Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 microM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2--5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.     

The kinetochore proteins Pcs1 and Mde4 and heterochromatin are required to prevent merotelic orientation

BACKGROUND: Accurate chromosome segregation depends on the establishment of correct-amphitelic-kinetochore orientation. Merotelic kinetochore orientation is an error that occurs when a single kinetochore attaches to microtubules emanating from opposite spindle poles, a condition that hinders segregation of the kinetochore to a spindle pole in anaphase. To avoid chromosome missegregation resulting from merotelic kinetochore orientation, cells have developed mechanisms to prevent or correct merotelic attachment. A protein called Pcs1 has been implicated in preventing merotelic attachment in mitosis and meiosis II in the fission yeast S. pombe. RESULTS: We report that Pcs1 forms a complex with a protein called Mde4. Both Pcs1 and Mde4 localize to the central core of centromeres. Deletion of mde4(+), like that of pcs1(+), causes the appearance of lagging chromosomes during the anaphases of mitotic and meiosis II cells. We provide evidence that the kinetochores of lagging chromosomes in both pcs1 and mde4 mutant cells are merotelically attached. In addition, we find that lagging chromosomes in cells with defective centromeric heterochromatin also display features consistent with merotelic attachment. CONCLUSIONS: We suggest that the Pcs1/Mde4 complex is the fission yeast counterpart of the budding yeast monopolin subcomplex Csm1/Lrs4, which promotes the segregation of sister kinetochores to the same pole during meiosis I. We propose that the Pcs1/Mde4 complex acts in the central kinetochore domain to clamp microtubule binding sites together, the centromeric heterochromatin coating the flanking domains provides rigidity, and both systems contribute to the prevention of merotelic attachment.     

Merotelic kinetochore attachment: causes and effects

Accurate chromosome segregation depends on the proper attachment of sister kinetochores to microtubules emanating from opposite spindle poles. Merotelic kinetochore orientation is an error in which a single kinetochore is attached to microtubules emanating from both spindle poles. Despite correction mechanisms, merotelically attached kinetochores can persist until anaphase, causing chromatids to lag on the mitotic spindle and hindering their timely segregation. Recent studies showing that merotelic kinetochore attachment represents a major mechanism of aneuploidy in mitotic cells and is the primary mechanism of chromosomal instability in cancer cells have underlined the importance of studying merotely. Here, we highlight recent progress in our understanding of how cells prevent and correct merotelic kinetochore attachments.     

Merotelic kinetochores in mammalian tissue cells

Merotelic kinetochore attachment is a major source of aneuploidy in mammalian tissue cells in culture. Mammalian kinetochores typically have binding sites for about 20-25 kinetochore microtubules. In prometaphase, kinetochores become merotelic if they attach to microtubules from opposite poles rather than to just one pole as normally occurs. Merotelic attachments support chromosome bi-orientation and alignment near the metaphase plate and they are not detected by the mitotic spindle checkpoint. At anaphase onset, sister chromatids separate, but a chromatid with a merotelic kinetochore may not be segregated correctly, and may lag near the spindle equator because of pulling forces toward opposite poles, or move in the direction of the wrong pole. Correction mechanisms are important for preventing segregation errors. There are probably more than 100 times as many PtK1 tissue cells with merotelic kinetochores in early mitosis, and about 16 times as many entering anaphase as the 1% of cells with lagging chromosomes seen in late anaphase. The role of spindle mechanics and potential functions of the Ndc80/Nuf2 protein complex at the kinetochore/microtubule interface is discussed for two correction mechanisms: one that functions before anaphase to reduce the number of kinetochore microtubules to the wrong pole, and one that functions after anaphase onset to move merotelic kinetochores based on the ratio of kinetochore microtubules to the correct versus incorrect pole.     

Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors

Merotelic kinetochore orientation is a misattachment in which a single kinetochore binds microtubules from both spindle poles rather than just one and can produce anaphase lagging chromosomes, a major source of aneuploidy. Merotelic kinetochore orientation occurs frequently in early mitosis, does not block chromosome alignment at the metaphase plate, and is not detected by the spindle checkpoint. However, microtubules to the incorrect pole are usually significantly reduced or eliminated before anaphase. We discovered that the frequency of lagging chromosomes in anaphase is very sensitive to partial inhibition of Aurora kinase activity by ZM447439 at a dose, 3 microM, that has little effect on histone phosphorylation, metaphase chromosome alignment, and cytokinesis in PtK1 cells. Partial Aurora kinase inhibition increased the frequency of merotelic kinetochores in late metaphase, and the fraction of microtubules to the incorrect pole. Measurements of fluorescence dissipation after photoactivation showed that kinetochore-microtubule turnover in prometaphase is substantially suppressed by partial Aurora kinase inhibition. Our results support a preanaphase correction mechanism for merotelic attachments in which correct plus-end attachments are pulled away from high concentrations of Aurora B at the inner centromere, and incorrect merotelic attachments are destabilized by being pulled toward the inner centromere.     

Laser microsurgery provides evidence for merotelic kinetochore attachments in fission yeast cells lacking Pcs1 or Clr4

In order to segregate chromosomes properly, the cell must prevent merotelic kinetochore attachment, an error that occurs when a single kinetochore is attached to microtubules emanating from both spindle poles. Merotelic kinetochore orientation represents a major mechanism of aneuploidy in mitotic mammalian cells and it is the primary mechanism of chromosome instability in cancer cells. Fission yeast mutants defective in putative microtubule-site clamp Pcs1/Mde4 or Clr4/Swi6-dependent centromeric heterochromatin display high frequencies of lagging chromosomes during anaphase. Here, we developed an assay based on laser microsurgery to show that the stretched morphology of lagging kinetochores in pcs1Δ and clr4Δ mutant cells is due to merotelic attachment. We further show that Mde4 is regulated by Cdc2 and that Cdc2 activity prevents precocious localization of Mde4 to the metaphase spindle. Finally, we show that Pcs1/Mde4 complex shares similar features with the conserved kinetochore complex Spc24/Spc25 suggesting that these two complexes may occupy a similar functional niche.     

Detection and Correction of Merotelic Kinetochore Orientation by Aurora B and its Partners

Merotelic kinetochore orientation is a kinetochore-microtubule mis-attachment in which a single kinetochore binds microtubules to both spindle poles, rather than just one. Merotelic attachments occur frequently in early mitosis and can induce anaphase lagging chromosomes and aneuploidy if not corrected before anaphase onset. Merotelic kinetochore orientation does not interfere with chromosome alignment at the metaphase plate and does not activate the mitotic spindle checkpoint. However, a correction mechanism for merotelic attachment reduces the number of merotelic kinetochores entering anaphase, thus preventing chromosome mis-segregation. Result from many different studies support the idea that Aurora B kinase plays a critical role in this merotelic correction mechanism by phosphorylating key substrates at the kinetochore and promoting turnover of kinetochore microtubules. In addition, recent studies are starting to identify the possible ‘sensors’ of the system that would be able to detect the mis-attachment and communicate this to Aurora B. Here, I review these studies and discuss a model for how merotelic kinetochore orientation could be detected and corrected before anaphase onset.     

Malorientation in half-bivalents at anaphase in crane fly spermatocytes following Colcemid treatment

Addition of Colcemid to the medium in which larvae of the crane fly Nephrotoma suturalis are cultivated induces a number of anomalous patterns of chromosome segregation. One of these is the anaphase lagging of autosomal half-bivalents. To investigate the cause of anaphase lagging, the orientation of sister kinetochores in Colcemidtreated spermatocytes having lagging half-bivalents was analyzed in serial sections. In contrast to nonlaggard halfbivalents that had pure syntelic orientation (sister kinetochores having all of their kinetochores microtubules (KMTs) extending to the same pole), six of the seven autosomal laggards that were selected for analysis had kinetochores with either amphitelic orientation (sister kinetochores each with a bundle of KMTs extending to opposite poles) or merotelic orientation (a single kinetochore having KMTs extending toward both poles). An additional laggard had syntelic orientation but two of the microtubules that were in its kinetochore fiber passed through the kinetochore and extended beyond it toward the equator. The bipolar malorientations observed in anaphase half-bivalents are interpreted to be a cause of the anaphase lagging induced by Colcemid treatment. Furthermore, it is hypothesized that such bipolar malorientations also may be stabilized at metaphase and thus explain the unusual tilting of metaphase bivalents commonly observed in Colcemid-treated cells.     

