Publications by authors named "Julian Haase"

22 Publications

  • Page 1 of 1

The Borealin dimerization domain interacts with Sgo1 to drive Aurora B-mediated spindle assembly.

Mol Biol Cell 2020 09 22;31(20):2207-2218. Epub 2020 Jul 22.

Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, NIH, Bethesda, MD 20892.

The chromosomal passenger complex (CPC), which includes the kinase Aurora B, is a master regulator of meiotic and mitotic processes that ensure the equal segregation of chromosomes. Sgo1 is thought to play a major role in the recruitment of the CPC to chromosomes, but the molecular mechanism and contribution of Sgo1-dependent CPC recruitment is currently unclear. Using egg extracts and biochemical reconstitution, we found that Sgo1 interacts directly with the dimerization domain of the CPC subunit Borealin. Borealin and the PP2A phosphatase complex can bind simultaneously to the coiled-coil domain of Sgo1, suggesting that Sgo1 can integrate Aurora B and PP2A activities to modulate Aurora B substrate phosphorylation. A Borealin mutant that specifically disrupts the Sgo1-Borealin interaction results in defects in CPC chromosomal recruitment and Aurora B-dependent spindle assembly, but not in spindle assembly checkpoint signaling at unattached kinetochores. These findings establish a direct molecular connection between Sgo1 and the CPC and have major implications for the different functions of Aurora B, which promote the proper interaction between spindle microtubules and chromosomes.
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http://dx.doi.org/10.1091/mbc.E20-05-0341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550704PMC
September 2020

Enrichment of Aurora B kinase at the inner kinetochore controls outer kinetochore assembly.

J Cell Biol 2019 10 16;218(10):3237-3257. Epub 2019 Sep 16.

Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD

Outer kinetochore assembly enables chromosome attachment to microtubules and spindle assembly checkpoint (SAC) signaling in mitosis. Aurora B kinase controls kinetochore assembly by phosphorylating the Mis12 complex (Mis12C) subunit Dsn1. Current models propose Dsn1 phosphorylation relieves autoinhibition, allowing Mis12C binding to inner kinetochore component CENP-C. Using egg extracts and biochemical reconstitution, we found that autoinhibition of the Mis12C by Dsn1 impedes its phosphorylation by Aurora B. Our data indicate that the INCENP central region increases Dsn1 phosphorylation by enriching Aurora B at inner kinetochores, close to CENP-C. Furthermore, centromere-bound CENP-C does not exchange in mitosis, and CENP-C binding to the Mis12C dramatically increases Dsn1 phosphorylation by Aurora B. We propose that the coincidence of Aurora B and CENP-C at inner kinetochores ensures the fidelity of kinetochore assembly. We also found that the central region is required for the SAC beyond its role in kinetochore assembly, suggesting that kinetochore enrichment of Aurora B promotes the phosphorylation of other kinetochore substrates.
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http://dx.doi.org/10.1083/jcb.201901004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781445PMC
October 2019

Stu2 uses a 15-nm parallel coiled coil for kinetochore localization and concomitant regulation of the mitotic spindle.

Mol Biol Cell 2018 02 29;29(3):285-294. Epub 2017 Nov 29.

Department of Biology, University of North Carolina, Chapel Hill, NC 27599

XMAP215/Dis1 family proteins are potent microtubule polymerases, critical for mitotic spindle structure and dynamics. While microtubule polymerase activity is driven by an N-terminal tumor overexpressed gene (TOG) domain array, proper cellular localization is a requisite for full activity and is mediated by a C-terminal domain. Structural insight into the C-terminal domain's architecture and localization mechanism remain outstanding. We present the crystal structure of the Stu2 C-terminal domain, revealing a 15-nm parallel homodimeric coiled coil. The parallel architecture of the coiled coil has mechanistic implications for the arrangement of the homodimer's N-terminal TOG domains during microtubule polymerization. The coiled coil has two spatially distinct conserved regions: CRI and CRII. Mutations in CRI and CRII perturb the distribution and localization of Stu2 along the mitotic spindle and yield defects in spindle morphology including increased frequencies of mispositioned and fragmented spindles. Collectively, these data highlight roles for the Stu2 dimerization domain as a scaffold for factor binding that optimally positions Stu2 on the mitotic spindle to promote proper spindle structure and dynamics.
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http://dx.doi.org/10.1091/mbc.E17-01-0057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996958PMC
February 2018

