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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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Downregulation of hCdc14B leads to multipolar mitotic spindles, lagging chromosomes, and binucleation.U2OS cells were transfected with pTER plasmids expressing two different hCdc14B-specific shRNAs (14B-sh-2, 14B-sh-3) or a control shRNA (Ctrl-sh). 72 h after transfection, cells were processed for immunofluorescence using anti-alpha-tubulin and Cy3-coupled anti-mouse antibodies (red), and CREST antiserum (kinetochores) and FITC-coupled anti-human antibodies (green). DNA was stained with Hoechst 33342. Representative examples of multipolar mitotic cells (A), bipolar mitotic cells with lagging anaphase chromosomes (arrow) (B), and binucleated cells (C) are shown. D. Frequencies of multipolar spindles and lagging anaphase chromosomes as visualized by staining with anti-alpha-tubulin and CREST. The numbers are derived from three independent experiments, each count comprising 60 mitotic cells. E. Western blot of hCdc14B after transfection with pTER-ctrl-shRNA (Ctrl-sh), pTER-Cdc14B-2 (14B-sh-2), and pTER-Cdc14B-3 (14B-sh-3) for 72 h. Decrease of hCdc14B expression was normalized to β-tubulin.
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pone-0014711-g004: Downregulation of hCdc14B leads to multipolar mitotic spindles, lagging chromosomes, and binucleation.U2OS cells were transfected with pTER plasmids expressing two different hCdc14B-specific shRNAs (14B-sh-2, 14B-sh-3) or a control shRNA (Ctrl-sh). 72 h after transfection, cells were processed for immunofluorescence using anti-alpha-tubulin and Cy3-coupled anti-mouse antibodies (red), and CREST antiserum (kinetochores) and FITC-coupled anti-human antibodies (green). DNA was stained with Hoechst 33342. Representative examples of multipolar mitotic cells (A), bipolar mitotic cells with lagging anaphase chromosomes (arrow) (B), and binucleated cells (C) are shown. D. Frequencies of multipolar spindles and lagging anaphase chromosomes as visualized by staining with anti-alpha-tubulin and CREST. The numbers are derived from three independent experiments, each count comprising 60 mitotic cells. E. Western blot of hCdc14B after transfection with pTER-ctrl-shRNA (Ctrl-sh), pTER-Cdc14B-2 (14B-sh-2), and pTER-Cdc14B-3 (14B-sh-3) for 72 h. Decrease of hCdc14B expression was normalized to β-tubulin.

Mentions: To investigate whether silencing of hCdc14B had perturbed spindle assembly or chromosome segregation, immunofluorescence microscopy was performed with CREST and anti-alpha-tubulin antibodies. Immunostaining of mitotic spindles in U2OS cells depleted of hCdc14B by hCdc14B-shRNA-2 or hCdc14B-shRNA-3 (Fig. 4E) revealed multi-polar spindles at frequencies that were significantly higher (24–28%) than in cells transfected with an unrelated Ctrl-shRNA (2.5%). Most of the aberrant spindles were tri- or tetrapolar (Fig. 4A and D), although some cells with even more than 5 spindle poles were observed. In addition, hCdc14B-depletion increased the number of bipolar anaphase cells with lagging chromosomes (18–25%), indicative of chromosome missegregation (Fig. 4B and D). Again, while frequency of binucleation was low in control cells, it was significantly elevated in hCdc14B-depleted cells (Fig. 4C). These analyses indicate that silencing of hCdc14B caused problems in proper segregation of chromosomes, which in turn may explain the delay in mid-mitosis observed in HeLa Kyoto cells depleted of hCdc14B (see Fig. 3), and caused defects in bipolar mitotic spindle assembly, which is consistent with previous studies demonstrating a role of hCdc14B in bundling of microtubules [28] and in centriole amplification [30].


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)

Downregulation of hCdc14B leads to multipolar mitotic spindles, lagging chromosomes, and binucleation.U2OS cells were transfected with pTER plasmids expressing two different hCdc14B-specific shRNAs (14B-sh-2, 14B-sh-3) or a control shRNA (Ctrl-sh). 72 h after transfection, cells were processed for immunofluorescence using anti-alpha-tubulin and Cy3-coupled anti-mouse antibodies (red), and CREST antiserum (kinetochores) and FITC-coupled anti-human antibodies (green). DNA was stained with Hoechst 33342. Representative examples of multipolar mitotic cells (A), bipolar mitotic cells with lagging anaphase chromosomes (arrow) (B), and binucleated cells (C) are shown. D. Frequencies of multipolar spindles and lagging anaphase chromosomes as visualized by staining with anti-alpha-tubulin and CREST. The numbers are derived from three independent experiments, each count comprising 60 mitotic cells. E. Western blot of hCdc14B after transfection with pTER-ctrl-shRNA (Ctrl-sh), pTER-Cdc14B-2 (14B-sh-2), and pTER-Cdc14B-3 (14B-sh-3) for 72 h. Decrease of hCdc14B expression was normalized to β-tubulin.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040744&req=5

pone-0014711-g004: Downregulation of hCdc14B leads to multipolar mitotic spindles, lagging chromosomes, and binucleation.U2OS cells were transfected with pTER plasmids expressing two different hCdc14B-specific shRNAs (14B-sh-2, 14B-sh-3) or a control shRNA (Ctrl-sh). 72 h after transfection, cells were processed for immunofluorescence using anti-alpha-tubulin and Cy3-coupled anti-mouse antibodies (red), and CREST antiserum (kinetochores) and FITC-coupled anti-human antibodies (green). DNA was stained with Hoechst 33342. Representative examples of multipolar mitotic cells (A), bipolar mitotic cells with lagging anaphase chromosomes (arrow) (B), and binucleated cells (C) are shown. D. Frequencies of multipolar spindles and lagging anaphase chromosomes as visualized by staining with anti-alpha-tubulin and CREST. The numbers are derived from three independent experiments, each count comprising 60 mitotic cells. E. Western blot of hCdc14B after transfection with pTER-ctrl-shRNA (Ctrl-sh), pTER-Cdc14B-2 (14B-sh-2), and pTER-Cdc14B-3 (14B-sh-3) for 72 h. Decrease of hCdc14B expression was normalized to β-tubulin.
Mentions: To investigate whether silencing of hCdc14B had perturbed spindle assembly or chromosome segregation, immunofluorescence microscopy was performed with CREST and anti-alpha-tubulin antibodies. Immunostaining of mitotic spindles in U2OS cells depleted of hCdc14B by hCdc14B-shRNA-2 or hCdc14B-shRNA-3 (Fig. 4E) revealed multi-polar spindles at frequencies that were significantly higher (24–28%) than in cells transfected with an unrelated Ctrl-shRNA (2.5%). Most of the aberrant spindles were tri- or tetrapolar (Fig. 4A and D), although some cells with even more than 5 spindle poles were observed. In addition, hCdc14B-depletion increased the number of bipolar anaphase cells with lagging chromosomes (18–25%), indicative of chromosome missegregation (Fig. 4B and D). Again, while frequency of binucleation was low in control cells, it was significantly elevated in hCdc14B-depleted cells (Fig. 4C). These analyses indicate that silencing of hCdc14B caused problems in proper segregation of chromosomes, which in turn may explain the delay in mid-mitosis observed in HeLa Kyoto cells depleted of hCdc14B (see Fig. 3), and caused defects in bipolar mitotic spindle assembly, which is consistent with previous studies demonstrating a role of hCdc14B in bundling of microtubules [28] and in centriole amplification [30].

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