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1.  Sister kinetochores are mechanically fused during meiosis I in yeast 
Science (New York, N.Y.)  2014;346(6206):248-251.
Production of healthy gametes requires a reductional meiosis I division in which replicated sister chromatids co-migrate, rather than separating as in mitosis or meiosis II. Fusion of sister kinetochores during meiosis I may underlie sister chromatid co-migration in diverse organisms, but direct evidence for such fusion has been lacking. Here we studied native kinetochore particles isolated from yeast using laser trapping and quantitative fluorescence microscopy. Meiosis I kinetochores formed stronger attachments and carried more microtubule-binding elements than kinetochores isolated from cells in mitosis or meiosis II. The meiosis I-specific monopolin complex was both necessary and sufficient to drive these modifications. Thus, kinetochore fusion directs sister chromatid co-migration, a conserved feature of meiosis that is fundamental to Mendelian inheritance.
PMCID: PMC4226495  PMID: 25213378
2.  Measuring kinetochore-microtubule interaction in vitro 
Methods in enzymology  2014;540:321-337.
Many proteins and protein complexes perform sophisticated, regulated functions in vivo. Many of these functions can be recapitulated using in vitro reconstitution, which serves as a means to establish unambiguous cause-effect relationships, for example between a protein and its phosphorylating kinase. Here, we describe a protocol to purify kinetochores, the protein complexes that attach chromosomes to microtubules during mitosis, and quantitatively assay their microtubule binding characteristics. Our assays, based on DIC imaging and laser trapping microscopy, are used to measure the attachment of microtubules to kinetochores and the load-bearing capabilities of those attachments. These assays provide a platform for studying kinase disruption of kinetochore-microtubule attachments, which is believed to be critical for correcting erroneous kinetochore-spindle attachments and thereby avoiding chromosome mis-segregation. The principles of our approach should be extensible to studies of a wide range of force-bearing interactions in biology.
PMCID: PMC4106247  PMID: 24630115
3.  Phosphoregulation of Spc105 by Mps1 and PP1 Regulates Bub1 Localization to Kinetochores 
Current biology : CB  2012;22(10):900-906.
Kinetochores are the macromolecular complexes that interact with microtubules to mediate chromosome segregation [1]. Accurate segregation requires that kinetochores make bioriented attachments to microtubules from opposite poles. Attachments between kinetochores and microtubules are monitored by the spindle checkpoint, a surveillance system that prevents anaphase until every pair of chromosomes makes proper bioriented attachments [2]. Checkpoint activity is correlated with the recruitment of checkpoint proteins to the kinetochore [1]. Mps1 is a conserved protein kinase that regulates segregation and the spindle checkpoint, but few of the targets that mediate its functions have been identified. Here, we show that Mps1 is the major kinase activity that co-purifies with budding yeast kinetochore particles and identify the conserved Spc105/KNL-1/Blinkin kinetochore protein as a substrate. Phosphorylation of conserved MELT motifs within Spc105 recruits the Bub1 protein to kinetochores and this is reversed by protein phosphatase I (PP1). Spc105 mutants lacking Mps1 phosphorylation sites are defective in the spindle checkpoint and exhibit growth defects. Together, these data identify Spc105 as a key target of the Mps1 kinase and show that the opposing activities of Mps1 and PP1 regulate the kinetochore localization of the Bub1 protein.
PMCID: PMC3723133  PMID: 22521787
4.  Reconstituting the kinetochore-microtubule interface: what, why, and how 
Chromosoma  2012;121(3):235-250.
The kinetochore is the proteinaceous complex that governs the movement of duplicated chromosomes by interacting with spindle microtubules during mitosis and meiosis. Faithful chromosome segregation requires that kinetochores form robust load-bearing attachments to the tips of dynamic spindle microtubules, correct microtubule attachment errors, and delay the onset of anaphase until all chromosomes have made proper attachments. To understand how this macromolecular machine operates to segregate duplicated chromosomes with exquisite accuracy, it is critical to reconstitute and study kinetochore-microtubule interactions in vitro using defined components. Here, we review the current status of reconstitution as well as recent progress in understanding the microtubule binding functions of kinetochores in vivo.