Distinct Roles of the Chromosomal Passenger Complex in the Detection of and Response to Errors in Kinetochore-Microtubule Attachment.

Dev Cell 2017 09;42(6):640-654.e5

Laboratory of Biochemistry & Molecular Biology, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address:

The chromosomal passenger complex (CPC) localizes to centromeres in early mitosis to activate its subunit Aurora B kinase. However, it is unclear whether centromeric CPC localization contributes to CPC functions beyond Aurora B activation. Here, we show that an activated CPC that cannot localize to centromeres supports functional assembly of the outer kinetochore but is unable to correct errors in kinetochore-microtubule attachment in Xenopus egg extracts. We find that CPC has two distinct roles at centromeres: one to selectively phosphorylate Ndc80 to regulate attachment and a second, conserved kinase-independent role in the proper composition of inner kinetochore proteins. Although a fully assembled inner kinetochore is not required for outer kinetochore assembly, we find it is essential to recruit tension indicators, such as BubR1 and 3F3/2, to erroneous attachments. We conclude centromeric CPC is necessary for tension-dependent removal of erroneous attachments and for the kinetochore composition required to detect tension loss.
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http://dx.doi.org/10.1016/j.devcel.2017.08.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260983PMC
September 2017

A Cohesin-Based Partitioning Mechanism Revealed upon Transcriptional Inactivation of Centromere.

PLoS Genet 2016 Apr 29;12(4):e1006021. Epub 2016 Apr 29.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Transcriptional inactivation of the budding yeast centromere has been a widely used tool in studies of chromosome segregation and aneuploidy. In haploid cells when an essential chromosome contains a single conditionally inactivated centromere (GAL-CEN), cell growth rate is slowed and segregation fidelity is reduced; but colony formation is nearly 100%. Pedigree analysis revealed that only 30% of the time both mother and daughter cell inherit the GAL-CEN chromosome. The reduced segregation capacity of the GAL-CEN chromosome is further compromised upon reduction of pericentric cohesin (mcm21∆), as reflected in a further diminishment of the Mif2 kinetochore protein at GAL-CEN. By redistributing cohesin from the nucleolus to the pericentromere (by deleting SIR2), there is increased presence of the kinetochore protein Mif2 at GAL-CEN and restoration of cell viability. These studies identify the ability of cohesin to promote chromosome segregation via kinetochore assembly, in a situation where the centromere has been severely compromised.
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http://dx.doi.org/10.1371/journal.pgen.1006021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4851351PMC
April 2016

How the kinetochore couples microtubule force and centromere stretch to move chromosomes.

Nat Cell Biol 2016 Apr 14;18(4):382-92. Epub 2016 Mar 14.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

The Ndc80 complex (Ndc80, Nuf2, Spc24 and Spc25) is a highly conserved kinetochore protein essential for end-on anchorage to spindle microtubule plus ends and for force generation coupled to plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex binds the kinetochore. The N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex. To determine how the microtubule- and MAP-binding domains of Ndc80 contribute to force production at the kinetochore in budding yeast, we have inserted a FRET tension sensor into the Ndc80 protein about halfway between its microtubule-binding and internal loop domains. The data support a mechanical model of force generation at metaphase where the position of the kinetochore relative to the microtubule plus end reflects the relative strengths of microtubule depolymerization, centromere stretch and microtubule-binding interactions with the Ndc80 and Dam1 complexes.
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http://dx.doi.org/10.1038/ncb3323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814359PMC
April 2016

Pat1 protects centromere-specific histone H3 variant Cse4 from Psh1-mediated ubiquitination.

