(A) Behavior of sister centromeres (white and red arrowheads) and the SPB (yellow) at MII in (top left and right) and (bottom left and right) mutants

The graph shows changes in the SPB-cen (D1 and D2) and SPB-SPB (D3) distances. PI, phase I; PII, phase II; PIII, phase III. Bar, 2 μm. (B) Mean distances of centromeres at phase II. More than eight pairs of centromeres were examined for each analysis. (C) Changes in spindle length at MII in the (left) and (right) mutants. Graphs show kinetics of two MII spindles in the same cell. Dotted lines in graphs show boundaries of the spindle phases. (D) Duration of the spindle phases at MII. (E) Duration of MI, MII, and the MI–MII interval. Wt, wild type. Error bars indicate standard deviation.<p><b>Copyright information:</b></p><p>Taken from "Spindle checkpoint activation at meiosis I advances anaphase II onset via meiosis-specific APC/C regulation"</p><p></p><p>The Journal of Cell Biology 2008;182(2):277-288.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483520.</p><p></p

A Taz1- and Microtubule-Dependent Regulatory Relationship between Telomere and Centromere Positions in Bouquet Formation Secures Proper Meiotic Divisions

<div><p>During meiotic prophase, telomeres cluster, forming the bouquet chromosome arrangement, and facilitate homologous chromosome pairing. In fission yeast, bouquet formation requires switching of telomere and centromere positions. Centromeres are located at the spindle pole body (SPB) during mitotic interphase, and upon entering meiosis, telomeres cluster at the SPB, followed by centromere detachment from the SPB. Telomere clustering depends on the formation of the microtubule-organizing center at telomeres by the linker of nucleoskeleton and cytoskeleton complex (LINC), while centromere detachment depends on disassembly of kinetochores, which induces meiotic centromere formation. However, how the switching of telomere and centromere positions occurs during bouquet formation is not fully understood. Here, we show that, when impaired telomere interaction with the LINC or microtubule disruption inhibited telomere clustering, kinetochore disassembly-dependent centromere detachment and accompanying meiotic centromere formation were also inhibited. Efficient centromere detachment required telomere clustering-dependent SPB recruitment of a conserved telomere component, Taz1, and microtubules. Furthermore, when artificial SPB recruitment of Taz1 induced centromere detachment in telomere clustering-defective cells, spindle formation was impaired. Thus, detachment of centromeres from the SPB without telomere clustering causes spindle impairment. These findings establish novel regulatory mechanisms, which prevent concurrent detachment of telomeres and centromeres from the SPB during bouquet formation and secure proper meiotic divisions.</p></div

Live cell analysis of Nuf2 localization in haploid meiotic cells.

<p>(A) Live cell analysis of Nuf2 localization in haploid wild-type and <i>bqt1Δ</i> cells. Cells were induced to enter meiosis by incubation in nitrogen-free medium at 30°C. After incubation for 4 h, they were observed every 10 min at 25°C. Each bar indicates Nuf2 localization in each meiotic cell. Blue and gray bars indicate periods when Nuf2 dots were present and absent, respectively, and red bars indicate the meiotic division stage. (B) Changes in Nuf2 localization in haploid wild-type and <i>bqt1Δ</i> cells during meiosis. Numbers show time from the start of meiosis I, and arrowheads show Nuf2 signals. White bars: 2 μm. (C) Nuf2 disappearance period relative to the start of meiosis I in haploid cells (grey bars). (D) Duration of Nuf2 disappearance in haploid meiosis. Numbers in parentheses and error bars indicate the number of examined cells and standard deviation, respectively.</p

Changes in chromosome positioning during meiosis and LINC-dependent telocentrosome formation.

