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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 mitotic cyclin CLB2 results in premature spindle elongation in swe1Δ mutants.A. Overexpression of CLB2 is toxic to swe1Δ mutants. WT and swe1Δ mutant cells with a control vector or PGALCLB1, PGALCLB2, PGALCLB3, and PGALCLB5 plasmids were grown to saturation in glucose medium, 10-fold diluted, and spotted onto glucose or galactose plates. The plates were scanned after incubation at 30°C for 3 days. B. Overexpression of CLB2 in swe1Δ mutant cells leads to premature spindle elongation. G1-arrested PDS1-Myc TUB1-GFP and swe1Δ PDS1-Myc TUB1-GFP cells with a vector or a PGALCLB2 plasmid were released into 30°C galactose medium to induce CLB2 overexpression. Cells were collected over time and fixed to examine GFP signal and for DAPI staining. Spindles longer than 3 µm were counted as elongated. The percentage of cells with an elongated spindle is shown in the left panel (n>100). The percentage of binucleate cells is shown in the middle panel and the spindle morphology in cells at 120 min time point is shown in the right panel. The arrow indicates a binucleate cell with premature spindle elongation. Scale bar, 5 µm. The budding index, FACS analysis and Pds1 protein level are shown in Figure S2. C. Live-cell imaging shows the premature spindle elongation in swe1Δ mutants overexpressing CLB2. swe1Δ TUB1-GFP cells with a vector or a PGALCLB2 plasmid were arrested in G1-phase in raffinose medium. After released into galactose medium for 1.5 hr, the cells were transferred to an agarose pad on a microscope slide to perform live-cell imaging at 25°C. Scale bar, 5 µm.
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pgen-1003319-g001: Overexpression of mitotic cyclin CLB2 results in premature spindle elongation in swe1Δ mutants.A. Overexpression of CLB2 is toxic to swe1Δ mutants. WT and swe1Δ mutant cells with a control vector or PGALCLB1, PGALCLB2, PGALCLB3, and PGALCLB5 plasmids were grown to saturation in glucose medium, 10-fold diluted, and spotted onto glucose or galactose plates. The plates were scanned after incubation at 30°C for 3 days. B. Overexpression of CLB2 in swe1Δ mutant cells leads to premature spindle elongation. G1-arrested PDS1-Myc TUB1-GFP and swe1Δ PDS1-Myc TUB1-GFP cells with a vector or a PGALCLB2 plasmid were released into 30°C galactose medium to induce CLB2 overexpression. Cells were collected over time and fixed to examine GFP signal and for DAPI staining. Spindles longer than 3 µm were counted as elongated. The percentage of cells with an elongated spindle is shown in the left panel (n>100). The percentage of binucleate cells is shown in the middle panel and the spindle morphology in cells at 120 min time point is shown in the right panel. The arrow indicates a binucleate cell with premature spindle elongation. Scale bar, 5 µm. The budding index, FACS analysis and Pds1 protein level are shown in Figure S2. C. Live-cell imaging shows the premature spindle elongation in swe1Δ mutants overexpressing CLB2. swe1Δ TUB1-GFP cells with a vector or a PGALCLB2 plasmid were arrested in G1-phase in raffinose medium. After released into galactose medium for 1.5 hr, the cells were transferred to an agarose pad on a microscope slide to perform live-cell imaging at 25°C. Scale bar, 5 µm.

Mentions: Clb2 is the major mitotic cyclin in budding yeast, but its overexpression from a galactose-inducible promoter does not cause obvious premature mitosis in wild-type (WT) cells. Because Swe1 kinase phosphorylates and inhibits mitotic CDK, it is possible that the presence of Swe1 prevents the hyper-activation of mitotic CDK after CLB2 overexpression. Therefore, we overexpressed CLB2 from a galactose-inducible promoter in swe1Δ mutant cells and examined the cell growth. The Western blotting result confirmed the high level expression of Clb2 protein after galactose induction (Figure S1A). Compared to the control cells, swe1Δ mutants with a PGALCLB2 plasmid showed obvious growth defect on galactose plates. Overexpression of Clb1, which is closely related to Clb2, also caused sick growth phenotype in swe1Δ cells, but overexpression of Clb3, Clb4, or S phase cyclin Clb5, Clb6 was not toxic (Figure 1A and S1B). Therefore overexpression of mitotic cyclins Clb1 and Clb2 is toxic to swe1Δ cells. To confirm that mitotic CDK is hyperactive in swe1Δ cells after CLB2 overexpression, we compared the phosphorylation kinetics of Pol12 in synchronized WT and swe1Δ cells overexpressing CLB2, as Pol12 is a known substrate of mitotic CDK required for DNA replication [24]. Our results showed that CLB2 overexpression induces premature Pol12 phosphorylation in both WT and swe1Δ cells based on the band-shift, and the phosphorylation became more significant in swe1Δ cells as indicated by the increased slow migrating band (Figure S1C). Therefore, the absence of Swe1 indeed causes hyper-activation of mitotic CDK after CLB2 overexpression.


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 mitotic cyclin CLB2 results in premature spindle elongation in swe1Δ mutants.A. Overexpression of CLB2 is toxic to swe1Δ mutants. WT and swe1Δ mutant cells with a control vector or PGALCLB1, PGALCLB2, PGALCLB3, and PGALCLB5 plasmids were grown to saturation in glucose medium, 10-fold diluted, and spotted onto glucose or galactose plates. The plates were scanned after incubation at 30°C for 3 days. B. Overexpression of CLB2 in swe1Δ mutant cells leads to premature spindle elongation. G1-arrested PDS1-Myc TUB1-GFP and swe1Δ PDS1-Myc TUB1-GFP cells with a vector or a PGALCLB2 plasmid were released into 30°C galactose medium to induce CLB2 overexpression. Cells were collected over time and fixed to examine GFP signal and for DAPI staining. Spindles longer than 3 µm were counted as elongated. The percentage of cells with an elongated spindle is shown in the left panel (n>100). The percentage of binucleate cells is shown in the middle panel and the spindle morphology in cells at 120 min time point is shown in the right panel. The arrow indicates a binucleate cell with premature spindle elongation. Scale bar, 5 µm. The budding index, FACS analysis and Pds1 protein level are shown in Figure S2. C. Live-cell imaging shows the premature spindle elongation in swe1Δ mutants overexpressing CLB2. swe1Δ TUB1-GFP cells with a vector or a PGALCLB2 plasmid were arrested in G1-phase in raffinose medium. After released into galactose medium for 1.5 hr, the cells were transferred to an agarose pad on a microscope slide to perform live-cell imaging at 25°C. Scale bar, 5 µm.
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Related In: Results  -  Collection

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

pgen-1003319-g001: Overexpression of mitotic cyclin CLB2 results in premature spindle elongation in swe1Δ mutants.A. Overexpression of CLB2 is toxic to swe1Δ mutants. WT and swe1Δ mutant cells with a control vector or PGALCLB1, PGALCLB2, PGALCLB3, and PGALCLB5 plasmids were grown to saturation in glucose medium, 10-fold diluted, and spotted onto glucose or galactose plates. The plates were scanned after incubation at 30°C for 3 days. B. Overexpression of CLB2 in swe1Δ mutant cells leads to premature spindle elongation. G1-arrested PDS1-Myc TUB1-GFP and swe1Δ PDS1-Myc TUB1-GFP cells with a vector or a PGALCLB2 plasmid were released into 30°C galactose medium to induce CLB2 overexpression. Cells were collected over time and fixed to examine GFP signal and for DAPI staining. Spindles longer than 3 µm were counted as elongated. The percentage of cells with an elongated spindle is shown in the left panel (n>100). The percentage of binucleate cells is shown in the middle panel and the spindle morphology in cells at 120 min time point is shown in the right panel. The arrow indicates a binucleate cell with premature spindle elongation. Scale bar, 5 µm. The budding index, FACS analysis and Pds1 protein level are shown in Figure S2. C. Live-cell imaging shows the premature spindle elongation in swe1Δ mutants overexpressing CLB2. swe1Δ TUB1-GFP cells with a vector or a PGALCLB2 plasmid were arrested in G1-phase in raffinose medium. After released into galactose medium for 1.5 hr, the cells were transferred to an agarose pad on a microscope slide to perform live-cell imaging at 25°C. Scale bar, 5 µm.
Mentions: Clb2 is the major mitotic cyclin in budding yeast, but its overexpression from a galactose-inducible promoter does not cause obvious premature mitosis in wild-type (WT) cells. Because Swe1 kinase phosphorylates and inhibits mitotic CDK, it is possible that the presence of Swe1 prevents the hyper-activation of mitotic CDK after CLB2 overexpression. Therefore, we overexpressed CLB2 from a galactose-inducible promoter in swe1Δ mutant cells and examined the cell growth. The Western blotting result confirmed the high level expression of Clb2 protein after galactose induction (Figure S1A). Compared to the control cells, swe1Δ mutants with a PGALCLB2 plasmid showed obvious growth defect on galactose plates. Overexpression of Clb1, which is closely related to Clb2, also caused sick growth phenotype in swe1Δ cells, but overexpression of Clb3, Clb4, or S phase cyclin Clb5, Clb6 was not toxic (Figure 1A and S1B). Therefore overexpression of mitotic cyclins Clb1 and Clb2 is toxic to swe1Δ cells. To confirm that mitotic CDK is hyperactive in swe1Δ cells after CLB2 overexpression, we compared the phosphorylation kinetics of Pol12 in synchronized WT and swe1Δ cells overexpressing CLB2, as Pol12 is a known substrate of mitotic CDK required for DNA replication [24]. Our results showed that CLB2 overexpression induces premature Pol12 phosphorylation in both WT and swe1Δ cells based on the band-shift, and the phosphorylation became more significant in swe1Δ cells as indicated by the increased slow migrating band (Figure S1C). Therefore, the absence of Swe1 indeed causes hyper-activation of mitotic CDK after CLB2 overexpression.

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