Cdc5 is required for phosphorylation, but not nuclear localisation, of Clb1 during meiosis I.

<p><i>P_CLB2CDC20 CLB1-myc₉</i> and <i>P_CLB2CDC5 P_CLB2CDC20 CLB1-myc₉</i> cells were induced to enter meiosis by transferring them to SPM. A) Samples were taken hourly throughout the time course for <i>in situ</i> immunofluorescence to determine Clb1 localisation (green-nuclear, red-cytoplasmic, blue-no signal). B) Samples were taken hourly from 4 hours for preparing whole cell extracts. Whole cell extracts were analysed by Western blotting using anti-myc (Clb1), anti-Cdc5 and anti-tubulin antibodies. Asterisk represents a cross-reactive band seen in anti-Cdc5 blots.</p

Clb1 modification and nuclear localisation are meiosis–specific.

<p><i>P_MET3CDC20 CLB1-myc₉</i> cells were arrested in metaphase by transferring them to YEPD medium with 0.15% methionine for 2.5 hours. Culture samples were taken for <i>in situ</i> immunofluorescence and for preparing whole cell extracts. Localisation of Clb1-Myc and spindle formation was examined by <i>in situ</i> immunofluorescence. A) Percentage of cells containing metaphase spindles following addition of methionine is indicated. B) Representative image of a cell arrested in metaphase displaying Clb1 localisation is shown. C) Localization of Clb1 during the experiment is graphically presented (green-nuclear, red-cytoplasmic, blue-no signal). D) Whole cell extracts were subjected to SDS-PAGE followed by Western analysis for assaying Clb1 phosphorylation. For comparison, whole cell extract from diploid <i>P_CLB2CDC20 CLB1-myc₉</i> cells after 8 hours in SPM was included in the gel.</p

CDK activity is required for Clb1 phosphorylation and nuclear localization during meiosis I.

<p>Cultures of <i>CDC28 P_CLB2CDC20 CLB1-myc₉</i> cells and <i>cdc28-as P_CLB2CDC20 CLB1-myc₉</i> cells were induced to enter meiosis by transferring them to SPM. 1-NM-PP1 (10 µM) was added to the cultures after 5, 6, 7 and 8 hours in SPM. As a control, an equivalent amount of DMSO was added to the cultures after 5 h in SPM. Samples were taken hourly from 5 hours onwards for whole cell extracts and for <i>in situ</i> immunofluorescence. Whole cell extracts were analysed by Western blotting using anti-myc and anti-tubulin antibodies. Localization of Clb1 was assayed by <i>in situ</i> immunofluorescence (green-nuclear, red-cytoplasmic, blue-no signal). Western blotting and Clb1 localization data for the three cultures DMSO/5 h, 1-NM-PP1/5 h and 1-NM-PP1/7 h are indicated in A, B and C respectively. Data for additional time points are indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079001#pone.0079001.s003" target="_blank">Figure S3</a>.</p

Increased nuclear localization of Clb1 promotes FEAR activation during meiosis.

<p>A) Diploid <i>CLB1, CLB1-NES</i> and <i>CLB1-NLS</i> strains bearing <i>spo12Δ</i> and <i>esp1-1</i> or their wild type alleles were allowed to sporulate. Percentage of cells forming dyads, tetrads and monads were calculated after 48 h. B) Cultures of <i>CLB1-ha PDS1-myc₁₈</i>, <i>CLB1-NES-ha PDS1-myc₁₈</i>, and <i>CLB1-NLS-ha PDS1-myc₁₈</i> cells were induced to enter meiosis by transferring them to SPM. Nucleolar separation in cells containing anaphase I and metaphase II spindles was assayed by immunofluorescence. Representative images of cells are shown on the right. C) Data obtained in B are presented graphically. D) Nucleolar separation and nucleolar release of Cdc14 in the sporulating cultures of <i>P_CLB2CDC20</i> (blue), <i>P_CLB2CDC20 P_CLB2CDC55</i> (red), <i>P_CLB2CDC20 P_CLB2CDC55 CLB1-ha</i> (green), <i>P_CLB2CDC20 P_CLB2CDC55 CLB1-NES-ha</i> (purple) and <i>P_CLB2CDC20 P_CLB2CDC55 CLB1-NLS-ha</i> (orange) strains were assayed by immunofluorescence and the data are graphically presented. E) Sample images of cells with nucleolar Cdc14 or Cdc14 released are shown on the right.</p

Fusion of NES/NLS sequences to Clb1 alters its nuclear localization without affecting its kinase activity.

