CDK-dependent nuclear localization of B-Cyclin Clb1 promotes FEAR activation during meiosis I in budding yeast

Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1's phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I

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

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

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

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