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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">eLife</journal-id><journal-id journal-id-type="hwp">elife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">00117</article-id><article-id pub-id-type="doi">10.7554/eLife.00117</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Cell biology</subject></subj-group><subj-group subj-group-type="heading"><subject>Genes and chromosomes</subject></subj-group></article-categories><title-group><article-title>Meiosis I chromosome segregation is established through regulation of microtubule–kinetochore interactions</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-1843"><name><surname>Miller</surname><given-names>Matthew P</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-1846"><name><surname>Ünal</surname><given-names>Elçin</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-1845"><name><surname>Brar</surname><given-names>Gloria A</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-1825"><name><surname>Amon</surname><given-names>Angelika</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Biology</institution>, <institution>Massachusetts Institute of Technology</institution>, <addr-line><named-content content-type="city">Cambridge</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Cellular and Molecular Pharmacology</institution>, <institution>University of California, San Francisco</institution>, <addr-line><named-content content-type="city">San Francisco</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Botchan</surname><given-names>Michael</given-names></name><role>Reviewing editor</role></contrib><aff><institution>University of California-Berkeley</institution>, <country>United States</country></aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>angelika@mit.edu</email></corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>18</day><month>12</month><year>2012</year></pub-date><pub-date pub-type="collection"><year>2012</year></pub-date><volume>1</volume><elocation-id>e00117</elocation-id><history><date date-type="received"><day>31</day><month>07</month><year>2012</year></date><date date-type="accepted"><day>18</day><month>10</month><year>2012</year></date></history><permissions><copyright-statement>© 2012, Miller et al</copyright-statement><copyright-year>2012</copyright-year><copyright-holder>Miller et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife00117.pdf"/><related-article ext-link-type="doi" id="ra1" related-article-type="commentary" xlink:href="10.7554/eLife.00386"/><abstract><object-id pub-id-type="doi">10.7554/eLife.00117.001</object-id><p>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.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.001">http://dx.doi.org/10.7554/eLife.00117.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.00117.002</object-id><title>eLife digest</title><p>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.</p><p>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.</p><p>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.</p><p>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.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.002">http://dx.doi.org/10.7554/eLife.00117.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>meiosis</kwd><kwd>cyclin-dependent kinase</kwd><kwd>tension</kwd><kwd>cohesin</kwd><kwd>chromosome segregation</kwd><kwd>kinetochore</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>S. cerevisiae</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>Howard Hughes Medical Institute</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Miller</surname><given-names>Matthew P</given-names></name><name><surname>Ünal</surname><given-names>Elçin</given-names></name><name><surname>Brar</surname><given-names>Gloria A</given-names></name><name><surname>Amon</surname><given-names>Angelika</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>GM62207</award-id><principal-award-recipient><name><surname>Miller</surname><given-names>Matthew P</given-names></name><name><surname>Ünal</surname><given-names>Elçin</given-names></name><name><surname>Brar</surname><given-names>Gloria A</given-names></name><name><surname>Amon</surname><given-names>Angelika</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>Jane Coffin Childs Memorial Fund</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Ünal</surname><given-names>Elçin</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>American Cancer Society</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Brar</surname><given-names>Gloria A</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Preventing premature interactions between microtubules and protein-based structures called kinetochores ensures that chromosomes are segregated by meiosis rather than mitosis in reproductive cells.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Cells have evolved intricate mechanisms to execute proper partitioning of the genetic material during cell division. This task is especially complex in meiosis, the cell division used by sexually reproducing organisms to generate gametes. The goal of meiosis is to reduce the genome content by half such that proper ploidy is maintained upon fusion of gametes. To achieve this, 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 segregate. Meiosis II resembles mitosis in that sister chromatids segregate from each other. The establishment of this specialized chromosome segregation pattern requires three changes that modulate how chromosomes interact with each other and with the microtubule cytoskeleton: (1) reciprocal recombination between homologous chromosomes, (2) the way linkages between sister chromatids, known as sister-chromatid cohesion, are removed from chromosomes and (3) the manner in which chromosomes attach to the meiotic spindle.</p><p>Homologous recombination is initiated by programmed double-strand breaks (DSBs), which are catalyzed by Spo11 following premeiotic DNA replication (<xref ref-type="bibr" rid="bib26">Keeney et al., 1997</xref>). Subsequent repair of DSBs by crossover recombination generates physical linkages between homologous chromosomes. This, in turn, allows homologs to attach to the meiosis I spindle such that each homolog interacts with microtubules emanating from opposite spindle poles. As a result, homologous chromosomes biorient on the meiosis I spindle. The spindle assembly checkpoint prevents the onset of chromosome segregation until this process is completed. Once each pair of homologs is bioriented, checkpoint signaling ceases and anaphase entry ensues. A ubiquitin ligase known as the anaphase promoting complex/cyclosome and its specificity factor Cdc20 (APC/C-Cdc20) targets Securin for degradation, relieving Separase inhibition (<xref ref-type="bibr" rid="bib14">Cohen-Fix et al., 1996</xref>; <xref ref-type="bibr" rid="bib12">Ciosk et al., 1998</xref>). Separase is a protease that cleaves the kleisin subunit of cohesin, the protein complex that mediates sister-chromatid cohesion (<xref ref-type="bibr" rid="bib53">Uhlmann et al., 1999</xref>, <xref ref-type="bibr" rid="bib54">2000</xref>; <xref ref-type="bibr" rid="bib45">Schleiffer et al., 2003</xref>). In meiosis I, cleavage of cohesin at chromosome arms allows homologs to segregate (<xref ref-type="bibr" rid="bib7">Buonomo et al., 2000</xref>). However, cohesin around the centromeres is protected from cleavage during meiosis I, which is essential for the accurate segregation of sister chromatids during meiosis II. Protection of centromeric cohesin is accomplished by preventing phosphorylation of Rec8, the meiosis-specific kleisin. This occurs, at least in part, by Sgo1 (MEI-S332)-dependent recruitment of the protein phosphatase PP2A to centromeric regions where it antagonizes Rec8 phosphorylation (<xref ref-type="bibr" rid="bib27">Kerrebrock et al., 1995</xref>; <xref ref-type="bibr" rid="bib23">Katis et al., 2004a</xref>; <xref ref-type="bibr" rid="bib29">Kitajima et al., 2004</xref>, <xref ref-type="bibr" rid="bib30">2006</xref>; <xref ref-type="bibr" rid="bib44">Riedel et al., 2006</xref>).</p><p>The third modification necessary to bring about the meiotic chromosome segregation pattern is the manner in which kinetochores attach to microtubules during meiosis I and meiosis II. In meiosis I, kinetochores of sister chromatid pairs (henceforth sister kinetochores) attach to microtubules emanating from the same spindle pole, a process called sister kinetochore coorientation. During meiosis II, as during mitosis, sister kinetochores attach to microtubules emanating from opposite spindle poles and are thus bioriented (reviewed in <xref ref-type="bibr" rid="bib36">Marston and Amon, 2004</xref>). In budding yeast, sister kinetochore coorientation is brought about by the monopolin complex, which consists of Mam1, Lrs4, Csm1 and the casein kinase 1, Hrr25 (<xref ref-type="bibr" rid="bib50">Toth et al., 2000</xref>; <xref ref-type="bibr" rid="bib43">Rabitsch et al., 2003</xref>; <xref ref-type="bibr" rid="bib42">Petronczki et al., 2006</xref>). Lrs4 and Csm1 localize to the nucleolus during interphase. During exit from pachytene, a stage of prophase I, Lrs4 and Csm1 associate with Mam1 and Hrr25 at kinetochores, a process that requires the Polo kinase Cdc5 (<xref ref-type="bibr" rid="bib13">Clyne et al., 2003</xref>; <xref ref-type="bibr" rid="bib32">Lee and Amon, 2003</xref>; <xref ref-type="bibr" rid="bib37">Matos et al., 2008</xref>). How the association of monopolin with kinetochores is coordinated with respect to kinetochore assembly and microtubule–kinetochore interactions during meiosis is not understood.</p><p>Cyclin-dependent kinases (CDKs) are the central regulators of the mitotic and meiotic divisions. In budding yeast, a single CDK associates with one of six B-type cyclins (Clb1-Clb6) (reviewed in <xref ref-type="bibr" rid="bib40">Morgan, 1997</xref>). In meiosis, Clb5- and Clb6-CDKs drive DNA replication and recombination, whereas Clb1-, Clb3- and Clb4-CDKs promote the meiotic nuclear divisions (reviewed in <xref ref-type="bibr" rid="bib36">Marston and Amon, 2004</xref>). Meiotic cyclin-CDK activity is regulated both at the transcriptional and translational level (<xref ref-type="bibr" rid="bib16">Grandin and Reed, 1993</xref>; <xref ref-type="bibr" rid="bib9">Carlile and Amon, 2008</xref>). Transcription of <italic>CLB1</italic>, <italic>CLB3</italic> and <italic>CLB4</italic> occurs only after exit from pachytene (<xref ref-type="bibr" rid="bib11">Chu and Herskowitz, 1998</xref>); <italic>CLB3</italic> is also translationally repressed during meiosis I, thus restricting Clb3-CDK activity to meiosis II (<xref ref-type="bibr" rid="bib9">Carlile and Amon, 2008</xref>). The major mitotic cyclin, <italic>CLB2</italic>, is not expressed during meiosis (<xref ref-type="bibr" rid="bib16">Grandin and Reed, 1993</xref>).</p><p>Here we investigate the importance of cyclin-CDK regulation in establishing the meiotic chromosome segregation pattern. We show that expression of a subset of cyclins during premeiotic S phase and early prophase I, defined as the prophase stages up to exit from pachytene, causes premature microtubule–kinetochore interactions. This, in turn, disrupts both sister kinetochore coorientation and protection of centromeric cohesin during meiosis I, revealing that the temporal control of microtubule–kinetochore interactions is essential for meiosis I chromosome morphogenesis. Furthermore, we define the mechanism by which premature microtubule–kinetochore interactions are prevented; through regulation of cyclin-CDK activity and of outer kinetochore assembly. Our results demonstrate that preventing premature microtubule–kinetochore interactions is essential for establishing a meiosis I-specific chromosome architecture and provide critical insights into how the mitotic chromosome segregation machinery is modulated to achieve a meiosis I-specific pattern of chromosome segregation.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Cyclin expression is sufficient to induce spindle formation and microtubule–kinetochore interactions</title><p>We previously reported that <italic>CLB3</italic> expression prior to meiosis I induces a change in the pattern of chromosome segregation such that sister chromatids, instead of homologous chromosomes, segregate during the first nuclear division (<xref ref-type="bibr" rid="bib9">Carlile and Amon, 2008</xref>). To determine how Clb-CDKs impact meiotic chromosome segregation and whether Clb-CDKs play redundant or specific roles in regulating this process, we examined the consequences of prematurely expressing <italic>CLB1</italic>, <italic>CLB3</italic>, <italic>CLB4</italic> or <italic>CLB5</italic>.</p><p>In our previous studies we expressed <italic>CLB3</italic> from the <italic>GAL1-10</italic> promoter driven by an estrogen inducible Gal4-ER fusion (<xref ref-type="bibr" rid="bib9">Carlile and Amon, 2008</xref>). Expression from the <italic>GAL1-10</italic> promoter led to Clb3 accumulation in meiosis I to levels that are comparable to those seen in meiosis II in wild-type cells (<xref ref-type="bibr" rid="bib9">Carlile and Amon, 2008</xref>). However, estrogen interferes with meiotic progression when added during early stages of sporulation (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). To circumvent this problem we utilized the copper-inducible <italic>CUP1</italic> promoter to drive Clb3 expression. Expression from the <italic>CUP1</italic> promoter led to approximately fivefold higher levels of Clb3 protein compared to expression from the <italic>GAL1-10</italic> promoter (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). To examine the consequences of the two <italic>CLB3</italic> constructs on chromosome segregation we used <italic>GAL-CLB3</italic> and <italic>CUP-CLB3</italic> strains in which one of the two homologs of chromosome III was marked by integrating a tandem array of tetO sequences ∼20 kb from CENIII (heterozygous LEU2-GFP dots). These cells also expressed a tetR-GFP fusion, which allowed visualization of the tetO arrays (<xref ref-type="bibr" rid="bib38">Michaelis et al., 1997</xref>). The analysis of GFP dot segregation during the first meiotic division revealed that despite the difference in Clb3 protein levels, the extent of sister chromatid segregation in meiosis I was similar between <italic>GAL-CLB3</italic> and <italic>CUP-CLB3</italic> cells (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). This finding indicates that expression of Clb3 from either the <italic>CUP1</italic> or <italic>GAL1-10</italic> promoter efficiently induces sister chromatid segregation during meiosis I. Furthermore, the timing of when Clb3 is expressed, rather than the amount of Clb3 present, appears to be the primary determinant of this phenotype. Based on this observation and the finding that all four cyclins showed equal expression when produced from the <italic>CUP1</italic> promoter (<xref ref-type="fig" rid="fig1">Figure 1D</xref>) we utilized the <italic>CUP1</italic> promoter for most subsequent analyses.</p><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.00117.003</object-id><label>Figure 1.</label><caption><title>Characterization of premature cyclin expression and corresponding total CDK activity.</title><p>(<bold>A</bold>) Wild-type (A4962) and <italic>GAL4-ER</italic> (A19151) cells were induced to sporulate. At the indicated time points, an aliquot was removed and treated with estradiol (1 μM). The percentage of cells that had sporulated after 24 hr was calculated as the sum of dyads, triads and tetrads divided by the total number of cells (n > 100 cells counted for each condition). (<bold>B</bold>) Wild-type (A18686), <italic>GAL-CLB3-3HA</italic> (A23084) and <italic>CUP-CLB3</italic> (A23086) cells also carrying the <italic>GAL4-ER</italic> fusion were induced to sporulate. After 3 hr, <italic>CLB3</italic> was induced. Each culture was treated with estradiol (1 μM) and CuSO<sub>4</sub> (50 μM). Cells were harvested after 1 hr of estradiol and CuSO<sub>4</sub> treatment for protein extraction. Levels of Clb3 were examined by Western blot analysis. A cross-reacting band was used as a loading reference. (<bold>C</bold>) Segregation of sister chromatids (equational division) using heterozygous GFP dots integrated at <italic>LEU2</italic> (∼20 kb from CENIII) was quantified in binucleate cells from wild-type (A18686), <italic>GAL-CLB3-3HA</italic> (A23084) and <italic>CUP-CLB3</italic> (A23086). Note that the samples were collected from the same experiment described in (<bold>B</bold>) at a time point when a fraction of the cells had completed meiosis I (6 hr 30 min and 7 hr after induction of sporulation) (n > 100 for each sample). Using a chi-square test (df 1), the fraction of binucleates that display a reductional or equational division was compared between wild-type and <italic>GAL-CLB3-3HA</italic> χ<sup>2</sup> = 166.4, p<0.0001 and between wild-type and <italic>CUP-CLB3</italic> χ<sup>2</sup> = 108.7, p<0.0001. (<bold>D</bold>) Wild-type or <italic>CUP-CLB-eGFP</italic> cells also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate. After 2 hr 15 min, cyclins were induced by addition of CuSO<sub>4</sub> (50 μM). Cells were released from the <italic>NDT80</italic> block at 4 hr 30 min post transfer to sporulation medium. Cyclin levels monitored by Western blot at the indicated time points in <italic>CUP-CLB1-eGFP</italic> (A28531), <italic>CUP-CLB3-eGFP</italic> (A28533), <italic>CUP-CLB4-eGFP</italic> (A28535) and <italic>CUP-CLB5-eGFP</italic> (A33199) cells. Pgk1 was used as a loading control. (<bold>E</bold>) Wild-type (A22678) cells carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 15 min after transfer into sporulation medium. Samples were taken at indicated time points to determine DNA content by flow cytometry. By 2 hr 15 min 43% of cells had a 4C DNA content. (<bold>F</bold>) Left: Wild-type (A28663), <italic>CUP-CLB1</italic> (A28665), <italic>CUP-CLB3</italic> (A28667), <italic>CUP-CLB4</italic> (A28669) and <italic>CUP-CLB5</italic> (A28671) cells carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 30 min after transfer into sporulation medium. In vitro kinase assays were performed with Cdc28-3V5 (Cdk1) immunoprecipitated from prophase I samples (collected 4 hr 30 min after sporulation induction, at the time of <italic>NDT80</italic> block-release) and metaphase I–anaphase I samples (collected 1 hr 30 min after release from the <italic>NDT80</italic> block). Right: specific activity was calculated by normalizing the amount of phosphorylated Histone H1 to the amount of immunoprecipitated Cdc28-3V5 using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.003">http://dx.doi.org/10.7554/eLife.00117.003</ext-link></p></caption><graphic xlink:href="elife00117f001"/></fig><p>Having established a system to effectively express various cyclins prior to meiosis I we next examined the consequences of their premature expression on meiosis I events. We first asked whether misexpression of various cyclins is sufficient to induce spindle formation in cells arrested in pachytene of prophase I, due to lack of the transcription factor Ndt80 (<xref ref-type="bibr" rid="bib58">Xu et al., 1995</xref>; <xref ref-type="bibr" rid="bib11">Chu and Herskowitz, 1998</xref>). We induced cyclin expression from the <italic>CUP1</italic> promoter 135 min after the induction of sporulation when typically 40–65% of the cells have replicated their DNA (<xref ref-type="fig" rid="fig1">Figure 1E</xref>; <xref ref-type="bibr" rid="bib4">Blitzblau et al., 2012</xref>) and examined spindle pole body (SPB, centrosome equivalent in budding yeast) separation and spindle morphology following induction. As expected, wild-type cells did not form spindles in the absence of <italic>NDT80</italic> function. Expression of <italic>CLB5</italic> from the <italic>CUP1</italic> promoter did not lead to SPB separation and spindle formation either, although expression of <italic>CLB5</italic> in the prophase I arrest led to a significant increase in total CDK activity (<xref ref-type="fig" rid="fig1 fig2">Figures 1F and 2A</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). In contrast, <italic>CUP-CLB1</italic>, <italic>CUP-CLB3</italic> and <italic>CUP-CLB4</italic> cells separated SPBs and formed bipolar spindles, shortly after copper addition (<xref ref-type="fig" rid="fig2">Figure 2A</xref> and <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). Similar results were observed in cells with intact <italic>NDT80</italic> (data not shown). We conclude that expression of <italic>CLB1</italic>, <italic>CLB3</italic> or <italic>CLB4</italic> is sufficient to promote bipolar spindle assembly in <italic>NDT80</italic>-depleted cells.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.00117.004</object-id><label>Figure 2.</label><caption><title>Premature expression of <italic>CLB1</italic> or <italic>CLB3</italic> causes sister kinetochore biorientation during prophase I and sister chromatid segregation in meiosis I.