<p>A) Schematic showing the variants of Clb1 constructed to alter its nuclear localization. Tandem copies of Nuclear Localization sequences (NLS) or Nuclear Export Sequences (NES) followed by a single copy of HA epitope was added the C-terminus of Clb1. B) Diploid cells bearing either wild type Clb1 or NES/NLS –tagged Clb1 and containing <i>CDC20</i> under the <i>CLB2</i> promoter were arrested in metaphase I by transferring them to SPM for 7 h. Clb1 localization was determined by immunofluorescence. Note that Clb1 is diffuse in the NES-tagged strain but nuclear in wild type/NLS-tagged strains. C) Cells bearing either wild type Clb1 or NES/NLS –tagged Clb1 and containing <i>CDC20</i> under the repressible <i>MET3</i> promoter were arrested in metaphase by Cdc20 depletion and Clb1localization was determined by immunofluorescence Notice that Clb1 is nuclear in the NLS-tagged strain but delocalized in wild type and NES-tagged strains (images of cells after 2 hours in methionine are shown). D) <i>CLB1-ha PDS1-myc₁₈ cdc28-as</i>, <i>CLB1-NES-ha PDS1-myc₁₈ cdc28-as</i> and <i>CLB1-NLS-ha PDS1-myc₁₈ cdc28-as</i> cells were induced to enter meiosis by transferring them to SPM. Samples were taken hourly to determine Clb1 localisation. E) Tagged Clb1 was immunoprecipitated from mitotic cultures of <i>P_CLB2CDC20 cdc28-as</i>, <i>CLB1-ha P_CLB2CDC20 cdc28-as</i>, <i>CLB1-NES-ha P_CLB2CDC20 cdc28-as</i>, and <i>CLB1-NLS-ha P_CLB2CDC20 cdc28-as</i> cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079001#pone.0079001.s004" target="_blank">Figure S4</a>). Purified Clb1 was incubated with 35 µM histone and 50 µM ATP (0.25 µCi/µL of γ-P³²-ATP) in kinase buffer for 40 minutes, with samples taken at 0, 10, 20 and 40 minutes in the presence or absence of the inhibitor 1-NM-PP1. Samples were analysed by SDS-PAGE and gels were dried, exposed to phosphorimager screen and the signals were quantified using IMAGE Quant.</p

Clb1 is phosphorylated and localizes to the nucleus during metaphase I.

<p>Cultures of <i>CLB1-myc₉</i>, <i>P_CLB2CDC55 CLB1-myc₉</i>, <i>P_CLB2CDC20 CLB1-myc₉</i> and <i>P_CLB2CDC20 P_CLB2CDC55 CLB1-myc₉</i> strains were induced to enter meiosis by transferring them to SPM. A) Modification of Clb1 was assayed by subjecting whole cell extracts from the above cultures to SDS-PAGE followed by Western analysis using an anti-myc antibody. Tubulin served as a loading control. B) Cells were fixed and examined for Clb1-Myc localisation by <i>in situ</i> immunofluorescence. Proportion of cells with nuclear Clb1 (green), cytoplasmic Clb1 (red) and no Clb1 signals (blue) are indicated. A representative image of a cell belonging to each of the three categories is shown below the graphs. C) Clb1 was immunoprecipitated from a culture of <i>P_CLB2CDC20 CLB1-myc₉ cdc28-as</i> cells following 8 hours into SPM and then were either mock treated or incubated with λ-phosphatase alone or with λ-phosphatase + phosphatase inhibitors at 30°C for 30 minutes. Samples were analysed by SDS-PAGE followed by Western blotting. Asterisk indicates a Clb1 cleavage fragment.</p

Regulation of entry into gametogenesis and quiescence by the Rim15-Endosulfine-PP2A^Cdc55 signalling module.

<p>Phosphorylation of endosulfine by Rim15 converts it into an inhibitor of PP2A^Cdc55 and thus leading to entry into gametogenesis and quiescence. Entry into quiescence is driven by activation of transcription factors Msn2, Msn4 and Gis1. PP2A^Cdc55 might inhibit a positive regulator of entry into gametogenesis and quiescence. Alternatively, PP2A^Cdc55 could inhibit entry into quiescence and gametogenesis by dephosphorylating distinct substrates.</p

Endosulfines are required for transcriptional induction of <i>IME1</i> caused by transfer of cells to sporulation medium.

