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Coordination of chromatid separation and spindle elongation by antagonistic activities of mitotic and S-phase CDKs.

Liang F, Richmond D, Wang Y - PLoS Genet. (2013)

Bottom Line: In contrast, mitotic CDK promotes spindle elongation by activating Cdc14 phosphatase, which reverses the protein phosphorylation imposed by S-phase CDK.Our data suggest that S-phase CDK negatively regulates spindle elongation partly through its phosphorylation of a spindle pole body (SPB) protein Spc110.We also show that hyperactive S-phase CDK compromises the microtubule localization of Stu2, a processive microtubule polymerase essential for spindle elongation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida, USA.

ABSTRACT
Because cohesion prevents sister-chromatid separation and spindle elongation, cohesion dissolution may trigger these two events simultaneously. However, the relatively normal spindle elongation kinetics in yeast cohesin mutants indicates an additional mechanism for the temporal control of spindle elongation. Here we show evidence indicating that S-phase CDK (cyclin dependent kinase) negatively regulates spindle elongation. In contrast, mitotic CDK promotes spindle elongation by activating Cdc14 phosphatase, which reverses the protein phosphorylation imposed by S-phase CDK. Our data suggest that S-phase CDK negatively regulates spindle elongation partly through its phosphorylation of a spindle pole body (SPB) protein Spc110. We also show that hyperactive S-phase CDK compromises the microtubule localization of Stu2, a processive microtubule polymerase essential for spindle elongation. Strikingly, we found that hyperactive mitotic CDK induces uncoupled spindle elongation and sister-chromatid separation in securin mutants (pds1Δ), and we speculate that asynchronous chromosome segregation in pds1Δ cells contributes to this phenotype. Therefore, the tight temporal control of spindle elongation and cohesin cleavage assure orchestrated chromosome separation and spindle elongation.

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Overexpression of CLB5 impairs the localization of Stu2 on spindle and cytoplasmic microtubules.A. stu2-10 temperature-sensitive mutants suppress the growth defect of swe1Δ cells overexpressing CLB2. The cell cultures with indicated genotypes were 10-fold diluted and then spotted onto glucose and galactose plates. The growth was examined after 4 day incubation at 25°C. B. Overexpression of CLB5 is toxic to stu2-10 mutants. The growth of WT and stu2-10 mutant cells with a control vector or PGALCLB5 plasmids at 25°C were examined as described in A. C. spc11018A91A mutants partially suppress the temperature sensitivity and spindle elongation defects of stu2-10 mutants. The growth of cells with the indicated genotypes at 25°C and 35°C is shown in the top panel. The cells with indicated genotypes were arrested at G1 phase at 25°C and then released into YPD medium at 35°C. The budding index and the percentage of cells with an elongated spindle (>3 µm) are shown in the bottom panel (n>100). D. Overexpression of CLB5 impairs the localization of Stu2 on spindle and cytoplasmic microtubules. WT and spc11018A91A cells with STU2-GFP TUB1-mApple harboring a control vector or a PGALCLB5 plasmid were arrested with 200 mM HU in raffinose medium for 2.5 hrs. After the cells were released into galactose medium for 1 hr, they were subjected to live-cell microscopy at 25°C. The Stu2 localization and spindle morphology are shown (a reprehensive from more than 10 cells). The scale bar is 5 µm.
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pgen-1003319-g005: Overexpression of CLB5 impairs the localization of Stu2 on spindle and cytoplasmic microtubules.A. stu2-10 temperature-sensitive mutants suppress the growth defect of swe1Δ cells overexpressing CLB2. The cell cultures with indicated genotypes were 10-fold diluted and then spotted onto glucose and galactose plates. The growth was examined after 4 day incubation at 25°C. B. Overexpression of CLB5 is toxic to stu2-10 mutants. The growth of WT and stu2-10 mutant cells with a control vector or PGALCLB5 plasmids at 25°C were examined as described in A. C. spc11018A91A mutants partially suppress the temperature sensitivity and spindle elongation defects of stu2-10 mutants. The growth of cells with the indicated genotypes at 25°C and 35°C is shown in the top panel. The cells with indicated genotypes were arrested at G1 phase at 25°C and then released into YPD medium at 35°C. The budding index and the percentage of cells with an elongated spindle (>3 µm) are shown in the bottom panel (n>100). D. Overexpression of CLB5 impairs the localization of Stu2 on spindle and cytoplasmic microtubules. WT and spc11018A91A cells with STU2-GFP TUB1-mApple harboring a control vector or a PGALCLB5 plasmid were arrested with 200 mM HU in raffinose medium for 2.5 hrs. After the cells were released into galactose medium for 1 hr, they were subjected to live-cell microscopy at 25°C. The Stu2 localization and spindle morphology are shown (a reprehensive from more than 10 cells). The scale bar is 5 µm.

