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.     

Anaphase spindle mechanics prevent mis-segregation of merotelically oriented chromosomes

Merotelic kinetochore orientation is a kinetochore misattachment in which a single kinetochore is attached to microtubules from both spindle poles instead of just one. It can be favored in specific circumstances, is not detected by the mitotic checkpoint, and induces lagging chromosomes in anaphase. In mammalian cells, it occurs at high frequency in early mitosis, but few anaphase cells show lagging chromosomes. We developed live-cell imaging methods to determine whether and how the mitotic spindle prevents merotelic kinetochores from producing lagging chromosomes. We found that merotelic kinetochores entering anaphase never lost attachment to the spindle poles; they remained attached to both microtubule bundles, but this did not prevent them from segregating correctly. The two microtubule bundles usually showed different fluorescence intensities, the brighter bundle connecting the merotelic kinetochore to the correct pole. During anaphase, the dimmer bundle lengthened much more than the brighter bundle as spindle elongation occurred. This resulted in correct segregation of the merotelically oriented chromosome. We propose a model based on the ratios of microtubules to the correct versus incorrect pole for how anaphase spindle dynamics and microtubule polymerization at kinetochores prevent potential segregation errors deriving from merotelic kinetochore orientation.     

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.     

Chromosome segregation: monopolin attracts condensin

To segregate chromosomes properly, the cell must prevent merotely, an error that occurs when a single kinetochore is attached to microtubules emanating from both spindle poles. Recent evidence suggests that cooperation between Pcs1/Mde4 and condensin complexes plays an important role in preventing merotely.     

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.     

A role for metaphase spindle elongation forces in correction of merotelic kinetochore attachments

During mitosis, equal segregation of chromosomes depends on proper kinetochore-microtubule attachments. Merotelic kinetochore orientation, in which a single kinetochore binds microtubules from both spindle poles [1], is a major cause of chromosome instability [2], which is commonly observed in solid tumors [3, 4]. Using the fission yeast Schizosaccharomyces pombe, we show that a proper force balance between kinesin motors on interpolar spindle microtubules is critical for correcting merotelic attachments. Inhibition of the plus-end-directed spindle elongation motors kinesin-5 (Cut7) and kinesin-6 (Klp9) reduces spindle length, tension at kinetochores, and the frequency of merotelic attachments. In contrast, merotely is increased by deletion of the minus-end-directed kinesin-14 (Klp2) or overexpression of Klp9. Also, Cdk1 regulates spindle elongation forces to promote merotelic correction by phosphorylating and inhibiting Klp9. The role of spindle elongation motors in merotelic correction is conserved, because partial inhibition of the human kinesin-5 homolog Eg5 using the drug monastrol reduces spindle length and lagging chromosome frequency in both normal (RPE-1) and tumor (CaCo-2) cells. These findings reveal unexpected links between spindle forces and correction of merotelic attachments and show that pharmacological manipulation of spindle elongation forces might be used to reduce chromosome instability in cancer cells.     

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.     

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.     

Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint

The spindle assembly checkpoint (SAC) is the major surveillance system that ensures that sister chromatids do not separate until all chromosomes are correctly bioriented during mitosis. Components of the checkpoint include Mad1, Mad2, Mad3 (BubR1), Bub3, and the kinases Bub1, Mph1 (Mps1), and Aurora B. Checkpoint proteins are recruited to kinetochores when individual kinetochores are not bound to spindle microtubules or not under tension. Kinetochore association of Mad2 causes it to undergo a conformational change, which promotes its association to Mad3 and Cdc20 to form the mitotic checkpoint complex (MCC). The MCC inhibits the anaphase-promoting complex/cyclosome (APC/C) until the checkpoint is satisfied. SAC silencing derepresses Cdc20-APC/C activity. This triggers the polyubiquitination of securin and cyclin, which promotes the dissolution of sister chromatid cohesion and mitotic progression. We, and others, recently showed that association of PP1 to the Spc7/Spc105/KNL1 family of kinetochore proteins is necessary to stabilize microtubule-kinetochore attachments and silence the SAC. We now report that phosphorylation of the conserved MELT motifs in Spc7 by Mph1 (Mps1) recruits Bub1 and Bub3 to the kinetochore and that this is required to maintain the SAC signal.     

Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore

Kinetochores attach the replicated chromosomes to the mitotic spindle and orchestrate their transmission to the daughter cells. Kinetochore-spindle binding and chromosome segregation are mediated by the multi-copy KNL1(Spc105), MIS12(Mtw1) and NDC80(Ndc80)?complexes that form the so-called KMN network. KMN-spindle attachment is regulated by the Aurora?B(Ipl1) and MPS1(Mps1)?kinases. It is unclear whether other mechanisms exist that support KMN activity during the cell cycle. Using budding yeast, we show that kinetochore protein Cnn1 localizes to the base of the Ndc80 complex and promotes a functionally competent configuration of the KMN network. Cnn1 regulates KMN activity in a spatiotemporal manner by inhibiting the interaction between its complexes. Cnn1 activity peaks in anaphase and is driven by the Cdc28, Mps1 and Ipl1 kinases.     

Spc24 and Stu2 promote spindle integrity when DNA replication is stalled

The kinetochore, a protein complex that links chromosomes to microtubules (MTs), is required to prevent spindle expansion during S phase in budding yeast, but the mechanism of how the kinetochore maintains integrity of the bipolar spindle before mitosis is not well understood. Here, we demonstrate that a mutation of Spc24, a component of the conserved Ndc80 kinetochore complex, causes lethality when cells are exposed to the DNA replication inhibitor hydroxyurea (HU) due to premature spindle expansion and segregation of incompletely replicated DNA. Overexpression of Stu1, a CLASP-related MT-associated protein or a truncated form of the XMAP215 orthologue Stu2 rescues spc24-9 HU lethality and prevents spindle expansion. Truncated Stu2 likely acts in a dominant-negative manner, because overexpression of full-length STU2 does not rescue spc24-9 HU lethality, and spindle expansion in spc24-9 HU-treated cells requires active Stu2. Stu1 and Stu2 localize to the kinetochore early in the cell cycle and Stu2 kinetochore localization depends on Spc24. We propose that mislocalization of Stu2 results in premature spindle expansion in S phase stalled spc24-9 mutants. Identifying factors that restrain spindle expansion upon inhibition of DNA replication is likely applicable to the mechanism by which spindle elongation is regulated during a normal cell cycle.     

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.     

Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1

The spindle checkpoint controls mitotic progression. Checkpoint proteins are temporally recruited to kinetochores, but their docking site is unknown. We show that a human kinetochore oncoprotein, AF15q14/blinkin, a member of the Spc105/Spc7/KNL-1 family, directly links spindle checkpoint proteins BubR1 and Bub1 to kinetochores and is required for spindle checkpoint and chromosome alignment. Blinkin RNAi causes accelerated mitosis due to a checkpoint failure and chromosome misalignment resulting from the lack of kinetochore and microtubule attachment. Blinkin RNAi phenotypes resemble the double RNAi phenotypes of Bub1 and BubR1 in living cells. While the carboxy domain associates with the c20orf172/hMis13 and DC8/hMis14 subunits of the hMis12 complex in the inner kinetochore, association of the amino and middle domain of blinkin with the TPR domains in the amino termini of BubR1 and Bub1 is essential for BubR1 and Bub1 to execute their distinct mitotic functions. Blinkin may be the center of the network for generating kinetochore-based checkpoint signaling.     

Changes in the localization of the Saccharomyces cerevisiae anaphase-promoting complex upon microtubule depolymerization and spindle checkpoint activation

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase in the ubiquitin-mediated proteolysis pathway (UMP). To understand how the APC/C was targeted to its substrates, we performed a detailed analysis of one of the APC/C components, Cdc23p. In live cells, Cdc23-GFP localized to punctate nuclear spots surrounded by homogenous nuclear signal throughout the cell cycle. These punctate spots colocalized with two outer kinetochore proteins, Slk19p and Okp1p, but not with the spindle pole body protein, Spc42p. In late anaphase, the Cdc23-GFP was also visualized along the length of the mitotic spindle. We hypothesized that spindle checkpoint activation may affect the APC/C nuclear spot localization. Localization of Cdc23-GFP was disrupted upon nocodazole treatment in the kinetochore mutant okp1-5 and in the cdc20-1 mutant. Cdc23-GFP nuclear spot localization was not affected in the ndc10-1 mutant, which is defective in spindle checkpoint function. Additional studies using a mad2Delta strain revealed a microtubule dependency of Cdc23-GFP spot localization, whether or not the checkpoint response was activated. On the basis of these data, we conclude that Cdc23p localization was dependent on microtubules and was affected by specific types of kinetochore disruption.     

