Loss of function of the Cik1/Kar3 motor complex results in chromosomes with syntelic attachment that are sensed by the tension checkpoint

The attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The spindle checkpoint can sense the lack of tension on chromosomes as well as detached kinetochores to prevent anaphase onset. In budding yeast Saccharomyces cerevisiae, tension checkpoint proteins Aurora/Ipl1 kinase and centromere-localized Sgo1 are required to sense the absence of tension but are dispensable for the checkpoint response to detached kinetochores. We have found that the loss of function of a motor protein complex Cik1/Kar3 in budding yeast leads to syntelic attachments. Inactivation of either the spindle or tension checkpoint enables premature anaphase entry in cells with dysfunctional Cik1/Kar3, resulting in co-segregation of sister chromatids. Moreover, the abolished Kar3-kinetochore interaction in cik1 mutants suggests that the Cik1/Kar3 complex mediates chromosome movement along microtubules, which could facilitate bipolar attachment. Therefore, we can induce syntelic attachments in budding yeast by inactivating the Cik1/Kar3 complex, and this approach will be very useful to study the checkpoint response to syntelic attachments.     

Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast

Sister kinetochores are bioriented toward the spindle poles in higher eukaryotic prometaphase before chromosome segregation. We show that, in budding yeast, the sister kinetochores are separated in the very early spindle, while the sister arms remain associated. Biorientation of the separated kinetochores is achieved already after replication. Mtw1p, a homolog of fission yeast Mis12 required for biorientation, locates at the centromeres in an Ndc10p-dependent manner. Mtw1p and the sequences 1.8 and 3.8 kb from CEN3 and CEN15, respectively, behave like the precociously separated kinetochores, whereas the sequences 23 and 35 kb distant from CEN3 and CEN5 previously used as the centromere markers behave like a part of the arm. Mtw1p and Ndc10p are identically located except for additional spindle localization of Ndc10p. A model explaining small centromeres and early spindle formation in budding yeast is proposed.     

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.     

Mps1 kinase promotes sister-kinetochore bi-orientation by a tension-dependent mechanism

Segregation of sister chromatids to opposite spindle poles during anaphase is dependent on the prior capture of sister kinetochores by microtubules extending from opposite spindle poles (bi-orientation). If sister kinetochores attach to microtubules from the same pole (syntelic attachment), the kinetochore-spindle pole connections must be re-oriented to be converted to proper bi-orientation. This re-orientation is facilitated by Aurora B kinase (Ipl1 in budding yeast), which eliminates kinetochore-spindle pole connections that do not generate tension. Mps1 is another evolutionarily conserved protein kinase, required for spindle-assembly checkpoint and, in some organisms, for duplication of microtubule-organizing centers. Separately from these functions, however, Mps1 has an important role in chromosome segregation. Here we show that, in budding yeast, Mps1 has a crucial role in establishing sister-kinetochore bi-orientation on the mitotic spindle. Failure in bi-orientation with inactive Mps1 is not due to a lack of kinetochore-spindle pole connections by microtubules, but due to a defect in properly orienting the connections. Mps1 promotes re-orientation of kinetochore-spindle pole connections and eliminates those that do not generate tension between sister kinetochores. We did not find evidence that Ipl1 regulates Mps1 or vice versa; therefore, they play similar, but possibly independent, roles in facilitating bi-orientation.     

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     

Tension-dependent regulation of microtubule dynamics at kinetochores can explain metaphase congression in yeast

During metaphase in budding yeast mitosis, sister kinetochores are tethered to opposite poles and separated, stretching their intervening chromatin, by singly attached kinetochore microtubules (kMTs). Kinetochore movements are coupled to single microtubule plus-end polymerization/depolymerization at kinetochore attachment sites. Here, we use computer modeling to test possible mechanisms controlling chromosome alignment during yeast metaphase by simulating experiments that determine the 1) mean positions of kinetochore Cse4-GFP, 2) extent of oscillation of kinetochores during metaphase as measured by fluorescence recovery after photobleaching (FRAP) of kinetochore Cse4-GFP, 3) dynamics of kMTs as measured by FRAP of GFP-tubulin, and 4) mean positions of unreplicated chromosome kinetochores that lack pulling forces from a sister kinetochore. We rule out a number of possible models and find the best fit between theory and experiment when it is assumed that kinetochores sense both a spatial gradient that suppresses kMT catastrophe near the poles and attachment site tension that promotes kMT rescue at higher amounts of chromatin stretch.     