PMCID: PMC3383909  PMID: 22289864
Chromosome segregation; mitosis; kinetochore; microtubules; in vitro reconstitution
5.  The structure of purified kinetochores reveals multiple microtubule attachment sites 
Chromosomes must be accurately partitioned to daughter cells to prevent aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are the macromolecular machines that segregate chromosomes by maintaining load-bearing attachments to the dynamic tips of microtubules. Here, we present the structure of isolated budding yeast kinetochore particles as visualized by electron microscopy (EM) and electron tomography of negatively stained preparations. The kinetochore appears as a ~126 nm particle containing a large central hub surrounded by multiple outer globular domains. In the presence of microtubules, some particles also have a ring that encircles the microtubule. Our data show that kinetochores bind to microtubules via multivalent attachments and lay the foundation to uncover the key mechanical and regulatory mechanisms by which kinetochores control chromosome segregation and cell division.
PMCID: PMC3443262  PMID: 22885327
6.  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.
PMCID: PMC3567142  PMID: 23408894
7.  An efficient purification system for native minichromosome from S. cerevisiae 
We have recently established a system for purifying minichromosome in a native state from S. cerevisiae. This system is extremely efficient, and a single-step purification yields samples with sufficient purity and quantity for mass spectrometry (MS) analysis of histones and non-histone proteins tightly associated with the minichromosome. The templates can also be used in various biochemical assays in vitro, such as transcription and recombination, and might be applicable to allow EM or other studies to be performed.
PMCID: PMC3243952  PMID: 22183591
chromatin; minichromosome; purification; histone modifications; episome; mass spectrometry; DNA replication; replication origin; TRP1-ARS1; FLAG tag
8.  An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain 
Molecular cell  2010;40(3):455-464.
Proper centromere function is critical to maintain genomic stability and to prevent aneuploidy, a hallmark of tumors and birth defects. A conserved feature of all eukaryotic centromeres is an essential histone H3 variant called CENP-A that requires a centromere targeting domain (CATD) for its localization. Although proteolysis prevents CENP-A from mislocalizing to euchromatin, regulatory factors have not been identified. Here, we identify an E3 ubiquitin ligase called Psh1 that leads to the degradation of Cse4, the budding yeast CENP-A homolog. Cse4 overexpression is toxic to psh1Δ cells and results in euchromatic localization. Strikingly, the Cse4 centromere targeting domain is a key regulator of its stability and helps Psh1 discriminate Cse4 from histone H3. Taken together, we propose that the CATD has a previously unknown role in maintaining the exclusive localization of Cse4 by preventing its mislocalization to euchromatin via Psh1-mediated degradation.
PMCID: PMC2995698  PMID: 21070971
9.  Tension directly stabilizes reconstituted kinetochore-microtubule attachments 
Nature  2010;468(7323):576-579.
Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes1,2. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore-microtubule attachments, which come under tension due to opposing forces exerted by microtubules3. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B4,5. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules6. Moreover, tension increases the lifetimes of the reconstituted attachments directly, via a catch bond-like mechanism that does not require Aurora B7-10. Based on these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.
PMCID: PMC3108429  PMID: 21107429
10.  Protein Phosphatase I Regulates Exit from the Spindle Checkpoint in Budding Yeast 
Current biology : CB  2009;19(14):1182-1187.
Accurate chromosome segregation depends on sister kinetochores coming under tension when they make bioriented attachments to microtubules from opposite poles. The spindle checkpoint halts the cell cycle in response to defects in generating proper attachments or tension on kinetochores [1, 2], although the precise signal that triggers the checkpoint is unclear because tension and attachment are coupled [3]. The target of the checkpoint is the Cdc20 protein that initiates the anaphase promoting complex (APC)-dependent degradation of the anaphase inhibitor Pds1/securin [4]. Although the molecular details of spindle checkpoint activation are still being elucidated, phosphorylation by at least four kinases is a crucial requirement [5]. However, less is known about the mechanisms that silence the checkpoint once kinetochores biorient. Here, we show that the catalytic subunit of the budding yeast protein phosphatase I, Glc7, regulates exit from the checkpoint. Glc7 overexpression prevents spindle checkpoint activation in response to both tension and attachment defects. Although glc7 mutant cells are able to efficiently release from a non-checkpoint-mediated metaphase arrest, they are uniquely sensitive to transient spindle checkpoint activation due to a failure in spindle checkpoint exit. We therefore propose that PP1 activity silences the checkpoint by reversing key phosphorylation events.
PMCID: PMC2731492  PMID: 19592248
11.  Pericentromeric Sister Chromatid Cohesion Promotes Kinetochore Biorientation 
Molecular Biology of the Cell  2009;20(17):3818-3827.