Bi-orienting chromosomes: acrobatics on the mitotic spindle

To maintain their genetic integrity, eukaryotic cells must segregate their chromosomes properly to opposite poles during mitosis. This process mainly depends on the forces generated by microtubules that attach to kinetochores. During prometaphase, kinetochores initially interact with a single microtubule that extends from a spindle pole and then move towards a spindle pole. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles (sister kinetochore bi-orientation). If sister kinetochores interact with microtubules in wrong orientation, this must be corrected before the onset of anaphase. Here, I discuss the processes leading to bi-orientation and the mechanisms ensuring this pivotal state that is required for proper chromosome segregation.     

Micromanipulation of Chromosomes in Mitotic Vertebrate Tissue Cells: Tension Controls the State of Kinetochore Movement

In mitotic vertebrate tissue cells, chromosome congression to the spindle equator in prometaphase and segregation to the poles in anaphase depend on the movements of kinetochores at their kinetochore microtubule attachment sites. To test if kinetochores sense tension to control their states of movement poleward (P) and away from the pole (AP), we applied an external force to the spindle in preanaphase newt epithelial cells by stretching chromosome arms with microneedles. For monooriented chromosomes (only one kinetochore fiber), an abrupt stretch of an arm away from the attached pole induced the single attached kinetochore to persist in AP movement at about 2 μm/min velocity, resulting in chromosome movement away from the pole. When the stretch was reduced or the needle removed, the kinetochore switched to P movement at about 2 μm/min and pulled the chromosome back to near the premanipulation position within the spindle. For bioriented chromosomes (sister kinetochores attached to opposite poles) near the spindle equator, stretching one arm toward a pole placed the kinetochore facing away from the direction of stretch under tension and the sister facing toward the stretch under reduced tension or compression. Kinetochores under increased tension exhibited prolonged AP movement while kinetochores under reduced tension or compression exhibited prolonged P movement, moving the centromeres at about 2 μm/min velocities off the metaphase plate in the direction of stretch. Removing the needle resulted in centromere movement back to near the spindle equator at similar velocities. These results show that tension controls the direction of kinetochore movement and associated kinetochore microtubule assembly/disassembly to position centromeres within the spindle of vertebrate tissue cells. High tension induces persistent AP movement while low tension induces persistent P movement. The velocity of P and AP movement appears to be load independent and governed by the molecular mechanisms which attach kinetochores to the dynamic ends of kinetochore microtubules.     

Immunofluorescence analysis of sucrose-induced changes in spindle morphology

Metaphase and anaphase PtK1 cells show spindle elongation without concomitant chromosome motion when treated with culture medium containing 0.5 M sucrose. Electron microscopy has shown sucrose-induced changes in microtubule (MT) organization, changes in trilaminar kinetochore structure, and specific kinetochore-MT associations which may account for these results. In this paper we employ double-label immunofluorescence techniques using antibodies against tubulin and the kinetochore to analyze changes in spindle microtubule and kinetochore distribution produced by sucrose treatment. Cells treated from prometaphase through anaphase with 0.5 M sucrose from 10 min to 2 h showed spindle elongation and a distinct rearrangement of spindle microtubules into bundles, with a pronounced increase in length of interpolar microtubule bundles. In sucrose-treated mitotic cells kinetochores remained as antigenically distinct structures, similar to those found in untreated interphase cells. Kinetochore determinants remained positioned within a diffuse chromatin mass, but the orientation of sister kinetochores to opposite spindle poles was lost. Instead, kinetochore pairs were found in lateral association with microtubule bundles, with several pairs of determinants associated with a single bundle in many instances. Cells released from 0.5 M sucrose treatment showed a return of the spindle to a pretreatment arrangement for both the microtubules and kinetochore determinants.     

