Disruption of astral microtubule contact with the cell cortex activates a Bub1, Bub3, and Mad3-dependent checkpoint in fission yeast

In animal and yeast cells, the mitotic spindle is aligned perpendicularly to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring. In fission yeast, spindle rotation is dependent upon the interaction of astral microtubules with the cortical actin cytoskeleton. In this article, we show that addition of Latrunculin A, which prevents spindle rotation, delays the separation of sister chromatids and anaphase promoting complex-mediated destruction of spindle-associated Securin and Cyclin B. Moreover, we find that whereas sister kinetochore pairs normally congress to the spindle midzone before anaphase onset, this congression is disrupted when astral microtubule contact with the actin cytoskeleton is disturbed. By analyzing the timing of kinetochore separation, we find that this anaphase delay requires the Bub3, Mad3, and Bub1 but not the Mad1 or Mad2 spindle assembly checkpoint proteins. In agreement with this, we find that Bub1 remains associated with kinetochores when spindles are mispositioned. These data indicate that, in fission yeast, astral microtubule contact with the medial cell cortex is monitored by a subset of spindle assembly checkpoint proteins. We propose that this checkpoint ensures spindles are properly oriented before anaphase takes place.     

Novel mad2 alleles isolated in a Schizosaccharomyces pombe gamma-tubulin mutant are defective in metaphase arrest activity, but remain functional for chromosome stability in unperturbed mitosis

A previously isolated fission yeast gamma-tubulin mutant containing apparently stabilized microtubules proliferated at an approximately identical rate as wild type, yet the mutant mitosis spindle dynamics were aberrant, particularly the kinetochore microtubule dynamics. Progression through mitosis in the mutant, however, resulted in mostly accurate chromosome segregation. In the absence of the spindle assembly checkpoint gene, mad2+, the spindle dynamics in the gamma-tubulin mutant were greatly compromised, leading to a high incidence of chromosome missegregation. Unlike in wild-type cells, green fluorescent protein (GFP)-tagged Mad2 protein often accumulated near one of the poles of an elongating spindle in the gamma-tubulin mutant. We isolated novel mad2 mutants that were defective in arresting mitotic progression upon gross perturbation of the spindle formation but remained functional for the viability of the gamma-tubulin mutant. Further, the mad2 mutations did not appreciably destabilize minichromosomes in unperturbed mitoses. When overexpressed ectopically, these mutant Mad2 proteins sequestered wild-type Mad2, preventing its function in mitotic checkpoint arrest, but not in minichromosome stability. These results indicated that the Mad2 functions required for checkpoint arrest and chromosome stability in unperturbed mitosis are genetically discernible. Immunoprecipitation studies demonstrated that GFP-fused mutant Mad2 proteins formed a Mad1-containing complex with altered stability compared to that formed with wild-type Mad2, providing clues to the novel mad2 mutant phenotype.     

Sgo1 is required for co-segregation of sister chromatids during achiasmate meiosis I

The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation, called meiosis I and meiosis II. While meiosis II is similar to mitosis in that sister kinetochores are bi-oriented and segregate to opposite poles, recombined homologous chromosomes segregate during the first meiotic division. Formation of chiasmata, mono-orientation of sister kinetochores and protection of centromeric cohesion are three major features of meiosis I chromosomes which ensure the reductional nature of chromosome segregation. Here we show that sister chromatids frequently segregate to opposite poles during meiosis I in fission yeast cells that lack both chiasmata and the protector of centromeric cohesion Sgo1. Our data are consistent with the notion that sister kinetochores are frequently bi-oriented in the absence of chiasmata and that Sgo1 prevents equational segregation of sister chromatids during achiasmate meiosis I.Key words: meiosis, chromosome segregation, recombination, kinetochore, Sgo1, fission yeast     

Lack of tension at kinetochores activates the spindle checkpoint in budding yeast

The spindle checkpoint delays the onset of anaphase until all pairs of sister chromatids are attached to the mitotic spindle. The checkpoint could monitor the attachment of microtubules to kinetochores, the tension that results from the two sister chromatids attaching to opposite spindle poles, or both. We tested the role of tension by allowing cells to enter mitosis without a prior round of DNA replication. The unreplicated chromatids are attached to spindle microtubules but are not under tension since they lack a sister chromatid that could attach to the opposite pole. Because the spindle checkpoint is activated in these cells, we conclude that the absence of tension at the yeast kinetochore is sufficient to activate the spindle checkpoint in mitosis.     

