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Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity.

Tumurbaatar I, Cizmecioglu O, Hoffmann I, Grummt I, Voit R - PLoS ONE (2011)

Bottom Line: Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known.Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis.Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology of the Cell II, German Cancer Research Centre, DKFZ-ZMBH Alliance, Heidelberg, Germany.

ABSTRACT
Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.

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Silencing of hCdc14B leads to enrichment of mitotic cells.HeLa Kyoto cells were reverse transfected twice with a control-siRNA pool (siCtrl) or siRNAs that target hCdc14A (si14A) [24, Reference S1] or hCdc14B (si14B-3). During the second transfection equal numbers of cells were seeded onto coverslips, and 24 h after the second transfection cells were subjected to immunofluorescence microscopy and analysis of hCdc14 expression. A, B. Representative immunofluorescence images of HeLa Kyoto cells transfected with siCtrl, si14A, or si14B-3. Mitotic cells were visualized by immunostaining with histone H3-phospho-Serine-10 (pSer10-H3) antibodies (red) (A), multinucleated cells by staining with anti-alpha-tubulin antibodies (red) (B). DNA was counterstained with Hoechst 33342. C. Frequencies of mitotic and multinucleated cells. The data shown here represent means (±SD) of three independent experiments derived from counting 400 cells each. D. hCdc14A and hCdc14B expression is reduced by the corresponding siRNAs. The graphs represent the mean levels (±SD) of hCdc14A-mRNA (light bar) and hCdc14B-mRNA (dark bar) normalized to β-actin-mRNA as determined by RT-qPCR in three independent experiments.
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pone-0014711-g002: Silencing of hCdc14B leads to enrichment of mitotic cells.HeLa Kyoto cells were reverse transfected twice with a control-siRNA pool (siCtrl) or siRNAs that target hCdc14A (si14A) [24, Reference S1] or hCdc14B (si14B-3). During the second transfection equal numbers of cells were seeded onto coverslips, and 24 h after the second transfection cells were subjected to immunofluorescence microscopy and analysis of hCdc14 expression. A, B. Representative immunofluorescence images of HeLa Kyoto cells transfected with siCtrl, si14A, or si14B-3. Mitotic cells were visualized by immunostaining with histone H3-phospho-Serine-10 (pSer10-H3) antibodies (red) (A), multinucleated cells by staining with anti-alpha-tubulin antibodies (red) (B). DNA was counterstained with Hoechst 33342. C. Frequencies of mitotic and multinucleated cells. The data shown here represent means (±SD) of three independent experiments derived from counting 400 cells each. D. hCdc14A and hCdc14B expression is reduced by the corresponding siRNAs. The graphs represent the mean levels (±SD) of hCdc14A-mRNA (light bar) and hCdc14B-mRNA (dark bar) normalized to β-actin-mRNA as determined by RT-qPCR in three independent experiments.

Mentions: Given that overexpression of hCdc14B impaired progression through mitosis, we next analyzed the mitotic phenotype caused by ablation of hCdc14B. Treatment of HeLa Kyoto cells with hCdc14A- or hCdc14B-specific siRNAs for 72 h reduced the level of hCdc14A- or hCdc14B-mRNAs to 35–60% (Fig. 2D). Immunofluorescence analysis of cycling cells treated with Cdc14A- or Cdc14B si-RNAs revealed that pSer10-H3-positive, mitotic cells were enriched up to 3-fold upon depletion of hCdc14B compared to cells treated with control siRNA or hCdc14A-specific siRNA (Fig. 2A and C). hCdc14A- and hCdc14B-specific siRNAs caused a drop in the overall cell number (data not shown), suggesting that both isoforms are required for proper cell proliferation. Consistent with results of Mailand et al. [24], the number of bi- and multinucleated cells was significantly increased in hCdc14A-deficient cells (Fig. 2B and C). Notably, binucleation was also observed upon knockdown of hCdc14B, albeit at lower frequency compared to hCdc14A-depleted cells (Fig. 2B and C). This suggests that knockdown of hCdc14A primarily perturbed cytokinesis without affecting progression through mitosis, whereas depletion of hCdc14B also impaired events required for progression through mitosis depletion increasing the amount of mitotic cells.


Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity.