Mentions: Our data suggest that the phosphorylation of SPB component Spc110 plays a role in the timing control of spindle elongation, and this phosphorylation is regulated by the balance of S-phase and mitotic CDKs. As a SPB component, however, it is likely that the phosphorylation of Spc110 regulates the spindle elongation via other microtubule-associated protein(s). Stu2 is the yeast homologue of the XMAP215 protein that binds to the microtubule plus-end [35], [36]. This protein is a processive microtubule polymerase essential for spindle elongation [37], [38]. One possibility is that the CDK activity controls the timing of spindle elongation by regulating the activity of Stu2. Interestingly, the temperature sensitive mutant stu2-10 dramatically suppressed the toxicity of CLB2 overexpression to swe1Δ mutant cells when incubated at 25°C (Figure 5A), indicating that intact Stu2 function is required for CLB2-induced premature spindle elongation. In contrast, stu2-10 mutant cells were more sensitive to CLB5 overexpression than WT cells (Figure 5B), indicating that Clb5 may negatively regulates Stu2 function. As we have showed that the phosphorylation of Spc110 by Clb5-Cdk1 plays a negative role in spindle elongation (Figure 4C and 4D), we further compared the growth and spindle elongation in stu2-10 and stu2-10 spc11018A91A at 35°C. The results showed that nonphosphorylatable spc110 mutant partially suppressed the temperature sensitivity and the spindle elongation defect of stu2-10 mutant cells (Figure 5C), suggesting that S-phase CDK-dependent Spc110 phosphorylation may down-regulate Stu2 function.


Coordination of chromatid separation and spindle elongation by antagonistic activities of mitotic and S-phase CDKs.

Liang F, Richmond D, Wang Y - PLoS Genet. (2013)

Overexpression of CLB5 impairs the localization of Stu2 on spindle and cytoplasmic microtubules.A. stu2-10 temperature-sensitive mutants suppress the growth defect of swe1Δ cells overexpressing CLB2. The cell cultures with indicated genotypes were 10-fold diluted and then spotted onto glucose and galactose plates. The growth was examined after 4 day incubation at 25°C. B. Overexpression of CLB5 is toxic to stu2-10 mutants. The growth of WT and stu2-10 mutant cells with a control vector or PGALCLB5 plasmids at 25°C were examined as described in A. C. spc11018A91A mutants partially suppress the temperature sensitivity and spindle elongation defects of stu2-10 mutants. The growth of cells with the indicated genotypes at 25°C and 35°C is shown in the top panel. The cells with indicated genotypes were arrested at G1 phase at 25°C and then released into YPD medium at 35°C. The budding index and the percentage of cells with an elongated spindle (>3 µm) are shown in the bottom panel (n>100). D. Overexpression of CLB5 impairs the localization of Stu2 on spindle and cytoplasmic microtubules. WT and spc11018A91A cells with STU2-GFP TUB1-mApple harboring a control vector or a PGALCLB5 plasmid were arrested with 200 mM HU in raffinose medium for 2.5 hrs. After the cells were released into galactose medium for 1 hr, they were subjected to live-cell microscopy at 25°C. The Stu2 localization and spindle morphology are shown (a reprehensive from more than 10 cells). The scale bar is 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3584997&req=5