Accurate chromosome segregation depends on sister kinetochores making bioriented attachments to microtubules from opposite poles. An essential regulator of biorientation is the Ipl1/Aurora B protein kinase that destabilizes improper microtubule–kinetochore attachments. To identify additional biorientation pathways, we performed a systematic genetic analysis between the ipl1-321 allele and all nonessential budding yeast genes. One of the mutants, mcm21Δ, precociously separates pericentromeres and this is associated with a defect in the binding of the Scc2 cohesin-loading factor at the centromere. Strikingly, Mcm21 becomes essential for biorientation when Ipl1 function is reduced, and this appears to be related to its role in pericentromeric cohesion. When pericentromeres are artificially tethered, Mcm21 is no longer needed for biorientation despite decreased Ipl1 activity. Taken together, these data reveal a specific role for pericentromeric linkage in ensuring kinetochore biorientation.
PMCID: PMC2735481  PMID: 19605555
12.  A pathway containing the Ipl1/Aurora protein kinase and the spindle midzone protein Ase1 regulates yeast spindle assembly 
Developmental cell  2007;13(3):433-445.
It is critical to elucidate the pathways that mediate spindle assembly and therefore ensure accurate chromosome segregation during cell division. Our studies of a unique allele of the budding yeast Ipl1/Aurora protein kinase revealed that it is required for centrosome-mediated spindle assembly in the absence of the BimC motor protein Cin8. In addition, we found that the Ase1 spindle midzone-associated protein is required for bipolar spindle assembly. The cin8 ipl1 and cin8 ase1 double mutant cells exhibit similar defects, and Ase1 overexpression completely restores spindle assembly in cin8 ipl1 strains. Consistent with the possibility that Ipl1 regulates Ase1, an ase1 mutant lacking the Ipl1 consensus phosphorylation sites cannot assemble spindles in the absence of Cin8. In addition, Ase1 phosphorylation and localization were altered in an ipl1 mutant. We therefore propose that Ipl1/Aurora and Ase1 constitute a previously unidentified spindle assembly pathway that becomes essential in the absence of Cin8.
PMCID: PMC2679386  PMID: 17765685
13.  Comment on “A Centrosome-Independent Role for γ-TuRC Proteins in the Spindle Assembly Checkpoint” 
Science (New York, N.Y.)  2007;316(5827):982.
Müller et al. (Reports, 27 October 2006, p. 654) showed that inhibition of the γ-tubulin ring complex (γ-TuRC) activates the spindle assembly checkpoint (SAC), which led them to suggest that γ-TuRC proteins play molecular roles in SAC activation. Because γ-TuRC inhibition leads to pleiotropic spindle defects, which are well known to activate kinetochore-derived checkpoint signaling, we believe that this conclusion is premature.
PMCID: PMC2590763  PMID: 17510347
14.  Glc7/Protein Phosphatase 1 Regulatory Subunits Can Oppose the Ipl1/Aurora Protein Kinase by Redistributing Glc7 
Molecular and Cellular Biology  2006;26(7):2648-2660.
Faithful chromosome segregation depends on the opposing activities of the budding yeast Glc7/PP1 protein phosphatase and Ipl1/Aurora protein kinase. We explored the relationship between Glc7 and Ipl1 and found that the phosphorylation of the Ipl1 substrate, Dam1, was altered by decreased Glc7 activity, whereas Ipl1 levels, localization, and kinase activity were not. These data strongly suggest that Glc7 ensures accurate chromosome segregation by dephosphorylating Ipl1 targets rather than regulating the Ipl1 kinase. To identify potential Glc7 and Ipl1 substrates, we isolated ipl1-321 dosage suppressors. Seven genes (SDS22, BUD14, GIP3, GIP4, SOL1, SOL2, and PEX31) encode newly identified ipl1 dosage suppressors, and all 10 suppressors encode proteins that physically interact with Glc7. The overexpression of the Gip3 and Gip4 suppressors altered Glc7 localization, indicating they are previously unidentified Glc7 regulatory subunits. In addition, the overexpression of Gip3 and Gip4 from the galactose promoter restored Dam1 phosphorylation in ipl1-321 mutant cells and caused wild-type cells to arrest in metaphase with unsegregated chromosomes, suggesting that Gip3 and Gip4 overexpression impairs Glc7's mitotic functions. We therefore propose that the overexpression of Glc7 regulatory subunits can titrate Glc7 away from relevant Ipl1 targets and thereby suppress ipl1-321 cells by restoring the balance of phosphatase/kinase activity.
PMCID: PMC1430313  PMID: 16537909
15.  De Novo Kinetochore Assembly Requires the Centromeric Histone H3 VariantD⃞ 
Molecular Biology of the Cell  2005;16(12):5649-5660.