Geometry and force behind kinetochore orientation: lessons from meiosis

During mitosis, replicated chromosomes (sister chromatids) become attached at the kinetochore by spindle microtubules emanating from opposite poles and segregate equationally. In the first division of meiosis, however, sister chromatids become attached from the same pole and co-segregate, whereas homologous chromosomes connected by chiasmata segregate to opposite poles. Disorder in this specialized chromosome attachment in meiosis is the leading cause of miscarriage in humans. Recent studies have elucidated the molecular mechanisms determining chromosome orientation, and consequently segregation, in meiosis. Comparative studies of meiosis and mitosis have led to the general principle that kinetochore geometry and tension exerted by microtubules synergistically generate chromosome 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.     

Chiasmata promote monopolar attachment of sister chromatids and their co-segregation toward the proper pole during meiosis I

The chiasma is a structure that forms between a pair of homologous chromosomes by crossover recombination and physically links the homologous chromosomes during meiosis. Chiasmata are essential for the attachment of the homologous chromosomes to opposite spindle poles (bipolar attachment) and their subsequent segregation to the opposite poles during meiosis I. However, the overall function of chiasmata during meiosis is not fully understood. Here, we show that chiasmata also play a crucial role in the attachment of sister chromatids to the same spindle pole and in their co-segregation during meiosis I in fission yeast. Analysis of cells lacking chiasmata and the cohesin protector Sgo1 showed that loss of chiasmata causes frequent bipolar attachment of sister chromatids during anaphase. Furthermore, high time-resolution analysis of centromere dynamics in various types of chiasmate and achiasmate cells, including those lacking the DNA replication checkpoint factor Mrc1 or the meiotic centromere protein Moa1, showed the following three outcomes: (i) during the pre-anaphase stage, the bipolar attachment of sister chromatids occurs irrespective of chiasma formation; (ii) the chiasma contributes to the elimination of the pre-anaphase bipolar attachment; and (iii) when the bipolar attachment remains during anaphase, the chiasmata generate a bias toward the proper pole during poleward chromosome pulling that results in appropriate chromosome segregation. Based on these results, we propose that chiasmata play a pivotal role in the selection of proper attachments and provide a backup mechanism that promotes correct chromosome segregation when improper attachments remain during anaphase I.     

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.     

Budding yeast mitotic chromosomes have an intrinsic bias to biorient on the spindle

BACKGROUND: Chromosomes must biorient on the mitotic spindle, with the two sisters attached to opposite spindle poles. The spindle checkpoint detects unattached chromosomes and monitors biorientation by detecting the lack of tension between two sisters attached to the same pole. After the spindle has been depolymerized and allowed to reform, budding yeast sgo1 mutants fail to biorient their sister chromatids and die as cells divide. RESULTS: In sgo1 mutants, chromosomes attach to microtubules normally but cannot reorient if both sisters attach to the same pole. The mutants' fate depends on the position of the spindle poles when the chromosomes attach to microtubules. If the poles have separated, sister chromatids biorient, but if the poles are still close, sister chromatids often attach to the same pole, missegregate, and cause cell death. CONCLUSIONS: These observations argue that budding yeast mitotic chromosomes have an intrinsic, geometric bias to biorient on the spindle. When the poles have already separated, attaching one kinetochore to one pole predisposes its sister to attach to the opposite pole, allowing the cells to segregate the chromosomes correctly. When the poles have not separated, the second kinetochore eventually attaches to either of the two poles randomly, causing orientation errors that are corrected in the wild-type but not in sgo1 mutants. In the absence of spindle damage, sgo1 cells divide successfully, suggesting that kinetochores only make stable attachments to microtubules after the cells have entered mitosis and separated their spindle poles.     

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.     

Bub1 and Bub3 promote the conversion from monopolar to bipolar chromosome attachment independently of shugoshin

The eukaryotic spindle assembly checkpoint (SAC) delays anaphase in the presence of chromosome attachment errors. Bub3 has been reported to be required for SAC activity in all eukaryotes examined so far. We find that Bub3, unlike its binding partner Bub1, is not essential for the SAC in fission yeast. As Bub3 is needed for the efficient kinetochore localization of Bub1, and of Mad1, Mad2 and Mad3, this implies that most SAC proteins do not need to be enriched at the kinetochores for the SAC to function. We find that Bub3 is also dispensable for shugoshin localization to the centromeres, which is the second known function of Bub1. Instead, Bub3, together with Bub1, has a specific function in promoting the conversion from chromosome mono‐orientation to bi‐orientation.     

