Interkinetochore Tension Research Articles

VTT-006, an anti-mitotic compound, binds to the Ndc80 complex and suppresses cancer cell growth in vitro

Hec1 (Highly expressed in cancer 1) resides in the outer kinetochore where it works to facilitate proper kinetochore-microtubule interactions during mitosis. Hec1 is overexpressed in various cancers and its expression shows correlation with high tumour grade and poor patient prognosis. Chemical perturbation of Hec1 is anticipated to impair kinetochore-microtubule binding, activate the spindle assembly checkpoint (spindle checkpoint) and thereby suppress cell proliferation. In this study, we performed high-throughput screen to identify novel small molecules that target the Hec1 calponin homology domain (CHD), which is needed for normal microtubule attachments. 4 million compounds were first virtually fitted against the CHD, and the best hit molecules were evaluated in vitro. These approaches led to the identification of VTT-006, a 1,2-disubstituted-tetrahydro-beta-carboline derivative, which showed binding to recombinant Ndc80 complex and modulated Hec1 association with microtubules in vitro. VTT-006 treatment resulted in chromosome congression defects, reduced chromosome oscillations and induced loss of inter-kinetochore tension. Cells remained arrested in mitosis with an active spindle checkpoint for several hours before undergoing cell death. VTT-006 suppressed the growth of several cancer cell lines and enhanced the sensitivity of HeLa cells to Taxol. Our findings propose that VTT-006 is a potential anti-mitotic compound that disrupts M phase, impairs kinetochore-microtubule interactions, and activates the spindle checkpoint.

Mechanobiology of the Mitotic Spindle.

The mitotic spindle is a microtubule-based assembly that separates the chromosomes during cell division. As the spindle is basically a mechanical micro machine, the understanding of its functioning is constantly motivating the development of experimental approaches based on mechanical perturbations, which are complementary to and work together with the classical genetics and biochemistry methods. Recent data emerging from these approaches in combination with theoretical modeling led to novel ideas and significant revisions of the basic concepts in the field. In this Perspective, we discuss the advances inthe understanding of spindle mechanics, focusing on microtubule forces that control chromosome movements.

Microtubule-interfering agents, spindle defects, and interkinetochore tension.

Microtubule-interfering agents have been very useful both as biological tools in studying mitosis and as chemotherapeutic agents against cancer. It remains poorly understood how these agents converge on the spindle assembly checkpoint (SAC) to halt mitotic progression, while inhibiting microtubule dynamics by different mechanisms. Cells arrested at mitosis by various microtubule-interfering agents exhibit strikingly different defects in the mitotic spindle. However, all the arrested cells possess the 3F3/2 phosphoepitope at the sister kinetochores of chromosomes, indicating the decrease of tension across the paired kinetochores. In addition, microtubule-interfering agents result in a comparable reduction in the distance between sister kinetochores, suggesting that these agents decrease interkinetochore tension to similar degrees. Here, we discuss recent progress that suggests impairment of kinetochore-microtubule attachment and reduction of interkinetochore tension as common mechanisms underlying the persistent SAC activation in response to diverse microtubule-interfering agents.

The nonmotor adaptor HMMR dampens Eg5-mediated forces to preserve the kinetics and integrity of chromosome segregation.

Mitotic spindle assembly and organization require forces generated by motor proteins. The activity of these motors is regulated by nonmotor adaptor proteins. However, there are limited studies reporting the functional importance of adaptors on the balance of motor forces and the promotion of faithful and timely cell division. Here we show that genomic deletion or small interfering RNA silencing of the nonmotor adaptor Hmmr/HMMR disturbs spindle microtubule organization and bipolar chromosome-kinetochore attachments with a consequent elevated occurrence of aneuploidy. Rescue experiments show a conserved motif in HMMR is required to generate interkinetochore tension and promote anaphase entry. This motif bears high homology with the kinesin Kif15 and is known to interact with TPX2, a spindle assembly factor. We find that HMMR is required to dampen kinesin Eg5-mediated forces through localizing TPX2 and promoting the formation of inhibitory TPX2-Eg5 complexes. In HMMR-silenced cells, K-fiber stability is reduced while the frequency of unattached chromosomes and the time needed for chromosome segregation are both increased. These defects can be alleviated in HMMR-silenced cells with chemical inhibition of Eg5 but not through the silencing of Kif15. Together, our findings indicate that HMMR balances Eg5--mediated forces to preserve the kinetics and integrity of chromosome segregation.