Mol Biol Cell 2015 Jun 1;26(11):2067-79. Epub 2015 Apr 1.

Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

Evolutionarily conserved histone H3 variant Cse4 and its homologues are essential components of specialized centromere (CEN)-specific nucleosomes and serve as an epigenetic mark for CEN identity and propagation. Cse4 is a critical determinant for the structure and function of the kinetochore and is required to ensure faithful chromosome segregation. The kinetochore protein Pat1 regulates the levels and spatial distribution of Cse4 at centromeres. Deletion of PAT1 results in altered structure of CEN chromatin and chromosome segregation errors. In this study, we show that Pat1 protects CEN-associated Cse4 from ubiquitination in order to maintain proper structure and function of the kinetochore in budding yeast. PAT1-deletion strains exhibit increased ubiquitination of Cse4 and faster turnover of Cse4 at kinetochores. Psh1, a Cse4-specific E3-ubiquitin ligase, interacts with Pat1 in vivo and contributes to the increased ubiquitination of Cse4 in pat1∆ strains. Consistent with a role of Psh1 in ubiquitination of Cse4, transient induction of PSH1 in a wild-type strain resulted in phenotypes similar to a pat1∆ strain, including a reduction in CEN-associated Cse4, increased Cse4 ubiquitination, defects in spatial distribution of Cse4 at kinetochores, and altered structure of CEN chromatin. Pat1 interacts with Scm3 and is required for its maintenance at kinetochores. In conclusion, our studies provide novel insights into mechanisms by which Pat1 affects the structure of CEN chromatin and protects Cse4 from Psh1-mediated ubiquitination for faithful chromosome segregation.
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http://dx.doi.org/10.1091/mbc.E14-08-1335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472017PMC
June 2015

Individual pericentromeres display coordinated motion and stretching in the yeast spindle.

J Cell Biol 2013 Nov 4;203(3):407-16. Epub 2013 Nov 4.

Department of Biology, 2 Department of Mathematics, and 3 Department Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

The mitotic segregation apparatus composed of microtubules and chromatin functions to faithfully partition a duplicated genome into two daughter cells. Microtubules exert extensional pulling force on sister chromatids toward opposite poles, whereas pericentric chromatin resists with contractile springlike properties. Tension generated from these opposing forces silences the spindle checkpoint to ensure accurate chromosome segregation. It is unknown how the cell senses tension across multiple microtubule attachment sites, considering the stochastic dynamics of microtubule growth and shortening. In budding yeast, there is one microtubule attachment site per chromosome. By labeling several chromosomes, we find that pericentromeres display coordinated motion and stretching in metaphase. The pericentromeres of different chromosomes exhibit physical linkage dependent on centromere function and structural maintenance of chromosomes complexes. Coordinated motion is dependent on condensin and the kinesin motor Cin8, whereas coordinated stretching is dependent on pericentric cohesin and Cin8. Linking of pericentric chromatin through cohesin, condensin, and kinetochore microtubules functions to coordinate dynamics across multiple attachment sites.
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http://dx.doi.org/10.1083/jcb.201307104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3824013PMC
November 2013

The spatial segregation of pericentric cohesin and condensin in the mitotic spindle.

Mol Biol Cell 2013 Dec 23;24(24):3909-19. Epub 2013 Oct 23.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280 Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280.

