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1.  Roles for the Conserved Spc105p/Kre28p Complex in Kinetochore-Microtubule Binding and the Spindle Assembly Checkpoint 
PLoS ONE  2009;4(10):e7640.
Background
Kinetochores attach sister chromatids to microtubules of the mitotic spindle and orchestrate chromosome disjunction at anaphase. Although S. cerevisiae has the simplest known kinetochores, they nonetheless contain ∼70 subunits that assemble on centromeric DNA in a hierarchical manner. Developing an accurate picture of the DNA-binding, linker and microtubule-binding layers of kinetochores, including the functions of individual proteins in these layers, is a key challenge in the field of yeast chromosome segregation. Moreover, comparison of orthologous proteins in yeast and humans promises to extend insight obtained from the study of simple fungal kinetochores to complex animal cell kinetochores.
Principal Findings
We show that S. cerevisiae Spc105p forms a heterotrimeric complex with Kre28p, the likely orthologue of the metazoan kinetochore protein Zwint-1. Through systematic analysis of interdependencies among kinetochore complexes, focused on Spc105p/Kre28p, we develop a comprehensive picture of the assembly hierarchy of budding yeast kinetochores. We find Spc105p/Kre28p to comprise the third linker complex that, along with the Ndc80 and MIND linker complexes, is responsible for bridging between centromeric heterochromatin and kinetochore MAPs and motors. Like the Ndc80 complex, Spc105p/Kre28p is also essential for kinetochore binding by components of the spindle assembly checkpoint. Moreover, these functions are conserved in human cells.
Conclusions/Significance
Spc105p/Kre28p is the last of the core linker complexes to be analyzed in yeast and we show it to be required for kinetochore binding by a discrete subset of kMAPs (Bim1p, Bik1p, Slk19p) and motors (Cin8p, Kar3p), all of which are nonessential. Strikingly, dissociation of these proteins from kinetochores prevents bipolar attachment, even though the Ndc80 and DASH complexes, the two best-studied kMAPs, are still present. The failure of Spc105 deficient kinetochores to bind correctly to spindle microtubules and to recruit checkpoint proteins in yeast and human cells explains the observed severity of missegregation phenotypes.
doi:10.1371/journal.pone.0007640
PMCID: PMC2764089  PMID: 19893618
2.  Monopolin Subunit Csm1 Associates with MIND Complex to Establish Monopolar Attachment of Sister Kinetochores at Meiosis I 
PLoS Genetics  2013;9(7):e1003610.
Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore.
Author Summary
All sexually reproducing organisms produce haploid gametes from diploid cells via meiosis. During meiosis, one round of DNA replication is followed by two rounds of nuclear division (called meiosis I and II). This is unlike mitotically proliferating cells wherein one round of DNA replication is followed by one round of nuclear division. During meiosis I, sister chromatids move towards the same spindle pole unlike in mitosis where they move towards opposite spindle poles. Poleward chromosome movement is achieved by association of kinetochores (a complex network of proteins assembled at centromeres on chromosomes) with microtubule ends emanating from spindle poles. The basis of the contrasting fate of sister chromatids during mitosis and meiosis I is best studied in budding yeast in which a protein complex called monopolin binds to sister kinetochores during meiosis I and ensures that they face the same spindle pole. But precisely how monopolin binds to kinetochores was unknown. In this study, we have identified a monopolin's receptor at the kinetochore. Disabling the receptor did not affect mitotic growth but severely compromised meiotic chromosome segregation. Cells lacking the monopolin receptor attempt to segregate sister chromatids towards opposite spindle poles during meiosis I with catastrophic genetic consequences.
doi:10.1371/journal.pgen.1003610
PMCID: PMC3701701  PMID: 23861669
3.  Tethering Sister Centromeres to Each Other Suggests the Spindle Checkpoint Detects Stretch within the Kinetochore 
PLoS Genetics  2014;10(8):e1004492.
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.
Author Summary
The spindle checkpoint monitors tension on chromosomes to distinguish between chromosomes that are correctly and incorrectly attached to the spindle. Tension is generated across a correctly attached chromosome as microtubules from opposite poles attach to and pull kinetochores apart, but are resisted by the cohesin that holds sister chromatids together. This tension generates separation between kinetochores as pericentric chromatin stretches and it also elongates the kinetochores. To monitor tension, the checkpoint could measure the separation between kinetochores or the stretch within them. We inhibited the ability of pericentric chromatin to stretch by tethering sister centromeres to each other, and we asked whether the resulting reduction in inter-kinetochore separation artificially activated the spindle checkpoint. Inhibiting inter-kinetochore separation does not delay anaphase, and the timing of mitosis was the same in cells with or without the spindle checkpoint, showing that the checkpoint is not activated. Inhibiting chromatin stretch does not alter the function of kinetochores as chromosomes are still segregated correctly, nor does it hinder the checkpoint. Cells whose sister kinetochores are held together can still activate the checkpoint in response to microtubule depolymerization. Our results indicate the spindle checkpoint does not monitor inter-kinetochore separation and likely monitors tension within kinetochores.
doi:10.1371/journal.pgen.1004492
PMCID: PMC4125069  PMID: 25101645
4.  BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin 
The Journal of Cell Biology  1990;111(6):2573-2586.
BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.
PMCID: PMC2116401  PMID: 2277073
5.  The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres 
PLoS Biology  2012;10(1):e1001250.
INCENP acts as a protein scaffold that integrates the functions of two crucial mitotic kinases, Aurora B and Polo, at centromeres of mitotic chromosomes.
The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. Our study concerns the coordination of the activities of these two kinases in vivo. We show that INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. We show here that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. Our results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation.
Author Summary
When cells divide, their chromosomes segregate to the two daughter cells on the mitotic spindle, a dynamic macromolecular scaffold composed of microtubules. Each chromosome consists of two sister chromatids. Microtubules attach to the chromatids at structures called kinetochores, which assemble at the surface of the constricted centromere region where the sister chromatids are most closely paired. To segregate correctly, sister kinetochores must attach to microtubules emanating from opposite spindle poles. Kinetochore attachment to microtubules occurs randomly and mistakes occur frequently. For example, both sister kinetochores may attach to one pole, or one kinetochore may attach to both poles simultaneously. Two protein kinases, Aurora B and Polo, have essential roles in regulating this process: Aurora B triggers the release of incorrect attachments and Polo strengthens the grip that correctly attached kinetochores have on microtubules. In this work, we have investigated the potential functional links between these two crucial enzymes at centromeres in cells of the fruitfly. We found that early in division, Aurora B and Polo both interact with a structural partner protein named INCENP at centromeres. This allows Aurora B to phosphorylate Polo, thereby activating it. We show that coordinating the activities of these two central mitotic kinases is crucial for successful cell division, and that this mechanism is conserved in human cells.
doi:10.1371/journal.pbio.1001250
PMCID: PMC3265468  PMID: 22291575
6.  Meiosis I chromosome segregation is established through regulation of microtubule–kinetochore interactions 
eLife  2012;1:e00117.
During meiosis, a single round of DNA replication is followed by two consecutive rounds of nuclear divisions called meiosis I and meiosis II. In meiosis I, homologous chromosomes segregate, while sister chromatids remain together. Determining how this unusual chromosome segregation behavior is established is central to understanding germ cell development. Here we show that preventing microtubule–kinetochore interactions during premeiotic S phase and prophase I is essential for establishing the meiosis I chromosome segregation pattern. Premature interactions of kinetochores with microtubules transform meiosis I into a mitosis-like division by disrupting two key meiosis I events: coorientation of sister kinetochores and protection of centromeric cohesin removal from chromosomes. Furthermore we find that restricting outer kinetochore assembly contributes to preventing premature engagement of microtubules with kinetochores. We propose that inhibition of microtubule–kinetochore interactions during premeiotic S phase and prophase I is central to establishing the unique meiosis I chromosome segregation pattern.
DOI: http://dx.doi.org/10.7554/eLife.00117.001
eLife digest
Diploid organisms contain two sets of chromosomes, one set inherited from the mother and the other from the father. Humans, for example, have 23 pairs of chromosomes, and the chromosomes within each pair are said to be homologous because they are similar to each other in a number of ways, including length and shape. When it comes time for one of these cells to duplicate, each chromosome is first replicated to generate a pair of identical chromosomes called sister chromatids, which subsequently separate in a cell division process known as mitosis to produce two identical daughter cells.
While most cells proliferate via mitotic cell division, the germ cells that generate gametes in the form of sperm or eggs undergo a different cell division known as meiosis. This process reduces the number of chromosomes by a factor of two, so that the original number of chromosomes is restored by the fusion of gametes during sexual reproduction. During meiotic cell division, a single round of DNA replication is followed by two consecutive rounds of nuclear division called meiosis I and meiosis II. During meiosis I, homologous chromosomes are separated. Subsequently, during meiosis II, the sister chromatids separate to produce a total of four products, each with half the number of chromosomes as the original cell.
The separation of homologous chromosomes or sister chromatids relies on them being pulled apart by microtubules. One end of each microtubule is attached to a protein-based structure called a kinetochore, which is assembled onto the centromere of each chromosome. The other end of each microtubule is attached to a structure that is called a centrosome in human cells and a spindle pole body in yeast cells. Human cells have two centrosomes, which reside on the opposite poles of the cell, and likewise for the spindle pole bodies in yeast cells. In mitotic cells and in meiosis II, microtubules attach to kinetochores in a way that means the sister chromatids are pulled apart. During meiosis I, on the other hand, they attach to kinetochores in a manner so the homologous chromosomes are pulled apart.
Miller et al. now show how the timing of the interaction between the kinetochore and microtubules is critical to ensure that the homologous chromosomes are separated during meiosis I. They found that premature interactions resulted in the separation of sister chromatids (as happens in mitosis) rather than the separation of homologous chromosomes, as is supposed to happen in meiosis I. They also showed that cells prevent such premature interactions by dismantling the outer regions of the kinetochore and reducing the levels of enzymes called CDKs in the cell. These results demonstrate that preventing premature microtubule–kinetochore interactions is essential for establishing a meiosis I-specific chromosome architecture, and they also provide fresh insights into how the molecular machinery that is responsible for mitotic chromosome segregation can be modulated to achieve meiosis.