pgen-1003319-g005: Overexpression of CLB5 impairs the localization of Stu2 on spindle and cytoplasmic microtubules.A. stu2-10 temperature-sensitive mutants suppress the growth defect of swe1Δ cells overexpressing CLB2. The cell cultures with indicated genotypes were 10-fold diluted and then spotted onto glucose and galactose plates. The growth was examined after 4 day incubation at 25°C. B. Overexpression of CLB5 is toxic to stu2-10 mutants. The growth of WT and stu2-10 mutant cells with a control vector or PGALCLB5 plasmids at 25°C were examined as described in A. C. spc11018A91A mutants partially suppress the temperature sensitivity and spindle elongation defects of stu2-10 mutants. The growth of cells with the indicated genotypes at 25°C and 35°C is shown in the top panel. The cells with indicated genotypes were arrested at G1 phase at 25°C and then released into YPD medium at 35°C. The budding index and the percentage of cells with an elongated spindle (>3 µm) are shown in the bottom panel (n>100). D. Overexpression of CLB5 impairs the localization of Stu2 on spindle and cytoplasmic microtubules. WT and spc11018A91A cells with STU2-GFP TUB1-mApple harboring a control vector or a PGALCLB5 plasmid were arrested with 200 mM HU in raffinose medium for 2.5 hrs. After the cells were released into galactose medium for 1 hr, they were subjected to live-cell microscopy at 25°C. The Stu2 localization and spindle morphology are shown (a reprehensive from more than 10 cells). The scale bar is 5 µm.
Mentions: Our data suggest that the phosphorylation of SPB component Spc110 plays a role in the timing control of spindle elongation, and this phosphorylation is regulated by the balance of S-phase and mitotic CDKs. As a SPB component, however, it is likely that the phosphorylation of Spc110 regulates the spindle elongation via other microtubule-associated protein(s). Stu2 is the yeast homologue of the XMAP215 protein that binds to the microtubule plus-end [35], [36]. This protein is a processive microtubule polymerase essential for spindle elongation [37], [38]. One possibility is that the CDK activity controls the timing of spindle elongation by regulating the activity of Stu2. Interestingly, the temperature sensitive mutant stu2-10 dramatically suppressed the toxicity of CLB2 overexpression to swe1Δ mutant cells when incubated at 25°C (Figure 5A), indicating that intact Stu2 function is required for CLB2-induced premature spindle elongation. In contrast, stu2-10 mutant cells were more sensitive to CLB5 overexpression than WT cells (Figure 5B), indicating that Clb5 may negatively regulates Stu2 function. As we have showed that the phosphorylation of Spc110 by Clb5-Cdk1 plays a negative role in spindle elongation (Figure 4C and 4D), we further compared the growth and spindle elongation in stu2-10 and stu2-10 spc11018A91A at 35°C. The results showed that nonphosphorylatable spc110 mutant partially suppressed the temperature sensitivity and the spindle elongation defect of stu2-10 mutant cells (Figure 5C), suggesting that S-phase CDK-dependent Spc110 phosphorylation may down-regulate Stu2 function.

Bottom Line: In contrast, mitotic CDK promotes spindle elongation by activating Cdc14 phosphatase, which reverses the protein phosphorylation imposed by S-phase CDK.Our data suggest that S-phase CDK negatively regulates spindle elongation partly through its phosphorylation of a spindle pole body (SPB) protein Spc110.We also show that hyperactive S-phase CDK compromises the microtubule localization of Stu2, a processive microtubule polymerase essential for spindle elongation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida, USA.

ABSTRACT
Because cohesion prevents sister-chromatid separation and spindle elongation, cohesion dissolution may trigger these two events simultaneously. However, the relatively normal spindle elongation kinetics in yeast cohesin mutants indicates an additional mechanism for the temporal control of spindle elongation. Here we show evidence indicating that S-phase CDK (cyclin dependent kinase) negatively regulates spindle elongation. In contrast, mitotic CDK promotes spindle elongation by activating Cdc14 phosphatase, which reverses the protein phosphorylation imposed by S-phase CDK. Our data suggest that S-phase CDK negatively regulates spindle elongation partly through its phosphorylation of a spindle pole body (SPB) protein Spc110. We also show that hyperactive S-phase CDK compromises the microtubule localization of Stu2, a processive microtubule polymerase essential for spindle elongation. Strikingly, we found that hyperactive mitotic CDK induces uncoupled spindle elongation and sister-chromatid separation in securin mutants (pds1Δ), and we speculate that asynchronous chromosome segregation in pds1Δ cells contributes to this phenotype. Therefore, the tight temporal control of spindle elongation and cohesin cleavage assure orchestrated chromosome separation and spindle elongation.

Show MeSH
Related in: MedlinePlus