Relaxation of interkinetochore tension after severing of a k-fiber depends on the length of the k-fiber stub

Relaxation of interkinetochore tension after severing of a k-fiber depends on the length of the k-fiber stub

JMJD5 (Jumonji Domain-containing 5) Associates with Spindle Microtubules and Is Required for Proper Mitosis

Precise mitotic spindle assembly is a guarantee of proper chromosome segregation during mitosis. Chromosome instability caused by disturbed mitosis is one of the major features of various types of cancer. JMJD5 has been reported to be involved in epigenetic regulation of gene expression in the nucleus, but little is known about its function in mitotic process. Here we report the unexpected localization and function of JMJD5 in mitotic progression. JMJD5 partially accumulates on mitotic spindles during mitosis, and depletion of JMJD5 results in significant mitotic arrest, spindle assembly defects, and sustained activation of the spindle assembly checkpoint (SAC). Inactivating SAC can efficiently reverse the mitotic arrest caused by JMJD5 depletion. Moreover, JMJD5 is found to interact with tubulin proteins and associate with microtubules during mitosis. JMJD5-depleted cells show a significant reduction of α-tubulin acetylation level on mitotic spindles and fail to generate enough interkinetochore tension to satisfy the SAC. Further, JMJD5 depletion also increases the susceptibility of HeLa cells to the antimicrotubule agent. Taken together, these results suggest that JMJD5 plays an important role in regulating mitotic progression, probably by modulating the stability of spindle microtubules.

Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores.

During metaphase, forces on kinetochores are exerted by k-fibres, bundles of microtubules that end at the kinetochore. Interestingly, non-kinetochore microtubules have been observed between sister kinetochores, but their function is unknown. Here we show by laser-cutting of a k-fibre in HeLa and PtK1 cells that a bundle of non-kinetochore microtubules, which we term ‘bridging fibre', bridges sister k-fibres and balances the interkinetochore tension. We found PRC1 and EB3 in the bridging fibre, suggesting that it consists of antiparallel dynamic microtubules. By using a theoretical model that includes a bridging fibre, we show that the forces at the pole and at the kinetochore depend on the bridging fibre thickness. Moreover, our theory and experiments show larger relaxation of the interkinetochore distance for cuts closer to kinetochores. We conclude that the bridging fibre, by linking sister k-fibres, withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape.

Borealin dimerization mediates optimal CPC checkpoint function by enhancing localization to centromeres and kinetochores.

The Chromosomal Passenger Complex (CPC) localizes to centromeres where it activates the mitotic checkpoint in response to inappropriate inter-kinetochore tension. This error correction function is essential for proper chromosome segregation. Here we define several critical features of CPC localization and function. First, the Borealin dimerization domain suppresses dynamic exchange at the centromere to allow optimal CPC function. Second, Borealin dimerization is essential to target a subpopulation of CPC proximal to the kinetochore when the mitotic spindle is disrupted. This subpopulation is also needed for full CPC checkpoint function. The existence of a pool of CPC at the kinetochore suggests that error correction is more complicated than predicted from the Aurora B phosphorylation gradient model. Finally, Haspin kinase plays a key role in maintaining the slowly exchanging centromere Borealin pool, while Aurora B and Mps1 play minimal roles in maintaining CPC localization once cells are in mitosis.

Novel pyrimidine-2,4-diamine derivative suppresses the cell viability and spindle assembly checkpoint activity by targeting Aurora kinases

Mitosis represents a clinically important determination point in the life cycle of proliferating cells. One potential drug target within the mitotic machinery is the spindle assembly checkpoint (SAC), an evolutionarily conserved signaling pathway that monitors the connections between microtubules (MTs) and chromosomes. Mistakes in SAC signaling may lead to cell division errors that can trigger elimination of cancer cells at M phase or soon after exit from mitosis. In this study, we describe the cellular effects of a novel pyrimidine-2,4-diamine derivative that we discovered to inhibit the activity of SAC. The compound caused rapid escape from the mitotic arrest induced by lack of interkinetochore tension but not by lack of MT-kinetochore attachments. In cycling cells, the compound disrupted the architecture of mitotic spindle that triggered a transient M-phase arrest that was rapidly followed by a forced mitotic exit. The premature termination of M phase was found to be a consequence of precocious inactivation of SAC caused by a direct inhibitory effect of the compound on Aurora B kinase in vitro and in cells. The compound also targets Aurora A kinase and tubulin in vitro and in cells, which can explain the observed spindle anomalies. The reduced activity of Aurora B kinase resulted in polyploidy and suppression of cancer cell viability. Our data suggest that this new pharmacophore possesses interesting anticancer properties that could be exploited in development of mitosis-targeting therapies.