DOI: http://dx.doi.org/10.7554/eLife.00117.002
doi:10.7554/eLife.00117
PMCID: PMC3525924  PMID: 23275833
meiosis; cyclin-dependent kinase; tension; cohesin; chromosome segregation; kinetochore; S. cerevisiae
7.  Loss of Function of the Cik1/Kar3 Motor Complex Results in Chromosomes with Syntelic Attachment That Are Sensed by the Tension Checkpoint 
PLoS Genetics  2012;8(2):e1002492.
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.
Author Summary
Chromosome bipolar attachment occurs when sister chromatids are attached by microtubules emanating from opposite spindle poles and is essential for faithful sister-chromatid segregation. Chromosomes are under tension once bipolar attachment is established. The absence of tension is sensed by the tension checkpoint that prevents chromosome segregation. The attachment of sister chromatids by microtubules from the same spindle pole generates syntelic attachment, which fails to generate tension on chromosomes. However, a reliable method to induce syntelic attachment is not available. Our findings indicate that the inactivation of the motor complex, Cik1/Kar3, results in chromosomes with syntelic attachment in budding yeast. In the absence of the tension checkpoint, yeast cells with dysfunctional Cik1/Kar3 enter anaphase, resulting in co-segregation of sister chromatids. Therefore, with this method we can experimentally induce syntelic attachment in yeast and investigate how cells respond to this incorrect attachment.
doi:10.1371/journal.pgen.1002492
PMCID: PMC3271067  PMID: 22319456
8.  Laterally attached kinetochores recruit the checkpoint protein Bub1, but satisfy the spindle checkpoint 
Cell cycle (Georgetown, Tex.)  2010;9(17):3619-3628.
Kinetochore attachment to the ends of dynamic microtubules is a conserved feature of mitotic spindle organization that is thought to be critical for proper chromosome segregation. Although kinetochores have been described to transition from lateral to end-on attachments, the phase of lateral attachment has been difficult to study in yeast due to its transient nature. We have previously described a kinetochore mutant, DAM1-765, which exhibits lateral attachments and misregulation of microtubule length. Here we show that the misregulation of microtubule length in DAM1-765 cells occurs despite localization of microtubule associated proteins Bik1, Stu2, Cin8, and Kip3 to microtubules. DAM1-765 kinetochores recruit the spindle checkpoint protein Bub1, however Bub1 localization to DAM1-765 kinetochores is not sufficient to cause a cell cycle arrest. Interestingly, the DAM1-765 mutation rescues the temperature sensitivity of a biorientation-deficient ipl1-321 mutant, and DAM1-765 chromosome loss rates are similar to wild-type cells. The spindle checkpoint in DAM1-765 cells responds properly to unattached kinetochores created by nocodazole treatment and loss of tension caused by a cohesin mutant. Progression of DAM1-765 cells through mitosis therefore suggests that satisfaction of the checkpoint depends more highly on biorientation of sister kinetochores than on achievement of a specific interaction between kinetochores and microtubule plus ends.
PMCID: PMC2963445  PMID: 20928940
spindle assembly checkpoint; kinetochore-microtubule attachments; biorientation; DAM1-765
9.  Laterally attached kinetochores recruit the checkpoint protein Bub1, but satisfy the spindle checkpoint 
Cell Cycle  2010;9(17):3619-3628.
Kinetochore attachment to the ends of dynamic microtubules is a conserved feature of mitotic spindle organization that is thought to be critical for proper chromosome segregation. Although kinetochores have been described to transition from lateral to end-on attachments, the phase of lateral attachment has been difficult to study in yeast due to its transient nature. We have previously described a kinetochore mutant, DAM1-765, which exhibits lateral attachments and misregulation of microtubule length. Here we show that the misregulation of microtubule length in DAM1-765 cells occurs despite localization of microtubule associated proteins Bik1, Stu2, Cin8 and Kip3 to microtubules. DAM1-765 kinetochores recruit the spindle checkpoint protein Bub1, however Bub1 localization to DAM1-765 kinetochores is not sufficient to cause a cell cycle arrest. Interestingly, the DAM1-765 mutation rescues the temperature sensitivity of a biorientationdeficient ipl1-321 mutant, and DAM1-765 chromosome loss rates are similar to wild-type cells. the spindle checkpoint in DAM1-765 cells responds properly to unattached kinetochores created by nocodazole treatment and loss of tension caused by a cohesin mutant. progression of DAM1-765 cells through mitosis therefore suggests that satisfaction of the checkpoint depends more highly on biorientation of sister kinetochores than on achievement of a specific interaction between kinetochores and microtubule plus ends.