<p>(A) Changes in chromosome positioning during meiosis. During mitotic interphase, centromeres (red circles) are located at the SPB, while telomeres (blue circles) are located away from it. When cells enter meiosis by cell conjugation, telomeres cluster at the SPB, which radiates microtubules, and centromeres become detached from it (Karyogamy). After nuclear fusion, the diploid nucleus becomes elongated and oscillates between the cell ends, led by the SPB (Horsetail stage). Thereafter, the nucleus remains in the middle of the cell with several microtubules extending in parallel to the long cell axis until meiotic divisions start (Post-horsetail stage). (B) Factors required for telomere-LINC interaction and telocentrosome formation. γ-TuC: γ-tubulin complex; NE: nuclear envelope.</p

Effects of MBC on centromere detachment and kinetochore disassembly.

<p>(A and B) Changes in the population of haploid cells with SPB-associated telomeres (A) or centromeres (B) during meiosis progression. SPB association of a portion of (Partial) or all (Full) Taz1/Cnp1 signals is shown by red or blue, respectively. (C) Centromere positioning in haploid cells. (D) Changes in the population of haploid cells containing Nuf2 signals during meiosis progression. Nuf2 localization is shown as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006304#pgen.1006304.g003" target="_blank">Fig 3D</a>. (E) Nuf2 localization in haploid cells. In (C) and (E), images show cells incubated in nitrogen-free medium for 5 h. White lines show cell outlines, and arrowheads show Cnp1 or Nuf2 signals co-localized with SPB signals. Bars: 5 μm. WT+DMSO: wild-type haploid cells treated with DMSO; WT+MBC: wild-type haploid cells treated with MBC.</p

Sister chromatid segregation in telomere clustering-defective cells.

<p>(A) Chromosome segregation patterns at meiosis I in <i>rec12Δ</i> diploid zygotes. (B and C) The frequencies of equational segregation of sister chromatids at meiosis I in <i>rec12Δ</i> (B) and <i>rec12Δ sgo1Δ</i> (C) diploid zygotes. (D) Chromosome segregation patterns at meiosis I in haploid cells. (E and F) Effects of <i>bqt1Δ</i> mutation (E) or MBC treatment (F) on sister chromatid segregation in haploid meiotic cells. Sister chromatid segregation was analyzed by visualizing the centromere-proximal region of chromosome II [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006304#pgen.1006304.ref040" target="_blank">40</a>]. +: no mutations otherwise depicted; DMSO: treated with DMSO; MBC: treated with MBC. Averages of three independent experiments are shown. Error bars indicate standard deviation. More than 60 cells were examined in each experiment. Lines indicate pairs of data that were statistically compared. *p<0.05; **p<0.005; ns: no significant difference (by the Student’s t-test).</p

Localization of Nuf2 in telomere clustering-defective cells.

<p>(A) Kinetochore disassembly and meiosis progression. Green dots indicate intact kinetochores. The intact kinetochores are absent during almost the entire period of karyogamy and the horsetail stage (Kinetochore disassembly stage). (B) Nuf2 localization. The karyogamy and horsetail stages were judged, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006304#pgen.1006304.g002" target="_blank">Fig 2C</a>. Arrowheads in the horsetail stage show SPB positions. (C) Population of cells containing Nuf2 signals. Karyogamy: karyogamy stage; Horsetail: the horsetail stage; Horsetail with Mrc1: the horsetail stage with nuclear Mrc1 signals. Numbers in parentheses show the number of examined cells. Bars show averages of three independent experiments except for those in “Horsetail with Mrc1”. More than 30 and 60 cells were examined in each experiment for karyogamy and the horsetail stage, respectively. Error bars indicate standard deviation. Lines indicate sets of data that were statistically compared. *p<i><</i>0.05; **p<i><</i>0.005; ***p<i><</i>0.0005; ns: no significant difference (by the Student’s t-test); nd: not detected. (D) Population of cells with different Nuf2 localization. At SPB: one SPB-associated signal; Off/at SPB: SPB-associated and SPB-dissociated signals; Off SPB: SPB-dissociated signals. Images in the box show the representative localization of the SPB and signals. (E) A Nuf2 signal dissociated from the centromere in the <i>taz1Δ</i> mutant (arrowhead). The percentage indicates the observation frequency of centromere-dissociated Nuf2 signals.</p

Effects of a Taz1 fragment on centromere positioning and kinetochore disassembly.