Evidence that the Ipl1-Sli15 (Aurora kinase-INCENP) complex promotes chromosome bi-orientation by altering kinetochore-spindle pole connections

How sister kinetochores attach to microtubules from opposite spindle poles during mitosis (bi-orientation) remains poorly understood. In yeast, the ortholog of the Aurora B-INCENP protein kinase complex (Ipl1-Sli15) may have a role in this crucial process, because it is necessary to prevent attachment of sister kinetochores to microtubules from the same spindle pole. We investigated IPL1 function in cells that cannot replicate their chromosomes but nevertheless duplicate their spindle pole bodies (SPBs). Kinetochores detach from old SPBs and reattach to old and new SPBs with equal frequency in IPL1+ cells, but remain attached to old SPBs in ipl1 mutants. This raises the possibility that Ipl1-Sli15 facilitates bi-orientation by promoting turnover of kinetochore-SPB connections until traction of sister kinetochores toward opposite spindle poles creates tension in the surrounding chromatin.     

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.     

The current view for the silencing of the spindle assembly checkpoint

Chromosome bipolar attachment is achieved when sister kinetochores are attached by microtubules emanating from opposite spindle poles, and this process is essential for faithful chromosome segregation during anaphase. A fundamental question in cell biology is how cells ensure that chromosome segregation only occurs after bipolar attachment. It is well documented that unattached kinetochores activate the spindle assembly checkpoint (SAC) to delay chromosome segregation. Therefore, the silencing of the SAC is thought to trigger anaphase onset, but how correct chromosome attachment is coupled with SAC silencing and the subsequent anaphase onset is poorly understood. The establishment of chromosome bipolar attachment not only results in the occupancy of kinetochores by microtubules but also applies tension on sister kinetochores. A long-standing debate is whether the kinetochore attachment (occupancy) or the tension silences the SAC. Recent work in budding yeast reveals the SAC silencing network SSN that prevents SAC silencing prior to tension generation at kinetochores. Therefore, this signaling pathway ensures that SAC silencing and the subsequent anaphase onset occur only after chromosome bipolar attachment applies tension on chromosomes. This review will summarize the recent advances in the understanding of the SAC silencing process.     

Analysis of kinesin motor function at budding yeast kinetochores

Accurate chromosome segregation during mitosis requires biorientation of sister chromatids on the microtubules (MT) of the mitotic spindle. Chromosome-MT binding is mediated by kinetochores, which are multiprotein structures that assemble on centromeric (CEN) DNA. The simple CENs of budding yeast are among the best understood, but the roles of kinesin motor proteins at yeast kinetochores have yet to be determined, despite evidence of their importance in higher eukaryotes. We show that all four nuclear kinesins in Saccharomyces cerevisiae localize to kinetochores and function in three distinct processes. Kip1p and Cin8p, which are kinesin-5/BimC family members, cluster kinetochores into their characteristic bilobed metaphase configuration. Kip3p, a kinesin-8,-13/KinI kinesin, synchronizes poleward kinetochore movement during anaphase A. The kinesin-14 motor Kar3p appears to function at the subset of kinetochores that become detached from spindle MTs. These data demonstrate roles for structurally diverse motors in the complex processes of chromosome segregation and reveal important similarities and intriguing differences between higher and lower eukaryotes.     

Repositioning of aurora B promoted by chiasmata ensures sister chromatid mono-orientation in meiosis I

During meiosis I, kinetochores of sister chromatids are juxtaposed or fused and mono-orient, while homologous chromosomes that are paired by chiasmata (bivalents) have to biorient. In the absence of chiasmata, biorientation of sister chromatids (univalents), which carries a risk of aneuploidy, has been occasionally detected in several species, including humans. We show in fission yeast that biorientation of fused sister kinetochores predominates during early prometaphase I. Without chiasmata, this undesirable biorientation of univalents persists and eventually evades the spindle assembly checkpoint, provoking abnormal anaphase. When univalents are connected by chiasmata or by an artificial tether, this erroneous attachment is converted to monopolar attachment and stabilized. This stabilization is apparently achieved by a chromosome configuration that brings kinetochores to the outer edge of the bivalent, while bringing Aurora B, a destabilizer of kinetochore-microtubule attachment, inward. Our results elucidate how chiasmata favor biorientation of bivalents over that of univalents at meiosis I.     

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.     