</title><p>Wild-type or <italic>CUP-CLB</italic> cells were induced to sporulate. After 2 hr 15 min, cyclins were induced by addition of CuSO<sub>4</sub> (50 μM). Cells were either arrested during prophase I or released from an <italic>NDT80</italic> block 4 hr 30 min after induction of sporulation. (<bold>A</bold>) Bipolar spindle formation determined in wild-type (A22678), <italic>CUP-CLB1</italic> (A27421), <italic>CUP-CLB3</italic> (A22702), <italic>CUP-CLB4</italic> (A27423) and <italic>CUP-CLB5</italic> (A27425) during prophase I (n = 100 per time point). Images on left show spindle formation in <italic>CUP-CLB</italic> cells 4 hr after induction of sporulation; in this and all subsequent Figures microtubules are shown in green and DNA in blue. The dotted line depicts the cell membrane. (<bold>B</bold>) Microtubule–kinetochore engagement monitored during prophase I, starting at 1 hr after CuSO<sub>4</sub> addition in wild-type (A30700), <italic>CUP-CLB1</italic> (A30702), <italic>CUP-CLB3</italic> (A30704), <italic>CUP-CLB4</italic> (A30707) and <italic>CUP-CLB5</italic> (A30708) by live cell microscopy. SPBs (marked by arrow) and heterozygous CENV-GFP dots are shown (arrowheads mark separated CENV dots). In this and all subsequent figures SPBs are in red, GFP dots are in green. (<bold>C</bold>) Top panel: representative images of wild-type (A30700) and <italic>CUP-CLB3</italic> (A30704). Bottom panel: separation of heterozygous CENV-GFP dots in prophase I-arrested cells quantified in wild-type (A22678), <italic>CUP-CLB1</italic> (A27421), <italic>CUP-CLB3</italic> (A22702), <italic>CUP-CLB4</italic> (A27423) and <italic>CUP-CLB5</italic> (A27425) by live cell microscopy (over the duration of 8 hr, n > 100) as described in the ‘Materials and methods’. The fraction of nuclei that display sister kinetochores as separate or together for each <italic>CUP-CLB</italic> strain was compared to wild-type using a chi-square test (df 1): <italic>CUP-CLB1</italic>, χ<sup>2</sup> = 40.77, p<0.0001; <italic>CUP-CLB3</italic>, χ<sup>2</sup> = 34.84, p<0.0001; <italic>CUP-CLB4</italic>, χ<sup>2</sup> = 0.1163, p=0.7330; <italic>CUP-CLB5</italic>, χ<sup>2</sup> = 1.418, p=0.2337. (<bold>D</bold>) Segregation of sister chromatids (equational division) using heterozygous CENV-GFP dots quantified in binucleates from wild-type (A22678), <italic>CUP-CLB1</italic> (A27421), <italic>CUP-CLB3</italic> (A22702), <italic>CUP-CLB4</italic> (A27423) and <italic>CUP-CLB5</italic> (A27425) (n = 100). The fraction of binucleates that display a reductional or equational division for each <italic>CUP-CLB</italic> strain was compared to wild-type using a chi-square test (df 1): <italic>CUP-CLB1</italic>, χ<sup>2</sup> = 45.13, p<0.0001; <italic>CUP-CLB3</italic>, χ<sup>2</sup> = 48.22, p<0.0001; <italic>CUP-CLB4</italic>, χ<sup>2</sup> = 1.020, p=0.3124; <italic>CUP-CLB5</italic>, χ<sup>2</sup> = 0, p=1. (<bold>E</bold>) Wild-type (A31019) and <italic>CUP-CLB3</italic> (A31021) cells monitored for segregation of heterozygous CENV-GFP dots with respect to Pds1 (Securin, red) degradation by live cell microscopy (n > 17). Time of Pds1 degradation set to t = 0, percent cells were plotted as a Kaplan–Meier curve. Note that for A31021, the analysis of cells that segregate sister chromatids in the first nuclear division is shown. Pds1 accumulation during prophase II is not observed using the Pds1-tdTomato construct, likely due to delayed maturation of the fluorophore (<xref ref-type="bibr" rid="bib24">Katis et al., 2010</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.004">http://dx.doi.org/10.7554/eLife.00117.004</ext-link></p></caption><graphic xlink:href="elife00117f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.005</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Spindle pole body separation in <italic>CUP-CLB</italic> cells.</title><p>Wild-type or <italic>CUP-CLB</italic> cells also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate. After 2 hr 15 min, cyclins were induced by addition of CuSO<sub>4</sub> (50 μM). Cells were arrested during prophase I and the percentage of cells with separated Spc42 foci (red dots) was determined at indicated time points in wild-type (A29581), <italic>CUP-CLB1</italic> (A29582), <italic>CUP-CLB3</italic> (A29583), <italic>CUP-CLB4</italic> (A29584) and <italic>CUP-CLB5</italic> (A29585) (n > 100 for each time point).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.005">http://dx.doi.org/10.7554/eLife.00117.005</ext-link></p></caption><graphic xlink:href="elife00117fs001"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.006</object-id><label>Figure 2—figure supplement 2.</label><caption><title>Homolog separation in <italic>CUP-CLB4</italic> cells.</title><p>Wild-type (A22688), <italic>CUP-CLB4</italic> (A32470) also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions or <italic>cdc20-mn</italic> (A15163) cells all carrying homozygous CENV GFP dots were induced to sporulate. After 2 hr 15 min, cyclins were induced by addition of CuSO<sub>4</sub> (50 μM). Cells were arrested either during prophase I (A22688, A324470) or metaphase I (A15163). Separated GFP foci (homologs separate) were analyzed 6 hr (prophase I-arrest) or 8 hr 30 min (metaphase I-arrest) after induction of sporulation (n > 100 for each time point). Using a chi-square test (df 1), the fraction of mononucleates that display homologs as together or separate during a prophase I arrest was compared between wild-type and <italic>CUP-CLB4</italic> χ<sup>2</sup> = 0.4422, p=0.5061.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.006">http://dx.doi.org/10.7554/eLife.00117.006</ext-link></p></caption><graphic xlink:href="elife00117fs002"/></fig><fig id="fig2s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.007</object-id><label>Figure 2—figure supplement 3.</label><caption><title>Chromosome III sister chromatid segregation in <italic>CUP-CLB3</italic> cells.</title><p>Wild-type (A18185) and <italic>CUP-CLB3</italic> (A22682) cells also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate. After 3 hr, <italic>CLB3</italic> was induced by addition of CuSO<sub>4</sub> (50 μM). At 6 hr, cells were released from the <italic>NDT80</italic> block. Subsequently, segregation of sister chromatids (equational division) using heterozygous GFP dots integrated at <italic>LEU2</italic> (∼20 kb from CENIII) was quantified in binucleate cells. The appearance of segregated sister chromatids in wild-type is likely due to recombination between <italic>LEU2</italic> and <italic>CEN3</italic>. Using a chi-square test (df 1), the fraction of binucleates that display a reductional or equational division was compared between wild-type and <italic>CUP-CLB3</italic> χ<sup>2</sup> = 35.65, p<0.0001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.007">http://dx.doi.org/10.7554/eLife.00117.007</ext-link></p></caption><graphic xlink:href="elife00117fs003"/></fig><fig id="fig2s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.008</object-id><label>Figure 2—figure supplement 4.</label><caption><title>Sister chromatid segregation in <italic>CUP-CLB3</italic> cells using dual-color marked chromosomes.</title><p>Wild-type (A27476) and <italic>CUP-CLB3</italic> (A27480) cells carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions and CENV-LacO/LacI-GFP on one homolog of chromosome V (green) and CENV-tetO/tetR-RFP on the other homolog of chromosome V (red) were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added at 2 hr 15 min. At 4 hr 30 min, cells were released from <italic>NDT80</italic> block and monitored by live cell microscopy starting 30 min after estradiol addition, and monitored every 15 min for 8 hr.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.008">http://dx.doi.org/10.7554/eLife.00117.008</ext-link></p></caption><graphic xlink:href="elife00117fs004"/></fig><fig id="fig2s5" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.009</object-id><label>Figure 2—figure supplement 5.</label><caption><title>Recombination in <italic>CUP-CLB3</italic> cells.</title><p>Left panel: wild-type (A21104) and <italic>GAL-CLB3</italic> (A21105) cells were induced to sporulate and estradiol (1 μM) was added 3 hr after transfer into sporulation medium. Genomic DNA was prepared and digested with XhoI and MluI and hybridized with Probe A. See <xref ref-type="bibr" rid="bib46">Storlazzi et al. (1995)</xref> for details. Right panel: recombination products were quantified as R2/P1. Note: A21104 and A21105 contain auxotrophies and have reduced meiotic kinetics relative to prototrophic strains.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.009">http://dx.doi.org/10.7554/eLife.00117.009</ext-link></p></caption><graphic xlink:href="elife00117fs005"/></fig><fig id="fig2s6" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.010</object-id><label>Figure 2—figure supplement 6.</label><caption><title>Localization of Rad51 in <italic>CUP-CLB3</italic> cells.</title><p>Wild-type (A22864) and <italic>CUP-CLB3</italic> (A22866) cells were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 3 hr after transfer into sporulation medium. Localization of the double-strand break repair protein Rad51 (green) was determined by nuclear spreads 4 hr after transfer to sporulation medium. DNA is shown in blue.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.010">http://dx.doi.org/10.7554/eLife.00117.010</ext-link></p></caption><graphic xlink:href="elife00117fs006"/></fig><fig id="fig2s7" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.011</object-id><label>Figure 2—figure supplement 7.</label><caption><title>Localization of Zip1 in <italic>CUP-CLB3</italic> cells.</title><p>Wild-type (A22836) and <italic>CUP-CLB3</italic> (A22838) cells were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 3 hr after transfer into sporulation medium. Localization of the synaptonemal complex component Zip1 (green) and the cohesin subunit Rec8-13myc (red) was determined by nuclear spreads 5 hr after transfer to sporulation medium. DNA is shown in blue.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.011">http://dx.doi.org/10.7554/eLife.00117.011</ext-link></p></caption><graphic xlink:href="elife00117fs007"/></fig><fig id="fig2s8" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.012</object-id><label>Figure 2—figure supplement 8.</label><caption><title>Preventing homologous recombination does not affect the phenotypes caused by premature <italic>CLB3</italic> expression.</title><p>Wild-type (A19396), <italic>GAL-CLB3</italic> (A19400), <italic>spo11∆</italic> (A21193) and <italic>spo11∆ GAL-CLB3</italic> (A21194) cells were induced to sporulate and estradiol (1 μM) was added 3 hr after transfer into sporulation medium. Subsequently, segregation of sister chromatids (equational division) was quantified using heterozygous CENV GFP dots in binucleate cells (n = 100). Note that <italic>CLB3-</italic>induced meiosis I sister chromatid segregation is higher in <italic>GAL-CLB3</italic> cells than in <italic>CUP-CLB3</italic> cells. This is presumably due to the more homogenous expression of <italic>CLB3</italic> in cells where expression is driven from the <italic>GAL1-10</italic> promoter. Using a chi-square test (df 1), the fraction of binucleates that display a reductional or equational division was compared between <italic>GAL-CLB3</italic> and <italic>GAL-CLB3 spo11∆</italic> χ<sup>2</sup> = 0.3072, p=0.5794.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.012">http://dx.doi.org/10.7554/eLife.00117.012</ext-link></p></caption><graphic xlink:href="elife00117fs008"/></fig></fig-group></p><p>Next, we determined whether expression of <italic>CLB1</italic>, <italic>CLB3</italic> or <italic>CLB4</italic> in pachytene-arrested cells also affects the manner in which chromosomes attach to the meiotic spindle using live-cell imaging. To this end we used strains carrying heterozygous CENV-GFP dots and an Spc42-mCherry fusion (Spc42 is an SPB component) to monitor the behavior of the marked centromere with respect to the spindle axis. In wild-type and <italic>CUP-CLB5</italic> cells, sister kinetochores remained closely associated with each other and did not appear to be tightly associated with SPBs, consistent with the observation that these cells failed to form a spindle. In contrast, we observed dynamic separation of heterozygous CENV-GFP dots upon expression of <italic>CLB1</italic> or <italic>CLB3</italic>, with sister kinetochores frequently splitting and coming together (<xref ref-type="fig" rid="fig2">Figure 2B,C</xref>). This observation is reminiscent of the behavior of bioriented sister chromatids during metaphase of mitosis (<xref ref-type="bibr" rid="bib41">Pearson et al., 2001</xref>).</p><p>Cells expressing <italic>CLB4</italic> did not show transient splitting of sister kinetochores in prophase I, indicating that chromosomes are either unable to attach to the spindle or that homologous chromosomes, instead of sister chromatids, are bioriented as occurs in wild-type cells during metaphase I. To distinguish between these possibilities, we examined the behavior of <italic>CUP-CLB4</italic> cells in which both homologs of chromosome V harbor CENV-GFP dots (henceforth homozygous CENV-GFP dots). Similar to wild-type, we observed that in <italic>CUP-CLB4</italic> cells the two CENV-GFP dots remained tightly associated in prophase I, indicating that the homologous chromosomes are paired and not attached to the prematurely formed spindle (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). Together, these results indicate that <italic>CUP-CLB1</italic>, <italic>CUP-CLB3</italic> or <italic>CUP-CLB4</italic> expression promotes bipolar spindle formation in pachytene-arrested cells, but only <italic>CLB1</italic> and <italic>CLB3</italic> expression can promote stable microtubule–kinetochore attachments sufficient to generate tension.</p><p>To determine whether different amounts of total CDK activity were responsible for the phenotypic differences of prematurely expressing Clb1 or Clb3 compared to Clb4, we measured total CDK activity (Cdc28 in budding yeast) using Histone H1 as a substrate. Cdc28-associated kinase activity was low during prophase I and increased more than 25-fold during metaphase I/anaphase I in wild-type cells (<xref ref-type="fig" rid="fig1">Figure 1F</xref>). Expression of all four cyclins led to a significant increase in total CDK activity in prophase I (<xref ref-type="fig" rid="fig1">Figure 1F</xref>), but importantly, the degree of increase did not correlate with the ability to induce sister chromatid splitting in the <italic>NDT80</italic> arrest. For example, Clb1 expression led to a similar increase in Cdc28-associated kinase activity as expression of Clb4, yet Clb1 induced sister chromatid splitting whereas Clb4 did not (<xref ref-type="fig" rid="fig1 fig2">Figures 1F and 2B,C</xref>). We conclude that the ability to induce sister chromatid splitting does not correlate with total CDK activity produced by the various <italic>CUP-CLB</italic> fusions. Furthermore, SPB separation and spindle formation are not sufficient to induce microtubule–kinetochore interactions. Events that can be triggered by Clb1 and Clb3, but not Clb4 are also necessary to promote attachments sufficient to generate tension. Determining why <italic>CLB4</italic> expressing cells fail to form productive microtubule–kinetochore interactions could provide important insights into substrate specificity of cyclin-CDK complexes.</p></sec><sec id="s2-2"><title>Expression of <italic>CLB3</italic> or <italic>CLB1</italic> during premeiotic S phase/prophase I causes sister chromatids to segregate during meiosis I</title><p>To determine the consequences of premature cyclin expression on meiosis I chromosome segregation, we examined the segregation of heterozygous CENV-GFP dots in cells that were reversibly arrested in pachytene using the <italic>NDT80</italic> block-release system. In this system, expression of <italic>NDT80</italic> is controlled by the <italic>GAL1-10</italic> promoter, which is regulated by an estrogen-inducible Gal4-ER fusion (<xref ref-type="bibr" rid="bib2">Benjamin et al., 2003</xref>; <xref ref-type="bibr" rid="bib9">Carlile and Amon, 2008</xref>). Cells were induced to sporulate and after 135 min, copper was added to induce cyclin expression. 4 hr 30 min after sporulation induction, estrogen was added to allow cells to synchronously proceed through the meiotic divisions. In wild-type, <italic>CUP-CLB4</italic> and <italic>CUP-CLB5</italic> cells, sister chromatids cosegregated in the first division, resulting in binucleate cells with a GFP dot in one of the two nuclei. In contrast, 39% of <italic>CUP-CLB1</italic> and 41% of <italic>CUP-CLB3</italic> cells segregated sister chromatids in the first division, as judged by the presence of binucleate cells with a GFP dot in each nucleus (<xref ref-type="fig" rid="fig2">Figure 2D</xref>). We observed a similar result for chromosome III and cells in which one copy of chromosome V was marked with a GFP dot and the other copy with an RFP dot (<xref ref-type="fig" rid="fig2s3 fig2s4">Figure 2—figure supplements 3 and 4</xref>).</p><p>To confirm that sister chromatids indeed split during meiosis I in cells expressing <italic>CLB3</italic> during prophase I, we examined when sister chromatid separation occurred with respect to Securin (Pds1 in budding yeast) degradation in <italic>CUP-CLB3</italic> cells. In wild-type cells harboring heterozygous CENV-GFP dots, Pds1 degradation was immediately followed by movement of the single GFP dot to one side of the cell, indicating that homologous chromosomes had segregated. Subsequently, these cells underwent meiosis II and sister chromatids segregated (median = 86 min after Pds1 degradation; <xref ref-type="fig" rid="fig2">Figure 2E</xref>). In contrast, <italic>CUP-CLB3</italic> cells segregated sister chromatids immediately after Pds1 degradation (median = 7 min after Pds1 degradation; <xref ref-type="fig" rid="fig2">Figure 2E</xref>). These results demonstrate that <italic>CUP-CLB3</italic> cells segregate sister chromatids during the first meiotic division. Thus, <italic>CUP-CLB3</italic> cells must be defective in two key aspects of meiosis I chromosome segregation: coorientation of sister kinetochores and maintenance of centromeric cohesion. We note that another essential aspect of meiosis I chromosome segregation, homologous recombination, was not affected by premature <italic>CLB3</italic> expression. We observed no major defects in DSB formation, synaptonemal complex assembly and generation of recombination products, nor did preventing homologous recombination affect the phenotypes caused by premature <italic>CLB3</italic> expression (<xref ref-type="fig" rid="fig2s5 fig2s6 fig2s7 fig2s8">Figure 2—figure supplements 5–8</xref>).</p></sec><sec id="s2-3"><title>Premature expression of <italic>CLB3</italic> interferes with monopolin localization</title><p>The finding that <italic>CUP-CLB1</italic> or <italic>CUP-CLB3</italic> cells segregate sister chromatids during meiosis I indicates that sister kinetochore coorientation is defective. To investigate this further, we examined monopolin localization in cells that segregate sister chromatids in meiosis I (<italic>CUP-CLB3</italic> cells) and cells that do not exhibit chromosome missegregation despite cyclin misexpression (<italic>CUP-CLB4</italic> cells). Colocalization of Lrs4 or Mam1 with the kinetochore component Ndc10 was dramatically reduced in <italic>CUP-CLB3</italic> but not <italic>CUP-CLB4</italic> cells (<xref ref-type="fig" rid="fig3">Figure 3A</xref> and <xref ref-type="fig" rid="fig3s1 fig3s2">Figure 3—figure supplements 1 and 2</xref>). Hyperphosphorylation of Lrs4, which correlates with monopolin function (<xref ref-type="bibr" rid="bib13">Clyne et al., 2003</xref>; <xref ref-type="bibr" rid="bib32">Lee and Amon, 2003</xref>; <xref ref-type="bibr" rid="bib37">Matos et al., 2008</xref>), was also significantly reduced in <italic>CUP-CLB3</italic>, but not in <italic>CUP-CLB4</italic> cells (<xref ref-type="fig" rid="fig3">Figure 3B</xref> and <xref ref-type="fig" rid="fig3s3">Figure 3—figure supplement 3</xref>). These results indicate that premature expression of <italic>CLB3</italic> prevents monopolin association with kinetochores.<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.00117.013</object-id><label>Figure 3.</label><caption><title>Premature <italic>CLB3</italic> expression disrupts monopolin function.</title><p>(<bold>A</bold>) Lrs4-13myc (green) localization relative to Ndc10-6HA (red) was determined in spread nuclei from wild-type (A9217), <italic>CUP-CLB3</italic> (A26278) and <italic>CUP-CLB4</italic> (A29643) harboring a Cdc20 depletion allele (<italic>cdc20-mn</italic>) were induced to undergo sporulation and arrested in metaphase I due to depletion of Cdc20. CuSO<sub>4</sub> was added at 3 hr after induction of sporulation (n > 40). The fraction of spread nuclei that display colocalized, partial or mislocalized Lrs4 with respect to Ndc10 was compared to wild-type using a chi-square test (df 2): <italic>CUP-CLB4</italic>, χ<sup>2</sup> = 1.136, p=0.5666; <italic>CUP-CLB3</italic>, χ<sup>2</sup> = 45.84, p<0.0001. (<bold>B</bold>) Western blots for Lrs4-13myc, Clb3 and Pgk1 from wild-type (A9217) and <italic>CUP-CLB3</italic> (A26278) cells. Cells were sporulated as described in (<bold>A</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.013">http://dx.doi.org/10.7554/eLife.00117.013</ext-link></p></caption><graphic xlink:href="elife00117f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.014</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Monopolin association with kinetochores is disrupted in <italic>CUP-CLB3</italic> but not in <italic>CUP-CLB4</italic> cells.