doi:10.4161/cc.9.17.12907
PMCID: PMC2963445  PMID: 20928940
spindle assembly checkpoint; kinetochore-microtubule attachments; biorientation; DAM1-765
10.  Chromosome segregation in plant meiosis 
Faithful chromosome segregation in meiosis is essential for ploidy stability over sexual life cycles. In plants, defective chromosome segregation caused by gene mutations or other factors leads to the formation of unbalanced or unreduced gametes creating aneuploid or polyploid progeny, respectively. Accurate segregation requires the coordinated execution of conserved processes occurring throughout the two meiotic cell divisions. Synapsis and recombination ensure the establishment of chiasmata that hold homologous chromosomes together allowing their correct segregation in the first meiotic division, which is also tightly regulated by cell-cycle dependent release of cohesin and monopolar attachment of sister kinetochores to microtubules. In meiosis II, bi-orientation of sister kinetochores and proper spindle orientation correctly segregate chromosomes in four haploid cells. Checkpoint mechanisms acting at kinetochores control the accuracy of kinetochore-microtubule attachment, thus ensuring the completion of segregation. Here we review the current knowledge on the processes taking place during chromosome segregation in plant meiosis, focusing on the characterization of the molecular factors involved.
doi:10.3389/fpls.2014.00279
PMCID: PMC4060054  PMID: 24987397
chromosome segregation; cohesion; kinetochore; meiosis; plant; recombination; spindle; synapsis
11.  The Mub1/Ubr2 Ubiquitin Ligase Complex Regulates the Conserved Dsn1 Kinetochore Protein 
PLoS Genetics  2013;9(2):e1003216.
The kinetochore is the macromolecular complex that assembles onto centromeric DNA and orchestrates the segregation of duplicated chromosomes. More than 60 components make up the budding yeast kinetochore, including inner kinetochore proteins that bind to centromeric chromatin and outer proteins that directly interact with microtubules. However, little is known about how these components assemble into a functional kinetochore and whether there are quality control mechanisms that monitor kinetochore integrity. We previously developed a method to isolate kinetochore particles via purification of the conserved Dsn1 kinetochore protein. We find that the Mub1/Ubr2 ubiquitin ligase complex associates with kinetochore particles through the CENP-CMif2 protein. Although Mub1/Ubr2 are not stable kinetochore components in vivo, they regulate the levels of the conserved outer kinetochore protein Dsn1 via ubiquitylation. Strikingly, a deletion of Mub1/Ubr2 restores the levels and viability of a mutant Dsn1 protein, reminiscent of quality control systems that target aberrant proteins for degradation. Consistent with this, Mub1/Ubr2 help to maintain viability when kinetochores are defective. Together, our data identify a previously unknown regulatory mechanism for the conserved Dsn1 kinetochore protein. We propose that Mub1/Ubr2 are part of a quality control system that monitors kinetochore integrity, thus ensuring genomic stability.
Author Summary
The flawless execution of cell division is essential to the survival of all organisms. The loss or gain of a single chromosome, the state called aneuploidy, is a hallmark of cancer cells and is the leading cause of spontaneous miscarriages and hereditary birth defects. Segregation is mediated by the kinetochore, the macromolecular complex that assembles on each chromosome and attaches to spindle microtubules to pull chromosomes to opposite poles when cells divide. It is therefore critical to understand how kinetochores are assembled and maintained. Here, we find that the levels of a conserved kinetochore protein are regulated by proteolysis. We propose that cells have quality control systems that ensure kinetochore integrity and thus genome stability.
doi:10.1371/journal.pgen.1003216
PMCID: PMC3567142  PMID: 23408894
12.  Nsk1 ensures accurate chromosome segregation by promoting association of kinetochores to spindle poles during anaphase B 
Molecular Biology of the Cell  2011;22(23):4486-4502.
Nsk1 is a novel fission yeast protein that binds the nucleolus during interphase and the nucleoplasm during early mitosis. After anaphase and following dephosphorylation by Clp1, Nsk1 binds the kinetochore–spindle pole junction and maintains accurate chromosome segregation by promoting the 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.
doi:10.1091/mbc.E11-07-0608
PMCID: PMC3226469  PMID: 21965289
13.  Drosophila CENP-A Mutations Cause a BubR1- Dependent Early Mitotic Delay without Normal Localization of Kinetochore Components 
PLoS Genetics  2006;2(7):e110.
The centromere/kinetochore complex plays an essential role in cell and organismal viability by ensuring chromosome movements during mitosis and meiosis. The kinetochore also mediates the spindle attachment checkpoint (SAC), which delays anaphase initiation until all chromosomes have achieved bipolar attachment of kinetochores to the mitotic spindle. CENP-A proteins are centromere-specific chromatin components that provide both a structural and a functional foundation for kinetochore formation. Here we show that cells in Drosophila embryos homozygous for null mutations in CENP-A (CID) display an early mitotic delay. This mitotic delay is not suppressed by inactivation of the DNA damage checkpoint and is unlikely to be the result of DNA damage. Surprisingly, mutation of the SAC component BUBR1 partially suppresses this mitotic delay. Furthermore, cid mutants retain an intact SAC response to spindle disruption despite the inability of many kinetochore proteins, including SAC components, to target to kinetochores. We propose that SAC components are able to monitor spindle assembly and inhibit cell cycle progression in the absence of sustained kinetochore localization.