In mitosis, the pericentromere is organized into a spring composed of cohesin, condensin, and a rosette of intramolecular chromatin loops. Cohesin and condensin are enriched in the pericentromere, with spatially distinct patterns of localization. Using model convolution of computer simulations, we deduce the mechanistic consequences of their spatial segregation. Condensin lies proximal to the spindle axis, whereas cohesin is radially displaced from condensin and the interpolar microtubules. The histone deacetylase Sir2 is responsible for the axial position of condensin, while the radial displacement of chromatin loops dictates the position of cohesin. The heterogeneity in distribution of condensin is most accurately modeled by clusters along the spindle axis. In contrast, cohesin is evenly distributed (barrel of 500-nm width × 550-nm length). Models of cohesin gradients that decay from the centromere or sister cohesin axis, as previously suggested, do not match experimental images. The fine structures of cohesin and condensin deduced with subpixel localization accuracy reveal critical features of how these complexes mold pericentric chromatin into a functional spring.
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http://dx.doi.org/10.1091/mbc.E13-06-0325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861086PMC
December 2013

A 3D map of the yeast kinetochore reveals the presence of core and accessory centromere-specific histone.

Curr Biol 2013 Oct 26;23(19):1939-44. Epub 2013 Sep 26.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA.

The budding yeast kinetochore is ~68 nm in length with a diameter slightly larger than a 25 nm microtubule. The kinetochores from the 16 chromosomes are organized in a stereotypic cluster encircling central spindle microtubules. Quantitative analysis of the inner kinetochore cluster (Cse4, COMA) reveals structural features not apparent in singly attached kinetochores. The cluster of Cse4-containing kinetochores is physically larger perpendicular to the spindle axis relative to the cluster of Ndc80 molecules. If there was a single Cse4 (molecule or nucleosome) at the kinetochore attached to each microtubule plus end, the cluster of Cse4 would appear geometrically identical to Ndc80. Thus, the structure of the inner kinetochore at the surface of the chromosomes remains unsolved. We have used point fluorescence microscopy and statistical probability maps to deduce the two-dimensional mean position of representative components of the yeast kinetochore relative to the mitotic spindle in metaphase. Comparison of the experimental images to three-dimensional architectures from convolution of mathematical models reveals a pool of Cse4 radially displaced from Cse4 at the kinetochore and kinetochore microtubule plus ends. The pool of displaced Cse4 can be experimentally depleted in mRNA processing pat1Δ or xrn1Δ mutants. The peripheral Cse4 molecules do not template outer kinetochore components. This study suggests an inner kinetochore plate at the centromere-microtubule interface in budding yeast and yields information on the number of Ndc80 molecules at the microtubule attachment site.
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http://dx.doi.org/10.1016/j.cub.2013.07.083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3796065PMC
October 2013

Bending the rules: widefield microscopy and the Abbe limit of resolution.

J Cell Physiol 2014 Feb;229(2):132-8

Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

One of the most fundamental concepts of microscopy is that of resolution-the ability to clearly distinguish two objects as separate. Recent advances such as structured illumination microscopy (SIM) and point localization techniques including photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) strive to overcome the inherent limits of resolution of the modern light microscope. These techniques, however, are not always feasible or optimal for live cell imaging. Thus, in this review, we explore three techniques for extracting high resolution data from images acquired on a widefield microscope-deconvolution, model convolution, and Gaussian fitting. Deconvolution is a powerful tool for restoring a blurred image using knowledge of the point spread function (PSF) describing the blurring of light by the microscope, although care must be taken to ensure accuracy of subsequent quantitative analysis. The process of model convolution also requires knowledge of the PSF to blur a simulated image which can then be compared to the experimentally acquired data to reach conclusions regarding its geometry and fluorophore distribution. Gaussian fitting is the basis for point localization microscopy, and can also be applied to tracking spot motion over time or measuring spot shape and size. All together, these three methods serve as powerful tools for high-resolution imaging using widefield microscopy.
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http://dx.doi.org/10.1002/jcp.24439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076117PMC
February 2014

Systematic triple-mutant analysis uncovers functional connectivity between pathways involved in chromosome regulation.

Cell Rep 2013 Jun 6;3(6):2168-78. Epub 2013 Jun 6.

Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Waltham, MA 02454, USA.

