fertility, especially on oocyte maturation and fertilization. In the present study, we reported that PTC exposure affects the oocyte developmental competence and oocyte fertilization ability to weaken female fertility. Firstly, PTC compromises oocyte development ability by disrupting spindle morphology and chromosome alignment, as well as decreasing acetylation level of α-tubulin and disrupting kinetochore-microtubule attachments. In addition, PTC compromises oocyte fertilization ability by weakening the sperm binding ability and impairing the dynamics of Juno, Cortical granule and Ovastacin. Finally, single-cell transcriptome analysis revealed that PTC exposure has potentially toxic effects on oocyte development and fertilization, which is caused by the mitochondrial dysfunction and the occurrence of oxidative stress and apoptosis. In summary, our results indicated that PTC exposure had potentially toxic effects on female fertility and led to poor oocyte quality in female mice.

Copper oxide nanoparticles impairs oocyte meiosis maturation by inducing mitochondrial dysfunction and oxidative stress

Copper oxides nanoparticles (CuO NPs) are widely used for a variety of industrial and life science applications. In addition to cause neurotoxicity, hepatotoxicity, immunotoxicity, CuO NPs have also been reported to adversely affect the reproductive system in animals; However, little is known about the effects and potential mechanism of CuO NPs exposure on oocyte quality, especially oocyte maturation. In the present study, we reported that CuO NPs exposure impairs the oocyte maturation by disrupting meiotic spindle assembly and chromosome alignment, as well as kinetochore-microtubule attachment. In addition, CuO NPs exposure also affects the acetylation level of α-tubulin in mice oocyte, which hence impairs microtubule dynamics and organization. Besides, CuO NPs exposure would result in the mis-localization of Juno and Ovastacin, which might be one of the critical factors leading to the failure of oocyte maturation. Finally, CuO NPs exposure impairs the mitochondrial distribution and induced high levels of ROS, which led to the accumulation of DNA damage and occurrence of apoptosis. In summary, our results indicated that CuO NPs exposure had potential toxic effects on female fertility and led to the poor oocyte quality in female mice.

Multimerization of a disordered kinetochore protein promotes accurate chromosome segregation by localizing a core dynein module.

Kinetochores connect chromosomes and spindle microtubules to maintain genomic integrity through cell division. Crosstalk between the minus-end directed motor dynein and kinetochore-microtubule attachment factors promotes accurate chromosome segregation by a poorly understood pathway. Here, we identify a linkage between the intrinsically disordered protein Spc105 (KNL1 orthologue) and dynein using an optogenetic oligomerization assay. Core pools of the checkpoint protein BubR1 and the adaptor complex RZZ contribute to the linkage. Furthermore, a minimal segment of Spc105 with a propensity to multimerize and which contains protein binding motifs is sufficient to link Spc105 to RZZ/dynein. Deletion of the minimal region from Spc105 compromises the recruitment of its binding partners to kinetochores and elevates chromosome missegregation due to merotelic attachments. Restoration of normal chromosome segregation and localization of BubR1 and RZZ requires both protein binding motifs and oligomerization of Spc105. Together, our results reveal that higher-order multimerization of Spc105 contributes to localizing a core pool of RZZ that promotes accurate chromosome segregation.

Fission yeast Wee1 is required for stable kinetochore-microtubule attachment.

Wee1 is a cell cycle regulator that phosphorylates Cdk1/Cdc2 and inhibits G2/M transition. Loss of Wee1 in fission yeast results in an early onset of mitosis. Interestingly, we found that cells lacking Wee1 require the functional spindle checkpoint for their viability. Genetic analysis indicated that the requirement is not attributable to the early onset of mitosis. Live-cell imaging revealed that some kinetochores are not attached or bioriented in the wee1 mutant. Furthermore, Mad2, a component of the spindle checkpoint known to recognize unattached kinetochores, accumulates in the vicinity of the spindle, representing activation of the spindle checkpoint in the mutant. It appears that the wee1 mutant cannot maintain stable kinetochore-microtubule attachment, and relies on the delay imposed by the spindle checkpoint for establishing biorientation of kinetochores. This study revealed a role of Wee1 in ensuring accurate segregation of chromosomes during mitosis, and thus provided a basis for a new principle of cancer treatment with Wee1 inhibitors.