Synopsis
Normal inheritance of genetic traits from one cell or organismal generation to the next depends on accurate chromosome replication and segregation. Defective chromosome segregation is associated with birth defects and cancer. The centromere is a single site on the chromosome that is responsible for assembling the kinetochore, which mediates chromosome attachment to the microtubule spindle and all chromosome movements. In addition, the spindle assembly checkpoint (SAC) ensures normal inheritance by delaying entry into anaphase when chromosome–spindle attachments are defective. Previous studies suggested that SAC function required kinetochore localization of key components. This study shows that elimination of a centromere-specific histone (CID) results in an early mitotic delay. Although this delay occurs earlier than the established time of SAC function (at the metaphase–anaphase transition), it depends on the presence of an essential SAC protein (BUBR1). Furthermore, the CID-mediated early mitotic delay occurs in the absence of kinetochore formation or localization of key SAC proteins. These results suggest that the fidelity of kinetochore–microtubule attachment is also monitored early in mitosis, and in the absence of kinetochore formation and localization of SAC components.
doi:10.1371/journal.pgen.0020110
PMCID: PMC1500813  PMID: 16839185
14.  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.
doi:10.1098/rstb.2004.1610
PMCID: PMC1569470  PMID: 15897180
mitosis; microtubule; kinetochore; aneuploidy; Ndc80; chromosome
15.  Dual Role of Topoisomerase II in Centromere Resolution and Aurora B Activity 
PLoS Biology  2008;6(8):e207.
Chromosome segregation requires sister chromatid resolution. Condensins are essential for this process since they organize an axial structure where topoisomerase II can work. How sister chromatid separation is coordinated with chromosome condensation and decatenation activity remains unknown. We combined four-dimensional (4D) microscopy, RNA interference (RNAi), and biochemical analyses to show that topoisomerase II plays an essential role in this process. Either depletion of topoisomerase II or exposure to specific anti-topoisomerase II inhibitors causes centromere nondisjunction, associated with syntelic chromosome attachments. However, cells degrade cohesins and timely exit mitosis after satisfying the spindle assembly checkpoint. Moreover, in topoisomerase II–depleted cells, Aurora B and INCENP fail to transfer to the central spindle in late mitosis and remain tightly associated with centromeres of nondisjoined sister chromatids. Also, in topoisomerase II–depleted cells, Aurora B shows significantly reduced kinase activity both in S2 and HeLa cells. Codepletion of BubR1 in S2 cells restores Aurora B kinase activity, and consequently, most syntelic attachments are released. Taken together, our results support that topoisomerase II ensures proper sister chromatid separation through a direct role in centromere resolution and prevents incorrect microtubule–kinetochore attachments by allowing proper activation of Aurora B kinase.
Author Summary
Successful cell division requires that chromosomes are properly condensed and that each sister chromatid is self-contained by the time the sister pairs are segregated into separate daughter cells. It is also essential that the kinetochores at the centromeres of each pair of sister chromatids bind microtubules from opposite spindle poles. Topoisomerase II is a highly conserved enzyme that removes interlinks from DNA and is known to be essential to proper chromosome segregation during cell division. In this work, we have used state-of-the-art four-dimensional fluorescent microscopy to follow progression through mitosis in living cells depleted of topoisomerase II. We find that when the enzyme is absent, the two sister centromeres do not separate, and chromosomes missegregate. Moreover, the inappropriate centromere structure that results prevents the correct activation of the Aurora B kinase, which forms part of a regulatory mechanism that monitors correct segregation of chromosomes; as a result, cells exit mitosis abnormally.
Analysis of cells lacking topoisomerase II reveals that the enzyme has an essential role in the segregation of chromosomes, and specifically centromeres, at anaphase-telophase of mitosis: it prevents non-disjunction and allows activation of the Aurora B kinase, so as to correct improper attachments between microtubules and the kinetochore.
doi:10.1371/journal.pbio.0060207
PMCID: PMC2525683  PMID: 18752348
16.  Two microtubule-associated proteins required for anaphase spindle movement in Saccharomyces cerevisiae [published erratum appears in J Cell Biol 1995 Oct;131(2):561] 
The Journal of Cell Biology  1995;130(6):1373-1385.
In many eucaryotic cells, the midzone of the mitotic spindle forms a distinct structure containing a specific set of proteins. We have isolated ASE1, a gene encoding a component of the Saccharomyces cerevisiae spindle midzone. Strains lacking both ASE1 and BIK1, which encodes an S. cerevisiae microtubule-associated protein, are inviable. The analysis of the phenotype of a bik1 ase1 conditional double mutant suggests that BIK1 and ASE1 are not required for the assembly of a bipolar spindle, but are essential for anaphase spindle elongation. The steady-state levels of Ase1p are regulated in a manner that is consistent with a function during anaphase: they are low in G1, accumulate to maximal levels after S phase and then drop as cells exit mitosis. Components of the spindle midzone may therefore be required in vivo for anaphase spindle movement. Additionally, anaphase spindle movement may depend on a dedicated set of genes whose expression is induced at G2/M.