Genetic interactions reveal the functional relationships between pairs of genes. In this study, we describe a method for the systematic generation and quantitation of triple mutants, termed triple-mutant analysis (TMA). We have used this approach to interrogate partially redundant pairs of genes in S. cerevisiae, including ASF1 and CAC1, two histone chaperones. After subjecting asf1Δ cac1Δ to TMA, we found that the Swi/Snf Rdh54 protein compensates for the absence of Asf1 and Cac1. Rdh54 more strongly associates with the chromatin apparatus and the pericentromeric region in the double mutant. Moreover, Asf1 is responsible for the synthetic lethality observed in cac1Δ strains lacking the HIRA-like proteins. A similar TMA was carried out after deleting both CLB5 and CLB6, cyclins that regulate DNA replication, revealing a strong functional connection to chromosome segregation. This approach can reveal functional redundancies that cannot be uncovered through traditional double-mutant analyses.
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http://dx.doi.org/10.1016/j.celrep.2013.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3718395PMC
June 2013

Pericentric chromatin loops function as a nonlinear spring in mitotic force balance.

J Cell Biol 2013 Mar;200(6):757-72

Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

The mechanisms by which sister chromatids maintain biorientation on the metaphase spindle are critical to the fidelity of chromosome segregation. Active force interplay exists between predominantly extensional microtubule-based spindle forces and restoring forces from chromatin. These forces regulate tension at the kinetochore that silences the spindle assembly checkpoint to ensure faithful chromosome segregation. Depletion of pericentric cohesin or condensin has been shown to increase the mean and variance of spindle length, which have been attributed to a softening of the linear chromatin spring. Models of the spindle apparatus with linear chromatin springs that match spindle dynamics fail to predict the behavior of pericentromeric chromatin in wild-type and mutant spindles. We demonstrate that a nonlinear spring with a threshold extension to switch between spring states predicts asymmetric chromatin stretching observed in vivo. The addition of cross-links between adjacent springs recapitulates coordination between pericentromeres of neighboring chromosomes.
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http://dx.doi.org/10.1083/jcb.201208163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601350PMC
March 2013

Bub1 kinase and Sgo1 modulate pericentric chromatin in response to altered microtubule dynamics.

Curr Biol 2012 Mar 23;22(6):471-81. Epub 2012 Feb 23.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA.

Background: Tension sensing of bioriented chromosomes is essential for the fidelity of chromosome segregation. The spindle assembly checkpoint (SAC) conveys lack of tension or attachment to the anaphase promoting complex. Components of the SAC (Bub1) phosphorylate histone H2A (S121) and recruit the protector of cohesin, Shugoshin (Sgo1), to the inner centromere. How the chromatin structural modifications of the inner centromere are integrated into the tension sensing mechanisms and the checkpoint are not known.

Results: We have identified a Bub1/Sgo1-dependent structural change in the geometry and dynamics of kinetochores and the pericentric chromatin upon reduction of microtubule dynamics. The cluster of inner kinetochores contract, whereas the pericentric chromatin and cohesin that encircle spindle microtubules undergo a radial expansion. Despite its increased spatial distribution, the pericentric chromatin is less dynamic. The change in dynamics is due to histone H2A phosphorylation and Sgo1 recruitment to the pericentric chromatin, rather than microtubule dynamics.

Conclusions: Bub1 and Sgo1 act as a rheostat to regulate the chromatin spring and maintain force balance. Through histone H2A S121 phosphorylation and recruitment of Sgo1, Bub1 kinase softens the chromatin spring in response to changes in microtubule dynamics. The geometric alteration of all 16 kinetochores and pericentric chromatin reflect global changes in the pericentromeric region and provide mechanisms for mechanically amplifying damage at a single kinetochore microtubule.
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http://dx.doi.org/10.1016/j.cub.2012.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3311747PMC
March 2012

Cohesin, condensin, and the intramolecular centromere loop together generate the mitotic chromatin spring.