Loss of KLP-19 polar ejection force causes misorientation and missegregation of holocentric chromosomes

Holocentric chromosomes assemble kinetochores along their length instead of at a focused spot. The elongated expanse of an individual holocentric kinetochore and its potential flexibility heighten the risk of stable attachment to microtubules from both poles of the mitotic spindle (merotelic attachment), and hence aberrant segregation of chromosomes. Little is known about the mechanisms that holocentric species have evolved to avoid this type of error. Our studies of the influence of KLP-19, an essential microtubule motor, on the behavior of holocentric Caenorhabditis elegans chromosomes suggest that it has a major role in combating merotelic attachments. Depletion of KLP-19, which associates with nonkinetochore chromatin, allows aberrant poleward chromosome motion during prometaphase, misalignment of holocentric kinetochores, and multiple anaphase chromosome bridges in all mitotic divisions. Time-lapse movies of GFP-labeled mono- and bipolar spindles demonstrate that KLP-19 generates a force on relatively stiff holocentric chromosomes that pushes them away from poles. We hypothesize that this polar ejection force minimizes merotelic misattachment by maintaining a constant tension on pole-kinetochore connections throughout prometaphase, tension that compels sister kinetochores to face directly toward opposite poles.     

Kinetochore capture and bi-orientation on the mitotic spindle

Kinetochores are large protein complexes that are formed on chromosome regions known as centromeres. For high-fidelity chromosome segregation, kinetochores must be correctly captured on the mitotic spindle before anaphase onset. During prometaphase, kinetochores are initially captured by a single microtubule that extends from a spindle pole and are then transported poleward along the microtubule. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles - this is known as bi-orientation. Here we discuss the molecular mechanisms of these processes, by focusing on budding yeast and drawing comparisons with other organisms.     

Orientation of nonrandomly segregating sex chromosomes in spermatocytes of the flea beetle, Alagoasa bicolor L.

In males of the flea beetle, Alagoasa bicolor L., spermatocytes have two achiasmate sex chromosomes, X and Y, each of which is approximately five times larger than the ten pairs of chiasmate autosomes. At metaphase I, these univalent sex chromosomes are located on a spindle domain separated from the autosomal spindle domain by a sheath of mitochondria. A single centriole pair is located at each pole of the spindle. In prometaphase I, each sex chromosome appears to maintain an attachment to both spindle poles via kinetochore microtubules (i.e., amphitelic orientation). Before anaphase I, this orientation changes to the syntelic orientation (both sister kinetochores connected to the same pole), perhaps by the release of microtubule attachments from the more distant pole by each of the chromosomes. The syntelic orientation just prior to anaphase I leaves each sex chromosome attached to the nearest pole via kinetochore microtubules, ensuring nonrandom segregation. As the sex chromosomes reorient, the autosomes follow in a sequential manner, starting with the bivalent closest to the sex spindle domain. We report here data that shed new light on the mechanism of this exceptional meiotic chromosome behavior.     

Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells

The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest-deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2-Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a t(1/2) of approximately 24-28 s. Cells entered anaphase approximately 10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.     

Cyclin B destruction triggers changes in kinetochore behavior essential for successful anaphase

Successful mitosis requires that anaphase chromosomes sustain a commitment to move to their assigned spindle poles. This requires stable spindle attachment of anaphase kinetochores. Prior to anaphase, stable spindle attachment depends on tension created by opposing forces on sister kinetochores [1]. Because tension is lost when kinetochores disjoin, stable attachment in anaphase must have a different basis. After expression of nondegradable cyclin B (CYC-B(S)) in Drosophila embryos, sister chromosomes disjoined normally but their anaphase behavior was abnormal [2]. Chromosomes exhibited cycles of reorientation from one pole to the other. Additionally, the unpaired kinetochores accumulated attachments to both poles (merotelic attachments), congressed (again) to a pseudometaphase plate, and reacquired associations with checkpoint proteins more characteristic of prometaphase kinetochores. Unpaired prometaphase kinetochores, which occurred in a mutant entering mitosis with unreplicated (unpaired) chromosomes, behaved just like the anaphase kinetochores at the CYC-B(S) arrest. Finally, the normal anaphase release of AuroraB/INCENP from kinetochores was blocked by CYC-B(S) expression and, reciprocally, was advanced in a CycB mutant. Given its established role in destabilizing kinetochore-microtubule interactions [3], Aurora B dissociation is likely to be key to the change in kinetochore behavior. These findings show that, in addition to loss of sister chromosome cohesion, successful anaphase requires a kinetochore behavioral transition triggered by CYC-B destruction.     