</title><p>Wild-type (A7450), <italic>CUP-CLB3</italic> (A28673) and <italic>CUP-CLB4</italic> (A28674) cells carrying the <italic>cdc20-mn</italic> allele were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Mam1-9myc (green) localization relative to Ndc10-6HA (red) was determined in spread nuclei from metaphase I-arrested cells (n > 40). DNA is shown in blue. Using a chi-square test (df 2) the fraction of spread nuclei that display colocalized, partial or mislocalized Mam1 with respect to Ndc10 was compared to wild-type: <italic>CUP-CLB4</italic>, χ<sup>2</sup> = 2.554, p=0.2788; <italic>CUP-CLB3</italic>, χ<sup>2</sup> = 39.31, p<0.0001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.014">http://dx.doi.org/10.7554/eLife.00117.014</ext-link></p></caption><graphic xlink:href="elife00117fs009"/></fig><fig id="fig3s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.015</object-id><label>Figure 3—figure supplement 2.</label><caption><title>Premature Clb3 expression does not interfere with Mam1 expression.</title><p>Wild-type (A7450), <italic>CUP-CLB3</italic> (A28673) and <italic>CUP-CLB4</italic> (A28674) cells carrying the <italic>cdc20-mn</italic> allele were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Mam1 protein levels were analyzed to determine whether premature Clb3 expression interferes with Mam1 expression. Pgk1 was used as a loading control.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.015">http://dx.doi.org/10.7554/eLife.00117.015</ext-link></p></caption><graphic xlink:href="elife00117fs010"/></fig><fig id="fig3s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.016</object-id><label>Figure 3—figure supplement 3.</label><caption><title>Lrs4 phosphorylation is not disrupted in <italic>CUP-CLB4</italic> cells.</title><p>Wild-type (A26277) and <italic>CUP-CLB4</italic> (A29643) cells carrying the <italic>cdc20-mn</italic> allele were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Levels of Lrs4, Clb3 and Pgk1 from cells arrested in metaphase I were examined by Western blot analysis.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.016">http://dx.doi.org/10.7554/eLife.00117.016</ext-link></p></caption><graphic xlink:href="elife00117fs011"/></fig></fig-group></p></sec><sec id="s2-4"><title>Centromeric cohesin is lost during meiosis I in <italic>CUP-CLB3</italic> cells</title><p>Sister chromatids segregate during meiosis I in <italic>CUP-CLB3</italic> cells, indicating that centromeric cohesin either fails to associate with chromosomes or is lost prematurely. To test the first possibility, we examined chromosome association of the cohesin subunit Rec8 and the cohesion maintenance factor Pds5 with chromosomes. Chromatin immunoprecipitation (ChIP) and chromosome spreads revealed that association of both proteins with chromosomes in <italic>CUP-CLB3</italic> cells was indistinguishable from that of wild-type cells during prophase I or metaphase I (<xref ref-type="fig" rid="fig4">Figure 4A</xref> and <xref ref-type="fig" rid="fig4s1 fig4s2">Figure 4—figure supplements 1 and 2</xref>). Thus, loading of cohesion factors onto chromosomes is not affected in <italic>CUP-CLB3</italic> cells.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.00117.017</object-id><label>Figure 4.</label><caption><title><italic>CLB3</italic> misexpression disrupts protection of centromeric cohesin.</title><p>Cyclin expression was induced after 2 hr 15 min (<bold>C</bold>) and (<bold>D</bold>), 2 hr 30 min (<bold>A</bold>), (<bold>B</bold>), (<bold>E</bold>), (<bold>F</bold>) and (<bold>H</bold>) or 3 hr (<bold>G</bold>) and (<bold>I</bold>) of sporulation. (<bold>A</bold>) Chromosomal association of Rec8-13myc was monitored by ChIP-chip in wild-type (A28716) and <italic>CUP-CLB3</italic> (A28718) during prophase I arrest. Centromere position is identified by a black circle. (<bold>B</bold>) Centromeric Rec8 localization was monitored in spread nuclei from wild-type (A28684), <italic>CUP-CLB3</italic> (A28685) and <italic>CUP-CLB4</italic> (A28686) cells carrying <italic>REC8-3HA</italic> (red) and <italic>NDC10-13myc</italic> (green) (n > 40). The fraction of spread nuclei that were Rec8 positive or negative was compared to wild-type using a chi-square test (df 1): <italic>CUP-CLB4</italic>, χ<sup>2</sup> = 0.001323, p=0.9710; <italic>CUP-CLB3</italic>, χ<sup>2</sup> = 32.79, p<0.0001. (<bold>C</bold>) Rec8 cleavage monitored by Western blot after release from an <italic>NDT80</italic> block (4 hr 30 min) in wild-type and <italic>CUP-CLB3</italic> carrying both a myc-tagged <italic>REC8</italic> allele as well as either HA-tagged <italic>REC8</italic> or <italic>rec8-29A</italic> allele (left to right: A29957, A29959, A29961, A29963). (<bold>D</bold>) Percentage of cells with short bipolar spindles was determined at indicated times in wild-type (A22804), <italic>CUP-CLB3</italic> (A29965), <italic>rec8-29A</italic> (A22803) and <italic>CUP-CLB3 rec8-29A</italic> (A29967) after release from an <italic>NDT80</italic> block (4 hr 30 min) (n = 100 per time point). (<bold>E</bold>) ChIP analysis for total Rec8, p-S179 Rec8 or p-S521 Rec8 from metaphase I-arrested (<italic>cdc20-mn</italic>) wild-type (A28681), <italic>CUP-CLB3</italic> (A28682) and Sgo1-depleted (<italic>sgo1-mn</italic>; A29994) cells. Relative occupancy at a chromosome arm site (c194) or at a centromeric site (CENV) was determined relative to a low binding region (c281). Error bars represent range (n = 2). (<bold>F</bold>) Chromosomal association of Sgo1-3V5 was monitored by ChIP-chip in wild-type (A29795) and <italic>CUP-CLB3</italic> (A29799) cells during prophase I-arrest. Centromere position is identified by a black circle. (<bold>G</bold>), (<bold>H</bold>) Localization of Sgo1-9myc (G, green) or Rts1-13myc (H, green) relative to Ndc10-6HA (red) determined by nuclear spreads in (<bold>G</bold>) wild-type (A22868) and <italic>CUP-CLB3</italic> (A22870) or (<bold>H</bold>) wild-type (A28329) and <italic>CUP-CLB3</italic> (A28330) during prophase I (n > 40). For (<bold>G</bold>), the fraction of spread nuclei that display colocalized or mislocalized Sgo1 relative to Ndc10 was compared between wild-type and <italic>CUP-CLB3</italic> using a chi-square test (df 1) χ<sup>2</sup> = 1.554, p=0.2125. For (<bold>H</bold>), the fraction of spread nuclei that display colocalized, partial or mislocalized Rts1 relative to Ndc10 was compared between wild-type and <italic>CUP-CLB3</italic> using a chi-square test (df 2) χ<sup>2</sup> = 3.712, p=0.1563. (<bold>I</bold>) Localization of Sgo1-9myc (green) in binucleates relative to Ndc10-6HA (red) determined by nuclear spreads from wild-type (A22868) and <italic>CUP-CLB3</italic> (A22870) (n > 40). The fraction of spread nuclei that were Sgo1 positive or negative was compared between wild-type and <italic>CUP-CLB3</italic> using a chi-square test (df 1) χ<sup>2</sup> = 23.92, p<0.0001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.017">http://dx.doi.org/10.7554/eLife.00117.017</ext-link></p></caption><graphic xlink:href="elife00117f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.018</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Chromosomal association of Rec8 in <italic>CUP-CLB3</italic> cells.</title><p>Wild-type (A26547) and <italic>CUP-CLB3</italic> (A26548) cells were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 3 hr after transfer into sporulation medium. Rec8-3HA localization (red) was determined in spread nuclei from prophase I cells. DNA is shown in blue.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.018">http://dx.doi.org/10.7554/eLife.00117.018</ext-link></p></caption><graphic xlink:href="elife00117fs012"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.019</object-id><label>Figure 4—figure supplement 2.</label><caption><title>Chromosomal association of Pds5 in <italic>CUP-CLB3</italic> cells.</title><p>Wild-type (A28681) and <italic>CUP-CLB3</italic> (A28682) cells carrying the <italic>cdc20-mn</italic> allele were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Pds5 localization was determined by ChIP-chip from metaphase I-arrested cells. Black balls depict centromere positions.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.019">http://dx.doi.org/10.7554/eLife.00117.019</ext-link></p></caption><graphic xlink:href="elife00117fs013"/></fig><fig id="fig4s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.020</object-id><label>Figure 4—figure supplement 3.</label><caption><title><italic>CUP-CLB3</italic> cells partially bypass the nuclear division delay of <italic>mam1∆</italic> cells.</title><p>Wild-type (A22678), <italic>CUP-CLB3</italic> (A22702), <italic>mam1∆</italic> (A31340) and <italic>mam1∆ CUP-CLB3</italic> (A31342) cells carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 15 min after transfer into sporulation medium. Cells were released from the <italic>NDT80</italic> block 4 hr 30 min after transfer into sporulation medium. The percentage of cells that had undergone one or two meiotic divisions was determined at the indicated time points (n = 100 per time point).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.020">http://dx.doi.org/10.7554/eLife.00117.020</ext-link></p></caption><graphic xlink:href="elife00117fs014"/></fig><fig id="fig4s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.021</object-id><label>Figure 4—figure supplement 4.</label><caption><title>Meiotic progression of the cells analyzed for Rec8 cleavage in <xref ref-type="fig" rid="fig4">Figure 4C</xref>.</title><p><italic>REC8-myc/REC8-HA</italic> (A29957), <italic>REC8-myc/rec8-29A-HA</italic> (A29961), <italic>REC8-myc/REC8-HA CUP-CLB3</italic> (A29959) and <italic>REC8-myc/rec8-29A-HA CUP-CLB3</italic> (A29963) cells carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 15 min after transfer into sporulation medium. Cells were released from the <italic>NDT80</italic> block 4 hr 30 min after transfer into sporulation medium. The percentage of cells in metaphase I (grey symbols), anaphase I (violet symbols), metaphase II (dark blue symbols) and anaphase II (green symbols) was determined at the indicated times (n = 100 per time point).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.021">http://dx.doi.org/10.7554/eLife.00117.021</ext-link></p></caption><graphic xlink:href="elife00117fs015"/></fig><fig id="fig4s5" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.022</object-id><label>Figure 4—figure supplement 5.</label><caption><title>Analysis of Rec8 cleavage in cells used for <xref ref-type="fig" rid="fig4">Figure 4D</xref>.</title><p>Wild-type (A22804), <italic>CUP-CLB3</italic> (A29965), <italic>rec8-29A</italic> (A22803) and <italic>rec8-29A CUP-CLB3</italic> (A29967) cells carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 15 min after transfer into sporulation medium. Cells were released from the <italic>NDT80</italic> block 4 hr 30 min after transfer into sporulation medium. Levels of full-length Rec8, cleaved Rec8, Clb3 and Pgk1 were monitored by Western blot.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.022">http://dx.doi.org/10.7554/eLife.00117.022</ext-link></p></caption><graphic xlink:href="elife00117fs016"/></fig><fig id="fig4s6" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.023</object-id><label>Figure 4—figure supplement 6.</label><caption><title>Meiotic progression of the cells analyzed for Rec8 cleavage in <xref ref-type="fig" rid="fig4">Figure 4D</xref>.</title><p>Wild-type (A22804), <italic>CUP-CLB3</italic> (A29965), <italic>rec8-29A</italic> (A22803) and <italic>rec8-29A CUP-CLB3</italic> (A29967) cells carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 15 min after transfer into sporulation medium. Cells were released from the <italic>NDT80</italic> block 4 hr 30 min after transfer into sporulation medium. The percentage of cells in metaphase I (grey symbols), anaphase I (violet symbols), metaphase II (dark blue symbols) and anaphase II (green symbols) was determined at the indicated times (n = 100 per time point).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.023">http://dx.doi.org/10.7554/eLife.00117.023</ext-link></p></caption><graphic xlink:href="elife00117fs017"/></fig><fig id="fig4s7" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.024</object-id><label>Figure 4—figure supplement 7.</label><caption><title>Chromosomal association of Sgo1 in <italic>CUP-CLB3</italic> cells.</title><p>Wild-type (A28712) and <italic>CUP-CLB3</italic> (A28713) cells carrying the <italic>cdc20-mn</italic> allele were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Sgo1-3V5 localization was determined by ChIP-chip, 7 hr after transfer into sporulation medium when cells were arrested in metaphase I. Arm peaks for Sgo1 correspond to cohesin-associated regions. The basis for Sgo1 enrichment at these sites is currently unclear. Black balls depict centromere positions.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.024">http://dx.doi.org/10.7554/eLife.00117.024</ext-link></p></caption><graphic xlink:href="elife00117fs018"/></fig><fig id="fig4s8" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.025</object-id><label>Figure 4—figure supplement 8.</label><caption><title>Localization of Rts1 in <italic>CUP-CLB3</italic> cells.</title><p>Wild-type (A28331) and <italic>CUP-CLB3</italic> (A28332) cells carrying the <italic>cdc20-mn</italic> allele were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Rts1-13myc (green) localization relative to Ndc10-6HA (red) was determined in spread nuclei from metaphase I-arrested cells (n > 40).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.025">http://dx.doi.org/10.7554/eLife.00117.025</ext-link></p></caption><graphic xlink:href="elife00117fs019"/></fig><fig id="fig4s9" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.026</object-id><label>Figure 4—figure supplement 9.</label><caption><title>Chromosomal association of Spo13 in <italic>CUP-CLB3</italic> cells.</title><p>(Left panel) wild-type (A30856), <italic>CUP-CLB3</italic> (A30858) and <italic>CUP-CLB4</italic> (A30860) cells carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 15 min after transfer into sporulation medium. Spo13-3V5 localization was determined by ChIP from prophase I-arrested cells. Relative occupancy at a centromeric site (CEN5) relative to a low binding region (HMR) was determined. Error bars represent the range (n = 2). (Right panel) wild-type (A30743), <italic>CUP-CLB3</italic> (A30745) and <italic>CUP-CLB4</italic> (A30747) cells carrying the <italic>cdc20-mn</italic> allele were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Spo13-3V5 localization was determined by ChIP 7 hr after transfer into sporulation medium when cells were arrested in metaphase I. Relative occupancy at a centromeric site (CENV) relative to a low binding region (HMR) was determined. Error bars represent the range (n = 2).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.026">http://dx.doi.org/10.7554/eLife.00117.026</ext-link></p></caption><graphic xlink:href="elife00117fs020"/></fig><fig id="fig4s10" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.027</object-id><label>Figure 4—figure supplement 10.</label><caption><title>Rts1 localization in binucleate <italic>CUP-CLB3</italic> cells.</title><p>Wild-type (A28329) and <italic>CUP-CLB3</italic> (A28330) cells were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Rts1-13myc (green) localization relative to Ndc10-6HA (red) was determined in spread nuclei from binucleates (n > 40). Using a chi-square test (df 2) the fraction of spread nuclei that display strong, weak or negative Rts1 with respect to Ndc10 was compared between wild-type and <italic>CUP-CLB3</italic> χ<sup>2</sup> = 54.49, p<0.0001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.027">http://dx.doi.org/10.7554/eLife.00117.027</ext-link></p></caption><graphic xlink:href="elife00117fs021"/></fig><fig id="fig4s11" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.028</object-id><label>Figure 4—figure supplement 11.</label><caption><title>Analysis of Rts1 localization in Rec8 phosphomimetic mutants.</title><p>Wild-type (A29645) and <italic>rec8-S136D S179D S197D T209D</italic> (A29647) cells were induced to sporulate and Rec8-3HA/rec8-4D-3HA or Rts1-3V5 localization relative to Ndc10-13myc was determined in spread nuclei from binucleates (n > 40). Characterization of <italic>rec8-S136D S179D S197D T209D</italic> has been described in <xref ref-type="bibr" rid="bib24">Katis et al. (2010)</xref>. Note that strains carrying this allele fail to maintain centromeric cohesin beyond metaphase I (bottom panel). These binucleates also have weak Rts1 staining (top panel), suggesting that Rts1 maintenance at centromeric regions in anaphase I depends on cohesin. For top panel, using a chi-square test (df 2) the fraction of spread nuclei that display strong, weak or negative Rts1 with respect to Ndc10 was compared between wild-type and <italic>CUP-CLB3</italic> χ<sup>2</sup> = 18.02, p=0.0001. For bottom panel, using a chi-square test (df 2) the fraction of spread nuclei that display strong, weak or negative Rec8 with respect to Ndc10 was compared between wild-type and <italic>CUP-CLB3</italic> χ<sup>2</sup> = 121.2, p<0.0001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.028">http://dx.doi.org/10.7554/eLife.00117.028</ext-link></p></caption><graphic xlink:href="elife00117fs022"/></fig></fig-group></p><p>To test the possibility that <italic>CUP-CLB3</italic> cells fail to maintain centromeric cohesion beyond anaphase I, we first determined the localization of the cohesin subunit Rec8 in cells that had progressed past metaphase I. Rec8 colocalized with the kinetochore component Ndc10 in binucleate wild-type and <italic>CUP-CLB4</italic> cells, demonstrating that centromeric cohesin is protected from removal until the onset of anaphase II. In contrast, Rec8 was not detected around centromeres in a substantial fraction of binucleate <italic>CUP-CLB3</italic> cells (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). Functional assays confirmed the defect in centromeric cohesion maintenance in <italic>CUP-CLB3</italic> cells. Although <italic>mam1∆</italic> cells biorient sister chromatids during meiosis I, they delay nuclear division until meiosis II due to the presence of centromeric cohesin (<xref ref-type="bibr" rid="bib50">Toth et al., 2000</xref>; <xref ref-type="bibr" rid="bib43">Rabitsch et al., 2003</xref>). The delay in nuclear division of a <italic>mam1∆</italic> was partially alleviated by the expression of <italic>CUP-CLB3</italic> (<xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3</xref>). This partial effect is likely due to not all <italic>CUP-CLB3</italic> cells losing centromeric cohesion prematurely in meiosis I (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). We conclude that both centromeric and arm cohesin are lost from chromosomes at the onset of anaphase I in <italic>CUP-CLB3</italic> cells.</p><p>Next, we investigated the cause of premature centromeric cohesin removal in <italic>CUP-CLB3</italic> cells. Cleavage of cohesin by separase requires the phosphorylation of Rec8 at multiple residues (<xref ref-type="bibr" rid="bib5">Brar et al., 2006</xref>; <xref ref-type="bibr" rid="bib24">Katis et al., 2010</xref>). A recessive allele of <italic>REC8</italic> in which 29 in vivo phosphorylation sites were mutated to alanine (<italic>rec8-29A</italic>) (<xref ref-type="bibr" rid="bib5">Brar et al., 2006</xref>) was not cleaved in <italic>CUP-CLB3</italic> cells, but wild-type Rec8 was (<xref ref-type="fig" rid="fig4">Figure 4C</xref> and <xref ref-type="fig" rid="fig4s4">Figure 4—figure supplement 4</xref>). Furthermore, the <italic>rec8-29A</italic> allele caused a similar metaphase I delay in wild-type and <italic>CUP-CLB3</italic> cells when expressed as the sole source of <italic>REC8</italic> (<xref ref-type="fig" rid="fig4">Figure 4D</xref> and <xref ref-type="fig" rid="fig4s5 fig4s6">Figure 4—figure supplements 5 and 6</xref>). We noticed that the Rec8 cleavage product was detected at lower levels in <italic>CUP-CLB3</italic> cells (<xref ref-type="fig" rid="fig4">Figure 4C</xref> and <xref ref-type="fig" rid="fig4s5">Figure 4—figure supplement 5</xref>). The cause of this reduction is currently unclear, but could indicate that in <italic>CUP-CLB3</italic> cells, cohesin removal also relies on a separase-independent pathway, that is the prophase removal pathway (<xref ref-type="bibr" rid="bib61">Yu and Koshland, 2005</xref>).</p><p>Our results demonstrate that Rec8 phosphorylation is required for cohesin removal in <italic>CUP-CLB3</italic> cells and suggest that the defect in centromeric cohesin protection may result from increased phosphorylation of centromeric Rec8. To test this possibility, we used phospho-specific antibodies against two in vivo phosphorylation sites of Rec8 (pS179 and pS521) (<xref ref-type="bibr" rid="bib5">Brar et al., 2006</xref>; <xref ref-type="bibr" rid="bib24">Katis et al., 2010</xref>; M. Attner personal communication, October 2011) and analyzed the relative enrichment of total Rec8 and phospho-Rec8 at CENV or at an arm cohesin binding site by ChIP in metaphase I-arrested cells. The two phospho-specific antibodies immunoprecipitated similar amounts of Rec8 in wild-type and <italic>CUP-CLB3</italic> cells at the arm site (<xref ref-type="fig" rid="fig4">Figure 4E</xref>), which is consistent with arm cohesin being primed for Separase cleavage. However, the amount of phosphorylated Rec8 was increased at the centromere in <italic>CUP-CLB3</italic> cells compared to wild-type cells, albeit not to the same extent as in cells depleted for Sgo1 (<italic>sgo1-mn</italic>), in which meiosis I centromeric-cohesin protection is completely defective (<xref ref-type="fig" rid="fig4">Figure 4E</xref>). We conclude that <italic>CUP-CLB3</italic> cells are compromised in preventing centromeric Rec8 phosphorylation during meiosis I.