Polo-like kinase-1 regulates kinetochore–microtubule dynamics and spindle checkpoint silencing

Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore-microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore-microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore-microtubule attachments and subsequently silencing the spindle checkpoint.

Error-prone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension

It is well established that chromosome segregation in female meiosis I (MI) is error-prone. The acentrosomal meiotic spindle poles do not have centrioles and are not anchored to the cortex via astral microtubules. By Cre recombinase-mediated removal in oocytes of the microtubule binding site of nuclear mitotic apparatus protein (NuMA), which is implicated in anchoring microtubules at poles, we determine that without functional NuMA, microtubules lose connection to MI spindle poles, resulting in highly disorganized early spindle assembly. Subsequently, very long spindles form with hyperfocused poles. The kinetochores of homologs make attachments to microtubules in these spindles but with reduced tension between them and accompanied by alignment defects. Despite this, the spindle assembly checkpoint is normally silenced and the advance to anaphase I and first polar body extrusion takes place without delay. Females without functional NuMA in oocytes are sterile, producing aneuploid eggs with altered chromosome number. These findings establish that in mammalian MI, the spindle assembly checkpoint is unable to sustain meiotic arrest in the presence of one or few misaligned and/or misattached kinetochores with reduced interkinetochore tension, thereby offering an explanation for why MI in mammals is so error-prone.

CENP-E Kinesin Interacts with SKAP Protein to Orchestrate Accurate Chromosome Segregation in Mitosis

Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochore. Although previous studies show that the mitotic kinesin CENP-E forms a link between attachment of the spindle microtubule to the kinetochore and the mitotic checkpoint signaling cascade, the molecular mechanism underlying dynamic kinetochore-microtubule interactions in mammalian cells remains elusive. Here, we identify a novel interaction between CENP-E and SKAP that functions synergistically in governing dynamic kinetochore-microtubule interactions. SKAP binds to the C-terminal tail of CENP-E in vitro and is essential for an accurate kinetochore-microtubule attachment in vivo. Immunoelectron microscopic analysis indicates that SKAP is a constituent of the kinetochore corona fibers of mammalian centromeres. Depletion of SKAP or CENP-E by RNA interference results in a dramatic reduction of inter-kinetochore tension, which causes chromosome mis-segregation with a prolonged delay in achieving metaphase alignment. Importantly, SKAP binds to microtubules in vitro, and this interaction is synergized by CENP-E. Based on these findings, we propose that SKAP cooperates with CENP-E to orchestrate dynamic kinetochore-microtubule interaction for faithful chromosome segregation.

The flavonoid eupatorin inactivates the mitotic checkpoint leading to polyploidy and apoptosis

The spindle assembly checkpoint (SAC) is a conserved mechanism that ensures the fidelity of chromosome distribution in mitosis by preventing anaphase onset until the correct bipolar microtubule–kinetochore attachments are formed. Errors in SAC function may contribute to tumorigenesis by inducing numerical chromosome anomalies (aneuploidy). On the other hand, total disruption of SAC can lead to massive genomic imbalance followed by cell death, a phenomena that has therapeutic potency. We performed a cell-based high-throughput screen with a compound library of 2000 bioactives for novel SAC inhibitors and discovered a plant-derived phenolic compound eupatorin (3′,5-dihydroxy-4′,6,7-trimethoxyflavone) as an anti-mitotic flavonoid. The premature override of the microtubule drug-imposed mitotic arrest by eupatorin is dependent on microtubule–kinetochore attachments but not interkinetochore tension. Aurora B kinase activity, which is essential for maintenance of normal SAC signaling, is diminished by eupatorin in cells and in vitro providing a mechanistic explanation for the observed forced mitotic exit. Eupatorin likely has additional targets since eupatorin treatment of pre-mitotic cells causes spindle anomalies triggering a transient M phase delay followed by impaired cytokinesis and polyploidy. Finally, eupatorin potently induces apoptosis in multiple cancer cell lines and suppresses cancer cell proliferation in organotypic 3D cell culture model.