Kinetochores mediate chromosome attachment to the mitotic spindle to ensure accurate chromosome segregation. Budding yeast is an excellent organism for kinetochore assembly studies because it has a simple defined centromere sequence responsible for the localization of >65 proteins. In addition, yeast is the only organism where a conditional centromere is available to allow studies of de novo kinetochore assembly. Using a conditional centromere, we found that yeast kinetochore assembly is not temporally restricted and can occur in both G1 phase and prometaphase. We performed the first investigation of kinetochore assembly in the absence of the centromeric histone H3 variant Cse4 and found that all proteins tested depend on Cse4 to localize. Consistent with this observation, Cse4-depleted cells had severe chromosome segregation defects. We therefore propose that yeast kinetochore assembly requires both centromeric DNA specificity and centromeric chromatin.
PMCID: PMC1289410  PMID: 16207811
16.  The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly 
The Journal of Cell Biology  2003;160(3):329-339.
Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1–GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.
PMCID: PMC2172676  PMID: 12566427
Ipl1/Aurora protein kinase; spindle; mitosis; microtubule; budding yeast
17.  Mutation of YCS4, a Budding Yeast Condensin Subunit, Affects Mitotic and Nonmitotic Chromosome Behavior 
Molecular Biology of the Cell  2002;13(2):632-645.
The budding yeast YCS4 gene encodes a conserved regulatory subunit of the condensin complex. We isolated an allele of this gene in a screen for mutants defective in sister chromatid separation or segregation. The phenotype of the ycs4-1 mutant is similar to topoisomerase II mutants and distinct from the esp1-1 mutant: the topological resolution of sister chromatids is compromised in ycs4-1 despite normal removal of cohesins from mitotic chromosomes. Consistent with a role in sister separation, YCS4 function is required to localize DNA topoisomerase I and II to chromosomes. Unlike its homologs in Xenopus and fission yeast, Ycs4p is associated with chromatin throughout the cell cycle; the only change in localization occurs during anaphase when the protein is enriched at the nucleolus. This relocalization may reveal the specific challenge that segregation of the transcriptionally hyperactive, repetitive array of rDNA genes can present during mitosis. Indeed, segregation of the nucleolus is abnormal in ycs4-1 at the nonpermissive temperature. Interrepeat recombination in the rDNA array is specifically elevated in ycs4-1 at the permissive temperature, suggesting that the Ycs4p plays a role at the array aside from its segregation. Furthermore, ycs4-1 is defective in silencing at the mating type loci at the permissive temperature. Taken together, our data suggest that there are mitotic as well as nonmitotic chromosomal abnormalities associated with loss of condensin function in budding yeast.
PMCID: PMC65655  PMID: 11854418
18.  The Yeast Centrin, Cdc31p, and the Interacting Protein Kinase, Kic1p, Are Required for Cell Integrity  
The Journal of Cell Biology  1998;143(3):751-765.
Cdc31p is the yeast homologue of centrin, a highly conserved calcium-binding protein of the calmodulin superfamily. Previously centrins have been implicated only in microtubule-based processes. To elucidate the functions of yeast centrin, we carried out a two-hybrid screen for Cdc31p-interacting proteins and identified a novel essential protein kinase of 1,080 residues, Kic1p (kinase that interacts with Cdc31p). Kic1p is closely related to S. cerevisiae Ste20p and the p-21– activated kinases (PAKs) found in a wide variety of eukaryotic organisms. Cdc31p physically interacts with Kic1p by two criteria; Cdc31p coprecipitated with GST–Kic1p and it bound to GST–Kic1p in gel overlay assays. Furthermore, GST–Kic1p exhibited in vitro kinase activity that was CDC31-dependent. Although kic1 mutants were not defective for spindle pole body duplication, they exhibited a variety of mutant phenotypes demonstrating that Kic1p is required for cell integrity. We also found that cdc31 mutants, previously identified as defective for spindle pole body duplication, exhibited lysis and morphological defects. The cdc31 kic1 double mutants exhibited a drastic reduction in the range of permissive temperature, resulting in a severe lysis defect. We conclude that Kic1p function is dependent upon Cdc31p both in vivo and in vitro. We postulate that Cdc31p is required both for SPB duplication and for cell integrity/morphogenesis, and that the integrity/morphogenesis function is mediated through the Kic1p protein kinase.
PMCID: PMC2148137  PMID: 9813095
microtubule organizing center; spindle pole body; budding; actin; cell wall

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