</p></sec><sec id="s2-5"><title>Sgo1-PP2A localization is not affected in <italic>CUP-CLB3</italic> cells</title><p>Sgo1-PP2A and the meiosis-specific protein Spo13 prevent centromeric Rec8 phosphorylation during meiosis I to protect this cohesin pool from cleavage. All three proteins localize to kinetochores during meiosis I, which is thought to be critical for their cohesin-protective function (<xref ref-type="bibr" rid="bib23">Katis et al., 2004a</xref>, <xref ref-type="bibr" rid="bib25">2004b</xref>; <xref ref-type="bibr" rid="bib29">Kitajima et al., 2004</xref>; <xref ref-type="bibr" rid="bib33">Lee et al., 2004</xref>; <xref ref-type="bibr" rid="bib30">Kitajima et al., 2006</xref>; <xref ref-type="bibr" rid="bib44">Riedel et al., 2006</xref>). Surprisingly, Sgo1, the PP2A regulatory subunit Rts1 and Spo13 localized normally in prophase I- and metaphase I-arrested <italic>CUP-CLB3</italic> cells (<xref ref-type="fig" rid="fig4">Figure 4F–H</xref> and <xref ref-type="fig" rid="fig4s7 fig4s8 fig4s9">Figure 4—figure supplements 7–9</xref>). We noticed a moderate reduction of Sgo1 and Rts1 at centromeres in binucleate <italic>CUP-CLB3</italic> cells (<xref ref-type="fig" rid="fig4">Figure 4I</xref> and <xref ref-type="fig" rid="fig4s10">Figure 4—figure supplement 10</xref>). However, this reduction during anaphase I is most likely a consequence rather than a cause of premature loss of centromeric cohesin. In cells expressing a phosphomimetic version of Rec8 (<italic>rec8-4D</italic>) that cannot be retained at centromeres beyond meiosis I, Rts1 localization is also reduced in anaphase I (<xref ref-type="fig" rid="fig4s11">Figure 4—figure supplement 11</xref>). It is thus unlikely that the reduction of Sgo1 and Rts1 at centromeres during anaphase I contributes to the premature loss of centromeric cohesin. These findings, together with our observation that centromeric Rec8 phosphorylation is increased in <italic>CUP-CLB3</italic> cells, indicate that Sgo1-PP2A function, but not localization, is impaired in <italic>CUP-CLB3</italic> cells.</p></sec><sec id="s2-6"><title>Modulating microtubule–kinetochore interactions affects monopolin-induced sister chromatid cosegregation during mitosis</title><p>How does premature expression of <italic>CLB3</italic> interfere with establishment of the meiosis I chromosome segregation pattern? The comparison of the effects caused by <italic>CLB3</italic> and <italic>CLB4</italic> misexpression provided insight into this question. Both cyclins induce spindle formation in prophase I. However, chromosomes are able to attach to this spindle and experience pulling forces only in <italic>CUP-CLB3</italic> cells. Thus, the ability to form tension-generating attachments (i.e. <italic>CUP-CLB1</italic> or <italic>CUP-CLB3</italic> cells) correlates with defects in meiosis I chromosome morphogenesis and segregation. This correlation suggests that premature microtubule–kinetochore engagement during premeiotic S phase/early prophase I is the underlying cause of chromosome missegregation in <italic>CUP-CLB3</italic> cells and predicts that tension generating microtubule–kinetochore attachments should inhibit meiosis I chromosome morphogenesis. Conversely, preventing them should enable building a proper meiosis I chromosome architecture.</p><p>We tested the first prediction using a previously described method in which monopolin-dependent sister kinetochore coorientation is induced during mitosis (<xref ref-type="bibr" rid="bib39">Monje-Casas et al., 2007</xref>). Overexpression of <italic>MAM1</italic> and <italic>CDC5</italic> upon a pheromone-induced G1 arrest is sufficient to induce cosegregation of sister chromatids in mitotic anaphase (<xref ref-type="bibr" rid="bib39">Monje-Casas et al., 2007</xref>, <xref ref-type="fig" rid="fig5">Figure 5A</xref>). However, when cells are allowed to form microtubule–kinetochore attachments prior to <italic>CDC5</italic> and <italic>MAM1</italic> expression, cosegregation of sister chromatids is prevented. We reversibly arrested cells in metaphase using a methionine repressible <italic>CDC20</italic> allele (<italic>MET-CDC20</italic>) and induced <italic>MAM1</italic> and <italic>CDC5</italic> expression after cells had arrested in metaphase and had formed microtubule–kinetochore interactions. Under these conditions, <italic>MAM1</italic> and <italic>CDC5</italic> expression did not induce sister chromatid cosegregation when cells were released into anaphase (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). Importantly, disrupting microtubule–kinetochore interactions by depolymerizing microtubules with nocodazole during the metaphase arrest resulted in robust cosegregation of sister chromatids in anaphase (48% cosegregation, <xref ref-type="fig" rid="fig5">Figure 5A</xref>). These results show that microtubule–kinetochore interactions modulate the ability of monopolin to induce sister chromatid cosegregation.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.00117.029</object-id><label>Figure 5.</label><caption><title>Transient disruption of microtubule–kinetochore interactions suppresses the chromosome segregation defects in <italic>CUP-CLB3</italic> cells.</title><p>(<bold>A</bold>) Wild-type (A10684) and <italic>GAL-CDC5 GAL-MAM1</italic> (A26546) cells, carrying a <italic>MET-CDC20</italic> allele and CENIV-GFP dots, were monitored for chromosome segregation in anaphase (see ‘Materials and methods’ for details). MT = microtubule, KT = kinetochore, (n = 100). The fraction of anaphase cells that segregate or cosegregate sister chromatids was compared between <italic>GAL-CDC5 GAL-MAM1</italic> condition (2) and <italic>GAL-CDC5 GAL-MAM1</italic> condition (3) using a chi-square test (df 1) χ<sup>2</sup> = 59.71, p<0.0001. (<bold>B</bold>) Schematic description of the experimental regime used for (<bold>C</bold>) through (<bold>H</bold>) see ‘Materials and methods’ for details. (<bold>C</bold>) Localization of Lrs4-13myc (green) in mononucleates relative to Ndc10-6HA (red) determined by nuclear spreads (n > 40) and (<bold>D</bold>) phosphorylation of Lrs4-13myc determined by gel mobility shift in wild-type (A29612), <italic>ndc80-1</italic> (A29614), <italic>CUP-CLB3</italic> (A29616) and <italic>CUP-CLB3 ndc80-1</italic> (A29618). For (<bold>C</bold>), using a chi-square test (df 2) the fraction of spread nuclei that display colocalized, partial or mislocalized Lrs4 with respect to Ndc10 was compared between wild-type and <italic>ndc80-1</italic> χ<sup>2</sup> = 0.9668, p=0.6167 and between <italic>CUP-CLB3</italic> and <italic>CUP-CLB3 ndc80-1</italic> χ<sup>2</sup> = 56.34, p<0.0001. (<bold>E</bold>) Localization of Rec8-13myc (green) in binucleates relative to Ndc10-6HA (red) determined by nuclear spreads in wild-type (A28716), <italic>ndc80-1</italic> (A28720), <italic>CUP-CLB3</italic> (A28718) and <italic>CUP-CLB3 ndc80-1</italic> (A28722) (n > 40). Using a chi-square test (df 1) the fraction of spread nuclei that were Rec8 positive or negative was compared between wild-type and <italic>ndc80-1</italic> χ<sup>2</sup> = 1.185, p=0.2764 and between <italic>CUP-CLB3</italic> and <italic>CUP-CLB3 ndc80-1</italic> χ<sup>2</sup> = 23.96, p<0.0001. (<bold>F</bold>) Segregation of sister chromatids using heterozygous CENV-GFP dots quantified in binucleates (n = 100) and (<bold>G</bold>) spore viability from wild-type (A22678), <italic>ndc80-1</italic> (A28621), <italic>CUP-CLB3</italic> (A22702) and <italic>CUP-CLB3 ndc80-1</italic> (A28623) (n = 40 tetrads for wild-type and <italic>ndc80-1</italic>, n > 60 tetrads for <italic>CUP-CLB3</italic> and <italic>CUP-CLB3 ndc80-1</italic>) (nonpermissive temperature >36°C). Using a chi-square test (df 1) the fraction of binucleates with a reductional or equational division was compared between <italic>CUP-CLB3</italic> and <italic>CUP-CLB3 ndc80-1</italic> χ<sup>2</sup> = 24.18, p<0.0001. (<bold>G</bold>) Segregation of chromosome V using homozygous CENV-GFP dots quantified in tetranucleates from wild-type (A22688), <italic>ndc80-1</italic> (A28625), <italic>CUP-CLB3</italic> (A22708) and <italic>CUP-CLB3 ndc80-1</italic> (A28627). Top panel: cells kept at 25°C for the duration of the experiment. Bottom panel: Cells treated as in (<bold>B</bold>) but monitored after meiosis II (n = 100).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.029">http://dx.doi.org/10.7554/eLife.00117.029</ext-link></p></caption><graphic xlink:href="elife00117f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.030</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Sporulation efficiency of <italic>ndc80-1</italic> mutants.</title><p>Wild-type (A22678) and <italic>ndc80-1</italic> (A28221) cells were induced to sporulate at 25°C. 2 hr 30 min after transfer into sporulation medium, cells were shifted to the indicated temperature and sporulation efficiency was determined after 24 hr.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.030">http://dx.doi.org/10.7554/eLife.00117.030</ext-link></p></caption><graphic xlink:href="elife00117fs023"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.031</object-id><label>Figure 5—figure supplement 2.</label><caption><title>Sister kinetochore coorientation in <italic>ndc80-1</italic> cells under a continuous inactivation regime at 34°C during a metaphase I arrest.</title><p>Wild-type (A7118), <italic>CUP-CLB3</italic> (A23074), <italic>ndc80-1</italic> (A29690) and <italic>ndc80-1 CUP-CLB3</italic> (A29692) cells also carrying the <italic>cdc20-mn</italic> allele, were induced to sporulate at 25°C. 2 hr 45 min after transfer into sporulation medium, CuSO<sub>4</sub> (50 μM) was added and concurrently, cultures were shifted to 34°C. The percentage of mononucleate cells with separated CENV-GFP dots was determined 7 hr 30 min after transfer into sporulation medium when cells were arrested in metaphase I (n = 100). The fraction of nuclei that display sister kinetochores as separate or together was compared between <italic>CUP-CLB3</italic> and <italic>CUP-CLB3 ndc80-1</italic> using a chi-square test (df 1) χ<sup>2</sup> = 7.228, p=0.0072.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.031">http://dx.doi.org/10.7554/eLife.00117.031</ext-link></p></caption><graphic xlink:href="elife00117fs024"/></fig><fig id="fig5s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.032</object-id><label>Figure 5—figure supplement 3.</label><caption><title>Sister kinetochore coorientation in <italic>ndc80-1</italic> cells after transient inactivation regime at 34°C during a metaphase I arrest.</title><p>Wild-type (A20958), <italic>CUP-CLB3</italic> (A23076), <italic>ndc80-1</italic> (A29718) and <italic>ndc80-1 CUP-CLB3</italic> (A29720) cells also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions and the <italic>cdc20-mn</italic> allele were induced to sporulate at 25°C. 2 hr 45 min after transfer into sporulation medium CuSO<sub>4</sub> (50 μM) was added and concurrently, cultures were shifted to 34°C. After 5 hr, when cells had arrested in the <italic>NDT80</italic> arrest, cells were released from the <italic>NDT80</italic> block and transferred to 25°C. The percentage of mononucleate cells with separated CENV-GFP dots was determined 7 hr 30 min after transfer into sporulation medium when cells were arrested in metaphase I (n = 100). The fraction of nuclei that display sister kinetochores as separate or together was compared between <italic>CUP-CLB3</italic> and <italic>CUP-CLB3 ndc80-1</italic> using a chi-square test (df 1) χ<sup>2</sup> = 5.007, p=0.0252.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.032">http://dx.doi.org/10.7554/eLife.00117.032</ext-link></p></caption><graphic xlink:href="elife00117fs025"/></fig><fig id="fig5s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.033</object-id><label>Figure 5—figure supplement 4.</label><caption><title>Meiosis I chromosome segregation in <italic>ndc80-1</italic> cells after a transient inactivation regime at 34°C.</title><p>Wild-type (A22678), <italic>ndc80-1</italic> (A28621), <italic>CUP-CLB3</italic> (A22702) and <italic>CUP-CLB3 ndc80-1</italic> (A28623) cells also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate at 25°C. 2 hr 45 min after transfer into sporulation medium CuSO<sub>4</sub> (50 μM) was added and concurrently, cultures were shifted to 34°C. After 5 hr, when cells had arrested in the <italic>NDT80</italic> block, cells were released and transferred to 25°C. The percentage of binucleate cells with segregated heterozygous CENV-GFP dots was determined 7 hr 30 min after transfer into sporulation medium (n = 100). Note that a greater suppression of meiosis I sister chromatid segregation was observed in <italic>ndc80-1 CUP-CLB3</italic> cells when cells were incubated at temperatures higher than 34°C (<xref ref-type="fig" rid="fig5">Figure 5F</xref> and data not shown). The fraction of binucleates that underwent reductional or equational division was compared between <italic>CUP-CLB3</italic> and <italic>CUP-CLB3 ndc80-1</italic> using a chi-square test (df 1) χ<sup>2</sup> = 5.776, p=0.0162.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.033">http://dx.doi.org/10.7554/eLife.00117.033</ext-link></p></caption><graphic xlink:href="elife00117fs026"/></fig><fig id="fig5s5" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.034</object-id><label>Figure 5—figure supplement 5.</label><caption><title>Transient disruption of microtubule–kinetochore interactions using <italic>dam1-1</italic> allele restores meiosis I chromosome segregation in <italic>CUP-CLB3</italic> cells.</title><p>Wild-type (A22678), <italic>dam1-1</italic> (A28311), <italic>CUP-CLB3</italic> (A22702) and <italic>CUP-CLB3 dam1-1</italic> (A28341) cells also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate at 25°C. 2 hr 45 min after transfer into sporulation medium CuSO<sub>4</sub> (50 μM) was added and concurrently, cultures were shifted to 34°C. After 5 hr, when cells had arrested in the <italic>NDT80</italic> block, cells were released and transferred to 25°C. The percentage of binucleate cells with segregated heterozygous CENV-GFP dots was determined 7 hr 30 min after transfer into sporulation medium (n = 100). The fraction of binucleates that underwent reductional or equational division was compared between <italic>CUP-CLB3</italic> and <italic>CUP-CLB3 dam1-1</italic> using a chi-square test (df 1) χ<sup>2</sup> = 16.77, p<0.0001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.034">http://dx.doi.org/10.7554/eLife.00117.034</ext-link></p></caption><graphic xlink:href="elife00117fs027"/></fig><fig id="fig5s6" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.035</object-id><label>Figure 5—figure supplement 6.</label><caption><title>Transient disruption of microtubule–kinetochore interactions by benomyl treatment restores meiosis I chromosome segregation in <italic>CUP-CLB3</italic> cells.</title><p>Wild-type (A22678) and <italic>CUP-CLB3</italic> (A22702) cells also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate at 30°C. 2 hr 15 min after transfer into sporulation medium CuSO<sub>4</sub> (50 μM) was added and concurrently, cells were treated with DMSO or benomyl (120 μg/ml). Cells were subsequently released from <italic>NDT80</italic> block 4 hr 30 min after transfer into sporulation medium and benomyl was washed out concomitant with <italic>NDT80-</italic>block release. The percentage of binucleate cells with segregated heterozygous CENV-GFP dots was determined 6 hr after transfer into sporulation medium (n = 100). See ‘Materials and methods’ for further details. The fraction of binucleates that underwent reductional or equational division was compared between <italic>CUP-CLB3</italic> + DMSO and <italic>CUP-CLB3</italic> + benomyl using a chi-square test (df 1) χ<sup>2</sup> = 32.12, p<0.0001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.035">http://dx.doi.org/10.7554/eLife.00117.035</ext-link></p></caption><graphic xlink:href="elife00117fs028"/></fig><fig id="fig5s7" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.036</object-id><label>Figure 5—figure supplement 7.</label><caption><title>Transient disruption of microtubule–kinetochore interactions during S phase/prophase I suppresses <italic>CUP-CLB3</italic>-induced meiosis I sister chromatid segregation in a spindle assembly checkpoint independent manner.</title><p>(Top panel) wild-type (A22678), <italic>mad3∆</italic> (A30386), <italic>ndc80-1</italic> (A28621), <italic>ndc80-1 mad3∆</italic> (A30390), <italic>CUP-CLB3</italic> (A22702), <italic>CUP-CLB3 mad3∆</italic> (A30388), <italic>CUP-CLB3 ndc80-1</italic> (A28623) and <italic>CUP-CLB3 ndc80-1 mad3∆</italic> (A30392) cells also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate at 25°C. 2 hr 45 min after transfer into sporulation medium CuSO<sub>4</sub> (50 μM) was added and concurrently, cultures were shifted to 36°C. Cells were subsequently released from <italic>NDT80</italic> block at 5 hr and transferred to 25°C. Percent binucleates with segregated heterozygous CENV-GFP dots was determined (n = 100). (Bottom panel) wild-type (A22688), <italic>mad3∆</italic> (A30638), <italic>ndc80-1</italic> (A28625), <italic>ndc80-1 mad3∆</italic> (A30642), <italic>CUP-CLB3</italic> (A22708), <italic>CUP-CLB3 mad3∆</italic> (A30640), <italic>CUP-CLB3 ndc80-1</italic> (A28627) and <italic>CUP-CLB3 ndc80-1 mad3∆</italic> (A30644) cells also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate at 25°C. 2 hr 45 min after transfer to sporulation medium CuSO<sub>4</sub> (50 μM) was added and concurrently, cultures were shifted to 36°C. After 5 hr, when cells had arrested in the <italic>NDT80</italic> block, cells were released and transferred to 25°C. The percentage of binucleate cells with segregated homozygous CENV-GFP dots was determined 7 hr 30 min after transfer into sporulation medium. Binucleate cells with GFP signal in only one of the two nuclei were categorized as having experienced a meiosis I non-disjunction event (n = 100). For top panel, using a chi-square test (df 1) the fraction of binucleates that underwent reductional or equational division was compared between <italic>CUP-CLB3</italic> and <italic>CUP-CLB3 mad3∆</italic> χ<sup>2</sup> = 0.1800, p=0.6714 and between <italic>CUP-CLB3 ndc80-1</italic> and <italic>CUP-CLB3 ndc80-1 mad3∆</italic> χ<sup>2</sup> = 0.02454, p=0.8755. For bottom panel, using a chi-square test (df 1) the fraction of binucleates that displayed MI nondisjunction or other was compared between <italic>CUP-CLB3</italic> and <italic>CUP-CLB3 mad3∆</italic> χ<sup>2</sup> = 1.228, p=0.2678 and between <italic>CUP-CLB3 ndc80-1</italic> and <italic>CUP-CLB3 ndc80-1 mad3∆</italic> χ<sup>2</sup> = 0.6486, p=0.4206.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.036">http://dx.doi.org/10.7554/eLife.00117.036</ext-link></p></caption><graphic xlink:href="elife00117fs029"/></fig><fig id="fig5s8" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.037</object-id><label>Figure 5—figure supplement 8.</label><caption><title>Transient disruption of microtubule–kinetochore interactions during S phase/prophase I restores meiotic chromosome segregation in <italic>CUP-CLB3</italic> cells in a spindle assembly checkpoint independent manner.</title><p>Wild-type (A22688), <italic>ndc80-1</italic> (A28625), <italic>CUP-CLB3</italic> (A22708), <italic>CUP-CLB3 ndc80-1</italic> (A28627), <italic>mad3∆</italic> (A30638), <italic>ndc80-1 mad3∆</italic> (A30642), <italic>CUP-CLB3 mad3∆</italic> (A30640) and <italic>CUP-CLB3 ndc80-1 mad3∆</italic> (A30644) cells also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate at 25°C. 2 hr 45 min after transfer to sporulation medium CuSO<sub>4</sub> (50 μM) was added and concurrently, cultures were shifted to 36°C. After 5 hr, when cells had arrested in the <italic>NDT80</italic> block, cells were released and transferred to 25°C. Segregation of homozygous CENV-GFP dots was determined in tetranucleates 12 hr after transfer into sporulation medium (n = 100).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.037">http://dx.doi.org/10.7554/eLife.00117.037</ext-link></p></caption><graphic xlink:href="elife00117fs030"/></fig><fig id="fig5s9" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.038</object-id><label>Figure 5—figure supplement 9.</label><caption><title>Transient <italic>ndc80-1</italic> inactivation does not alter in vitro Cdk1 activity.</title><p>Wild-type (A25508), <italic>ndc80-1</italic> (A33203), <italic>CUP-CLB3</italic> (A33201) and <italic>CUP-CLB3 ndc80-1</italic> (A33205) cells also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate at 25°C. 2 hr 45 min after transfer to sporulation medium CuSO<sub>4</sub> (50 μM) was added and concurrently, cultures were shifted to 35°C. Samples were harvested 5 hr post transfer to sporulation medium, when cells were arrested in the <italic>NDT80</italic> block. In vitro kinase assays were performed with Cdc28-3V5 (Cdk1) immunoprecipitated from prophase I-arrested samples. Amounts of phosphorylated Histone H1 and immunoprecipitated Cdc28-3V5 are shown.