Kinetochore microtubule dynamics and attachment stability are regulated by Hec1

Mitotic cells face the challenging tasks of linking kinetochores to growing and shortening microtubules and actively regulating these dynamic attachments to produce accurate chromosome segregation. We report here that Ndc80/Hec1 functions in regulating kinetochore microtubule plus-end dynamics and attachment stability. Microinjection of an antibody to the N terminus of Hec1 suppresses both microtubule detachment and microtubule plus-end polymerization and depolymerization at kinetochores of PtK1 cells. Centromeres become hyperstretched, kinetochore fibers shorten from spindle poles, kinetochore microtubule attachment errors increase, and chromosomes severely mis-segregate. The N terminus of Hec1 is phosphorylated by Aurora B kinase in vitro, and cells expressing N-terminal nonphosphorylatable mutants of Hec1 exhibit an increase in merotelic attachments, hyperstretching of centromeres, and errors in chromosome segregation. These findings reveal a key role for the Hec1 N terminus in controlling dynamic behavior of kinetochore microtubules.     

Bipolar spindle attachments affect redistributions of ZW10, a Drosophila centromere/kinetochore component required for accurate chromosome segregation

Previous efforts have shown that mutations in the Drosophila ZW10 gene cause massive chromosome missegregation during mitotic divisions in several tissues. Here we demonstrate that mutations in ZW10 also disrupt chromosome behavior in male meiosis I and meiosis II, indicating that ZW10 function is common to both equational and reductional divisions. Divisions are apparently normal before anaphase onset, but ZW10 mutants exhibit lagging chromosomes and irregular chromosome segregation at anaphase. Chromosome missegregation during meiosis I of these mutants is not caused by precocious separation of sister chromatids, but rather the nondisjunction of homologs. ZW10 is first visible during prometaphase, where it localizes to the kinetochores of the bivalent chromosomes (during meiosis I) or to the sister kinetochores of dyads (during meiosis II). During metaphase of both divisions, ZW10 appears to move from the kinetochores and to spread toward the poles along what appear to be kinetochore microtubules. Redistributions of ZW10 at metaphase require bipolar attachments of individual chromosomes or paired bivalents to the spindle. At the onset of anaphase I or anaphase II, ZW10 rapidly relocalizes to the kinetochore regions of the separating chromosomes. In other mutant backgrounds in which chromosomes lag during anaphase, the presence or absence of ZW10 at a particular kinetochore predicts whether or not the chromosome moves appropriately to the spindle poles. We propose that ZW10 acts as part of, or immediately downstream of, a tension-sensing mechanism that regulates chromosome separation or movement at anaphase onset.     

Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 "dynamitin" protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.     

Merotelic kinetochore orientation, aneuploidy, and cancer

Accurate chromosome segregation in mitosis is crucial to maintain a diploid chromosome number. A majority of cancer cells are aneuploid and chromosomally unstable, i.e. they tend to gain and lose chromosomes at each mitotic division. Chromosome mis-segregation can arise when cells progress through mitosis with mis-attached kinetochores. Merotelic kinetochore orientation, a type of mis-attachment in which a single kinetochore binds microtubules from two spindle poles rather than just one, can represent a particular threat for dividing cells, as: (i) it occurs frequently in early mitosis; (ii) it is not detected by the spindle assembly checkpoint (unlike other types of mis-attachments); (iii) it can lead to chromosome mis-segregation, and, hence, aneuploidy. A number of studies have recently started to unveil the cellular and molecular mechanisms involved in merotelic kinetochore formation and correction. Here, I review these studies and discuss the relevance of merotelic kinetochore orientation in cancer cell biology.