PMCID: PMC2120566  PMID: 7559759
17.  Ska3 Is Required for Spindle Checkpoint Silencing and the Maintenance of Chromosome Cohesion in Mitosis 
Current biology : CB  2009;19(17):1467-1472.
SUMMARY
Balanced chromosome segregation in mitosis requires synchronous chromatid separation at anaphase and the precise coordination of anaphase with cytokinesis and mitotic exit. The mitotic spindle checkpoint monitors proper attachment and/or tension induced by microtubule binding to sister kinetochores. Within each cell, once all chromosomes achieve bipolar attachment to the spindle poles and align at the metaphase plate, the spindle checkpoint is silenced triggering anaphase onset, cytokinesis, and mitotic exit. We used a bioinformatics approach to identify a candidate protein, C13orf3/Ska3, predicted to function in mitosis. Cells in which Ska3 expression was reduced by RNAi achieved metaphase alignment but were unable to silence the spindle checkpoint and enter normal anaphase. After hours of metaphase arrest, chromatids separated but retained robust attachment to spindle microtubules. These cells remained checkpoint arrested with strong accumulation of the checkpoint protein Bub1 at kinetochores. During normal mitosis Ska3 protein accumulated on kinetochores in prometaphase after nuclear envelope breakdown. This kinetochore localization of Ska3 was dependent on Shugoshin (Sgo1), the “guardian spirit” of chromatid cohesion. In contrast, Sgo1, which accumulates at the centromeres in early prophase, was not dependent on Ska3. Although Ska3 is required for maintenance of sister chromatid cohesion and is dependent upon Sgo1, cells with reduced Sgo1 show a stronger premature chromatid separation phenotype than those with reduced Ska3. We hypothesize that Ska3 functions as a component of a network that coordinates checkpoint signaling from the microtubule binding sites within a kinetochore by laterally linking the individual binding sites. We suggest that this network plays a major role in silencing the spindle checkpoint when chromosomes are aligned at metaphase to allow timely anaphase onset and mitotic exit.
doi:10.1016/j.cub.2009.07.017
PMCID: PMC2783354  PMID: 19646878
18.  Regulation of Microtubule Dynamics by Bim1 and Bik1, the Budding Yeast Members of the EB1 and CLIP-170 Families of Plus-End Tracking Proteins 
Molecular Biology of the Cell  2010;21(12):2013-2023.
Bim1 promotes microtubule assembly in vitro, primarily by decreasing the frequency of catastrophes. In contrast, Bik1 inhibits microtubule assembly by slowing growth and, consequently, promoting catastrophes. These proteins interact to form a complex that affects microtubule dynamics in much the same way as Bim1 alone.
Microtubule dynamics are regulated by plus-end tracking proteins (+TIPs), which bind microtubule ends and influence their polymerization properties. In addition to binding microtubules, most +TIPs physically associate with other +TIPs, creating a complex web of interactions. To fully understand how +TIPs regulate microtubule dynamics, it is essential to know the intrinsic biochemical activities of each +TIP and how +TIP interactions affect these activities. Here, we describe the activities of Bim1 and Bik1, two +TIP proteins from budding yeast and members of the EB1 and CLIP-170 families, respectively. We find that purified Bim1 and Bik1 form homodimers that interact with each other to form a tetramer. Bim1 binds along the microtubule lattice but with highest affinity for the microtubule end; however, Bik1 requires Bim1 for localization to the microtubule lattice and end. In vitro microtubule polymerization assays show that Bim1 promotes microtubule assembly, primarily by decreasing the frequency of catastrophes. In contrast, Bik1 inhibits microtubule assembly by slowing growth and, consequently, promoting catastrophes. Interestingly, the Bim1-Bik1 complex affects microtubule dynamics in much the same way as Bim1 alone. These studies reveal new activities for EB1 and CLIP-170 family members and demonstrate how interactions between two +TIP proteins influence their activities.
doi:10.1091/mbc.E10-02-0083
PMCID: PMC2883945  PMID: 20392838
19.  The Regulation of Microtubule Dynamics in Saccharomyces cerevisiae by Three Interacting Plus-End Tracking Proteins 
Molecular Biology of the Cell  2006;17(6):2789-2798.