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.038">http://dx.doi.org/10.7554/eLife.00117.038</ext-link></p></caption><graphic xlink:href="elife00117fs031"/></fig></fig-group></p></sec><sec id="s2-7"><title>Transient disruption of microtubule–kinetochore interactions restores meiosis I chromosome segregation in <italic>CUP-CLB3</italic> cells</title><p>If the defects in sister kinetochore coorientation and centromeric cohesin maintenance of <italic>CUP-CLB3</italic> cells are caused by premature microtubule–kinetochore interactions, proper meiosis I chromosome morphogenesis should be restored by transiently disrupting microtubule–kinetochore interactions. To test this, we used a temperature sensitive allele of <italic>NDC80</italic> (<italic>ndc80-1</italic>), which encodes a component of the outer kinetochore. <italic>ndc80-1</italic> cells grow and sporulate normally at 25°C, but fail to undergo any nuclear divisions at temperatures above 34°C (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>).</p><p>We first asked whether disrupting microtubule–kinetochore interactions suppresses the kinetochore localization defect of monopolin in <italic>CUP-CLB3</italic> cells. Using the <italic>NDT80</italic> block-release system, we induced cells to sporulate at 25°C. After 165 min, we induced cyclin expression and concurrently transferred cells to 34°C to inactivate the <italic>ndc80-1</italic> allele. Cells were then incubated for an additional 135 min to arrest them in the <italic>NDT80-</italic>depletion block. We then transferred cells to the permissive temperature and released them from the <italic>NDT80</italic> block into a metaphase I-arrest by depleting <italic>CDC20</italic> (<italic>cdc20-mn</italic>) (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). Under these conditions, wild-type and <italic>ndc80-1</italic> cells arrested in metaphase I with the monopolin subunit Lrs4 localized to kinetochores, while <italic>CUP-CLB3</italic> cells showed a defect in Lrs4 localization (<xref ref-type="fig" rid="fig5">Figure 5C</xref>). Remarkably, <italic>CUP-CLB3 ndc80-1</italic> cells showed near wild-type levels of Lrs4 association with kinetochores (<xref ref-type="fig" rid="fig5">Figure 5C</xref>). Transient inactivation of <italic>Ndc80</italic> also restored Lrs4 phosphorylation in <italic>CUP-CLB3</italic> cells (<xref ref-type="fig" rid="fig5">Figure 5D</xref>). Our results demonstrate that premature microtubule–kinetochore interactions prevent sister kinetochore coorientation by disrupting proper localization of the monopolin complex. The finding that transient disruption of microtubule–kinetochore interactions also suppresses the Lrs4 phosphorylation defect of <italic>CUP-CLB3</italic> cells, furthermore suggests that Lrs4 hyperphosphorylation occurs not at the time of nucleolar release, but once Lrs4 localizes to kinetochores.</p><p>We next asked whether transient inactivation of microtubule–kinetochore interactions also suppresses the premature loss of centromeric cohesin observed in <italic>CUP-CLB3</italic> cells. We used a similar protocol to the one described above, except cells were not arrested in metaphase I following release from the <italic>NDT80</italic> block, but were allowed to proceed into anaphase I to examine Rec8 localization. Remarkably, disrupting microtubule–kinetochore interactions at the time of Clb3 expression caused a considerable increase in the percentage of <italic>CUP-CLB3</italic> cells that retained Rec8 around centromeres during anaphase I (<xref ref-type="fig" rid="fig5">Figure 5E</xref>).</p><p>Finally, restoring centromeric cohesin protection and sister kinetochore coorientation to <italic>CUP-CLB3</italic> cells by transient inactivation of <italic>Ndc80</italic> restored homolog segregation during meiosis I (<xref ref-type="fig" rid="fig5">Figure 5F</xref> and <xref ref-type="fig" rid="fig5s2 fig5s3 fig5s4">Figure 5—figure supplements 2–4</xref>). Similar results were obtained with a temperature sensitive allele of the gene encoding the outer kinetochore component Dam1 (<italic>dam1-1</italic>) or by disrupting microtubule–kinetochore interactions by benomyl treatment (<xref ref-type="fig" rid="fig5s5 fig5s6">Figure 5—figure supplements 5 and 6</xref>). We further observed a striking improvement in overall chromosome segregation and spore viability in <italic>CUP-CLB3 ndc80-1</italic> compared to <italic>CUP-CLB3</italic> cells (<xref ref-type="fig" rid="fig5">Figure 5G,H</xref>). The suppression of chromosome missegregation in <italic>CUP-CLB3 ndc80-1</italic> cells did not depend on the spindle assembly checkpoint, because deletion of <italic>MAD3</italic> had no discernable effect on the extent of <italic>ndc80-1</italic> mediated suppression (<xref ref-type="fig" rid="fig5s7 fig5s8">Figure 5—figure supplements 7 and 8</xref>), nor was it due to the <italic>ndc80-1</italic> allele lowering Clb3-CDK activity (<xref ref-type="fig" rid="fig5s9">Figure 5—figure supplement 9</xref>). In summary, our results demonstrate that the defects associated with <italic>CUP-CLB3</italic> cells are due to premature microtubule–kinetochore interactions. Our results further suggest that preventing microtubule–kinetochore interactions during premeiotic S phase and prophase I is necessary to establish a meiosis I-specific chromosome architecture.</p></sec><sec id="s2-8"><title>The outer kinetochore is disassembled during premeiotic S phase and prophase I</title><p>Our results demonstrate that preventing premature interactions of microtubules with kinetochores is essential for establishing a meiosis I chromosome architecture. This occurs, at least in part, by restricting Clb-CDK activity during premeiotic S phase and prophase I. Are additional mechanisms in place to prevent premature microtubule–kinetochore interactions? Insight into this question came from the variability in <italic>CUP-CLB3</italic>-associated phenotypes.</p><p>We initially noticed that the timing of <italic>CLB3</italic> induction had an impact on the extent of sister chromatid segregation in meiosis I, especially in experiments that employed the <italic>NDT80</italic> block-release system. To investigate this further, we expressed <italic>CLB3</italic> at different times after induction of sporulation. We observed that the extent of meiosis I sister chromatid segregation declined as <italic>CLB3</italic> was expressed later during the <italic>NDT80</italic> block (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). One possibility is that <italic>CLB3</italic>-induced sister chromatid segregation depends on additional factors that become limiting. Kinetochore components are good candidates for such additional factors, because previous studies in fission yeast demonstrated that a subset of outer kinetochore components dissociates from the kinetochore during prophase I (<xref ref-type="bibr" rid="bib1">Asakawa et al., 2005</xref>).<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.00117.039</object-id><label>Figure 6.</label><caption><title>Meiosis I sister chromatid segregation correlates with presence of outer kinetochore components.</title><p>(<bold>A</bold>) Schematic description of the experimental regime and segregation of sister chromatids using heterozygous CENV-GFP dots quantified in binucleates from wild-type (A22678) and <italic>CUP-CLB3</italic> (A29406) after cyclin induction at 2 hr 15 min, 3 hr, 4 hr and 4 hr 30 min post transfer to sporulation medium. Cells released from <italic>NDT80</italic>-block at 4 hr 30 min (n = 100). Using a chi-square test (df 1), the fraction of binucleates that display a reductional or equational division was compared between wild-type and <italic>CUP-CLB3</italic> for each induction time point: (2:15), χ<sup>2</sup> = 58.00, p<0.0001; (3:00), χ<sup>2</sup> = 14.46, p=0.0001; (4:00), χ<sup>2</sup> = 1.020, p=0.3124; (4:30), χ<sup>2</sup> = 0.3384, p=0.5607. (<bold>B</bold>) Cluster analysis of kinetochore components from the indicated time points. Further details are in the ‘Materials and methods’ and in <xref ref-type="bibr" rid="bib6">Brar et al. (2012)</xref>. Inner kinetochore = Cse4 nucleosomes, Cbf3, Ctf19 complexes and Mif2. Outer kinetochore = Spc105, Mis12, Ndc80 and DASH complexes. Fold induction is calculated by dividing the average expression from metaphase I—anaphase I by the average expression from DNA replication-prophase I. (<bold>C</bold>) Ordered plot for mRNA-seq and ribosome footprinting data for <italic>NDC80</italic> and (<bold>D</bold>) <italic>HSK3</italic> at the indicated stages. Dotted line indicates time of release from <italic>NDT80</italic> block. Further details are in the ‘Materials and methods’ and in <xref ref-type="bibr" rid="bib6">Brar et al. (2012)</xref>. (<bold>E</bold>) Western blot for Ndc80-3V5 and Pgk1 from A30340 cells and (<bold>F</bold>) Hsk3-3V5 and Pgk1 from A31861 cells. Cells induced to sporulate and released from <italic>NDT80</italic> block at 4 hr 30 min.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.039">http://dx.doi.org/10.7554/eLife.00117.039</ext-link></p></caption><graphic xlink:href="elife00117f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.040</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Schematic representation of the kinetochore–microtubule interface.</title><p>Components of the kinetochore subcomplexes are grouped in color coded boxes. Note that the schematic representation is not drawn to scale.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.040">http://dx.doi.org/10.7554/eLife.00117.040</ext-link></p></caption><graphic xlink:href="elife00117fs032"/></fig><fig id="fig6s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.041</object-id><label>Figure 6—figure supplement 2.</label><caption><title>Meiotic cluster analysis of kinetochore components.</title><p>Cluster analysis of kinetochore components from the indicated time points. Further details are in ‘Materials and methods’ and in <xref ref-type="bibr" rid="bib6">Brar et al. (2012)</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.041">http://dx.doi.org/10.7554/eLife.00117.041</ext-link></p></caption><graphic xlink:href="elife00117fs033"/></fig><fig id="fig6s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.042</object-id><label>Figure 6—figure supplement 3.</label><caption><title>Meiotic expression of DASH complex subunits.</title><p>Ordered plot of mRNA-seq and ribosome footprinting data for the DASH complex components at indicated stages of sporulation. Dotted line indicates time of release from <italic>NDT80</italic> block. See <xref ref-type="bibr" rid="bib6">Brar et al. (2012)</xref> for details.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.042">http://dx.doi.org/10.7554/eLife.00117.042</ext-link></p></caption><graphic xlink:href="elife00117fs034"/></fig><fig id="fig6s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.043</object-id><label>Figure 6—figure supplement 4.</label><caption><title>Meiotic expression of Ndc80 complex subunits.</title><p>Ordered plot of mRNA-seq and ribosome footprinting data for the Ndc80 complex at indicated stages. Dotted line indicates time of release from <italic>NDT80</italic> block. See <xref ref-type="bibr" rid="bib6">Brar et al. (2012)</xref> for details.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.043">http://dx.doi.org/10.7554/eLife.00117.043</ext-link></p></caption><graphic xlink:href="elife00117fs035"/></fig><fig id="fig6s5" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.044</object-id><label>Figure 6—figure supplement 5.</label><caption><title>Meiotic expression of Mif2.</title><p>Ordered plot of the mRNA-seq and ribosome footprinting data for Mif2 at the indicated stages. Dotted line indicates time of release from the <italic>NDT80</italic> block. See <xref ref-type="bibr" rid="bib6">Brar et al. (2012)</xref> for details.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.044">http://dx.doi.org/10.7554/eLife.00117.044</ext-link></p></caption><graphic xlink:href="elife00117fs036"/></fig><fig id="fig6s6" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.045</object-id><label>Figure 6—figure supplement 6.</label><caption><title>Meiotic expression of KNL-1 complex subunits.</title><p>Ordered plot of the mRNA-seq and ribosome footprinting data for KNL-1 complex subunits (Spc105 complex) at the indicated stages. Dotted line indicates time of release from the <italic>NDT80</italic> block. See <xref ref-type="bibr" rid="bib6">Brar et al. (2012)</xref> for details.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.045">http://dx.doi.org/10.7554/eLife.00117.045</ext-link></p></caption><graphic xlink:href="elife00117fs037"/></fig><fig id="fig6s7" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.046</object-id><label>Figure 6—figure supplement 7.</label><caption><title>Meiotic expression of Mis12 complex subunits.</title><p>Ordered plot of the mRNA-seq and ribosome footprinting data for Mis12 complex subunits at the indicated stages. Dotted line indicates time of release from <italic>NDT80</italic> block. See <xref ref-type="bibr" rid="bib6">Brar et al. (2012)</xref> for details.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.046">http://dx.doi.org/10.7554/eLife.00117.046</ext-link></p></caption><graphic xlink:href="elife00117fs038"/></fig><fig id="fig6s8" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.047</object-id><label>Figure 6—figure supplement 8.</label><caption><title>Meiotic expression of Ctf19 complex subunits.</title><p>Ordered plot for mRNA-seq and ribosome footprinting data for Ctf19 complex at indicated stages. Dotted line indicates time of release from the <italic>NDT80</italic> block. See <xref ref-type="bibr" rid="bib6">Brar et al. (2012)</xref> for details.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.047">http://dx.doi.org/10.7554/eLife.00117.047</ext-link></p></caption><graphic xlink:href="elife00117fs039"/></fig><fig id="fig6s9" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.048</object-id><label>Figure 6—figure supplement 9.</label><caption><title>Meiotic expression of Cbf3 complex subunits.</title><p>Ordered plot of the mRNA-seq and ribosome footprinting data for Cbf3 complex subunits at the indicated stages. Dotted line indicates time of release from the <italic>NDT80</italic> block. See <xref ref-type="bibr" rid="bib6">Brar et al. (2012)</xref> for details.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.048">http://dx.doi.org/10.7554/eLife.00117.048</ext-link></p></caption><graphic xlink:href="elife00117fs040"/></fig><fig id="fig6s10" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.049</object-id><label>Figure 6—figure supplement 10.</label><caption><title>Meiotic expression of Histone subunits.</title><p>Ordered plot for mRNA-seq and ribosome footprinting data for the histones at indicated stages. Dotted line indicates time of release from <italic>NDT80</italic> block See (<xref ref-type="bibr" rid="bib6">Brar et al., 2012</xref>) for details.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.049">http://dx.doi.org/10.7554/eLife.00117.049</ext-link></p></caption><graphic xlink:href="elife00117fs041"/></fig><fig id="fig6s11" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00117.050</object-id><label>Figure 6—figure supplement 11.</label><caption><title>Meiotic expression of Dam1 and Ask1.</title><p><italic>DAM1-3V5</italic> (A28898) cells carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusion and <italic>ASK1-13myc</italic> (A29161) carrying <italic>ndt80∆</italic> were induced to sporulate and were either released from (left panel) or arrested in (right panel) the <italic>NDT80</italic> block. Levels of Dam1-3V5, Ask1-13myc, Kar2 and Pgk1 were monitored by Western blot analysis. Pgk1 and Kar2 served as loading controls.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.050">http://dx.doi.org/10.7554/eLife.00117.050</ext-link></p></caption><graphic xlink:href="elife00117fs042"/></fig></fig-group></p><p>Using a high-resolution ribosome profiling dataset (<xref ref-type="bibr" rid="bib6">Brar et al., 2012</xref>), we examined the timing of synthesis of all kinetochore components during meiotic progression by cluster analysis. This analysis revealed two major expression classes, one included kinetochore components that peak in expression prior to or during prophase I (early class), and the other contained components that instead show peak expression during the meiotic divisions (late class). The early class was enriched for inner kinetochore components (16 of 23), while the late class included primarily outer kinetochore components (13 of 18) (<xref ref-type="fig" rid="fig6">Figure 6B</xref>, <xref ref-type="fig" rid="fig6s1 fig6s10 fig6s2 fig6s3 fig6s4 fig6s5 fig6s6 fig6s7 fig6s8 fig6s9">Figure 6—figure supplements 1–10</xref>). The inner kinetochore binds to the centromere and generates a platform for the assembly of the outer kinetochore, which mediates microtubule attachments (<xref ref-type="bibr" rid="bib49">Tanaka, 2010</xref>). The temporal difference in expression suggests that the inner kinetochore is assembled prior to the meiotic divisions, while the outer kinetochore is constructed only as cells enter the meiotic divisions.</p><p>Among the outer kinetochore components that displayed peak synthesis during the divisions, <italic>NDC80</italic> and a subunit of the DASH complex, <italic>HSK3,</italic> displayed the most differential expression prior to meiosis I and during meiosis I, with a 9.02 and 2.64-fold induction, respectively (<xref ref-type="fig" rid="fig6">Figure 6B–D</xref>). This decline in Ndc80 expression is consistent with a previous study in fission yeast, showing that Ndc80 becomes undetectable at kinetochores during prophase I (<xref ref-type="bibr" rid="bib1">Asakawa et al., 2005</xref>). Analysis of Ndc80 protein levels provided an explanation for why cells upregulate the synthesis of outer kinetochore components during entry into meiosis I. Ndc80 levels declined during premeiotic S phase and became undetectable during late prophase I (<xref ref-type="fig" rid="fig6">Figure 6E</xref>). Importantly, the ability of <italic>CUP-CLB3</italic> to induce sister-chromatid segregation during meiosis I tightly correlated with Ndc80 protein levels; as Ndc80 protein declines, so does <italic>CLB3-</italic>induced meiosis I sister chromatid segregation (compare <xref ref-type="fig" rid="fig6">Figure 6A,E</xref>).</p><p>Hsk3 protein levels were also low until meiosis I entry (<xref ref-type="fig" rid="fig6">Figure 6F</xref>), but not all outer kinetochore components exhibited this decline in protein levels. For example, Ask1, a subunit of the DASH complex, was present throughout prophase I and levels of another DASH complex component, Dam1, increased during prophase I (<xref ref-type="fig" rid="fig6s11">Figure 6—figure supplement 11</xref>). Our findings indicate that the assembly of the outer kinetochore is restricted prior to <italic>NDT80</italic> expression and pachytene exit due to low levels of a subset of outer kinetochore components.</p></sec><sec id="s2-9"><title>Expression of <italic>NDC80</italic> and <italic>HSK3</italic> during premeiotic S phase/prophase I enhances <italic>CLB3</italic>-induced meiosis I sister chromatid segregation</title><p>To determine whether reduced expression of the outer kinetochore components Ndc80 and Hsk3 contributes to preventing premature microtubule–kinetochore engagement, we examined the consequences of expressing the two genes from the <italic>CUP1</italic> promoter (<xref ref-type="fig" rid="fig7">Figure 7</xref>). We first assessed whether expression of the two proteins allows for the recruitment of the DASH complex to kinetochores, which occurs via delivery through microtubules and can thus be used as a means of assessing end-on attachment of kinetochores (<xref ref-type="bibr" rid="bib10">Cheeseman et al., 2001</xref>; <xref ref-type="bibr" rid="bib49">Tanaka, 2010</xref>). Cells were induced to sporulate and after 4 hr, a time when Ndc80 levels are normally diminished, we induced the expression of <italic>CLB3</italic>, <italic>NDC80</italic> and/or <italic>HSK3</italic>. Whereas expression of either gene alone caused only a few cells to recruit Ask1 to kinetochores, cells simultaneously expressing <italic>NDC80</italic>, <italic>HSK3</italic> and <italic>CLB3</italic> during prophase I showed colocalization between Ask1 and the inner kinetochore component Ndc10, to an equal or greater extent than what was observed in metaphase I-arrested wild-type cells (<xref ref-type="fig" rid="fig8">Figure 8A,B</xref>). The difference in Ask1 localization was not due to a difference in <italic>ASK1</italic> expression (<xref ref-type="fig" rid="fig8">Figure 8C</xref>). In addition, induction of <italic>CLB3</italic> under the conditions mentioned above gave rise to bipolar spindles that appeared fragile with a weakened midzone. In contrast, consistent with stable microtubule–kinetochore interactions, coexpression of <italic>CLB3</italic>, <italic>HSK3</italic> and <italic>NDC80</italic> resulted in the formation of robust bipolar spindles (<xref ref-type="fig" rid="fig8">Figure 8D,E</xref>). Importantly, the expression of <italic>NDC80</italic> and/or <italic>HSK3</italic> during an <italic>NDT80</italic> block caused a considerable increase in meiosis I sister chromatid segregation in <italic>CUP-CLB3</italic> cells (<xref ref-type="fig" rid="fig8">Figure 8F</xref>). Furthermore, under conditions in which <italic>CLB3</italic> expression alone failed to induce meiosis I sister chromatid segregation, expression of <italic>CLB3</italic> together with <italic>NDC80</italic> and <italic>HSK3</italic> caused a substantial increase in meiosis I sister chromatid segregation (<xref ref-type="fig" rid="fig8">Figure 8G</xref>). This occurred even when cells were maintained in a prolonged <italic>NDT80</italic> block prior to expression of <italic>CLB3</italic>, <italic>NDC80</italic> and <italic>HSK3</italic> (<xref ref-type="fig" rid="fig9">Figure 9</xref>), ruling out the possibility that the expression of <italic>NDT80</italic> targets, such as <italic>CDC5</italic>, early during sporulation contributes to sister chromatid segregation during meiosis I. We conclude that limiting outer kinetochore assembly is an additional mechanism to prevent microtubule–kinetochore interactions during premeiotic S phase and prophase I.