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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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Mitotic CDK promotes spindle elongation through FEAR pathway.A. FEAR and cdc14-1 mutants suppress the growth defects in swe1Δ cells overexpressing CLB2. A control vector or a PGALCLB2 plasmid was transformed into WT, swe1Δ, swe1Δ net1-6Cdk, and swe1Δ spo12Δ cells. The growth of the transformants on 30°C glucose and galactose plates was examined as described in Figure 1A. The growth of WT, swe1Δ, swe1Δ cdc14-1 with a vector control of PGALCLB2 plasmid on glucose and galactose plates at 25°C is also shown. B. FEAR mutants suppress the binucleate phenotype in swe1Δ cells overexpressing CLB2. Cells with the indicated genotypes were grown to mid-log phase in raffinose medium and then shifted to galactose medium. The cells were collected after 4 hr incubation at 30°C and fixed for DAPI staining. The percentage of binucleate cells is shown. The experiments were repeated for 3 times and at least 100 cells were counted for each sample. C. cdc14-1 mutants suppress the binucleate phenotype in swe1Δ cells overexpressing CLB2. WT, swe1Δ and cdc14-1 swe1Δ cells with a control vector or a PGALCLB2 plasmid were grown to mid-log phase in raffinose medium and then switched to galactose medium. After 4 hr incubation at 37°C, the cells were fixed for DAPI staining. The percentage of binucleate cells from three independent experiments is shown (n>100). D. FEAR mutants suppress the premature spindle elongation in swe1Δ cells overexpressing CLB2. cdc15-2 swe1Δ and cdc15-2 swe1Δ net1-6Cdk cells with a control vector or a PGALCLB2 plasmid were arrested in G1 phase in raffinose medium at 25°C and then released into galactose medium at 37°C. The budding index and the percentage of cells with an elongated spindle are shown in the left panels (n>100). The spindle morphology after 120 min release from G1 is shown in the right panel. The scale bar is 5 µm. E. Overexpression of CLB2 in swe1Δ mutant results in premature Cdc14 release. G1-arrested CDC14-5GFP, swe1Δ CDC14-5GFP and swe1Δ net1-6Cdk CDC14-5GFP cells with a vector or a PGALCLB2 plasmid were released into 30°C galactose medium containing 20 µg/ml nocodazole (NOC). Cells were collected over time and fixed to examine Cdc14 localization. The Cdc14 localization in cells at 3 hr time point is shown in the left panel. The percentage of cells with released Cdc14 was counted over time (right panel, n>100). Scale bar, 5 µm.
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pgen-1003319-g002: Mitotic CDK promotes spindle elongation through FEAR pathway.A. FEAR and cdc14-1 mutants suppress the growth defects in swe1Δ cells overexpressing CLB2. A control vector or a PGALCLB2 plasmid was transformed into WT, swe1Δ, swe1Δ net1-6Cdk, and swe1Δ spo12Δ cells. The growth of the transformants on 30°C glucose and galactose plates was examined as described in Figure 1A. The growth of WT, swe1Δ, swe1Δ cdc14-1 with a vector control of PGALCLB2 plasmid on glucose and galactose plates at 25°C is also shown. B. FEAR mutants suppress the binucleate phenotype in swe1Δ cells overexpressing CLB2. Cells with the indicated genotypes were grown to mid-log phase in raffinose medium and then shifted to galactose medium. The cells were collected after 4 hr incubation at 30°C and fixed for DAPI staining. The percentage of binucleate cells is shown. The experiments were repeated for 3 times and at least 100 cells were counted for each sample. C. cdc14-1 mutants suppress the binucleate phenotype in swe1Δ cells overexpressing CLB2. WT, swe1Δ and cdc14-1 swe1Δ cells with a control vector or a PGALCLB2 plasmid were grown to mid-log phase in raffinose medium and then switched to galactose medium. After 4 hr incubation at 37°C, the cells were fixed for DAPI staining. The percentage of binucleate cells from three independent experiments is shown (n>100). D. FEAR mutants suppress the premature spindle elongation in swe1Δ cells overexpressing CLB2. cdc15-2 swe1Δ and cdc15-2 swe1Δ net1-6Cdk cells with a control vector or a PGALCLB2 plasmid were arrested in G1 phase in raffinose medium at 25°C and then released into galactose medium at 37°C. The budding index and the percentage of cells with an elongated spindle are shown in the left panels (n>100). The spindle morphology after 120 min release from G1 is shown in the right panel. The scale bar is 5 µm. E. Overexpression of CLB2 in swe1Δ mutant results in premature Cdc14 release. G1-arrested CDC14-5GFP, swe1Δ CDC14-5GFP and swe1Δ net1-6Cdk CDC14-5GFP cells with a vector or a PGALCLB2 plasmid were released into 30°C galactose medium containing 20 µg/ml nocodazole (NOC). Cells were collected over time and fixed to examine Cdc14 localization. The Cdc14 localization in cells at 3 hr time point is shown in the left panel. The percentage of cells with released Cdc14 was counted over time (right panel, n>100). Scale bar, 5 µm.