Tumurbaatar I, Cizmecioglu O, Hoffmann I, Grummt I, Voit R - PLoS ONE (2011)

Silencing of hCdc14B leads to enrichment of mitotic cells.HeLa Kyoto cells were reverse transfected twice with a control-siRNA pool (siCtrl) or siRNAs that target hCdc14A (si14A) [24, Reference S1] or hCdc14B (si14B-3). During the second transfection equal numbers of cells were seeded onto coverslips, and 24 h after the second transfection cells were subjected to immunofluorescence microscopy and analysis of hCdc14 expression. A, B. Representative immunofluorescence images of HeLa Kyoto cells transfected with siCtrl, si14A, or si14B-3. Mitotic cells were visualized by immunostaining with histone H3-phospho-Serine-10 (pSer10-H3) antibodies (red) (A), multinucleated cells by staining with anti-alpha-tubulin antibodies (red) (B). DNA was counterstained with Hoechst 33342. C. Frequencies of mitotic and multinucleated cells. The data shown here represent means (±SD) of three independent experiments derived from counting 400 cells each. D. hCdc14A and hCdc14B expression is reduced by the corresponding siRNAs. The graphs represent the mean levels (±SD) of hCdc14A-mRNA (light bar) and hCdc14B-mRNA (dark bar) normalized to β-actin-mRNA as determined by RT-qPCR in three independent experiments.
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Related In: Results  -  Collection

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pone-0014711-g002: Silencing of hCdc14B leads to enrichment of mitotic cells.HeLa Kyoto cells were reverse transfected twice with a control-siRNA pool (siCtrl) or siRNAs that target hCdc14A (si14A) [24, Reference S1] or hCdc14B (si14B-3). During the second transfection equal numbers of cells were seeded onto coverslips, and 24 h after the second transfection cells were subjected to immunofluorescence microscopy and analysis of hCdc14 expression. A, B. Representative immunofluorescence images of HeLa Kyoto cells transfected with siCtrl, si14A, or si14B-3. Mitotic cells were visualized by immunostaining with histone H3-phospho-Serine-10 (pSer10-H3) antibodies (red) (A), multinucleated cells by staining with anti-alpha-tubulin antibodies (red) (B). DNA was counterstained with Hoechst 33342. C. Frequencies of mitotic and multinucleated cells. The data shown here represent means (±SD) of three independent experiments derived from counting 400 cells each. D. hCdc14A and hCdc14B expression is reduced by the corresponding siRNAs. The graphs represent the mean levels (±SD) of hCdc14A-mRNA (light bar) and hCdc14B-mRNA (dark bar) normalized to β-actin-mRNA as determined by RT-qPCR in three independent experiments.
Mentions: Given that overexpression of hCdc14B impaired progression through mitosis, we next analyzed the mitotic phenotype caused by ablation of hCdc14B. Treatment of HeLa Kyoto cells with hCdc14A- or hCdc14B-specific siRNAs for 72 h reduced the level of hCdc14A- or hCdc14B-mRNAs to 35–60% (Fig. 2D). Immunofluorescence analysis of cycling cells treated with Cdc14A- or Cdc14B si-RNAs revealed that pSer10-H3-positive, mitotic cells were enriched up to 3-fold upon depletion of hCdc14B compared to cells treated with control siRNA or hCdc14A-specific siRNA (Fig. 2A and C). hCdc14A- and hCdc14B-specific siRNAs caused a drop in the overall cell number (data not shown), suggesting that both isoforms are required for proper cell proliferation. Consistent with results of Mailand et al. [24], the number of bi- and multinucleated cells was significantly increased in hCdc14A-deficient cells (Fig. 2B and C). Notably, binucleation was also observed upon knockdown of hCdc14B, albeit at lower frequency compared to hCdc14A-depleted cells (Fig. 2B and C). This suggests that knockdown of hCdc14A primarily perturbed cytokinesis without affecting progression through mitosis, whereas depletion of hCdc14B also impaired events required for progression through mitosis depletion increasing the amount of mitotic cells.

Bottom Line: Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known.Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis.Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology of the Cell II, German Cancer Research Centre, DKFZ-ZMBH Alliance, Heidelberg, Germany.

ABSTRACT
Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.

Show MeSH
Related in: MedlinePlus