J Cell Biol 2011 Jun;193(7):1167-80

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Sister chromatid cohesion provides the mechanistic basis, together with spindle microtubules, for generating tension between bioriented chromosomes in metaphase. Pericentric chromatin forms an intramolecular loop that protrudes bidirectionally from the sister chromatid axis. The centromere lies on the surface of the chromosome at the apex of each loop. The cohesin and condensin structural maintenance of chromosomes (SMC) protein complexes are concentrated within the pericentric chromatin, but whether they contribute to tension-generating mechanisms is not known. To understand how pericentric chromatin is packaged and resists tension, we map the position of cohesin (SMC3), condensin (SMC4), and pericentric LacO arrays within the spindle. Condensin lies proximal to the spindle axis and is responsible for axial compaction of pericentric chromatin. Cohesin is radially displaced from the spindle axis and confines pericentric chromatin. Pericentric cohesin and condensin contribute to spindle length regulation and dynamics in metaphase. Together with the intramolecular centromere loop, these SMC complexes constitute a molecular spring that balances spindle microtubule force in metaphase.
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http://dx.doi.org/10.1083/jcb.201103138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3216333PMC
June 2011

Systematic exploration of essential yeast gene function with temperature-sensitive mutants.

Nat Biotechnol 2011 Apr 27;29(4):361-7. Epub 2011 Mar 27.

Banting and Best Department of Medical Research, The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (∼45%) of the 1,101 essential yeast genes, with ∼30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.
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http://dx.doi.org/10.1038/nbt.1832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286520PMC
April 2011

Function and assembly of DNA looping, clustering, and microtubule attachment complexes within a eukaryotic kinetochore.

Mol Biol Cell 2009 Oct 5;20(19):4131-9. Epub 2009 Aug 5.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA.

The kinetochore is a complex protein-DNA assembly that provides the mechanical linkage between microtubules and the centromere DNA of each chromosome. Centromere DNA in all eukaryotes is wrapped around a unique nucleosome that contains the histone H3 variant CENP-A (Cse4p in Saccharomyces cerevisiae). Here, we report that the inner kinetochore complex (CBF3) is required for pericentric DNA looping at the Cse4p-containing nucleosome. DNA within the pericentric loop occupies a spatially confined area that is radially displaced from the interpolar central spindle. Microtubule-binding kinetochore complexes are not involved in pericentric DNA looping but are required for the geometric organization of DNA loops around the spindle microtubules in metaphase. Thus, the mitotic segregation apparatus is a composite structure composed of kinetochore and interpolar microtubules, the kinetochore, and organized pericentric DNA loops. The linkage of microtubule-binding to centromere DNA-looping complexes positions the pericentric chromatin loops and stabilizes the dynamic properties of individual kinetochore complexes in mitosis.
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http://dx.doi.org/10.1091/mbc.e09-05-0359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2754927PMC
October 2009

Chromosome congression by Kinesin-5 motor-mediated disassembly of longer kinetochore microtubules.

Cell 2008 Nov;135(5):894-906

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.

During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression.
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http://dx.doi.org/10.1016/j.cell.2008.09.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2683758PMC
November 2008

Pericentric chromatin is organized into an intramolecular loop in mitosis.

Curr Biol 2008 Jan;18(2):81-90

Department of Biology, 623 Fordham Hall CB#3280, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280, USA.

Background: Cohesin proteins link sister chromatids and provide the basis for tension between bioriented sister chomatids in mitosis. Cohesin is concentrated at the centromere region of the chromosome despite the fact that sister centromeres can be separated by 800 nm in vivo. The function of cohesin at sites of separated DNA is unknown.

Results: We provide evidence that the kinetochore promotes the organization of pericentric chromatin into a cruciform in mitosis such that centromere-flanking DNA adopts an intramolecular loop, whereas sister-chromatid arms are paired intermolecularly. Visualization of cohesin subunits by fluorescence microscopy revealed a cylindrical structure that encircles the central spindle and spans the distance between sister kinetochores. Kinetochore assembly at the apex of the loop initiates intrastrand loop formation that extends approximately 25 kb (12.5 kb on either side of the centromere). Two centromere loops (one from each sister chromatid) are stretched between the ends of sister-kinetochore microtubules along the spindle axis. At the base of the loop there is a transition to intermolecular sister-chromatid pairing.