The molecular basis of monopolin recruitment to the kinetochore

The monopolin complex is a multifunctional molecular crosslinker, which in S. pombe binds and organises mitotic kinetochores to prevent aberrant kinetochore-microtubule interactions. In the budding yeast S. cerevisiae, whose kinetochores bind a single microtubule, the monopolin complex crosslinks and mono-orients sister kinetochores in meiosis I, enabling the biorientation and segregation of homologs. Here, we show that both the monopolin complex subunit Csm1 and its binding site on the kinetochore protein Dsn1 are broadly distributed throughout eukaryotes, suggesting a conserved role in kinetochore organisation and function. We find that budding yeast Csm1 binds two conserved motifs in Dsn1, one (termed Box 1) representing the ancestral, widely conserved monopolin binding motif and a second (termed Box 2-3) with a likely role in enforcing specificity of sister kinetochore crosslinking. We find that Box 1 and Box 2-3 bind the same conserved hydrophobic cavity on Csm1, suggesting competition or handoff between these motifs. Using structure-based mutants, we also find that both Box 1 and Box 2-3 are critical for monopolin function in meiosis. We identify two conserved serine residues in Box 2-3 that are phosphorylated in meiosis and whose mutation to aspartate stabilises Csm1-Dsn1 binding, suggesting that regulated phosphorylation of these residues may play a role in sister kinetochore crosslinking specificity. Overall, our results reveal the monopolin complex as a broadly conserved kinetochore organiser in eukaryotes, which budding yeast have co-opted to mediate sister kinetochore crosslinking through the addition of a second, regulatable monopolin binding interface.

     

Kinetochore rearrangement in meiosis II requires attachment to the spindle

The distinctive behaviors of chromosomes in mitosis and meiosis depend upon differences in kinetochore position. Kinetochore position is well established except for a critical transition between meiosis I and meiosis II. We examined kinetochore position during the transition and compared it with the position of kinetochores in mitosis. Immunofluorescence staining using the 3F3/2 antibody showed that in mitosis in grasshopper cells, as in other organisms, kinetochores are positioned on opposite sides of the two sister chromatids. In meiosis I, sister kinetochores are positioned side by side. At nuclear envelope breakdown in meiosis II, sister kinetochores are still side by side, but are separated by the time all chromosomes have fully attached in metaphase II. Micromanipulation experiments reveal that this switch from side-by-side to separated sister kinetochores requires attachment to the spindle. Moreover, it is irreversible, as chromosomes detached from a metaphase II spindle retain separate kinetochores. How this critical separation of sister kinetochores occurs in meiosis is uncertain, but clearly it is not built into the chromosome before nuclear envelope breakdown, as it is in mitosis.     

Nsk1 ensures accurate chromosome segregation by promoting association of kinetochores to spindle poles during anaphase B

Type 1 phosphatase (PP1) antagonizes Aurora B kinase to stabilize kinetochore-microtubule attachments and to silence the spindle checkpoint. We screened for factors that exacerbate the growth defect of Δdis2 cells, which lack one of two catalytic subunits of PP1 in fission yeast, and identified Nsk1, a novel protein required for accurate chromosome segregation. During interphase, Nsk1 resides in the nucleolus but spreads throughout the nucleoplasm as cells enter mitosis. Following dephosphorylation by Clp1 (Cdc14-like) phosphatase and at least one other phosphatase, Nsk1 localizes to the interface between kinetochores and the inner face of the spindle pole body during anaphase. In the absence of Nsk1, some kinetochores become detached from spindle poles during anaphase B. If this occurs late in anaphase B, then the sister chromatids of unclustered kinetochores segregate to the correct daughter cell. These unclustered kinetochores are efficiently captured, retrieved, bioriented, and segregated during the following mitosis, as long as Dis2 is present. However, if kinetochores are detached from a spindle pole early in anaphase B, then these sister chromatids become missegregated. These data suggest Nsk1 ensures accurate chromosome segregation by promoting the tethering of kinetochores to spindle poles during anaphase B.     

The 'anaphase problem': how to disable the mitotic checkpoint when sisters split

Two closely connected mechanisms safeguard the fidelity of chromosome segregation in eukaryotic cells. The mitotic checkpoint monitors the attachment of kinetochores to microtubules and delays anaphase onset until all sister kinetochores have become attached to opposite poles. In addition, an error correction mechanism destabilizes erroneous attachments that do not lead to tension at sister kinetochores. Aurora B kinase, the catalytic subunit of the CPC (chromosomal passenger complex), acts as a sensor and effector in both pathways. In this review we focus on a poorly understood but important aspect of mitotic control: what prevents the mitotic checkpoint from springing into action when sister centromeres are split and tension is suddenly lost at anaphase onset? Recent work has shown that disjunction of sister chromatids, in principle, engages the mitotic checkpoint, and probably also the error correction mechanism, with potentially catastrophic consequences for cell division. Eukaryotic cells have solved this 'anaphase problem' by disabling the mitotic checkpoint at the metaphase-to-anaphase transition. Checkpoint inactivation is in part due to the reversal of Cdk1 (cyclin-dependent kinase 1) phosphorylation of the CPC component INCENP (inner centromere protein; Sli15 in budding yeast), which causes the relocation of the CPC from centromeres to the spindle midzone. These findings highlight principles of mitotic checkpoint control: when bipolar chromosome attachment is reached in mitosis, the checkpoint is satisfied, but still active and responsive to loss of tension. Mitotic checkpoint inactivation at anaphase onset is required to prevent checkpoint re-engagement when sister chromatids split.     