Microtubule plus-end tracking proteins (+TIPs) are a diverse group of molecules that regulate microtubule dynamics and interactions of microtubules with other cellular structures. Many +TIPs have affinity for each other but the functional significance of these associations is unclear. Here we investigate the physical and functional interactions among three +TIPs in S. cerevisiae, Stu2, Bik1, and Bim1. Two-hybrid, coimmunoprecipitation, and in vitro binding assays demonstrate that they associate in all pairwise combinations, although the interaction between Stu2 and Bim1 may be indirect. Three-hybrid assays indicate that these proteins compete for binding to each other. Thus, Stu2, Bik1, and Bim1 interact physically but do not appear to be arranged in a single unique complex. We examined the functional interactions among pairs of proteins by comparing cytoplasmic and spindle microtubule dynamics in cells lacking either one or both proteins. On cytoplasmic microtubules, Stu2 and Bim1 act cooperatively to regulate dynamics in G1 but not in preanaphase, whereas Bik1 acts independently from Stu2 and Bim1. On kinetochore microtubules, Bik1 and Bim1 are redundant for regulating dynamics, whereas Stu2 acts independently from Bik1 and Bim1. These results indicate that interactions among +TIPS can play important roles in the regulation of microtubule dynamics.
doi:10.1091/mbc.E05-09-0892
PMCID: PMC1474793  PMID: 16571681
20.  Mps1 Kinase Promotes Sister-Kinetochore Bi-orientation by a Tension-Dependent Mechanism 
Current Biology  2007;17(24):2175-2182.
Summary
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 [1, 2]. This re-orientation is facilitated by Aurora B kinase (Ipl1 in budding yeast), which eliminates kinetochore-spindle pole connections that do not generate tension [3–6]. Mps1 is another evolutionarily conserved protein kinase, required for spindle-assembly checkpoint and, in some organisms, for duplication of microtubule-organizing centers [7]. Separately from these functions, however, Mps1 has an important role in chromosome segregation [8]. 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.
doi:10.1016/j.cub.2007.11.032
PMCID: PMC2515371  PMID: 18060784
CELLCYCLE; CELLBIO
21.  Mps1 Kinase Promotes Sister-Kinetochore Bi-orientation by a Tension-Dependent Mechanism 
Current biology : CB  2007;17(24):2175-2182.
Summary
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 [1, 2]. This re-orientation is facilitated by Aurora B kinase (Ipl1 in budding yeast), which eliminates kinetochore-spindle pole connections that do not generate tension [3-6]. Mps1 is another evolutionarily conserved protein kinase, required for spindle-assembly checkpoint and, in some organisms, for duplication of microtubule-organizing centers [7]. Separately from these functions, however, Mps1 has an important role in chromosome segregation [8]. 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.
doi:10.1016/j.cub.2007.11.032
PMCID: PMC2515371  PMID: 18060784
22.  The CLIP-170 Homologue Bik1p Promotes the Phosphorylation and Asymmetric Localization of Kar9pD⃞ 
Molecular Biology of the Cell  2006;17(1):178-191.
Accurate positioning of the mitotic spindle in Saccharomyces cerevisiae is coordinated with the asymmetry of the two poles and requires the microtubule-to-actin linker Kar9p. The asymmetric localization of Kar9p to one spindle pole body (SPB) and microtubule (MT) plus ends requires Cdc28p. Here, we show that the CLIP-170 homologue Bik1p binds directly to Kar9p. In the absence of Bik1p, Kar9p localization is not restricted to the daughter-bound SPB, but it is instead found on both SPBs. Kar9p is hypophosphorylated in bik1Δ mutants, and Bik1p binds to both phosphorylated and unphosphorylated isoforms of Kar9p. Furthermore, the two-hybrid interaction between full-length KAR9 and the cyclin CLB5 requires BIK1. The binding site of Clb5p on Kar9p maps to a short region within the basic domain of Kar9p that contains a conserved phosphorylation site, serine 496. Consistent with this, Kar9p is found on both SPBs in clb5Δ mutants at a frequency comparable with that seen in kar9-S496A strains. Together, these data suggest that Bik1p promotes the phosphorylation of Kar9p on serine 496, which affects its asymmetric localization to one SPB and associated cytoplasmic MTs. These findings provide further insight into a mechanism for directing centrosomal inheritance.
doi:10.1091/mbc.E05-06-0565
PMCID: PMC1345657  PMID: 16236795
23.  Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast 
Molecular Biology of the Cell  2011;22(9):1473-1485.
Members of chromosome passenger complex BIR1 and SLI15 suppress the chromosome segregation defect of bub1Δ and sgo1Δ. Neither Bub1 nor Sgo1 is required for CPC activity. This study found genetic interaction between Mps1 and Sgo1. Mps1 governs localization of Sgo1. Bub1, Sgo1, and Mps1 function in parallel to the CPC in correction of syntelic attachments.