Conclusions: The C loop conformation reveals the structural basis for sister-kinetochore clustering in budding yeast and for kinetochore biorientation and thus resolves the paradox of maximal interstrand separation in regions of highest cohesin concentration.
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http://dx.doi.org/10.1016/j.cub.2007.12.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2238682PMC
January 2008

The microtubule-based motor Kar3 and plus end-binding protein Bim1 provide structural support for the anaphase spindle.

J Cell Biol 2008 Jan 7;180(1):91-100. Epub 2008 Jan 7.

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.

In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and approximately 8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs.
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http://dx.doi.org/10.1083/jcb.200710164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2213616PMC
January 2008

FluoroSim: A Visual Problem-Solving Environment for Fluorescence Microscopy.

Eurographics Workshop Vis Comput Biomed 2008 Jan;2008:151-158

Department of Computer Science, UNC Chapel Hill, USA.

Fluorescence microscopy provides a powerful method for localization of structures in biological specimens. However, aspects of the image formation process such as noise and blur from the microscope's point-spread function combine to produce an unintuitive image transformation on the true structure of the fluorescing molecules in the specimen, hindering qualitative and quantitative analysis of even simple structures in unprocessed images. We introduce FluoroSim, an interactive fluorescence microscope simulator that can be used to train scientists who use fluorescence microscopy to understand the artifacts that arise from the image formation process, to determine the appropriateness of fluorescence microscopy as an imaging modality in an experiment, and to test and refine hypotheses of model specimens by comparing the output of the simulator to experimental data. FluoroSim renders synthetic fluorescence images from arbitrary geometric models represented as triangle meshes. We describe three rendering algorithms on graphics processing units for computing the convolution of the specimen model with a microscope's point-spread function and report on their performance. We also discuss several cases where the microscope simulator has been used to solve real problems in biology.
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http://dx.doi.org/10.2312/VCBM/VCBM08/151-158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860625PMC
January 2008

The yeast DNA damage checkpoint proteins control a cytoplasmic response to DNA damage.

Proc Natl Acad Sci U S A 2007 Jul 22;104(27):11358-63. Epub 2007 Jun 22.

Rosenstiel Center and Department of Biology, Brandeis University, Waltham, MA 02454-9110, USA.

A single HO endonuclease-induced double-strand break (DSB) is sufficient to activate the DNA damage checkpoint and cause Saccharomyces cells to arrest at G(2)/M for 12-14 h, after which cells adapt to the presence of the DSB and resume cell cycle progression. The checkpoint signal leading to G(2)/M arrest was previously shown to be nuclear-limited. Cells lacking ATR-like Mec1 exhibit no DSB-induced cell cycle delay; however, cells lacking Mec1's downstream protein kinase targets, Rad53 or Chk1, still have substantial G(2)/M delay, as do cells lacking securin, Pds1. This delay is eliminated only in the triple mutant chk1Delta rad53Delta pds1Delta, suggesting that Rad53 and Chk1 control targets other than the stability of securin in enforcing checkpoint-mediated cell cycle arrest. The G(2)/M arrest in rad53Delta and chk1Delta revealed a unique cytoplasmic phenotype in which there are frequent dynein-dependent excursions of the nucleus through the bud neck, without entering anaphase. Such excursions are infrequent in wild-type arrested cells, but have been observed in cells defective in mitotic exit, including the semidominant cdc5-ad mutation. We suggest that Mec1-dependent checkpoint signaling through Rad53 and Chk1 includes the repression of nuclear movements that are normally associated with the execution of anaphase.
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http://dx.doi.org/10.1073/pnas.0609636104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1896138PMC
July 2007