Cohesins determine the attachment manner of kinetochores to spindle microtubules at meiosis I in fission yeast

During mitosis, sister kinetochores attach to microtubules that extend to opposite spindle poles (bipolar attachment) and pull the chromatids apart at anaphase (equational segregation). A multisubunit complex called cohesin, including Rad21/Scc1, plays a crucial role in sister chromatid cohesion and equational segregation at mitosis. Meiosis I differs from mitosis in having a reductional pattern of chromosome segregation, in which sister kinetochores are attached to the same spindle (monopolar attachment). During meiosis, Rad21/Scc1 is largely replaced by its meiotic counterpart, Rec8. If Rec8 is inactivated in fission yeast, meiosis I is shifted from reductional to equational division. However, the reason rec8Delta cells undergo equational rather than random division has not been clarified; therefore, it has been unclear whether equational segregation is due to a loss of cohesin in general or to a loss of a specific requirement for Rec8. We report here that the equational segregation at meiosis I depends on substitutive Rad21, which relocates to the centromeres if Rec8 is absent. Moreover, we demonstrate that even if sufficient amounts of Rad21 are transferred to the centromeres at meiosis I, thereby establishing cohesion at the centromeres, rec8Delta cells never recover monopolar attachment but instead secure bipolar attachment. Thus, Rec8 and Rad21 define monopolar and bipolar attachment, respectively, at meiosis I. We conclude that cohesin is a crucial determinant of the attachment manner of kinetochores to the spindle microtubules at meiosis I in fission yeast.     

Chromosome bi-orientation on the mitotic spindle

For proper chromosome segregation, sister kinetochores must attach to microtubules extending from opposite spindle poles prior to anaphase onset. This state is called sister kinetochore bi-orientation or chromosome bi-orientation. The mechanism ensuring chromosome bi-orientation lies at the heart of chromosome segregation, but is still poorly understood. Recent evidence suggests that mal-oriented kinetochore-to-pole connections are corrected in a tension-dependent mechanism. The cohesin complex and the Ipl1/Aurora B protein kinase seem to be key regulators for this correction. In this article, I discuss how cells ensure sister kinetochore bi-orientation for all chromosomes, mainly focusing on our recent findings in budding yeast.     

Molecular analysis of kinetochore-microtubule attachment in budding yeast

The complex series of movements that mediates chromosome segregation during mitosis is dependent on the attachment of microtubules to kinetochores, DNA-protein complexes that assemble on centromeric DNA. We describe the use of live-cell imaging and chromatin immunoprecipitation in S. cerevisiae to identify ten kinetochore subunits, among which are yeast homologs of microtubule binding proteins in animal cells. By analyzing conditional mutations in several of these proteins, we show that they are required for the imposition of tension on paired sister kinetochores and for correct chromosome movement. The proteins include both molecular motors and microtubule associated proteins (MAPs), implying that motors and MAPs function together in binding chromosomes to spindle microtubules.     

Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I

During meiosis, two rounds of chromosome segregation occur after a single round of DNA replication, producing haploid progeny from diploid progenitors. Three innovations in chromosome behaviour during meiosis I accomplish this unique division. First, crossovers between maternal and paternal sister chromatids (detected cytologically as chiasmata) bind replicated maternal and paternal chromosomes together. Second, sister kinetochores attach to microtubules from the same pole (mono-polar orientation), causing maternal and paternal centromere pairs (and not sister chromatids) to be separated. Third, sister chromatid cohesion near centromeres is preserved at anaphase I when cohesion along chromosome arms is destroyed. The finding that destruction of mitotic cohesion is regulated by Polo-like kinases prompted us to investigate the meiotic role of the yeast Polo-like kinase Cdc5. We show here that cells lacking Cdc5 synapse homologues and initiate recombination normally, but fail to efficiently resolve recombination intermediates as crossovers. They also fail to properly localize the Lrs4 (ref. 3) and Mam1 (ref. 4) monopolin proteins, resulting in bipolar orientation of sister kinetochores. Cdc5 is thus required both for the formation of chiasmata and for cosegregation of sister centromeres at meiosis I.