Mentions: One of the substrates of mitotic CDK is the nucleolar Cdc14-binding protein Net1, whose phosphorylation triggers the dissociation of Cdc14 from Net1 and the release of Cdc14 from the nucleolus. It is possible that hyperactive mitotic CDK stimulates spindle elongation by activating FEAR. Because the replacement of 6 CDK phosphorylation sites in Net1 with alanine generates net1-6Cdk mutant, which prevents FEAR activation [14], we first compared the growth of swe1Δ and swe1Δ net1-6Cdk cells after CLB2 overexpression. The swe1Δ net1-6Cdk cells grew much better than the single mutant cells after CLB2 overexpression. Another FEAR mutant spo12Δ showed an even stronger suppression of the sick growth phenotype of swe1Δ (Figure 2A). The nuclear morphology was also compared in swe1Δ, swe1Δ spo12Δ and swe1Δ net1-6Cdk mutant cells overexpressing CLB2, both spo12Δ and net1-6Cdk mutants suppressed the formation of binucleate cells (Figure 2B), suggesting that the activation of FEAR pathway contributes to the growth defect in swe1Δ cells overexpressing CLB2. To directly determine if the toxicity of CLB2 overexpression to swe1Δ cells is due to hyperactive Cdc14, we examined the growth of swe1Δ cdc14-1 cells overexpressing CLB2. We found that cdc14-1 swe1Δ cells with PGALCLB2 plasmids grew better than swe1Δ cells on galactose plates at 25°C (Figure 2A). In addition, the cdc14-1 mutant partially suppressed binucleate phenotype of swe1Δ cells (Figure 2C). We reason that the incomplete suppression is due to the presence of partial functional Cdc14 in cdc14-1 mutant. Therefore, CLB2 overexpression likely induces premature spindle elongation by activating Cdc14.


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)

Mitotic CDK promotes spindle elongation through FEAR pathway.A. FEAR and cdc14-1 mutants suppress the growth defects in swe1Δ cells overexpressing CLB2. A control vector or a PGALCLB2 plasmid was transformed into WT, swe1Δ, swe1Δ net1-6Cdk, and swe1Δ spo12Δ cells. The growth of the transformants on 30°C glucose and galactose plates was examined as described in Figure 1A. The growth of WT, swe1Δ, swe1Δ cdc14-1 with a vector control of PGALCLB2 plasmid on glucose and galactose plates at 25°C is also shown. B. FEAR mutants suppress the binucleate phenotype in swe1Δ cells overexpressing CLB2. Cells with the indicated genotypes were grown to mid-log phase in raffinose medium and then shifted to galactose medium. The cells were collected after 4 hr incubation at 30°C and fixed for DAPI staining. The percentage of binucleate cells is shown. The experiments were repeated for 3 times and at least 100 cells were counted for each sample. C. cdc14-1 mutants suppress the binucleate phenotype in swe1Δ cells overexpressing CLB2. WT, swe1Δ and cdc14-1 swe1Δ cells with a control vector or a PGALCLB2 plasmid were grown to mid-log phase in raffinose medium and then switched to galactose medium. After 4 hr incubation at 37°C, the cells were fixed for DAPI staining. The percentage of binucleate cells from three independent experiments is shown (n>100). D. FEAR mutants suppress the premature spindle elongation in swe1Δ cells overexpressing CLB2. cdc15-2 swe1Δ and cdc15-2 swe1Δ net1-6Cdk cells with a control vector or a PGALCLB2 plasmid were arrested in G1 phase in raffinose medium at 25°C and then released into galactose medium at 37°C. The budding index and the percentage of cells with an elongated spindle are shown in the left panels (n>100). The spindle morphology after 120 min release from G1 is shown in the right panel. The scale bar is 5 µm. E. Overexpression of CLB2 in swe1Δ mutant results in premature Cdc14 release. G1-arrested CDC14-5GFP, swe1Δ CDC14-5GFP and swe1Δ net1-6Cdk CDC14-5GFP cells with a vector or a PGALCLB2 plasmid were released into 30°C galactose medium containing 20 µg/ml nocodazole (NOC). Cells were collected over time and fixed to examine Cdc14 localization. The Cdc14 localization in cells at 3 hr time point is shown in the left panel. The percentage of cells with released Cdc14 was counted over time (right panel, n>100). Scale bar, 5 µm.