<p>(A) Schematic diagrams of Taz1, Taz1<i>Δ</i>myb, and Taz1<i>Δ</i>myb-Sad1. Black and blue numbers indicate amino acid numbers of Taz1 and Sad1, respectively. TRFH: TRF homology domain; RBM: Rap1-binding motif; DD: dimerization domain; Myb; Myb DNA-binding domain; NLS: nuclear localization sequence; TM: transmembrane domain; SUN: SUN domain [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006304#pgen.1006304.ref057" target="_blank">57</a>] (<a href="http://www.pombase.org/spombe/result/SPBC12D12.01" target="_blank">http://www.pombase.org/spombe/result/SPBC12D12.01</a>). Note that Sad1 lacks its N-terminal 21 amino acids. (B) Intracellular localization of Taz1<i>Δ</i>myb (left) and Taz1<i>Δ</i>myb-Sad1 (right) during karyogamy. Arrowheads show co-localization of SPB and Taz1<i>Δ</i>myb/Taz1<i>Δ</i>myb-Sad1 signals, and arrows show SPB-dissociated Taz1<i>Δ</i>myb-Sad1 signals. (C and D) Population of cells containing SPB-associated centromeres (C) or Nuf2 signals (D) during karyogamy. Averages of three independent experiments are shown. At least 30 cells were examined in each experiment. Error bars: standard deviation. Shaded bars indicate the data adopted from Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006304#pgen.1006304.g002" target="_blank">2D</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006304#pgen.1006304.g003" target="_blank">3C</a>. Lines indicate sets of data that were statistically compared. *p<0.05, **p<i><</i>0.005, ***p<0.0005; ns: no significant difference (p>0.05) (Student’s t-test). In all experiments, the fusion gene of Taz1<i>Δ</i>myb or Taz1<i>Δ</i>myb-Sad1 was integrated at the <i>aur1</i>^<i>+</i> locus on the chromosome and expressed under the <i>taz1</i>^<i>+</i> promoter.</p

Regulation of centromere detachment from the SPB and its role in meiosis.

<p>(A) Regulation of centromere detachment from the SPB. Telomere clustering causes SPB recruitment of Taz1, and SPB-recruited Taz1 promotes centromere detachment from the SPB. SPB- and telocentrosome-nucleated microtubules, which drive telomere clustering, also promote centromere detachment. Ndc80C: Ndc80 complex; Mis12C: Mis12 complex. (B) A role of telomere-dependent regulation of centromere detachment in meiosis. During mitosis centromeres are attached to the SPB, while telomeres are located away from it (Rabl). Centromeres become detached from the SPB after clustering of telomeres at the SPB, forming the bouquet chromosome arrangement (Bouquet). When telomere clustering is impaired, the Taz1- and/or microtubule-dependent regulatory mechanism inhibits centromere detachment, preventing concomitant detachment of centromeres and telomeres from the SPB, which is harmful for spindle formation.</p

Taz1-independent inhibition of centromere detachment from the SPB and kinetochore disassembly by microtubule disruption.

<p>(A) Effects of Taz1Δmyb-Sad1 on centromere (Cnp1) and Nuf2 (Nuf2) localization in <i>mto1Δ</i> (<i>mto1Δ</i>) or MBC-treated wild-type (WT+MBC) cells. Data of <i>mto1Δ</i> or MBC-treated wild-type cells without Taz1Δmyb-Sad1 expression (+None) are adopted from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006304#pgen.1006304.g004" target="_blank">Fig 4</a>. +None: without Taz1Δmyb-Sad1 expression; +Taz1Δmyb-Sad1: with Taz1Δmyb-Sad1 expression. (B) Effects of MBC on centromere (Cnp1) and Nuf2 (Nuf2) localization in <i>bqt1Δ</i> cells with (<i>bqt1Δ+</i>Taz1Δmyb-Sad1) or without (<i>bqt1Δ</i>) Taz1Δmyb-Sad1 expression. +DMSO: treated with DMSO; +MBC: treated with MBC.</p