Merotelic kinetochore attachment in oocyte meiosis II causes sister chromatids segregation errors in aged mice

Mammalian oocyte chromosomes undergo 2 meiotic divisions to generate haploid gametes. The frequency of chromosome segregation errors during meiosis I increase with age. However, little attention has been paid to the question of how aging affects sister chromatid segregation during oocyte meiosis II. More importantly, how aneuploid metaphase II (MII) oocytes from aged mice evade the spindle assembly checkpoint (SAC) mechanism to complete later meiosis II to form aneuploid embryos remains unknown. Here, we report that MII oocytes from naturally aged mice exhibited substantial errors in chromosome arrangement and configuration compared with young MII oocytes. Interestingly, these errors in aged oocytes had no impact on anaphase II onset and completion as well as 2-cell formation after parthenogenetic activation. Further study found that merotelic kinetochore attachment occurred more frequently and could stabilize the kinetochore-microtubule interaction to ensure SAC inactivation and anaphase II onset in aged MII oocytes. This orientation could persist largely during anaphase II in aged oocytes, leading to severe chromosome lagging and trailing as well as delay of anaphase II completion. Therefore, merotelic kinetochore attachment in oocyte meiosis II exacerbates age-related genetic instability and is a key source of age-dependent embryo aneuploidy and dysplasia.     

The budding yeast proteins Spc24p and Spc25p interact with Ndc80p and Nuf2p at the kinetochore and are important for kinetochore clustering and checkpoint control

Here, we show that the budding yeast proteins Ndc80p, Nuf2p, Spc24p and Spc25p interact at the kinetochore. Consistently, Ndc80p, Nuf2p, Spc24p and Spc25p associate with centromere DNA in chromatin immunoprecipitation experiments, and SPC24 interacts genetically with MCM21 encoding a kinetochore component. Moreover, although conditional lethal spc24-2 and spc25-7 cells form a mitotic spindle, the kinetochores remain in the mother cell body and fail to segregate the chromosomes. Despite this defect in chromosome segregation, spc24-2 and spc25-7 cells do not arrest in metaphase in response to checkpoint control. Furthermore, spc24-2 cells showed a mitotic checkpoint defect when microtubules were depolymerized with nocodazole, indicating that Spc24p has a function in checkpoint control. Since Ndc80p, Nuf2p and Spc24p are conserved proteins, it is likely that similar complexes are part of the kinetochore in other organisms.     

Perturbation of Spc25 expression affects meiotic spindle organization,chromosome alignment and spindle assembly checkpoint in mouse oocytes.

Spc25 is a component of the Ndc80 complex which consists of Ndc80, Nuf2, Spc24, and Spc25. Previous work has shown that Spc25 is involved in regulation of kinetochore microtubule attachment and the spindle assembly checkpoint in mitosis. The roles of Spc25 in meiosis remain unknown. Here, we report its expression, localization and functions in mouse oocyte meiosis. The Spc25 mRNA level gradually increased from the GV to MI stage, but decreased by MII during mouse oocyte meiotic maturation. Immunofluorescent staining showed that Spc25 was restricted to the germinal vesicle, and associated with chromosomes during all stages after GVBD. Overexpression of Spc25 by mRNA injection resulted in oocyte meiotic arrest, chromosome misalignment and spindle disruption. Conversely, Spc25 RNAi by siRNA injection resulted in precocious polar body extrusion and caused severe chromosome misalignment and aberrant spindle formation. Our data suggest that Spc25 is required for chromosome alignment, spindle formation, and proper spindle checkpoint signaling during meiosis.     

Phosphoregulation of Spc105 by Mps1 and PP1 regulates Bub1 localization to kinetochores

Kinetochores are the macromolecular complexes that interact with microtubules to mediate chromosome segregation. Accurate segregation requires that kinetochores make bioriented attachments to microtubules from opposite poles. Attachments between kinetochores and microtubules are monitored by the spindle checkpoint, a surveillance system that prevents anaphase until every pair of chromosomes makes proper bioriented attachments. Checkpoint activity is correlated with the recruitment of checkpoint proteins to the kinetochore. Mps1 is a conserved protein kinase that regulates segregation and the spindle checkpoint, but few of the targets that mediate its functions have been identified. Here, we show that Mps1 is the major kinase activity that copurifies with budding yeast kinetochore particles and identify the conserved Spc105/KNL-1/blinkin kinetochore protein as a substrate. Phosphorylation of conserved MELT motifs within Spc105 recruits the Bub1 protein to kinetochores, and this is reversed by protein phosphatase I (PP1). Spc105 mutants lacking Mps1 phosphorylation sites are defective in the spindle checkpoint and exhibit growth defects. Together, these data identify Spc105 as a key target of the Mps1 kinase and show that the opposing activities of Mps1 and PP1 regulate the kinetochore localization of the Bub1 protein.