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pgen-1003319-g002: Mitotic CDK promotes spindle elongation through FEAR pathway.A. FEAR and cdc14-1 mutants suppress the growth defects in swe1Δ cells overexpressing CLB2. A control vector or a PGALCLB2 plasmid was transformed into WT, swe1Δ, swe1Δ net1-6Cdk, and swe1Δ spo12Δ cells. The growth of the transformants on 30°C glucose and galactose plates was examined as described in Figure 1A. The growth of WT, swe1Δ, swe1Δ cdc14-1 with a vector control of PGALCLB2 plasmid on glucose and galactose plates at 25°C is also shown. B. FEAR mutants suppress the binucleate phenotype in swe1Δ cells overexpressing CLB2. Cells with the indicated genotypes were grown to mid-log phase in raffinose medium and then shifted to galactose medium. The cells were collected after 4 hr incubation at 30°C and fixed for DAPI staining. The percentage of binucleate cells is shown. The experiments were repeated for 3 times and at least 100 cells were counted for each sample. C. cdc14-1 mutants suppress the binucleate phenotype in swe1Δ cells overexpressing CLB2. WT, swe1Δ and cdc14-1 swe1Δ cells with a control vector or a PGALCLB2 plasmid were grown to mid-log phase in raffinose medium and then switched to galactose medium. After 4 hr incubation at 37°C, the cells were fixed for DAPI staining. The percentage of binucleate cells from three independent experiments is shown (n>100). D. FEAR mutants suppress the premature spindle elongation in swe1Δ cells overexpressing CLB2. cdc15-2 swe1Δ and cdc15-2 swe1Δ net1-6Cdk cells with a control vector or a PGALCLB2 plasmid were arrested in G1 phase in raffinose medium at 25°C and then released into galactose medium at 37°C. The budding index and the percentage of cells with an elongated spindle are shown in the left panels (n>100). The spindle morphology after 120 min release from G1 is shown in the right panel. The scale bar is 5 µm. E. Overexpression of CLB2 in swe1Δ mutant results in premature Cdc14 release. G1-arrested CDC14-5GFP, swe1Δ CDC14-5GFP and swe1Δ net1-6Cdk CDC14-5GFP cells with a vector or a PGALCLB2 plasmid were released into 30°C galactose medium containing 20 µg/ml nocodazole (NOC). Cells were collected over time and fixed to examine Cdc14 localization. The Cdc14 localization in cells at 3 hr time point is shown in the left panel. The percentage of cells with released Cdc14 was counted over time (right panel, n>100). Scale bar, 5 µm.
Mentions: One of the substrates of mitotic CDK is the nucleolar Cdc14-binding protein Net1, whose phosphorylation triggers the dissociation of Cdc14 from Net1 and the release of Cdc14 from the nucleolus. It is possible that hyperactive mitotic CDK stimulates spindle elongation by activating FEAR. Because the replacement of 6 CDK phosphorylation sites in Net1 with alanine generates net1-6Cdk mutant, which prevents FEAR activation [14], we first compared the growth of swe1Δ and swe1Δ net1-6Cdk cells after CLB2 overexpression. The swe1Δ net1-6Cdk cells grew much better than the single mutant cells after CLB2 overexpression. Another FEAR mutant spo12Δ showed an even stronger suppression of the sick growth phenotype of swe1Δ (Figure 2A). The nuclear morphology was also compared in swe1Δ, swe1Δ spo12Δ and swe1Δ net1-6Cdk mutant cells overexpressing CLB2, both spo12Δ and net1-6Cdk mutants suppressed the formation of binucleate cells (Figure 2B), suggesting that the activation of FEAR pathway contributes to the growth defect in swe1Δ cells overexpressing CLB2. To directly determine if the toxicity of CLB2 overexpression to swe1Δ cells is due to hyperactive Cdc14, we examined the growth of swe1Δ cdc14-1 cells overexpressing CLB2. We found that cdc14-1 swe1Δ cells with PGALCLB2 plasmids grew better than swe1Δ cells on galactose plates at 25°C (Figure 2A). In addition, the cdc14-1 mutant partially suppressed binucleate phenotype of swe1Δ cells (Figure 2C). We reason that the incomplete suppression is due to the presence of partial functional Cdc14 in cdc14-1 mutant. Therefore, CLB2 overexpression likely induces premature spindle elongation by activating Cdc14.

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