METTL16-mediated N6-methyladenosine modification of Soga1 enables proper chromosome segregation and chromosomal stability in colorectal cancer.

N6-methyladenosine (m6A) is the most prevalent internal modification in mammalian messenger RNAs and is associated with numerous biological processes. However, its role in chromosomal instability remains to be established. Here, we report that an RNA m6A methyltransferase, METTL16, plays an indispensable role in the progression of chromosome segregation and is required to preserve chromosome stability in colorectal cancer (CRC) cells. Depletion or inhibition of the methyltransferase activity of METTL16 results in abnormal kinetochore-microtubule attachment during mitosis, leading to delayed mitosis, lagging chromosomes, chromosome mis-segregation and chromosomal instability. Mechanistically, METTL16 exerts its oncogenic effects by enhancing the expression of suppressor of glucose by autophagy 1 (Soga1) in an m6A-dependent manner. CDK1 phosphorylates Soga1, thereby triggering its direct interaction with the polo box domain of PLK1. This interaction facilitates PLK1 activation and promotes mitotic progression. Therefore, targeting the METTL16-Soga1 pathway may provide a potential treatment strategy against CRC because of its essential role in maintaining chromosomal stability.

Structural mechanism of outer kinetochore Dam1-Ndc80 complex assembly on microtubules.

Kinetochores couple chromosomes to the mitotic spindle to segregate the genome during cell division. An error correction mechanism drives the turnover of kinetochore-microtubule attachments until biorientation is achieved. The structural basis for how kinetochore-mediated chromosome segregation is accomplished and regulated remains an outstanding question. In this work, we describe the cryo-electron microscopy structure of the budding yeast outer kinetochore Ndc80 and Dam1 ring complexes assembled onto microtubules. Complex assembly occurs through multiple interfaces, and a staple within Dam1 aids ring assembly. Perturbation of key interfaces suppresses yeast viability. Force-rupture assays indicated that this is a consequence of impaired kinetochore-microtubule attachment. The presence of error correction phosphorylation sites at Ndc80-Dam1 ring complex interfaces and the Dam1 staple explains how kinetochore-microtubule attachments are destabilized and reset.

CRMP5 participates in oocyte meiosis by regulating spastin to correct microtubule-kinetochore misconnection.

Our previous studies have suggested that spastin, which aggregates on spindle microtubules in oocytes, may promote the assembly of mouse oocyte spindles by cutting microtubules. This action may be related to CRMP5, as knocking down CRMP5 results in reduced spindle microtubule density and maturation defects in oocytes. In this study, we found that, after knocking down CRMP5 in oocytes, spastin distribution shifted from the spindle to the spindle poles and errors in microtubule-kinetochore attachment appeared in oocyte spindles. However, CRMP5 did not interact with the other two microtubule-severing proteins, katanin-like-1 (KATNAL1) and fidgetin-like-1 (FIGNL1), which aggregate at the spindle poles. We speculate that, in oocytes, due to the reduction of spastin distribution on chromosomes after knocking down CRMP5, microtubule-kinetochore errors cannot be corrected through severing, resulting in meiotic division abnormalities and maturation defects in oocytes. This finding provides new insights into the regulatory mechanisms of spastin in oocytes and important opportunities for the study of meiotic division mechanisms.

High-Salt Diet Causes Defective Oocyte Maturation and Embryonic Development to Impair Female Fertility in Mice.

High salinity has been reported to induce many human disorders in tissues and organs to interfere with their normal physiological functions. However, it is unknown how salinity affects the development of female germ cells. This study suggests that a high-salt diet (HSD) may weaken oocyte quality to impair female fertility in mice and investigates the underlying mechanisms. C57BL/6 female mice are fed with a regular diet (Control) or a high-salt diet (HSD). Oocyte maturation, fertilization rate, embryonic development, and female fertility are evaluated. In addition, the spindle organization, actin polymerization, and kinetochore-microtubule attachment of oocytes are examined in both groups. Moreover, single-cell transcriptome data are used to demonstrate how HSD alters the transcript levels of genes. The observations confirm that HSD leads to female subfertility due to the deterioration of oocyte and embryo quality. The mechanism underlying reveals HSD compromises the oocytes' autophagy, apoptosis level, and mitochondrial function. The work illustrates that a high concentration of salt diet results in oocyte meiotic arrest, fertilization failure, and early developmental defection that embryos undergo to reduce female fertility in mice by perturbing the level of autophagy and apoptosis, mitochondrial function in oocytes.

A bifunctional kinase-phosphatase module balances mitotic checkpoint strength and kinetochore-microtubule attachment stability.

Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore-microtubule error-correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of these processes are set by a kinase-phosphatase pair (PLK1-PP2A) that engage in negative feedback from adjacent phospho-binding motifs on the BUB complex. Uncoupling this feedback to skew the balance towards PLK1 produces a strong checkpoint, hypostable microtubule attachments and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, hyperstable microtubule attachments and chromosome segregation errors. These phenotypes are associated with altered BUB complex recruitment to KNL1-MELT motifs, implicating PLK1-PP2A in controlling auto-amplification of MELT phosphorylation. In support, KNL1-BUB disassembly becomes contingent on PLK1 inhibition when KNL1 is engineered to contain excess MELT motifs. This elevates BUB-PLK1/PP2A complex levels on metaphase kinetochores, stabilises kinetochore-microtubule attachments, induces chromosome segregation defects and prevents KNL1-BUB disassembly at anaphase. Together, these data demonstrate how a bifunctional PLK1/PP2A module has evolved together with the MELT motifs to optimise BUB complex dynamics and ensure accurate chromosome segregation.

Carbendazim exposure inhibits mouse oocytes meiotic maturation in vitro by destroying spindle assembly.

Successful fertilization and early embryonic development heavily depend on the quality of the oocytes. Carbendazim (CBZ), a broad-spectrum fungicide, is widely available in the environment and has adverse effects on organisms. The present study focused on exploring the potential reproductive toxicity of CBZ exposure by investigating its effects on the maturation of mouse oocytes. The results demonstrated that although no disruptions were observed in the G2/M stage transition for meiosis resumption, CBZ did hinder the polar body extrusion (PBE) occurring during oocyte maturation. Cell cycle distribution analysis revealed that CBZ exposure interfered with the meiotic process, causing oocytes to be arrested at the metaphase I (MI) stage. The subsequent investigation highlighted that CBZ exposure impeded the spindle assembly and chromosomal alignment, which was linked to a decline in the level of p-MAPK. Additionally, CBZ exposure adversely affected the kinetochore-microtubule (K-MT) attachment, leading to the persistent activation of the spindle-assembly checkpoint (SAC). The study further noticed a substantial rise in the acetylation of α-tubulin and a reduction in spindle microtubule stability in CBZ-treated oocytes. In addition, the distribution pattern of estrogen receptor alpha (ERα) was altered in oocytes treated with CBZ, with abnormal aggregation on the spindles. CBZ exposure also resulted in altered histone modifications. A notable finding from this research was that the meiotic maturation of some oocytes remained unaffected even after CBZ treatment. However, during the ensuing metaphase II (MII) stage, these oocytes displayed anomalies in their spindle morphology and chromosome arrangement and diminished ability to bind to the sperm. The observations made in this study underscore the potential for CBZ to disrupt the meiotic maturation of oocytes, leading to a decline in the overall quality of oocytes.

CENP-E activation by Aurora A and B controls kinetochore fibrous corona disassembly

Accurate chromosome segregation in mitosis depends on multiprotein structures called kinetochores that are built on the centromeric region of sister chromatids and serve to capture mitotic spindle microtubules. In early mitosis, unattached kinetochores expand a crescent-shaped structure called fibrous corona whose function is to facilitate initial kinetochore-microtubule attachments and chromosome transport by microtubules. Subsequently, the fibrous corona must be timely disassembled to prevent segregation errors. Although recent studies provided new insights on the molecular content and mechanism of fibrous corona assembly, it remains unknown what triggers the disassembly of the outermost and dynamic layer of the kinetochore. Here, we show that Aurora A and B kinases phosphorylate CENP-E to release it from an autoinhibited state. At kinetochores, Aurora B phosphorylates CENP-E to prevent its premature removal together with other corona proteins by dynein. At the spindle poles, Aurora A phosphorylates CENP-E to promote chromosome congression and prevent accumulation of corona proteins at the centrosomes, allowing for their intracellular redistribution. Thus, we propose the Aurora A/B-CENP-E axis as a critical element of the long-sought-for mechanism of fibrous corona disassembly that is essential for accurate chromosome segregation.