HURP Regulates Chromosome Congression by Modulating Kinesin Kif18A Function

Chromosome biorientation and congression during mitosis require precise control of microtubule dynamics [1-4]. Thedynamics of kinetochore microtubules (K-MTs) are regulated by a variety of microtubule-associated proteins (MAPs) [4-9]. Recently, a MAP known as HURP (hepatoma upregulated protein) was identified [10-12]. During mitosis, Ran-guanosine 5'-triphosphate (RanGTP) releases HURP from the importin β inhibitory complex and allows it to localize to the kinetochore fiber (k-fiber) [12, 13]. HURP stabilizes k-fibers and promotes chromosome congression [12, 14, 15]. However, the molecular mechanism underlying the role of HURP in regulating chromosome congression remains elusive. Here, we show that overexpression of the N-terminal microtubule binding domain (1-278 aa, HURP(278)) of HURP induces a series of mitotic defects that mimic the effects of Kif18A depletion. In addition, coimmunoprecipitation and bimolecular fluorescence complementation assays identify Kif18A as a novel interaction partner of HURP. Furthermore, quantitative results from live-cell imaging analyses illustrate that HURP regulates Kif18A localization and dynamics at the plus end of K-MTs. Lastly, misaligned chromosomes in HURP(278)-overexpressing cells can be partially rescued by the overexpression of Kif18A. Our results demonstrate in part the regulatory mechanism for Kif18A during chromosome congression and provide new insights into the mechanism of chromosome movement at the metaphase plate.

DDA3 associates with MCAK and controls chromosome congression

DDA3 regulates spindle microtubule (MT) dynamics and chromosome movement in mitosis through its interaction with and subsequent recruitment of Kif2a, a minus end-MT depolymerase. Depletion of DDA3 causes a hyper-stabilization of spindle MT, a loss of inter-kinetochore tension, and a defect in chromosome congression, leading to unaligned chromosomes at metaphase. We report here that DDA3 is also localized at kinetochores and interacts with MCAK. Furthermore, CENP-E, a plus end-motor protein, accumulates at kinetochores in unaligned chromosomes in mitotic cells depleted of DDA3. On the other hand, the localization of chromosomal passenger complex (CPC) and the kinase activity of Aurora B are normal in DDA3-depleted cells. We conclude that MCAK and CENP-E are involved in DDA3-mediated chromosome congression.

Molecular-Mechanical Model of Kinetochore-Microtubule Interactions Identifies Flexibility of the Kinetochore Mesh as a Key Determinant of Errorless Bi-Orientation

The accuracy of chromosome segregation relies on the remarkable ability of mitotic kinetochores to bi-orient, whereby sister kinetochores form microtubule attachments to opposing spindle poles. Since the probability of forming erroneous attachments vastly exceeds the chance of attaching correctly, several mechanisms have been proposed to explain how kinetochores avoid and resolve these errors. Here, we use quantitative molecular-mechanical modeling of the kinetochore-microtubule interface to evaluate these factors and determine their respective roles. Our analysis defines several key features that ensure expedient error correction. First, geometric constraints, wich bias orientation of paired sister kinetochores such that they preferentially face opposite poles, contribute to proper attachments, but they are not sufficient to provide error-free segregation. Second, two aspects of Aurora B kinase-dependent regulation play significant and distinct roles in establishing and maintaining correct microtubule-attachments: 1) Its ability to promote the rapid turn-over of all kinetochore-microtubule attachments, not just those that are inappropriately attached, and 2) phosphor-regulation of microtubule affinity of spatially-distributed factors in a manner that depends on inter-kinetochore tension. However, a combination of geometric constraints and the error-resolving activities of Aurora B are not sufficient for a fully robust error correction. To solve this problem we hypothesize that the individual microtubule binding sites behave semi-autonomously such that intra-kinetochore tension arises locally in a flexible kinetochore meshwork. Indeed, when such a meshwork is added to the model, kinetochore bi-orientation and expedient error correction occur in a highly reproducible, deterministic way over a significant range of system parameters and even when cells are challenged by error-inducing treatments. Our work has generated the first comprehensive quantitative model to explain spatiotemporal self-organization during chromosome segregation and has provided a solid molecular-mechanical basis for the error correction mechanism.