</p><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.00117.051</object-id><label>Figure 7.</label><caption><title>Characterization of <italic>NDC80</italic> and <italic>HSK3</italic> overexpression.</title><p>(<bold>A</bold>) <italic>CUP-NDC80-3V5</italic> (A30342) and <italic>CUP-HSK3-3HA</italic> (A32060) cells also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate. After 2 hr 30 min CuSO<sub>4</sub> (50 μM) was added and cells were subsequently released from <italic>NDT80</italic> block 4 hr 30 min after transfer into sporulation medium. The levels of Ndc80-3V5, Hsk3-3HA and Pgk1 were monitored by Western blot. (<bold>B</bold>) <italic>CUP-NDC80-3V5 CUP-CLB3</italic> (A31949), <italic>CUP-NDC80-3V5 CUP-HSK3</italic> (A31951) and <italic>CUP-NDC80-3V5 CUP-HSK3 CUP-CLB3</italic> (A31953) cells were induced to sporulate. 4 hr after transfer into sporulation medium CuSO<sub>4</sub> (50 μM) was added, and localization of Ndc80-3V5 (green) relative to Ndc10-6HA (red) was determined by nuclear spreads 5 hr after transfer into sporulation medium.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.051">http://dx.doi.org/10.7554/eLife.00117.051</ext-link></p></caption><graphic xlink:href="elife00117f007"/></fig><fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.00117.052</object-id><label>Figure 8.</label><caption><title>Expression of <italic>NDC80</italic> and <italic>HSK3</italic> in prophase I enhances Clb3-CDK-induced meiosis I sister chromatid segregation.</title><p>For (<bold>A</bold>)–(<bold>E</bold>), wild-type (A31945), <italic>CUP-CLB3</italic> (A31947), <italic>CUP-NDC80 CUP-HSK3</italic> (A31951), <italic>CUP-NDC80 CUP-CLB3</italic> (A31949), <italic>CUP-NDC80 CUP-HSK3 CUP-CLB3</italic> (A31953) and <italic>cdc20-mn</italic> (A31955) cells were induced to sporulate and CuSO<sub>4</sub> (50 μM) was added at 4 hr after sporulation induction. (<bold>A</bold>) Representative images and (<bold>B</bold>) quantification of Ask1-13myc (green) in mononucleates relative to Ndc10-6HA (red) determined by nuclear spreads prepared after 1 hr of CuSO<sub>4</sub> induction (n > 40 except for A31955 [n = 28]). For (<bold>B</bold>), using a chi-square test (df 2) the fraction of spread nuclei that display colocalized, partial or mislocalized Ask1 with respect to Ndc10 was compared between <italic>CUP-CLB3</italic> and <italic>CUP-CLB3 CUP-NDC80 CUP-HSK3</italic> χ<sup>2</sup> = 51.49, p<0.0001. (<bold>C</bold>) Western blots of Ask1-13myc and Pgk1. (<bold>D</bold>) Bipolar spindle morphology and (<bold>E</bold>) left panel, total (robust + fragile) bipolar spindle formation, and right panel, robust bipolar spindle formation determined at the indicated time points (see ‘Materials and methods’ for further description) (n = 100 per time point). Note: <italic>CUP-NDC80 CUP-HSK3</italic> (dark blue) data points occluded by wild-type (grey) data points. (<bold>F</bold>), (<bold>G</bold>) Segregation of sister chromatids using heterozygous CENV-GFP dots quantified in binucleates from wild-type (A30340), <italic>CUP-NDC80</italic> (A30342), <italic>CUP-HSK3</italic> (A31849), <italic>CUP-NDC80 CUP-HSK3</italic> (A31855), <italic>CUP-CLB3</italic> (A31847), <italic>CUP-CLB3 CUP-NDC80</italic> (A31853), <italic>CUP-CLB3 CUP-HSK3</italic> (A31851) and <italic>CUP-CLB3 CUP-NDC80 CUP-HSK3</italic> (A31857) (early induction = 2:15 hr, late induction= 4:30 hr after induction of sporulation; release from <italic>NDT80</italic> block at 4:30 hr) (n = 100). For (<bold>F</bold>), using a chi-square test (df 1) the fraction of binucleates with a reductional or equational division was compared between <italic>CUP-CLB3</italic> and <italic>CUP-CLB3 CUP-NDC80 CUP-HSK3</italic> χ<sup>2</sup> = 22.28, p<0.0001. For (<bold>G</bold>), using a chi-square test (df 1) the fraction of binucleates with a reductional or equational division was compared between <italic>CUP-CLB3</italic> and <italic>CUP-CLB3 CUP-NDC80 CUP-HSK3</italic> χ<sup>2</sup> = 102.7, p<0.0001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.052">http://dx.doi.org/10.7554/eLife.00117.052</ext-link></p></caption><graphic xlink:href="elife00117f008"/></fig><fig id="fig9" position="float"><object-id pub-id-type="doi">10.7554/eLife.00117.053</object-id><label>Figure 9.</label><caption><title><italic>CUP-CLB3 CUP-NDC80 CUP-HSK3</italic>-induced meiosis I sister chromatid segregation is independent of the length of the prophase I arrest.</title><p>(<bold>A</bold>) Schematic description of the experimental regime used in (<bold>B</bold>) and (<bold>C</bold>). (<bold>B</bold>), (<bold>C</bold>) Wild-type (A22678), <italic>CUP-CLB3</italic> (A22702) and <italic>CUP-CLB3 CUP-NDC80 CUP-HSK3</italic> (A31857) cells also carrying the <italic>GAL4-ER</italic> and <italic>GAL-NDT80</italic> fusions were induced to sporulate. Cells were released from the <italic>NDT80</italic> block and concurrently <italic>pCUP1</italic>-dependent expression was induced at either 6 hr, 7 hr or 8 hr post transfer to sporulation medium (by addition of 1 μM estradiol and 50 μM CuSO<sub>4</sub> respectively). Samples were taken at the indicated time points to determine DNA content (<bold>B</bold>) and the percentage of binucleate cells with segregated sister chromatids (<bold>C</bold>). For (<bold>C</bold>), using a chi-square test (df 1), the fraction of binucleates that display a reductional or equational division in <italic>CUP-CLB3 CUP-NDC80 CUP-HSK3</italic> cells was compared between 6 hr and 7 hr induction χ<sup>2</sup> = 0.3212, p=0.5709 and between 6 hr and 8 hr induction χ<sup>2</sup> = 0.1831, p=0.6687.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.053">http://dx.doi.org/10.7554/eLife.00117.053</ext-link></p></caption><graphic xlink:href="elife00117f009"/></fig></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>The specialized chromosome segregation pattern in meiosis likely evolved through modifications of the mitotic cell division program. We find that preventing microtubule–kinetochore interactions during premeiotic S phase and prophase I is essential for transforming mitosis into meiosis I. Meiosis I chromosome morphogenesis, including the assembly of cohesin protective structures around centromeres and sister kinetochore coorientation, occurs during prophase I. We propose that when microtubules interact with kinetochores prior to completion of this remodeling process, they establish a default attachment, biorientation, which is incompatible with establishing sister kinetochore coorientation and a cohesin protective domain around centromeres (<xref ref-type="fig" rid="fig10">Figure 10</xref>). Our findings reveal a novel regulatory event that is essential for accurate meiosis I chromosome segregation and demonstrate that temporal restriction of microtubule–kinetochore interactions is instrumental in transforming mitosis into meiosis.</p><fig id="fig10" position="float"><object-id pub-id-type="doi">10.7554/eLife.00117.054</object-id><label>Figure 10.</label><caption><title>Model for temporal regulation of microtubule–kinetochore interactions during meiosis.</title><p>(<bold>A</bold>) As prophase I progresses, the propensity of sister chromatids to biorient decreases and the ability to coorient sister chromatids increases. (<bold>B</bold>) Top panel: inhibiting Clb-CDK activity and outer kinetochore (KT) assembly during prophase I establishes a meiosis I-specific chromosome segregation pattern by allowing sister kinetochore coorientation and protection of centromeric cohesin. Bottom panel: disrupting the regulation of microtubule–kinetochore (MT–KT) interactions causes sister chromatid segregation in meiosis I.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.054">http://dx.doi.org/10.7554/eLife.00117.054</ext-link></p></caption><graphic xlink:href="elife00117f010"/></fig><sec id="s3-1"><title>The effects of premature microtubule–kinetochore engagement on meiosis I chromosome morphogenesis</title><p>Transcriptional and translational controls restrict <italic>CLB3</italic> expression to meiosis II (<xref ref-type="bibr" rid="bib9">Carlile and Amon, 2008</xref>). Eliminating both, by placing the gene under the control of the <italic>GAL1-10</italic> promoter or the <italic>CUP1</italic> promoter has dramatic effects on meiosis I chromosome segregation. <italic>CLB3</italic> expression from the <italic>GAL1-10</italic> promoter, which leads to Clb3 levels comparable to those seen for wild-type cells in meiosis II, causes a significant suppression of the meiosis I chromosome segregation pattern. This defect is not further enhanced by overexpression of the protein (by expression from the <italic>CUP1</italic> promoter), which further indicates that this phenotype does not emanate from expressing exceedingly high levels of the cyclin, but is a consequence of premature expression.</p><p>The consequences of premature <italic>CLB3</italic> expression are dramatic. It leads to premature microtubule–kinetochore interactions and prevents coorientation factors from associating with kinetochores. The observation that the transient disruption of microtubule–kinetochore interactions, either by inactivating the outer kinetochore or by microtubule depolymerization, allowed coorientation factors to associate with kinetochores, despite <italic>CLB3</italic> misexpression, led us to conclude that it is premature microtubule–kinetochore interactions that interfere with the establishment of sister kinetochore coorientation during meiosis I. It is currently unclear how preexisting microtubule–kinetochore interactions prevent monopolin association with kinetochores. Precocious attachment of microtubules to kinetochores could occlude the monopolin complex from binding to kinetochores. Alternatively, tension between sister kinetochores generated from stable microtubule–kinetochore interactions could induce a conformational change at the kinetochore and/or pericentric chromatin such that coorientation factors can no longer associate with the kinetochore.</p><p>In addition to preventing sister kinetochore coorientation, premature expression of <italic>CLB3</italic> interferes with protecting centromeric cohesin from removal during meiosis I. The same logic as outlined for coorientation factors applies to the conclusion that it is Clb3-CDK mediated premature microtubule–kinetochore interactions that lead to loss of centromeric cohesin protection in <italic>CUP-CLB3</italic> cells; disrupting microtubule–kinetochore interactions by various means restores stepwise loss of cohesin in <italic>CUP-CLB3</italic> cells. A simple interpretation of this result is that the centromeric-cohesin protective domain can be disrupted by tension between sister kinetochores at any meiotic stage prior to anaphase I. This does not appear to be the case. In cells lacking the coorientation factor <italic>MAM1</italic>, sister kinetochores come under tension in metaphase I, yet in these cells centromeric cohesin is not removed prematurely (<xref ref-type="bibr" rid="bib50">Toth et al., 2000</xref> and <xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3</xref>). Thus, the timing of microtubule–kinetochore interactions is of importance. It is tempting to speculate that the establishment of the centromeric-cohesin protective domain, which occurs during prophase I or perhaps even earlier, is sensitive to premature microtubule–kinetochore interactions and/or tension that promote biorientation of sister kinetochores. However, once this domain is established, its maintenance during meiosis I can no longer be disrupted by tension between sister kinetochores.</p><p>How premature microtubule–kinetochore interactions affect the centromeric cohesin protection machinery is not yet known. A defect in localization of the protective machinery to kinetochores does not appear to be the cause of this defect. Sgo1 and PP2A localize normally to kinetochores in <italic>CUP-CLB3</italic> cells. Therefore, lack of cohesin protection upon premature microtubule–kinetochore engagement must either result from a defect in an unknown cohesin protection pathway or from a decrease in the activity of Sgo1 and/or PP2A. Premature association of kinetochores with microtubules could result in the untimely recruitment of a factor (e.g. Clb-CDKs themselves) to the pericentromere that inhibits the cohesin protective machinery. Alternatively, microtubule–kinetochore engagement could directly affect the activity of the protective machinery. Two mechanisms have been previously proposed whereby tension modulates the activity of the cohesin protective machinery. In mammalian cells, tension spatially separates centromeric cohesin from Sgo1-PP2A, perhaps leading to loss of protection (<xref ref-type="bibr" rid="bib34">Lee et al., 2008</xref>). Tension has also been proposed to cause a deformation in PP2A, thus inhibiting its catalytic activity (<xref ref-type="bibr" rid="bib17">Grinthal et al., 2010</xref>). Irrespective of whether it is tension-dependent perturbation of Sgo1-PP2A and/or recruitment of inhibitory factors, it is clear that premature microtubule–kinetochore engagement is a bona fide modulator of the cohesin protective machinery.</p></sec><sec id="s3-2"><title>Regulated kinetochore assembly contributes to preventing microtubule–kinetochore interactions</title><p>Cyclin-CDKs regulate multiple aspects of microtubule–kinetochore dynamics. Cyclin-CDKs promote centrosome separation and bipolar spindle assembly (<xref ref-type="bibr" rid="bib15">Fitch et al., 1992</xref>), kinetochore maturation (<xref ref-type="bibr" rid="bib20">Holt et al., 2009</xref>) and chromosomal passenger complex localization (<xref ref-type="bibr" rid="bib52">Tsukahara et al., 2010</xref>). Given the importance of preventing premature microtubule–kinetochore engagement to meiosis I chromosome morphogenesis, it is not surprising that cyclin-CDK activity is regulated at multiple levels in budding yeast; transcription of <italic>CLB1</italic>, <italic>CLB3</italic> and <italic>CLB4</italic> is not activated until cells exit pachytene (<xref ref-type="bibr" rid="bib11">Chu and Herskowitz, 1998</xref>) and <italic>CLB3</italic> translation is restricted to meiosis II (<xref ref-type="bibr" rid="bib9">Carlile and Amon, 2008</xref>).</p><p>Cyclin-CDK activity is also tightly regulated in other eukaryotes. Metazoan oocytes arrest for an extended period of time in prophase I. Multiple mechanisms keep cyclin-CDK activity low to maintain this arrest (reviewed in <xref ref-type="bibr" rid="bib56">Von Stetina and Orr-Weaver, 2011</xref>). Similar regulation is observed in <italic>D. melanogaster</italic> and <italic>C. elegans</italic>. Remarkably, inappropriate activation of <italic>Cyclin A</italic> or cyclin E during prophase I in fruit flies and worms, respectively, results in a mitosis-like division (<xref ref-type="bibr" rid="bib47">Sugimura and Lilly, 2006</xref>; <xref ref-type="bibr" rid="bib3">Biedermann et al., 2009</xref>). Thus, restricting cyclin-CDK activity during premeiotic S phase and prophase I also appears to be required to establish a meiosis I-specific chromosome architecture in higher eukaryotes.</p><p>Restriction of cyclin-CDK activity during premeiotic S phase and prophase I appears to be the major mechanism preventing premature microtubule–kinetochore interactions, but our data indicate that regulation of outer kinetochore assembly serves as an additional mechanism to prevent this from occurring. <italic>CUP-CLB3</italic> can only induce meiosis I sister chromatid segregation when expressed during premeiotic S phase/early prophase I, but fails to do so when expressed during late prophase I. This difference is likely due to the outer kinetochore being present only until early prophase I. When Ndc80, Hsk3 and Clb3 are coexpressed during late prophase I, sister chromatid segregation occurs in meiosis I. This result demonstrates that the presence of Clb3-CDKs alone during late prophase I is not sufficient to cause meiosis I sister chromatid segregation but that outer kinetochore components must also be expressed. Whether outer kinetochore disassembly solely occurs to prevent microtubule kinetochore interactions remains to be determined. Outer kinetochore disassembly could also serve additional purposes during prophase I such as enabling telomere-mediated chromosome movements. Further study of the kinetochore assembly/disassembly cycle during meiosis will provide insights into the full impact of kinetochore regulation on meiotic chromosome segregation.</p><p>In budding yeast, two essential components of the outer kinetochore, Ndc80 and Hsk3, are downregulated during prophase I. In <italic>S. pombe</italic>, Ndc80 and its binding partner Nuf2 dissociate from kinetochores in prophase I (<xref ref-type="bibr" rid="bib1">Asakawa et al., 2005</xref>) raising the interesting possibility that deconstruction of the outer kinetochore is a conserved feature of meiotic prophase I. This dissociation depends on the mating pheromone signaling pathway (<xref ref-type="bibr" rid="bib1">Asakawa et al., 2005</xref>). Intriguingly, ectopic induction of meiosis without mating pheromone signaling (i.e. in <italic>pat1</italic> mutants), results in segregation of sister chromatids instead of homologous chromosomes in meiosis I (<xref ref-type="bibr" rid="bib59">Yamamoto and Hiraoka, 2003</xref>; <xref ref-type="bibr" rid="bib60">Yamamoto et al., 2004</xref>). Perhaps this change in the pattern of chromosome segregation in <italic>pat1</italic> mutants arises from premature microtubule–kinetochore interactions due to a defect in outer kinetochore disassembly. Interestingly, in mouse oocytes, the Ndc80 complex is recruited to chromosomes only after nuclear envelope breakdown (<xref ref-type="bibr" rid="bib48">Sun et al., 2011</xref>), raising the possibility that outer kinetochore assembly is also prevented in meiotic prophase I in vertebrates.</p></sec><sec id="s3-3"><title>Concluding remarks</title><p>Proper segregation of the genome during gametogenesis is critical for the proliferation of sexually reproducing species. Errors in chromosome segregation during meiosis result in aneuploidy, the leading cause of birth defects and miscarriages in humans (<xref ref-type="bibr" rid="bib18">Hassold and Hunt, 2001</xref>). Thus, it is crucial to understand how accurate meiotic chromosome segregation is achieved. We discovered that the establishment of a meiosis-specific chromosome segregation pattern depends on the regulation of microtubule–kinetochore interactions. This is achieved by regulating cyclin-CDK activity as well as assembly of the outer kinetochore. There is evidence for similar regulatory events across different organisms (<xref ref-type="bibr" rid="bib1">Asakawa et al., 2005</xref>; <xref ref-type="bibr" rid="bib47">Sugimura and Lilly, 2006</xref>; <xref ref-type="bibr" rid="bib3">Biedermann et al., 2009</xref>; <xref ref-type="bibr" rid="bib56">Von Stetina and Orr-Weaver, 2011</xref>), suggesting that temporal restriction of microtubule-kinetochore interactions is an evolutionarily conserved event required to execute proper meiotic chromosome segregation.</p></sec></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Strains and plasmids</title><p>Strains used in this study are described in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref> and are derivatives of SK1 (all meiosis experiments) or W303 (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). <italic>GAL-NDT80</italic> and <italic>GAL4-ER</italic> constructs are described in <xref ref-type="bibr" rid="bib2">Benjamin et al. (2003)</xref>. <italic>CUP-CLB1, CUP-CLB3, CUP-CLB4, CUP-CLB5, SPC42-mCherry, SGO1-3V5, RTS1-13myc, RTS1-3V5, HSK3-3V5, NDC80-3V5, ASK1-13myc, CUP-NDC80-3V5, CUP-HSK3, mam1∆, SPO13-3V5, mad3∆, DAM1-3V5, CUP-HSK3-3HA</italic> were constructed by PCR-based methods described in <xref ref-type="bibr" rid="bib35">Longtine et al. (1998)</xref>. Primer sequences for strain constructions are available upon request. <italic>ndc80-1</italic> and <italic>dam1-1</italic> are described in <xref ref-type="bibr" rid="bib57">Wigge et al. (1998)</xref>; <xref ref-type="bibr" rid="bib22">Jones et al. (1999)</xref> and SK1 strains carrying these alleles were constructed via backcrossing (>9X). CENV-LacO was constructed by cloning a CENV homology region with XhoI restriction sites into the SalI cut plasmid pCM40 (gift from Doug Koshland) and integrated near <italic>CDEIII</italic> (<1 kb) by BamHI digest. pHG40 carrying <italic>CUP1</italic> promoter was a gift from Hong-Guo Yu. 3V5 tagging plasmids were provided by Vincent Guacci.</p></sec><sec id="s4-2"><title>Sporulation conditions</title><p>Strains were grown to saturation in YPD at room temperature, diluted in BYTA (1% yeast extract, 2% tryptone, 1% potassium acetate, 50 mM potassium phthalate) to OD<sub>600</sub> = 0.25, and grown overnight at 30°C (room temperature for <italic>ndc80-1</italic> and <italic>dam1-1</italic> experiments). Cells were resuspended in sporulation medium (0.3% potassium acetate [pH 7], 0.02% raffinose) to OD<sub>600</sub> = 1.85 and sporulated at 30°C unless otherwise indicated. <italic>GAL-NDT80 GAL4-ER</italic> strains were released from the <italic>NDT80</italic> block by the addition of 1 μM β-estradiol (5 mM stock in ethanol; Sigma E2758-1G, St. Louis, MO) at 4 hr 30 min unless otherwise indicated. Note: strains released from <italic>NDT80</italic> block at 4 hr 30 min are prototrophic and have accelerated meiotic kinetics relative to strains containing auxotrophies. Strains with <italic>CUP1</italic> promoter driven alleles were induced by addition of CuSO<sub>4</sub> (50 μM final concentration; 100 mM stock made from anhydrous powder [FW = 159.6 g/mol]; Mallinckrodt, Hazelwood, MO) at indicated times.