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.     

Mitotic chromosome biorientation in fission yeast is enhanced by dynein and a minus-end-directed, kinesin-like protein

Chromosome biorientation, the attachment of sister kinetochores to sister spindle poles, is vitally important for accurate chromosome segregation. We have studied this process by following the congression of pole-proximal kinetochores and their subsequent anaphase segregation in fission yeast cells that carry deletions in any or all of this organism's minus end-directed, microtubule-dependent motors: two related kinesin 14s (Pkl1p and Klp2p) and dynein. None of these deletions abolished biorientation, but fewer chromosomes segregated normally without Pkl1p, and to a lesser degree without dynein, than in wild-type cells. In the absence of Pkl1p, which normally localizes to the spindle and its poles, the checkpoint that monitors chromosome biorientation was defective, leading to frequent precocious anaphase. Ultrastructural analysis of mutant mitotic spindles suggests that Pkl1p contributes to error-free biorientation by promoting normal spindle pole organization, whereas dynein helps to anchor a focused bundle of spindle microtubules at the pole.     

Tethering Sister Centromeres to Each Other Suggests the Spindle Checkpoint Detects Stretch within the Kinetochore

The spindle checkpoint ensures that newly born cells receive one copy of each chromosome by preventing chromosomes from segregating until they are all correctly attached to the spindle. The checkpoint monitors tension to distinguish between correctly aligned chromosomes and those with both sisters attached to the same spindle pole. Tension arises when sister kinetochores attach to and are pulled toward opposite poles, stretching the chromatin around centromeres and elongating kinetochores. We distinguished between two hypotheses for where the checkpoint monitors tension: between the kinetochores, by detecting alterations in the distance between them, or by responding to changes in the structure of the kinetochore itself. To distinguish these models, we inhibited chromatin stretch by tethering sister chromatids together by binding a tetrameric form of the Lac repressor to arrays of the Lac operator located on either side of a centromere. Inhibiting chromatin stretch did not activate the spindle checkpoint; these cells entered anaphase at the same time as control cells that express a dimeric version of the Lac repressor, which cannot cross link chromatids, and cells whose checkpoint has been inactivated. There is no dominant checkpoint inhibition when sister kinetochores are held together: cells expressing the tetrameric Lac repressor still arrest in response to microtubule-depolymerizing drugs. Tethering chromatids together does not disrupt kinetochore function; chromosomes are successfully segregated to opposite poles of the spindle. Our results indicate that the spindle checkpoint does not monitor inter-kinetochore separation, thus supporting the hypothesis that tension is measured within the kinetochore.     

Dynein light intermediate chain 1 is required for progress through the spindle assembly checkpoint

The spindle assembly checkpoint monitors microtubule attachment to kinetochores and tension across sister kinetochores to ensure accurate division of chromosomes between daughter cells. Cytoplasmic dynein functions in the checkpoint, apparently by moving critical checkpoint components off kinetochores. The dynein subunit required for this function is unknown. Here we show that human cells depleted of dynein light intermediate chain 1 (LIC1) delay in metaphase with increased interkinetochore distances; dynein remains intact, localised and functional. The checkpoint proteins Mad1/2 and Zw10 localise to kinetochores under full tension, whereas BubR1 is diminished at kinetochores. Metaphase delay and increased interkinetochore distances are suppressed by depletion of Mad1, Mad2 or BubR1 or by re‐expression of wtLIC1 or a Cdk1 site phosphomimetic LIC1 mutant, but not Cdk1‐phosphorylation‐deficient LIC1. When the checkpoint is activated by microtubule depolymerisation, Mad1/2 and BubR1 localise to kinetochores. We conclude that a Cdk1 phosphorylated form of LIC1 is required to remove Mad1/2 and Zw10 but not BubR1 from kinetochores during spindle assembly checkpoint silencing.     

Cryo-electron tomography: The challenge of doing structural biology in situ

The mitotic segregation apparatus composed of microtubules and chromatin functions to faithfully partition a duplicated genome into two daughter cells. Microtubules exert extensional pulling force on sister chromatids toward opposite poles, whereas pericentric chromatin resists with contractile springlike properties. Tension generated from these opposing forces silences the spindle checkpoint to ensure accurate chromosome segregation. It is unknown how the cell senses tension across multiple microtubule attachment sites, considering the stochastic dynamics of microtubule growth and shortening. In budding yeast, there is one microtubule attachment site per chromosome. By labeling several chromosomes, we find that pericentromeres display coordinated motion and stretching in metaphase. The pericentromeres of different chromosomes exhibit physical linkage dependent on centromere function and structural maintenance of chromosomes complexes. Coordinated motion is dependent on condensin and the kinesin motor Cin8, whereas coordinated stretching is dependent on pericentric cohesin and Cin8. Linking of pericentric chromatin through cohesin, condensin, and kinetochore microtubules functions to coordinate dynamics across multiple attachment sites.     