The conserved mitotic kinase Bub1 performs multiple functions that are only partially characterized. Besides its role in the spindle assembly checkpoint and chromosome alignment, Bub1 is crucial for the kinetochore recruitment of multiple proteins, among them Sgo1. Both Bub1 and Sgo1 are dispensable for growth of haploid and diploid budding yeast, but they become essential in cells with higher ploidy. We find that overexpression of SGO1 partially corrects the chromosome segregation defect of bub1Δ haploid cells and restores viability to bub1Δ tetraploid cells. Using an unbiased high-copy suppressor screen, we identified two members of the chromosomal passenger complex (CPC), BIR1 (survivin) and SLI15 (INCENP, inner centromere protein), as suppressors of the growth defect of both bub1Δ and sgo1Δ tetraploids, suggesting that these mutants die due to defects in chromosome biorientation. Overexpression of BIR1 or SLI15 also complements the benomyl sensitivity of haploid bub1Δ and sgo1Δ cells. Mutants lacking SGO1 fail to biorient sister chromatids attached to the same spindle pole (syntelic attachment) after nocodazole treatment. Moreover, the sgo1Δ cells accumulate syntelic attachments in unperturbed mitoses, a defect that is partially corrected by BIR1 or SLI15 overexpression. We show that in budding yeast neither Bub1 nor Sgo1 is required for CPC localization or affects Aurora B activity. Instead we identify Sgo1 as a possible partner of Mps1, a mitotic kinase suggested to have an Aurora B–independent function in establishment of biorientation. We found that Sgo1 overexpression rescues defects caused by metaphase inactivation of Mps1 and that Mps1 is required for Sgo1 localization to the kinetochore. We propose that Bub1, Sgo1, and Mps1 facilitate chromosome biorientation independently of the Aurora B–mediated pathway at the budding yeast kinetochore and that both pathways are required for the efficient turnover of syntelic attachments.
doi:10.1091/mbc.E10-08-0673
PMCID: PMC3084670  PMID: 21389114
24.  Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling 
eLife  2013;2:e01030.
Regulation of macromolecular interactions by phosphorylation is crucial in signaling networks. In the spindle assembly checkpoint (SAC), which enables errorless chromosome segregation, phosphorylation promotes recruitment of SAC proteins to tensionless kinetochores. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on the kinetochore subunit Spc105/Knl1. The phosphorylated MELT motifs (MELTP) then promote recruitment of downstream signaling components. How MELTP motifs are recognized is unclear. In this study, we report that Bub3, a 7-bladed β-propeller, is the MELTP reader. It contains an exceptionally well-conserved interface that docks the MELTP sequence on the side of the β-propeller in a previously unknown binding mode. Mutations targeting the Bub3 interface prevent kinetochore recruitment of the SAC kinase Bub1. Crucially, they also cause a checkpoint defect, showing that recognition of phosphorylated targets by Bub3 is required for checkpoint signaling. Our data provide the first detailed mechanistic insight into how phosphorylation promotes recruitment of checkpoint proteins to kinetochores.
DOI: http://dx.doi.org/10.7554/eLife.01030.001
eLife digest
The cell cycle is the process by which a cell divides to produce two near-identical daughter cells. Two crucial parts of the cell cycle are the duplication of the chromosomes in the original cell, and the segregation of these chromosomes between the two daughter cells. These and other parts of the cell cycle are strictly regulated to prevent errors, which can lead to cancer and other diseases.
After chromosome duplication has taken place, the pairs of identical chromosomes, known as sister chromatids, remain tightly bound to each other. These sister chromatids line up in the middle of the cell, with protein filaments called microtubules connecting them to a bipolar structure called the spindle. For the cell to divide correctly, the sister chromatids in each pair must be connected to opposite poles of the spindle. A signalling network known as the spindle assembly checkpoint (SAC) ensures that the sister chromatids have enough time to line up correctly and to correct possible problems. Once everything is in place, the SAC releases its ‘break’, and the microtubules then pull the sister chromatids away from each other. This way, each daughter cell receives the same complement of chromosomes that was present in the mother cell.
The microtubules are not directly attached to the sister chromatids but to protein complexes called kinetochores that assemble on each sister chromatid. In particular, each microtubule binds to a very large protein complex called the KMN network. Knl1, which is part of this network, recruits two SAC proteins–Bub1 and Bub3–to the kinetochore. It is known that a phosphate group is added to Knl1 when the SAC is active, and that Knl1 can only recruit Bub1 and Bub3 after it has been phosphorylated. However, the details of the interactions between Knl1, Bub1 and Bub3 are not understood, and it is not clear whether these interactions are essential for the SAC.
Now Primorac et al. have shown that Bub3 binds directly to Knl1 through a region that contains multiple MELT motifs (where M, E, L and T are all amino acids), and that this interaction only happens if these ‘MELT repeats’ have been phosphorylated. Moreover, once bound to the Knl1, Bub3 then recruits Bub1 to the kinetochore. By showing that the recognition of phosphorylated Knl1 by the Bub1-Bub3 complex has a central role in the spindle assembly checkpoint, these results highlight the importance of phosphorylation as a way of regulating the timing of events during the cell cycle.
DOI: http://dx.doi.org/10.7554/eLife.01030.002
doi:10.7554/eLife.01030
PMCID: PMC3779320  PMID: 24066227
spindle assembly checkpoint; kinetochore; Bub3; Bub1; Mad3; Knl1; S. cerevisiae
25.  Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle 
The Journal of Cell Biology  1994;127(5):1301-1310.
To test the popular but unproven assumption that the metaphase-anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK1 cells. We found that the time from nuclear envelope breakdown to anaphase onset is linearly related (r2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays anaphase onset. We also found that anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle. We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered anaphase and completed normal poleward chromosome motion (anaphase A) up to 3 h after the treatment--well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.
PMCID: PMC2120267  PMID: 7962091

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