</p></sec><sec id="s4-3"><title>Transient inactivation of the <italic>ndc80-1</italic> or <italic>dam1-1</italic> alleles</title><p>Wild-type, <italic>ndc80-1</italic> or <italic>dam1-1</italic> cells carrying <italic>GAL-NDT80 GAL4-ER</italic> were induced to sporulate at room temperature (permissive temperature). After 2 hr 45 min, cyclin expression was induced by addition of 50 μM CuSO<sub>4</sub> and cells were concurrently shifted to the semi-permissive (34°C) or non-permissive (>35.5°C) temperature and allowed to arrest in pachytene. Cells were then transferred to the permissive temperature and released from the <italic>NDT80</italic> block by addition of 1 μM β-estradiol into either a metaphase I arrest (by depleting Cdc20) or allowed to proceed through the meiotic divisions.</p></sec><sec id="s4-4"><title>Benomyl treatment of meiotic cultures</title><p>Wild-type or <italic>CUP-CLB3</italic> cells carrying the <italic>GAL-NDT80 GAL4-ER</italic> constructs were induced to sporulate at 30°C. 2 hr 15 min after transfer into sporulation medium, cells were filtered and transferred to medium containing CuSO<sub>4</sub> (50 μM) and either 0.4% DMSO or benomyl (120 μg/ml). After an additional 2 hr 15 min incubation, benomyl was washed out by filtering and washing cells with 10 volumes of sterile dH<sub>2</sub>0 containing 0.4% DMSO. Cells were subsequently resuspended in sporulation medium containing 1 μM β-estradiol to release from <italic>NDT80</italic> block. The efficacy of benomyl treatment was confirmed by spindle morphology. See <xref ref-type="bibr" rid="bib19">Hochwagen et al. (2005)</xref> for further technical details regarding benomyl resuspension in sporulation medium.</p></sec><sec id="s4-5"><title>Mitotic induction of monopolin</title><p><italic>MAT</italic><bold>a</bold> haploid cells carrying the <italic>MET-CDC20</italic> or <italic>MET-CDC20 GAL-CDC5 GAL-MAM1</italic> fusions and CENIV-GFP dots cultured in complete synthetic medium without methionine (CSM-MET) containing 2% raffinose were arrested in G1 with 5 μg/ml α-factor. For <xref ref-type="fig" rid="fig5">Figure 5A</xref> condition (1), cells were treated with galactose (to induce Cdc5 and Mam1 production) for 1 hr prior to α-factor release. When arrest was complete, cells were released into rich medium (YEP) with 2% raffinose lacking pheromone and containing 2% galactose, 1% DMSO and 8 mM methionine (to repress Cdc20 production). 8 mM methionine was added every hour to maintain metaphase arrest. When metaphase arrest was complete, cells were released into CSM-MET medium, containing 2% dextrose, 1% DMSO and 5 μg/ml α-factor. For condition (2), G1 arrested cells were released into YEP medium with 2% raffinose, lacking pheromone, containing 8 mM methionine and 1% DMSO. 8 mM methionine was added every hour to maintain the metaphase arrest. After 2 hr, cells were treated with 2% galactose for 1 hr and were subsequently released into CSM-MET medium, containing 2% dextrose, 1% DMSO and 5 μg/ml α-factor. For condition (3), G1 arrested cells were released into YEP medium with 2% raffinose, lacking pheromone, containing 8 mM methionine and 15 μg/ml nocodazole in DMSO. 8 mM methionine was added every hour to maintain the metaphase arrest. After 2 hr, cells were treated with 2% galactose for 1 hr and were subsequently released into CSM-MET medium, containing 2% dextrose, 1% DMSO and 5 μg/ml α-factor. Samples were taken every 15 min after release from metaphase arrest to determine GFP dot segregation in anaphase.</p></sec><sec id="s4-6"><title>Indirect immunofluorescence</title><p>Indirect immunofluorescence was performed as described in <xref ref-type="bibr" rid="bib28">Kilmartin and Adams (1984)</xref>. Spindle morphologies were classified as follows: metaphase I or metaphase I-like spindles were defined as a short, bipolar spindle spanning a single DAPI mass. Anaphase I spindles were defined as an elongated spindle spanning two distinct DAPI masses. Metaphase II spindles were defined as two short, bipolar spindles, each spanning a DAPI mass. Anaphase II spindles were defined as two elongated spindles, each spanning two distinct DAPI masses (four DAPI masses total). For <xref ref-type="fig" rid="fig8">Figure 8D,E</xref>, robust bipolar spindle was classified as a short, thick, bipolar spindle with equal intensity tubulin staining across the entire length of the spindle. A fragile spindle was classified as a short bipolar spindle with lower intensity tubulin staining in the middle of the spindle axis.</p></sec><sec id="s4-7"><title>Live cell imaging</title><p>Cells were induced to sporulate and CuSO<sub>4</sub> was added at the indicated times. After 30–60 min post CuSO<sub>4</sub> induction, cells were layered on a Concanavalin A (2 mg/ml; stock solution 20 mg/ml diluted in 50 mM CaCl<sub>2</sub>, 50 mM MnSO<sub>4</sub>) coated cover slip and assembled into an FCS2 fluidic chamber (Bioptechs Inc. Butler, PA). Sporulation medium was heated to 30°C, aerated using an aquarium air pump (Petco Animal Supplies, Inc. Cambridge, MA) and was perfused into the fluidic chamber using a peristaltic pump (Gilson Inc., Middleton, WI) with a flow rate of 4–7 ml/h. Alternatively, cells were induced to sporulate as above and transferred to a microfluidic chamber (CellASIC Corp. Hayward, CA). Cells were imaged using a Zeiss Axio Observer-Z1 with a 100× objective (NA = 1.45), equipped with a Hamamatsu ORCA-ER digital camera. 11 Z-stacks (1 micron apart) were acquired and maximally projected. Metamorph software was used for image acquisition and processing. Images for <xref ref-type="fig" rid="fig2">Figure 2B</xref> was processed using Metamorph deconvolution software. For <xref ref-type="fig" rid="fig2">Figure 2C</xref>, a cell was scored as harboring a separated pair of sister kinetochores if the heterozygous CENV-GFP dot signal underwent transient splitting for at least two time points for the duration of the movie.</p></sec><sec id="s4-8"><title>GFP-dot and Spc42-mCherry cell fixation conditions</title><p>An aliquot of cells was fixed with 3.7% formaldehyde in 100 mM phosphate buffer (pH 6.4) for 10–15 min. Cells were washed once with 100 mM phosphate, 1.2 M sorbitol buffer (pH 7.5) and permeabilized with 1% Triton X-100 stained with 0.05 μg/ml 4′,6-diamidino-2-phenylindole (DAPI). Cells were imaged using a Zeiss Axioplan 2 microscope or a Zeiss Axio Observer-Z1 with a 100× objective (NA = 1.45), equipped with a Hamamatsu ORCA-ER digital camera. Openlab or Metamorph software was used for image acquisition and processing.</p></sec><sec id="s4-9"><title>Chromosome spreads</title><p>4 OD<sub>600</sub> units of cells were harvested and spheroplasted with 0.1 mg/ml zymolyase 100T (Seikagaku Corp, Japan) and 15 mM DTT in solution 1 (2% potassium acetate, 0.8% sorbitol) for 10–13 min at 37°C. Ice-cold solution 2 (100 mM MES [pH 6.4], 1 mM EDTA, 0.5 mM MgCl<sub>2</sub>, 1 M sorbitol) was added to stop spheroplasting and cells were centrifuged at 2500 rpm for 2–3 min. The supernatant was discarded and the pellet was gently resuspended in 100–200 μl of solution 2. 15 μl of the resuspension was spread onto a glass slide. Subsequently, 30 μl of fixative solution (4% paraformaldehyde, 3.4% sucrose), 60 μl of 1% lipsol and 60 μl of fixative solution were added on top of cell suspension and spread using a glass rod seven to ten times back and forth. The slides were dried for at least 2 hr at room temperature, rehydrated in PBS pH 7.4, blocked with 0.2% gelatin, 0.5% BSA in PBS, and stained as described in the ‘Antibody’ section. For quantifications of spread nuclei, images were acquired using a Zeiss Axioplan 2 microscope or a Zeiss Axio Observer-Z1 with a 100× objective (NA = 1.45), equipped with a Hamamatsu ORCA-ER digital camera. Openlab or Metamorph software was used for image acquisition and processing. 40–100 spread nuclei were counted for each sample, except for strain A31955 in <xref ref-type="fig" rid="fig8">Figure 8B</xref> (n = 28). Two proteins were identified as colocalized in spread nuclei when more than 90% of foci overlapped. They were defined as partially colocalized when the overlap between foci was approximately 50% and as mislocalized when the overlap was negligible.</p></sec><sec id="s4-10"><title>In vitro kinase assay</title><p>In vitro kinase assays were performed as described in <xref ref-type="bibr" rid="bib9">Carlile and Amon (2008)</xref> with the following modifications: 1 mg of total protein was incubated with 40 μl of 50% slurry anti-V5 agarose affinity gel (Sigma, St. Louis, MO) for 2 hr at 4°C. One half of the immunoprecipitate was used for the in vitro kinase assay, while the other half was used for Western blotting to detect Cdc28-3V5.</p></sec><sec id="s4-11"><title>Western blot analysis</title><p>For immunoblot analysis, ∼10 OD<sub>600</sub> units of cells were harvested and treated with 5% trichloroacetic acid for at least 10 min at 4°C. The acid was washed away with acetone and the cell pellet was subsequently dried. The cell pellet was pulverized with glass beads in 100 μL of lysis buffer (50 mM Tris–HCl at pH 7.5, 1 mM EDTA, 2.75 mM DTT, complete protease inhibitor cocktail [Roche, Basel, Switzerland]) using a bead-beater (Biospec Products, Inc. Bartlesville, OK). 3× SDS sample buffer was added and the cell homogenates were boiled. Standard procedures for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were followed (<xref ref-type="bibr" rid="bib31">Laemmli, 1970</xref>; <xref ref-type="bibr" rid="bib51">Towbin et al., 1979</xref>; <xref ref-type="bibr" rid="bib8">Burnette, 1981</xref>). A nitrocellulose membrane (VWR International LLC, Radnor, PA) was used to transfer proteins from polyacrylamide gels. Antibody dilutions are described in the ‘Antibody’ section.</p></sec><sec id="s4-12"><title>Flow cytometry</title><p>1 ml aliquot of a meiotic culture was spun down and the pellet was re-suspended in 70% ethanol and fixed for at least 60 min. Ethanol was removed and the cell pellet was washed with 50 mM sodium citrate, pH 7 and sonicated for 6 s at 50% output. The sample was subsequently incubated with 0.25 mg/ml Ribonuclease A (Sigma, St. Louis, MO) in 50 mM sodium citrate overnight at 37°C, washed once with 50 mM sodium citrate and re-suspended in 50 mM sodium citrate with either 1 µM Sytox Green (Molecular Probes, Carlsbad, CA) or 16 µg/ml propidium iodide (Sigma). Samples were analyzed using FACSCalibur (Becton Dickenson Co. Franklin Lakes, NJ).</p></sec><sec id="s4-13"><title>Chromatin immunoprecipitation</title><p>400 OD<sub>600</sub> units of cells were fixed for 15 min at room temperature in 1% formaldehyde. The formaldehyde was quenched by addition of 125 mM glycine. Samples were processed as previously described (<xref ref-type="bibr" rid="bib55">Vader et al., 2011</xref>). Before immunoprecipitation, 120th of the sample was removed as the input sample. The antibodies used for immunoprecipitation are described in the ‘Antibody’ section. For ChIP-chip, samples were processed and analyzed as described in <xref ref-type="bibr" rid="bib55">Vader et al. (2011)</xref>. For qPCR analysis, DNA was amplified using SYBR Premix ExTaq Perfect Real Time Kit (Takara Bio Inc. Otsu, Shiga, Japan). PCR reactions were 40 cycles of 95°C, 20 s; 55°C, 30 s; 72°C, 30 s using a Roche LightCycler 480 II (Roche, Basel, Switzerland). The following primers were used (5′–3′):<list list-type="simple"><list-item><p><bold>CENV F:</bold> CTT GTT TAG TGC AAG CCA CTG TT</p></list-item><list-item><p><bold>CENV R:</bold> CCG CAT TTC CTT GAT TTA CTG TC</p></list-item><list-item><p><bold>c281 F:</bold> CAA CGA ACC GTG GGA ACG TTA TAG</p></list-item><list-item><p><bold>c281 R:</bold> GAA ACT TTC CTG GTA CCT TCT GC</p></list-item><list-item><p><bold>c194 F:</bold> GCT GAA AGC ATG CCA CTG TA</p></list-item><list-item><p><bold>c194 R:</bold> GGT GTT CCT GCT TCG TTG TTA G</p></list-item><list-item><p><bold>HMR F:</bold> ACG ATC CCC GTC CAA GTT ATG</p></list-item><list-item><p><bold>HMR R:</bold> CTT CAA AGG AGT CTT AAT TTC CCT G</p></list-item></list></p></sec><sec id="s4-14"><title>Recombination southern</title><p>∼20 OD<sub>600</sub> units of cells were harvested and treated with sodium azide (0.1% final concentration). Cells were pelleted and snap frozen in liquid nitrogen. Genomic DNA was extracted as follows: Cells were washed once in TE and spheroplasted with 1/100 volume of beta-mercaptoethanol and 250 μg/ml zymolyase T100 in spheroplasting buffer (1 M sorbitol, 42 mM K<sub>2</sub>HPO<sub>4</sub>, 8 mM KH<sub>2</sub>PO<sub>4</sub>, 5 mM EDTA) for 30 min at 37°C on a rotating rack. 100 μl preheated (65°C) lysis buffer (1:1 mix of 1 M Tris pH 8 and 0.5 M EDTA, 2.5–3% SDS) was added and mixed by inverting. 15 μl proteinase K (18 ± 4 mg/ml PCR grade solution; Roche, Basel, Switzerland) was added and incubated at 65°C for ∼1.5 hr. Subsequently, 150 μl 5 M potassium acetate was added, mixed by inverting and transferred to 4°C for 10 min. Samples were centrifuged at 4°C for 20 min and 650 μl of supernatant was transferred into a 2 ml tube containing 750 μl 100% ethanol, avoiding as much of the white fluff as possible. Samples were mixed by inverting and left at 4°C for 10 min. Nucleic acid was precipitated at 15,000 rpm for 10 min, 4°C. Samples were subsequently resuspended in TE and treated with RNase A (50 μg/ml; Roche), for 15–20 min at 37°C and kept at 4°C overnight. DNA was extracted with phenol/chloroform/isopropanol and was resuspended in 125 μl TE. XhoI-MluI digested DNA fragments were separated on 0.6% agarose gel in 1× TBE and transferred onto Hybond-XL plus membranes (GE Healthcare Biosciences, Pittsburgh, PA) by alkaline transfer. Southern blotting was performed as previously described (<xref ref-type="bibr" rid="bib21">Hunter and Kleckner, 2001</xref>).</p></sec><sec id="s4-15"><title>Antibodies</title><sec id="s4-15-1"><title>Indirect immunofluorescence</title><p>Spindle morphology was determined using a rat anti-tubulin antibody (Serotec, Kidlington, UK) used at a dilution of 1:100, and anti-rat FITC antibodies (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) used at a dilution of 1:100–200.</p></sec><sec id="s4-15-2"><title>Western blotting</title><p>Lrs4-13myc, Rec8-13myc, Ask1-13myc and Mam1-9myc were detected using a mouse anti-myc antibody (Covance, Princeton, NJ) at a 1:500 dilution. Rec8-3HA and Hsk3-3HA were detected using a mouse anti-HA antibody (HA.11; Covance) at a 1:1000 dilution. Hsk3-3V5, Ndc80-3V5 and Dam1-3V5 were detected using a mouse anti-V5 antibody (Invitrogen, Carlsbad, CA) at a 1:2000 dilution. Pgk1 was detected using a mouse anti-Pgk1 antibody (Molecular Probes, Carlsbad, CA) at a 1:10,000 dilution. Clb3 was detected using a rabbit anti-Clb3 antibody (Sc7167; Santa Cruz Biotechnology Inc. Santa Cruz, CA) at a 1:500 dilution. Kar2 was detected using a rabbit anti-Kar2 antibody (kindly provided by Mark Rose) at a 1:200,000 dilution. The secondary antibodies used were a sheep anti-mouse antibody conjugated to horseradish peroxidase (HRP) (GE Healthcare Biosciences, Pittsburgh, PA) at a 1:5000 dilution or a goat anti-rabbit antibody conjugated to HRP (BioRad, Hercules, CA) at a 1:10,000 dilution. Antibodies were detected using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA).</p></sec><sec id="s4-15-3"><title>Chromatin immunoprecipitation</title><p>Rec8-3HA was immunoprecipitated using 2–5 μg of rat anti-HA antibody (3F10; Roche, Basel, Switzerland) in combination with 50 μl of 50% slurry Protein G beads (Roche). Rec8-13myc was immunoprecipitated using 2–5 μg of mouse anti-myc antibody (9E11) in combination with 50 μl of 50% slurry Protein G beads (Roche). Sgo1-3V5 and Spo13-3V5 were immunoprecipitated with 40–50 μl of 50% slurry anti-V5 agarose affinity gel (Sigma, St. Louis, MO). Pds5 was immunoprecipitated using 1.3μl of rabbit anti-Pds5 antibody (kindly provided by Vincent Guacci) in combination with 50 μl of 50% slurry Protein A beads (Roche). Phosphorylated Rec8 was immunoprecipitated using 2 μg of rabbit anti-phospho-S179 Rec8 or rabbit anti-phospho-S521 Rec8 in combination with 50 μl of 50% slurry Protein A beads (Roche).</p></sec><sec id="s4-15-4"><title>Chromosome spreads</title><p>Lrs4-13myc, Ndc10-13myc, Sgo1-9myc, Rts1-13myc, Rec8-13myc, Ask1-13myc, and Mam1-9myc were detected using a preabsorbed rabbit anti-myc antibody (Gramsch, Schwabhausen, Germany) at a 1:400 dilution. Ndc10-6HA and Rec8-3HA were detected using either a preabsorbed mouse anti-HA antibody (HA.11; Covance, Princeton, NJ) or a rat anti-HA antibody (3F10; Roche, Basel, Switzerland) at a 1:400 dilution. Ndc80-3V5 was detected using a mouse anti-V5 antibody (Invitrogen, Carlsbad, CA) at a 1:400 dilution. Zip1 was detected using y-300 rabbit antibody (Santa Cruz Biotechnology Inc. Santa Cruz, CA) at a 1:400 dilution. Rad51 was detected using y-180 rabbit IgG (Santa Cruz Biotechnology Inc.) at a 1:400 dilution. Secondary antibodies used were preabsorbed anti-rabbit FITC antibody (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA), preabsorbed anti-rat CY3 antibody (Jackson ImmunoResearch Laboratories, Inc.) or preabsorbed anti-mouse CY3 antibody (Jackson ImmunoResearch Laboratories, Inc.) at a 1:400–1:800 dilution.</p></sec></sec><sec id="s4-16"><title>Cluster analysis and ordered plots for mRNA-seq and ribosome footprinting data</title><p>Cluster analysis of the ribosome footprinting data for the kinetochore components listed in <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref> was performed using Cluster 3.0. Genes were clustered by hierarchical average based on Spearman correlation using mean centered arrays. Clustering data (<xref ref-type="fig" rid="fig6">Figure 6B</xref>, <xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2</xref>) were visualized using Java Treeview. Note that ribosome footprints are normalized such that the sum of expression across the time course is equivalent for each gene. For plots in <xref ref-type="fig" rid="fig6">Figure 6C,D</xref> and <xref ref-type="fig" rid="fig6s3 fig6s4 fig6s5 fig6s6 fig6s7 fig6s8 fig6s9 fig6s10">Figure 6—figure supplements 3–10</xref>, mRNA-seq and ribosome footprinting data were plotted for indicated genes based on the dataset from <xref ref-type="bibr" rid="bib6">Brar et al. (2012)</xref>. The meiotic stages plotted on the x-axis are in the following order: vegetative (gb15 exponential and A14201 exponential), meiotic entry (1, A, B and D), DNA replication (E and F), recombination (G and I), prophase I (3 and 4), metaphase I (5 and 6), anaphase I (7 and 8), metaphase II (9 and 10), anaphase II (11, 12 and 13) and spore formation (15 and 18). The detailed explanation of the above letter and number codes can be found in <xref ref-type="bibr" rid="bib6">Brar et al. (2012)</xref>.</p></sec><sec id="s4-17"><title>Statistical analysis</title><p>Chi-square (χ<sup>2</sup>) tests were performed using GraphPad Prism 6.0 software with two-tailed P values and 95% confidence intervals. Corresponding degrees of freedom (df), χ<sup>2</sup> and P values are shown in the figure legends.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We are grateful to Hong-Guo Yu, Vincent Guacci, and Wolfgang Zachariae for reagents, Hannah Blitzblau, Gerben Vader, Jingxun Chen, Kristin Kuhn, Kristin Knouse, Ann Thompson, André and Charles Felts for technical assistance, Steve Bell, Leon Chan, Dean Dawson, Doug Koshland, Andrew Murray, Terry Orr-Weaver and members of the Amon lab for their critical reading of this manuscript.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors have declared that no competing interests exist</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>MPM: Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>EÜ: Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article;</p></fn><fn fn-type="con" id="con3"><p>GAB: Acquisition of data, Analysis and interpretation of data, Drafting or revising the article;</p></fn><fn fn-type="con" id="con4"><p>AA: Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.00117.055</object-id><label>Supplementary file 1.</label><caption><p>Strains used in this study.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00117.055">http://dx.doi.org/10.7554/eLife.00117.055</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife00117s001.xlsx"/></supplementary-material></sec><sec sec-type="datasets"><title>Major datasets</title><p>The following datasets were generated</p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro1"><name><surname>Miller</surname><given-names>MP</given-names></name>, <name><surname>Ünal</surname><given-names>E</given-names></name>, <name><surname>Brar</surname><given-names>GA</given-names></name>, <name><surname>Amon</surname><given-names>A</given-names></name>, <year>2012</year><x>, </x><source>Mapping of cohesion factor association sites in <italic>S. cerevisiae</italic>—Meiosis I chromosome segregation is established through regulation of microtubule–kinetochore interactions</source><x>, </x><object-id pub-id-type="art-access-id">GSE41339</object-id><x>; </x><ext-link ext-link-type="uri" 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States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://www.elifesciences.org/the-journal/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for choosing to send your work entitled “Meiosis I Chromosome Segregation is Established by Inhibiting Microtubule-Kinetochore Interactions in Prophase I” for consideration at <italic>eLife</italic>. Your article has been evaluated by a Senior Editor and 3 reviewers, one of whom is a member of <italic>eLife's</italic> Board of Reviewing Editors.