The NDC80 complex proteins Nuf2 and Hec1 make distinct contributions to kinetochore–microtubule attachment in mitosis

Successful mitosis requires that kinetochores stably attach to the plus ends of spindle microtubules. Central to generating these attachments is the NDC80 complex, made of the four proteins Spc24, Spc25, Nuf2, and Hec1/Ndc80. Structural studies have revealed that portions of both Hec1 and Nuf2 N termini fold into calponin homology (CH) domains, which are known to mediate microtubule binding in certain proteins. Hec1 also contains a basic, positively charged stretch of amino acids that precedes its CH domain, referred to as the "tail." Here, using a gene silence and rescue approach in HeLa cells, we show that the CH domain of Hec1, the CH domain of Nuf2, and the Hec1 tail each contributes to kinetochore-microtubule attachment in distinct ways. The most severe defects in kinetochore-microtubule attachment were observed in cells rescued with a Hec1 CH domain mutant, followed by those rescued with a Hec1 tail domain mutant. Cells rescued with Nuf2 CH domain mutants, however, generated stable kinetochore-microtubule attachments but failed to generate wild-type interkinetochore tension and failed to enter anaphase in a timely manner. These data suggest that the CH and tail domains of Hec1 generate essential contacts between kinetochores and microtubules in cells, whereas the Nuf2 CH domain does not.

Release of Mps1 from kinetochores is crucial for timely anaphase onset

Mps1 kinase activity is required for proper chromosome segregation during mitosis through its involvements in microtubule-chromosome attachment error correction and the mitotic checkpoint. Mps1 dynamically exchanges on unattached kinetochores but is largely removed from kinetochores in metaphase. Here we show that Mps1 promotes its own turnover at kinetochores and that removal of Mps1 upon chromosome biorientation is a prerequisite for mitotic checkpoint silencing. Inhibition of Mps1 activity increases its half-time of recovery at unattached kinetochores and causes accumulation of Mps1 protein at these sites. Strikingly, preventing dissociation of active Mps1 from kinetochores delays anaphase onset despite normal chromosome attachment and alignment, and high interkinetochore tension. This delay is marked by continued recruitment of Mad1 and Mad2 to bioriented chromosomes and is attenuated by Mad2 depletion, indicating chronic engagement of the mitotic checkpoint in metaphase. We propose that release of Mps1 from kinetochores is essential for mitotic checkpoint silencing and a fast metaphase-to-anaphase transition.

Mitotic Kinesin CENP-E Promotes Microtubule Plus-End Elongation

Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis, including establishment of microtubule (MT)-chromosome linkage and movement of mono-oriented chromosomes on kinetochore microtubules for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus ends. A human CENP-E construct was engineered, expressed, and purified, and it yielded theCENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end-directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity-marked MTs exhibited CENP-E-promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 μm/30 min. Immunolocalization studies revealed that 80.8% of plus-end-elongated MTs showed CENP-E at the MT plus end. The time dependence of CENP-E-promoted MT elongation in solution best fit a single exponential function (k(obs) = 5.1 s(-1)), which is indicative of a mechanism in which α,β-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E, as part of its function in chromosome kinetochore-MT linkage, plays a direct role in MT elongation.

SKAP associates with kinetochores and promotes the metaphase-to-anaphase transition

The spindle assembly checkpoint (SAC) controls the anaphase onset by preventing premature chromosome segregation until bipolar microtubule (MT) attachment and inter-kinetochore tension are fully established for every kinetochore pair. Once the SAC is off, activation of the Anaphase-Promoting Complex/Cyclosome, a ubiquitin ligase, leads to the degradation of securin and cyclin B, the activation of separase and the initiation of anaphase. We report here the identification and characterization of a G2-induced gene, SKAP, as a regulator for the anaphase onset. SKAP localizes to spindle MTs and kinetochores in mitosis. Depletion of SKAP does not activate the SAC, but substantially increases the duration of metaphase, delays the activation of separase, and decreases the fidelity of chromosome segregation. Our study identifies SKAP as a novel regulator of the metaphase-to-anaphase transition and demonstrates that mis-regulation of the separase activation results in a reduced fidelity of chromosome segregation and a reduced genomic stability independent of the SAC.