<p>A) Wild-type and <i>igo1Δ igo2Δ</i> cells were induced to enter meiosis by transferring them to sporulation medium (SPM). Total RNA was prepared from cells after 0, 2, 4, 6 and 8 hours following transfer to SPM and <i>IME1</i> transcript levels were assayed by quantitative RT-PCR. The <i>IME1</i> transcript levels were normalized with respect to <i>ACT1</i> mRNA and expressed relative to normalized <i>IME1</i> transcript levels in mitotically grown cells. B) Wild-type and <i>igo1Δ igo2Δ</i> cells containing P<i>_GAL</i>-<i>ha_3-IME1 GAL4-ER</i> were induced to sporulate. Either β-estradiol or ethanol was added to the cultures at t = 0 h. After 24 hours, sporulation efficiency was measured (n = 200). C) Aliquots of cells in B were collected after 0, 2, 4, 6 and 8 hours following addition of β-estradiol/ethanol and total cell extracts were prepared. Immunoblotting was performed using an anti-HA antibody and anti-tubulin antibody. The lane labelled C on the western image on the right contained extracts from β-estradiol treated wild type cells (t = 4 hours) and served as a positive control. D) Aliquots of cells in B were collected after 0, 2, 4, 6 and 8 hours following addition of β-estradiol and total RNA from the cells was prepared. <i>IME1</i> mRNA levels were quantified by quantitative RT-PCR and normalized with respect to <i>ACT1</i> mRNA. In the graph, <i>IME1</i> mRNA levels are expressed relative to <i>IME1</i> mRNA levels in cells before induction of <i>IME1</i> expression (t = 0 h).</p

Igo1 associates and inhibits the phosphatase activity of PP2A^Cdc55 in a phosphorylation dependent manner.

<p>A) Either MBP or MBP fused to Igo1, Igo-S64A and Igo1-S64D were purified from bacteria and equal amount of protein was incubated with yeast cell expressing Cdc55-TAP. The proteins bound to the beads were run on 10% SDS-PAGE and probed with anti-TAP antibody. The MBP purified proteins were visualized by coomassie staining of SDS-PAGE gels. WCE denotes whole cell extracts. B) GST-Rim15 and GST-Rim15-kd were purified from yeast cells. Equal amounts of MBP fused Igo1, Igo1-S64A and Igo1-S64D were subjected to in vitro phosphorylation using GST-Rim15 and GST-Rim15-kd respectively. MBP fused proteins were then pulled down with amylose beads and mixed with soluble protein extracts from a yeast strain expressing Cdc55-TAP. The proteins bound to the beads were analysed by western blotting using an anti-TAP antibody. Purified MBP-tagged proteins were visualized by coomassie staining of SDS-PAGE gels. Phosphorylation of Igo1 by Rim15 at S-64 was assayed using a phospho-specific antibody. WCE denotes whole cell extracts. C) TAP eluates from <i>CDC55</i>-TAP and untagged strains were analysed by silver staining. D) Phosphatase activity of TAP eluates from <i>CDC55</i>-TAP and untagged strains was measured using a colorimetric assay (Millipore). E) Phospho-mimetic mutant of Igo1 (Igo1S64D) inhibits the phosphatase activity of PP2A^Cdc55. Purified Cdc55 was incubated with equal amount (25 µg) of MBP-Igo1, MBP-Igo1-S64A and MBP-Igo1-S64D respectively. MBP was used as a control. The mixture was then incubated with 500 µM phosphopeptide (Millipore). The release of free phosphate was measured by colorimetric assay (Millipore).</p

Absence of Cdc55 suppresses the gametogenesis- and quiescence- entry defect of endosulfine mutants.

<p>A) Wild-type, <i>igo1Δ igo2Δ</i>, <i>P_CLB2CDC55</i> and <i>igo1Δ igo2Δ P_CLB2CDC55</i>, <i>igo1Δ igo2Δ P_CLB2CDC55 net1-6Cdk</i>, <i>igo1Δ igo2Δ P_CLB2CDC55 NET1</i>, <i>rim15Δ</i>, <i>rim15Δ P_CLB2CDC55</i>, <i>rim15Δ P_CLB2CDC55 net1-6Cdk</i>, <i>rim15Δ P_CLB2CDC55 NET1</i>, <i>rts1Δ</i> and <i>igo1Δ igo2Δ rts1Δ</i> cells were incubated on sporulation plates for 24 hours and number of sporulated (includes monad, dyad, triads/tetrads) and unsporulated cells were counted using a light microscope. B) Wild-type, <i>igo1Δ igo2Δ</i> and <i>igo1Δ igo2Δ P_CLB2CDC55</i> cells were induced to enter meiosis by transferring them to SPM. Pre-meiotic DNA replication in the cultures was assayed by flow cytometry. C) Analysis of expression of Cdc5. Whole-cell extracts of hourly culture in SPM was prepared by TCA method. Protein samples were run on 10% SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-Cdc5 and anti-Cdc28 antibody respectively. D) Wild-type, <i>igo1Δ igo2Δ</i>, <i>cdc55Δ</i> and <i>igo1Δ igo2Δ cdc55Δ</i> cells expressing Hsp26-ha3 were grown to log phase at 30°C, rapamycin (final concentration 200 ng/ml) was added to each culture and samples were collected at indicated times. Total cell extracts were prepared by TCA method. Proteins were run on 12% SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-HA and anti-Cdc28 antibodies.</p