</p><p>The Reviewing Editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing Editor has assembled the following comments based on the reviewers' reports. Our goal is to provide the essential revision requirements as a single set of instructions, so that you have a clear view of the revisions that are necessary for us to publish your work.</p><p><bold>General assessment and substantive concerns to be addressed during revision:</bold></p><p>Amon and colleagues previously showed that aberrant expression of Clb3 in meiotic prophase I induces sister segregation in the first meiotic division. Here, Miller et al. show that this is due to early development of bipolar spindles and present evidence that premature kinetochore-microtubule attachments disrupt normal morphogenesis of kinetochores and pericentric cohesion. That these connections must be at least transiently disrupted to allow development of monopolar kinetochores and in particular a protective cohesin domain formed around the centromeres is an important and new discovery. This is a typical tour de force effort from the Amon lab, with cleverly designed and well-executed experiments and several novel observations. The data are for the most part impeccable. However, the simple message is perhaps lost by emphasis on other points that are either overstated or speculative without careful comment to these issues. Furthermore, certain novelty claims are unwarranted and need rewriting (see point 1). Substantial points that must be addressed in a revision are given below in no particular order with regard to significance.</p><p>1. The paper of Asakawa et al. (MBC 2005) should be discussed earlier and more comprehensively. Presenting this information in the discussion section as it now reads is confusing. This paper demonstrated meiosis-specific disassembly of the outer kinetochore (the <italic>S. pombe</italic> Nif2-Ndc80 complex) and indicated that this is required for reductional segregation at MI. Moreover, Asakawa also showed that this process requires the Pat1 kinase, the Mei2 regulator of meiotic transcript stability, and mating-pheromone signaling. The current study in this regard is an advance in important ways, that this is critical in part for connections, but does not supersede it.</p><p>2. It's surprising that there is no figure at the beginning of the Results showing westerns of CLB overexpression. It is important to see timing and know how the overexpression compares to the levels that normally form in WT. There are such figures for Clb3 later, e.g., in panels of Fig 2B and Fig S2B, but there is no explicit discussion of what's seen with respect to timing or levels, and the other CLBs are not shown. It would be interesting to know if the differences in phenotypes detected for the specific cyclins were because of kinase protein levels or activities? One of the most interesting points developed in the present story was that Clb4 expression was only capable of disrupting SPB but not cohesion – is this specificity or levels?</p><p>3. Most of the data are superb, but the recombination Southern and quantification are not (fig S1E): FigureS1E shows that recombination is not normal in GAL-CLB3 cells - the crossovers form earlier (at least 1 hr and with biphasic kinetics) and at lower levels than seen in wild type. However, the authors claim that recombination is normal when CLB3 is prematurely expressed. This is a contradiction that needs to be addressed. In fact, it is hard to believe that the quantification is accurate since it indicates that products are showing up at high levels already at 3 hr in the GAL-CLB3 strain, even though no products are obvious on the Southern at this time point. Also, it seems a bit odd to include DSBs along with recombinant DNA molecules as “recombination products”.</p><p>4. We are concerned about the physiological relevance of a number of experiments. While the phenomena described here are fascinating, how relevant is the pathology associated with very early, very high expression of Clb3 for understanding wild-type meiosis? This needs some discussion and balance with regard to proof versus speculation. The introduction section does not make a link to how over-expression might be used to learn about the normal process. Further some conclusions remain speculative with the approach and should be stated as such.</p><p>A) For example, with respect to the physiological role of outer-kinetochore disassembly during prophase, the authors' interpretation is that this process is redundant with suppression of cyclins in preventing premature kinetochore-spindle connections. But it seems that prevention of prophase spindle assembly completely overrides any need to disassemble the outer kinetochore; bipolar spindles never form in prophase I (Figure 1).</p><p>B) Moreover, artificial assembly of the outer kinetochore in prophase I (CUP-NDC80 CUP-HSK3) does not influence MI segregation unless CLB3 is also artificially expressed. This is an important paradox that must be addressed. It remains completely unproven whether the physiological role of outer kinetochore disassembly in prophase I is to prevent premature spindle-kinetochore connections. It seems equally possible that SPB-kinetochore connections must be dissociated for normal execution of prophase I events, e.g. telomere-nuclear envelope attachment, chromosome movement, etc.</p><p>5. For most experiments, CUP-CLB3 is induced so early that cells may still be in S phase. FACs analysis for representative time courses should be shown. The induction of Ndt80 expression at 4.5 hrs is also questionably early - has a majority of the culture reached the ndt80 arrest point at 4.5 hrs? The concern is that early, non-physiological expression of Cdc5 is also occurring in these experiments and may actually be required for the observed phenomena. What happens if Ndt80 is expressed later? There isn't a single time-course in this paper to demonstrate when MI occurs in a typical wild-type culture. The time-course in FigureS1E shows that crossovers don't form until 7 hrs making it unlikely that cells were in mid/late pachytene at 4.5 hrs.</p><p>6. Similarly it is an overstatement to write categorically that the work shows two independent mechanisms for timely engagement of the microtubules with the kinetochore. Manipulating the synthesis of the outer kinetochore proteins enhances the Cdk effects, but perhaps this is because the unknown kinase targets are not completely modified? One might suggest that the transcription or translation of the kinetochore proteins is regulated by a repressor whose activity is ablated by Cdk function.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.00117.057</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1. The paper of Asakawa et al. (MBC 2005) should be discussed earlier and more comprehensively. Presenting this information in the discussion section as it now reads is confusing. This paper demonstrated meiosis-specific disassembly of the outer kinetochore (the S. pombe Nif2-Ndc80 complex) and indicated that this is required for reductional segregation at MI. Moreover, Asakawa also showed that this process requires the Pat1 kinase, the Mei2 regulator of meiotic transcript stability, and mating-pheromone signaling. The current study in this regard is an advance in important ways, that this is critical in part for connections, but does not supersede it</italic>.</p><p>We have included a detailed discussion of the Asakawa paper in the Results section.</p><p><italic>2. It's surprising that there is no figure at the beginning of the Results showing westerns of CLB overexpression. It is important to see timing and know how the overexpression compares to the levels that normally form in WT. There are such figures for Clb3 later, e.g., in panels of Fig 2B and Fig S2B, but there is no explicit discussion of what's seen with respect to timing or levels, and the other CLBs are not shown. It would be interesting to know if the differences in phenotypes detected for the specific cyclins were because of kinase protein levels or activities? One of the most interesting points developed in the present story was that Clb4 expression was only capable of disrupting SPB but not cohesion – is this specificity or levels</italic>?</p><p>Several points are of importance here. First, Clb3 produced from the <italic>GAL1-10</italic> promoter (and driven by estrogen induction) is expressed at similar levels as endogenous Clb3. This was demonstrated in Carlile and Amon (2008) and is now mentioned in the text. Thus, when Clb3 is driven from the <italic>GAL1-10</italic> promoter the protein is not overexpressed, just prematurely expressed. Expression from the <italic>CUP1</italic> promoter results in a 5-fold overexpression compared to the <italic>GAL1-10</italic> promoter. This is now shown in Figure 1B. Importantly, the degree of expression has no effect on the phenotype. The percentage of cells segregating sister chromatids during meiosis I is similar in <italic>CUP-CLB3</italic> and <italic>GAL-CLB3</italic> cells (shown in Figure 1C). This result indicates that degree of ectopic expression is irrelevant for the phenotype. The only aspect that matters is <italic>premature</italic> expression. This is now explained in detail in the text.</p><p>The reader may further wonder why we did not use the <italic>GAL-CLB3</italic> construct but instead used the <italic>CUP-CLB3</italic> fusion for most of our experiments, if the former only leads to premature expression but the latter to premature expression and overexpression. This is now also explained. Figure 1A shows that estrogen addition during early stages of sporulation interferes with meiotic progression, precluding the analysis of the meiotic products.</p><p>Finally, as requested by the reviewers we have compared protein levels and kinase activity of the various cyclins expressed from the <italic>CUP1</italic> promoter. Figure 1D shows that the various Clb cyclins are expressed at similar levels when driven from the <italic>CUP1</italic> promoter. Figure 1F shows that there is no correlation between the amount of kinase activity produced by the various cyclins expressed from the <italic>CUP1</italic> promoter and the ability to induce sister chromatid segregation during meiosis I. Thus cyclin specificity, not quantity, is responsible for the differences in phenotype.</p><p><italic>3. Most of the data are superb, but the recombination Southern and quantification are not (fig S1E): FigureS1E shows that recombination is not normal in GAL-CLB3 cells - the crossovers form earlier (at least 1 hr and with biphasic kinetics) and at lower levels than seen in wild type. However, the authors claim that recombination is normal when CLB3 is prematurely expressed. This is a contradiction that needs to be addressed. In fact, it is hard to believe that the quantification is accurate since it indicates that products are showing up at high levels already at 3 hr in the GAL-CLB3 strain, even though no products are obvious on the Southern at this time point. Also, it seems a bit odd to include DSBs along with recombinant DNA molecules as “recombination products”</italic>.</p><p>We re-quantified the recombination products by taking the ratio of R2 over P1. The new graph displayed in Figure 2 – Figure Supplement 5 shows no significant impact of prematurely expressed <italic>CLB3</italic> on recombination.</p><p><italic>4. We are concerned about the physiological relevance of a number of experiments. While the phenomena described here are fascinating, how relevant is the pathology associated with very early, very high expression of Clb3 for understanding wild-type meiosis? This needs some discussion and balance with regard to proof versus speculation. The introduction section does not make a link to how over-expression might be used to learn about the normal process. Further some conclusions remain speculative with the approach and should be stated as such</italic>.</p><p>The reviewers are concerned that our analyses solely rely on experiments that overexpress <italic>CLB</italic>s and that the “pathology” described here has no bearing on the effects of <italic>CLB</italic>s in wild-type meiosis. We fundamentally disagree with this assessment.</p><p>First, as described in Carlile and Amon (2008) and now explicitly mentioned in the revised manuscript, expression of <italic>CLB3</italic> from the <italic>GAL1-10</italic> promoter does not lead to non-physiological levels of Clb3. The levels produced from the <italic>GAL1-10</italic> promoter during meiosis I are the same as observed in wild-type cells during meiosis II. More importantly, Figure 1C shows that amount of premature Clb3 expression does not impact the phenotype. The degree of meiosis I sister chromatid segregation is similar in <italic>GAL-CLB3</italic> and <italic>CUP-CLB3</italic> cells. This is discussed extensively in the revised manuscript.</p><p>Second, one must ask whether there is a method other than expressing <italic>CLB3</italic> from a heterologous promoter to determine why so many mechanisms, namely transcriptional and translational inhibition, are in place to keep Clb3-CDKs low during meiosis I. To our knowledge, expression from a heterologous promoter is the only way to effectively override such control mechanisms and examine the consequences of loss of these controls.</p><p><italic>A) For example, with respect to the physiological role of outer-kinetochore disassembly during prophase, the authors' interpretation is that this process is redundant with suppression of cyclins in preventing premature kinetochore-spindle connections. But it seems that prevention of prophase spindle assembly completely overrides any need to disassemble the outer kinetochore; bipolar spindles never form in prophase I (Figure 1)</italic>.</p><p>Of course, without a spindle chromosomes cannot segregate. As spindle formation is a prerequisite for the observed phenotype, Clb-CDK expression is an absolute must. However three observations indicate that both, a cyclin-CDK induced bipolar spindle and a functional kinetochore must be present during prophase I in order to cause premature microtubule-kinetochore interactions that ultimately result in meiosis I chromosome segregation defects.</p><p>First, <italic>CLB3</italic>-induced meiosis I chromosome segregation defects are completely dependent on a functional kinetochore during prophase I (Figure 5 and Figure 5 – Figure Supplement 5). Second, whereas early <italic>CLB3</italic> misexpression causes defects in meiosis I chromosome segregation, <italic>CLB3</italic> misexpression during late prophase I has no effect on chromosome segregation (Figure 6A). This difference is likely due to the outer kinetochore being present during premeiotic S phase and early prophase I but not late prophase I (Figure 6E). Most importantly, during late prophase I, only both, <italic>CLB3</italic> and outer kinetochore component expression, are able to induce meiosis I sister chromatid segregation (Figure 8G, Figure 9). This result demonstrates that the presence of cyclin alone during late prophase I is not sufficient to cause sister chromatid segregation during meiosis I, and that both pathways must be repressed during prophase I to establish the meiosis I-specific chromosome segregation pattern.</p><p>These arguments have been added to the discussion to ensure that the reader understands the contribution of Clb-CDK induced premature spindle formation and outer kinetochore assembly to the observed phenotype.</p><p><italic>B) Moreover, artificial assembly of the outer kinetochore in prophase I (CUP-NDC80 CUP-HSK3) does not influence MI segregation unless CLB3 is also artificially expressed. This is an important paradox that must be addressed</italic>.</p><p>Ectopic expression of Ndc80 and Hsk3 alone does not affect meiosis I chromosome segregation, but neither does <italic>CUP-CLB3</italic> when expressed only during late prophase I. The only point we are trying to make here is that in addition to formation of the spindle early in prophase I, a functional kinetochore must be present to observe a phenotype. Our data clearly show that outer kinetochores are disassembled during prophase I and that this disassembly precludes <italic>CUP-CLB3</italic> from interfering with meiosis I chromosome segregation. As mentioned above, we extended the discussion of these points to make this conclusion clearer.</p><p><italic>It remains completely unproven whether the physiological role of outer kinetochore disassembly in prophase I is to prevent premature spindle-kinetochore connections. It seems equally possible that SPB-kinetochore connections must be dissociated for normal execution of prophase I events, e.g. telomere-nuclear envelope attachment, chromosome movement, etc</italic>.</p><p>It is entirely possible that disassembly of the outer kinetochore serves additional purposes. This possibility is now mentioned in the Discussion.</p><p><italic>5. For most experiments, CUP-CLB3 is induced so early that cells may still be in S phase. FACs analysis for representative time courses should be shown. The induction of Ndt80 expression at 4.5 hrs is also questionably early - has a majority of the culture reached the ndt80 arrest point at 4.5 hrs? The concern is that early, non-physiological expression of Cdc5 is also occurring in these experiments and may actually be required for the observed phenomena. What happens if Ndt80 is expressed later? There isn't a single time-course in this paper to demonstrate when MI occurs in a typical wild-type culture. The time-course in FigureS1E shows that crossovers don't form until 7 hrs making it unlikely that cells were in mid/late pachytene at 4.5 hrs</italic>.</p><p>New Figure 1E shows progression of wild-type cells through sporulation. By 2:15 hours, 43.2 percent of cells have replicated their DNA, by 4.5 hours all cells are in G2. Using very similar conditions a recent publication by Blitzblau et al. (PLOS Genetics 2012) has reported even faster kinetics with bulk DNA replication being completed by 3 hours. Sporulation kinetics vary significantly between experiments and in earlier experiments we observed divisions as early as 4 hrs in an unperturbed meiosis. These fast kinetics are most likely due to the fact that most of the strains we used for these studies were prototrophs. It should also be noted that the strains used in old Figure S1E (new Figure 2 – Figure Supplement 5) are not prototrophic which results in slower meiotic kinetics. However the reviewers are correct in that not all cells have replicated their DNA yet by 2:15 hours. Thus we modified the text to indicate that the bulk of cells are in S phase or early prophase I.</p><p>The reviewers were also concerned that premature expression of <italic>NDT80</italic> and its target <italic>CDC5</italic>, 4.5 hours after transfer into sporulation medium could contribute to the observed phenotypes. We have addressed this concern by examining chromosome segregation when <italic>NDT80</italic> was expressed substantially later, 6, 7 or 8 hours after induction of sporulation. At these times all cells had arrested in pachytene as judged by FACS analysis. The data in new Figure 9 show that concomitant expression of <italic>NDT80</italic> from the <italic>GAL1-10</italic> promoter and <italic>CLB3</italic>, <italic>NDC80</italic> and <italic>HSK3</italic> from the <italic>CUP1</italic> promoter after 6, 7, or 8 hours in sporulation medium leads to a similar phenotype as expression of the genes after 4.5 hours in sporulation medium. Thus, expression of <italic>NDT80</italic> early during meiosis, 4.5 hours after transfer into sporulation medium, is not responsible for the observed phenotypes. Furthermore, meiosis I sister chromatid segregation is also seen in cells lacking the <italic>NDT80</italic> block release system (Carlile and Amon, 2008, Figure 1C).</p><p><italic>6. Similarly it is an overstatement to write categorically that the work shows two independent mechanisms for timely engagement of the microtubules with the kinetochore. Manipulating the synthesis of the outer kinetochore proteins enhances the Cdk effects, but perhaps this is because the unknown kinase targets are not completely modified? One might suggest that the transcription or translation of the kinetochore proteins is regulated by a repressor whose activity is ablated by Cdk function</italic>.</p><p>The reviewers’ assessment is only accurate when Clb3-CDKs are expressed during premeiotic S phase/early prophase I. As mentioned above, when the outer kinetochore is fully disassembled, by late prophase I, Clb3-CDK misexpression alone is insufficient to cause MI sister chromatid segregation (Figure 6A, 8G, and Figure 9).</p><p>A scenario where outer kinetochore assembly is under Clb-CDK control is possible, but unlikely. Such a mechanism is not expected to be sensitive to when Clb3-CDKs are expressed during prophase I.</p></body></sub-article></article> |