Bi-orienting chromosomes on the mitotic spindle

For the proper segregation of sister chromatids before cell division, each sister kinetochore must attach to microtubules that extend to opposite spindle poles. This process is called bipolar microtubule attachment or chromosome bi-orientation. The mechanism for chromosome bi-orientation lies at the heart of chromosome segregation, but is still poorly understood. Recent studies suggest that cells can promote bi-orientation by re-orienting kinetochore-spindle pole connections.     

Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation

Accurate chromosome segregation depends on biorientation, whereby sister chromatids attach to microtubules from opposite spindle poles. The spindle-assembly checkpoint is a surveillance mechanism in eukaryotes that inhibits anaphase until all chromosomes have bioriented. In present models, the recruitment of the spindle-assembly checkpoint protein Mad2, through Mad1, to non-bioriented kinetochores is needed to stop cell-cycle progression. However, it is unknown whether Mad1-Mad2 targeting to kinetochores is sufficient to block anaphase. Furthermore, it is unclear whether regulators of biorientation (for example, Aurora kinases) have checkpoint functions downstream of Mad1-Mad2 recruitment or whether they act upstream to quench the primary error signal. Here, we engineered a Mad1 construct that localizes to bioriented kinetochores. We show that the kinetochore localization of Mad1 is sufficient for a metaphase arrest that depends on Mad1-Mad2 binding. By uncoupling the checkpoint from its primary error signal, we show that Aurora, Mps1 and BubR1 kinases, but not Polo-like kinase, are needed to maintain checkpoint arrest when Mad1 is present on kinetochores. Together, our data suggest a model in which the biorientation errors, which recruit Mad1-Mad2 to kinetochores, may be signalled not only through Mad2 template dynamics, but also through the activity of widely conserved kinases, to ensure the fidelity of cell division.     

A Highlights from MBoC Selection: Ipl1/Aurora-B is necessary for kinetochore restructuring in meiosis I in Saccharomyces cerevisiae

In mitosis, the centromeres of sister chromosomes are pulled toward opposite poles of the spindle. In meiosis I, the opposite is true: the sister centromeres move together to the same pole, and the homologous chromosomes are pulled apart. This change in segregation patterns demands that between the final mitosis preceding meiosis and the first meiotic division, the kinetochores must be restructured. In budding yeast, unlike mammals, kinetochores are largely stable throughout the mitotic cycle. In contrast, previous work with budding and fission yeast showed that some outer kinetochore proteins are lost in early meiosis. We use quantitative mass spectrometry methods and imaging approaches to explore the kinetochore restructuring process that occurs in meiosis I in budding yeast. The Ndc80 outer kinetochore complex, but not other subcomplexes, is shed upon meiotic entry. This shedding is regulated by the conserved protein kinase Ipl1/Aurora-B and promotes the subsequent assembly of a kinetochore that will confer meiosis-specific segregation patterns on the chromosome.     

A stochastic model of kinetochore-microtubule attachment accurately describes fission yeast chromosome segregation

In fission yeast, erroneous attachments of spindle microtubules to kinetochores are frequent in early mitosis. Most are corrected before anaphase onset by a mechanism involving the protein kinase Aurora B, which destabilizes kinetochore microtubules (ktMTs) in the absence of tension between sister chromatids. In this paper, we describe a minimal mathematical model of fission yeast chromosome segregation based on the stochastic attachment and detachment of ktMTs. The model accurately reproduces the timing of correct chromosome biorientation and segregation seen in fission yeast. Prevention of attachment defects requires both appropriate kinetochore orientation and an Aurora B-like activity. The model also reproduces abnormal chromosome segregation behavior (caused by, for example, inhibition of Aurora B). It predicts that, in metaphase, merotelic attachment is prevented by a kinetochore orientation effect and corrected by an Aurora B-like activity, whereas in anaphase, it is corrected through unbalanced forces applied to the kinetochore. These unbalanced forces are sufficient to prevent aneuploidy.