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

Tibbles KL, Sarkar S, Novak B, Arumugam P - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Warwick, Coventry, United Kingdom.

ABSTRACT
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.

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Increased nuclear localization of Clb1 promotes FEAR activation during meiosis.A) Diploid CLB1, CLB1-NES and CLB1-NLS strains bearing spo12Δ and esp1-1 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 CLB1-ha PDS1-myc18, CLB1-NES-ha PDS1-myc18, and CLB1-NLS-ha PDS1-myc18 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 PCLB2CDC20 (blue), PCLB2CDC20 PCLB2CDC55 (red), PCLB2CDC20 PCLB2CDC55 CLB1-ha (green), PCLB2CDC20 PCLB2CDC55 CLB1-NES-ha (purple) and PCLB2CDC20 PCLB2CDC55 CLB1-NLS-ha (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.
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pone-0079001-g007: Increased nuclear localization of Clb1 promotes FEAR activation during meiosis.A) Diploid CLB1, CLB1-NES and CLB1-NLS strains bearing spo12Δ and esp1-1 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 CLB1-ha PDS1-myc18, CLB1-NES-ha PDS1-myc18, and CLB1-NLS-ha PDS1-myc18 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 PCLB2CDC20 (blue), PCLB2CDC20 PCLB2CDC55 (red), PCLB2CDC20 PCLB2CDC55 CLB1-ha (green), PCLB2CDC20 PCLB2CDC55 CLB1-NES-ha (purple) and PCLB2CDC20 PCLB2CDC55 CLB1-NLS-ha (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.

Mentions: Activation of FEAR during meiosis I but not during mitosis is sufficient to cause disassembly of anaphase spindles. We therefore tested whether nuclear localisation of Clb1 amplifies FEAR during meiosis. We first tested whether FEAR mutants show a genetic interaction with Clb1 localisation mutants (Figure 7A). spo12Δ and esp1-2 mutant cells do not activate FEAR efficiently during meiosis I and fail to disassemble anaphase I spindles [22]. The mutant cells undergo two divisions on the same spindle forming dyads. CLB1-NLS-ha but not CLB1-NES-ha partially suppressed the dyad phenotype of both spo12Δ and esp1-2 mutant strains (Figure 7A). This is consistent with the possibility that increased nuclear localization of Clb1 promotes FEAR activation during meiosis I.


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

Tibbles KL, Sarkar S, Novak B, Arumugam P - PLoS ONE (2013)

Increased nuclear localization of Clb1 promotes FEAR activation during meiosis.A) Diploid CLB1, CLB1-NES and CLB1-NLS strains bearing spo12Δ and esp1-1 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 CLB1-ha PDS1-myc18, CLB1-NES-ha PDS1-myc18, and CLB1-NLS-ha PDS1-myc18 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 PCLB2CDC20 (blue), PCLB2CDC20 PCLB2CDC55 (red), PCLB2CDC20 PCLB2CDC55 CLB1-ha (green), PCLB2CDC20 PCLB2CDC55 CLB1-NES-ha (purple) and PCLB2CDC20 PCLB2CDC55 CLB1-NLS-ha (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.
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Related In: Results  -  Collection

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pone-0079001-g007: Increased nuclear localization of Clb1 promotes FEAR activation during meiosis.A) Diploid CLB1, CLB1-NES and CLB1-NLS strains bearing spo12Δ and esp1-1 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 CLB1-ha PDS1-myc18, CLB1-NES-ha PDS1-myc18, and CLB1-NLS-ha PDS1-myc18 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 PCLB2CDC20 (blue), PCLB2CDC20 PCLB2CDC55 (red), PCLB2CDC20 PCLB2CDC55 CLB1-ha (green), PCLB2CDC20 PCLB2CDC55 CLB1-NES-ha (purple) and PCLB2CDC20 PCLB2CDC55 CLB1-NLS-ha (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.
Mentions: Activation of FEAR during meiosis I but not during mitosis is sufficient to cause disassembly of anaphase spindles. We therefore tested whether nuclear localisation of Clb1 amplifies FEAR during meiosis. We first tested whether FEAR mutants show a genetic interaction with Clb1 localisation mutants (Figure 7A). spo12Δ and esp1-2 mutant cells do not activate FEAR efficiently during meiosis I and fail to disassemble anaphase I spindles [22]. The mutant cells undergo two divisions on the same spindle forming dyads. CLB1-NLS-ha but not CLB1-NES-ha partially suppressed the dyad phenotype of both spo12Δ and esp1-2 mutant strains (Figure 7A). This is consistent with the possibility that increased nuclear localization of Clb1 promotes FEAR activation during meiosis I.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Warwick, Coventry, United Kingdom.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus