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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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S-phase CDK negatively regulates spindle elongation.A. Overexpression of S-phase cyclin CLB5 leads to accumulation of cells with a short spindle. TUB1-GFP cells with a control vector or a PGALCLB5 plasmid were grown in raffinose medium to log phase and then shifted into 30°C galactose medium for 4 hrs. The budding index and the percentage of cells with a short (<3 µm) or long spindle are shown in the left panel. The spindle morphology in some representative cells (4 hr in galactose) is shown in the right panel. The experiment was repeated 3 times. The scale bar is 5 µm. B. Overexpression of CLB5 blocks spindle elongation in cells released from HU arrest. The G1-arrested cells with the indicated genotypes were released into 200 mM HU medium and incubated at 30°C for 2 hr. After HU was washed off, the cells were released into 30°C galactose medium and collected over time to examine spindle morphology. The percentage of cells with elongated spindle (>3 µm) is shown in the left panel (n>100). The spindle morphology in some representative cells at time 0 and 2.5 hr is shown in the right panel. Scale bar, 5 µm. C. Deletion of PDS1 gene does not suppress the spindle elongation defects in cells with high levels of Clb5. TUB1-GFP cells with the indicated genotypes were grown in raffinose medium and then shifted to 25°C galactose medium for 4 hr. The experiment was repeated 3 times and the percentage of large budded cells with a short or long spindle is shown in the left panel. The spindle morphology in some representative cells is shown in the right panel. Scale bar, 5 µm.
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pgen-1003319-g003: S-phase CDK negatively regulates spindle elongation.A. Overexpression of S-phase cyclin CLB5 leads to accumulation of cells with a short spindle. TUB1-GFP cells with a control vector or a PGALCLB5 plasmid were grown in raffinose medium to log phase and then shifted into 30°C galactose medium for 4 hrs. The budding index and the percentage of cells with a short (<3 µm) or long spindle are shown in the left panel. The spindle morphology in some representative cells (4 hr in galactose) is shown in the right panel. The experiment was repeated 3 times. The scale bar is 5 µm. B. Overexpression of CLB5 blocks spindle elongation in cells released from HU arrest. The G1-arrested cells with the indicated genotypes were released into 200 mM HU medium and incubated at 30°C for 2 hr. After HU was washed off, the cells were released into 30°C galactose medium and collected over time to examine spindle morphology. The percentage of cells with elongated spindle (>3 µm) is shown in the left panel (n>100). The spindle morphology in some representative cells at time 0 and 2.5 hr is shown in the right panel. Scale bar, 5 µm. C. Deletion of PDS1 gene does not suppress the spindle elongation defects in cells with high levels of Clb5. TUB1-GFP cells with the indicated genotypes were grown in raffinose medium and then shifted to 25°C galactose medium for 4 hr. The experiment was repeated 3 times and the percentage of large budded cells with a short or long spindle is shown in the left panel. The spindle morphology in some representative cells is shown in the right panel. Scale bar, 5 µm.

Mentions: Overexpression of CLB5 slows cell growth, suggesting that hyperactive Clb5-Cdk1 may have a negative effect on the cell cycle (Figure 1A). To test if hyperactive S-phase CDK inhibits spindle elongation, we examined the spindle structure in WT cells overexpressing CLB5. After incubation in galactose medium for 4 hrs, more yeast cells with a PGALCLB5 plasmid arrested with a large bud and a very short spindle structure (Figure 3A). Because these spindles are very short, one explanation is that the failure of SPB separation contributes to the spindle elongation defect. Therefore, we examined the spindle elongation kinetics in cells overexpressing CLB5 after release from hydroxyurea (HU) arrest. HU blocks DNA synthesis and HU-arrested cells have a short spindle with separated SPBs [30]. After HU wash off, CLB5 overexpression also caused a clear spindle elongation delay as indicated by the accumulation of cells with a bar-like short spindle structure (Figure 3B). This result suggests that the short spindle observed in cells with hyperactive S-phase CDK is not due to SPB separation defect. As we have shown that CLB5 overexpression blocks nuclear division in FEAR mutants, such as slk19Δ and spo12Δ [19], we further examined the spindle elongation kinetics in spo12Δ mutants with and without CLB5 overexpression after HU release. Obviously, spindle elongation was largely blocked by CLB5 overexpression in spo12Δ mutant cells (Figure 3B).


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)

S-phase CDK negatively regulates spindle elongation.A. Overexpression of S-phase cyclin CLB5 leads to accumulation of cells with a short spindle. TUB1-GFP cells with a control vector or a PGALCLB5 plasmid were grown in raffinose medium to log phase and then shifted into 30°C galactose medium for 4 hrs. The budding index and the percentage of cells with a short (<3 µm) or long spindle are shown in the left panel. The spindle morphology in some representative cells (4 hr in galactose) is shown in the right panel. The experiment was repeated 3 times. The scale bar is 5 µm. B. Overexpression of CLB5 blocks spindle elongation in cells released from HU arrest. The G1-arrested cells with the indicated genotypes were released into 200 mM HU medium and incubated at 30°C for 2 hr. After HU was washed off, the cells were released into 30°C galactose medium and collected over time to examine spindle morphology. The percentage of cells with elongated spindle (>3 µm) is shown in the left panel (n>100). The spindle morphology in some representative cells at time 0 and 2.5 hr is shown in the right panel. Scale bar, 5 µm. C. Deletion of PDS1 gene does not suppress the spindle elongation defects in cells with high levels of Clb5. TUB1-GFP cells with the indicated genotypes were grown in raffinose medium and then shifted to 25°C galactose medium for 4 hr. The experiment was repeated 3 times and the percentage of large budded cells with a short or long spindle is shown in the left panel. The spindle morphology in some representative cells is shown in the right panel. Scale bar, 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003319-g003: S-phase CDK negatively regulates spindle elongation.A. Overexpression of S-phase cyclin CLB5 leads to accumulation of cells with a short spindle. TUB1-GFP cells with a control vector or a PGALCLB5 plasmid were grown in raffinose medium to log phase and then shifted into 30°C galactose medium for 4 hrs. The budding index and the percentage of cells with a short (<3 µm) or long spindle are shown in the left panel. The spindle morphology in some representative cells (4 hr in galactose) is shown in the right panel. The experiment was repeated 3 times. The scale bar is 5 µm. B. Overexpression of CLB5 blocks spindle elongation in cells released from HU arrest. The G1-arrested cells with the indicated genotypes were released into 200 mM HU medium and incubated at 30°C for 2 hr. After HU was washed off, the cells were released into 30°C galactose medium and collected over time to examine spindle morphology. The percentage of cells with elongated spindle (>3 µm) is shown in the left panel (n>100). The spindle morphology in some representative cells at time 0 and 2.5 hr is shown in the right panel. Scale bar, 5 µm. C. Deletion of PDS1 gene does not suppress the spindle elongation defects in cells with high levels of Clb5. TUB1-GFP cells with the indicated genotypes were grown in raffinose medium and then shifted to 25°C galactose medium for 4 hr. The experiment was repeated 3 times and the percentage of large budded cells with a short or long spindle is shown in the left panel. The spindle morphology in some representative cells is shown in the right panel. Scale bar, 5 µm.
Mentions: Overexpression of CLB5 slows cell growth, suggesting that hyperactive Clb5-Cdk1 may have a negative effect on the cell cycle (Figure 1A). To test if hyperactive S-phase CDK inhibits spindle elongation, we examined the spindle structure in WT cells overexpressing CLB5. After incubation in galactose medium for 4 hrs, more yeast cells with a PGALCLB5 plasmid arrested with a large bud and a very short spindle structure (Figure 3A). Because these spindles are very short, one explanation is that the failure of SPB separation contributes to the spindle elongation defect. Therefore, we examined the spindle elongation kinetics in cells overexpressing CLB5 after release from hydroxyurea (HU) arrest. HU blocks DNA synthesis and HU-arrested cells have a short spindle with separated SPBs [30]. After HU wash off, CLB5 overexpression also caused a clear spindle elongation delay as indicated by the accumulation of cells with a bar-like short spindle structure (Figure 3B). This result suggests that the short spindle observed in cells with hyperactive S-phase CDK is not due to SPB separation defect. As we have shown that CLB5 overexpression blocks nuclear division in FEAR mutants, such as slk19Δ and spo12Δ [19], we further examined the spindle elongation kinetics in spo12Δ mutants with and without CLB5 overexpression after HU release. Obviously, spindle elongation was largely blocked by CLB5 overexpression in spo12Δ mutant cells (Figure 3B).

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