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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 CLB2 in pds1Δ mutants causes uncoupled sister-chromatid separation and spindle elongation.A. Saturated cells with the indicated genotypes were examined for the growth on glucose and galactose plates after 4 day incubation at 25°C. B. Cells with the indicated genotypes were grown to mid-log phase in raffinose medium and then shifted to galactose medium. Cells were collected over time and the viability was examined using platting efficiency (n>300). C. WT, pds1Δ, and pds1Δ spo12Δ cells in CEN4-GFP TUB1-mCherry background with a vector or a PGALCLB2 plasmid were arrested in G1-phase and then released into 25°C galactose medium to induce CLB2 overexpression. Cells were collected at 160 and 180 min and fixed to examine chromosome segregation. The spindle morphology and CEN4-GFP segregation in some representative cells at 180 min are shown in the left panel. The arrow indicates a cell with an elongated spindle and mis-segregated CEN4-GFP. The average percentage of cells with mis-segregated CEN4-GFP at 160 and 180 min from three experiments is shown in the right panel (n>100). Scale bar, 5 µm. D. WT and pds1Δ cells in CEN4-GFP TUB1-mCherry background with a vector or PGALCLB2 plasmid were grown to mid-log phase in raffinose medium and then shifted to galactose medium. The cells were collected after 4 hr incubation at 25°C and fixed for DAPI staining. The segregation of CEN4-GFP as well as the spindle and nuclear morphology in some representative cells is shown. The scale bar is 5 µm. E. WT, pds1Δ, and pds1Δ spo12Δ cells in TEL5-GFP NUF2-mCherry background were treated as described in C. The experiment was repeated for 3 times. The segregation of telomere of chromosome V (TEL5-GFP) in cells at 180 min is shown in the left panel. The arrow indicates a cell with unseparated TEL5. The average percentage of cells with two Nuf2 foci and one TEL5-GFP dot at 160 and 180 min is shown in the right panel after three repeats (n>100). Scale bar, 5 µm.
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pgen-1003319-g006: Overexpression of CLB2 in pds1Δ mutants causes uncoupled sister-chromatid separation and spindle elongation.A. Saturated cells with the indicated genotypes were examined for the growth on glucose and galactose plates after 4 day incubation at 25°C. B. Cells with the indicated genotypes were grown to mid-log phase in raffinose medium and then shifted to galactose medium. Cells were collected over time and the viability was examined using platting efficiency (n>300). C. WT, pds1Δ, and pds1Δ spo12Δ cells in CEN4-GFP TUB1-mCherry background with a vector or a PGALCLB2 plasmid were arrested in G1-phase and then released into 25°C galactose medium to induce CLB2 overexpression. Cells were collected at 160 and 180 min and fixed to examine chromosome segregation. The spindle morphology and CEN4-GFP segregation in some representative cells at 180 min are shown in the left panel. The arrow indicates a cell with an elongated spindle and mis-segregated CEN4-GFP. The average percentage of cells with mis-segregated CEN4-GFP at 160 and 180 min from three experiments is shown in the right panel (n>100). Scale bar, 5 µm. D. WT and pds1Δ cells in CEN4-GFP TUB1-mCherry background with a vector or PGALCLB2 plasmid were grown to mid-log phase in raffinose medium and then shifted to galactose medium. The cells were collected after 4 hr incubation at 25°C and fixed for DAPI staining. The segregation of CEN4-GFP as well as the spindle and nuclear morphology in some representative cells is shown. The scale bar is 5 µm. E. WT, pds1Δ, and pds1Δ spo12Δ cells in TEL5-GFP NUF2-mCherry background were treated as described in C. The experiment was repeated for 3 times. The segregation of telomere of chromosome V (TEL5-GFP) in cells at 180 min is shown in the left panel. The arrow indicates a cell with unseparated TEL5. The average percentage of cells with two Nuf2 foci and one TEL5-GFP dot at 160 and 180 min is shown in the right panel after three repeats (n>100). Scale bar, 5 µm.

Mentions: Securin Pds1 binds to and inhibits separase Esp1, whose activity is required for the cleavage of cohesion Scc1/Mcd1 and the subsequent anaphase onset [2]. Thus, cell cycle-regulated Pds1 protein levels control the timing of cohesion cleavage and anaphase onset. A previous study showed that deletion of a nonessential gene CDH1 caused lethality in pds1Δ cells and expression of an extra copy of SWE1 suppressed this lethality [39]. Because CDH1 encodes an APC activator required for Clb2 degradation, it is possible that the high Clb2 levels contribute to the synthetic lethality of cdh1 pds1 mutants. Therefore, we examined the growth of pds1Δ cells overexpressing CLB2 and found that overexpression of CLB2 was very toxic to pds1Δ cells (Figure 6A). One possibility is that high levels of mitotic CDK cause chromosome biorientation defects [40], which require the intact spindle assembly checkpoint for survival. However, overexpression of CLB2 was not toxic to checkpoint mutant mad2Δ (Figure S9), excluding the possibility that the checkpoint defect in pds1Δ contributes to the lethality. After CLB2 expression for 4 hrs, 68% of pds1Δ cells lost viability (Figure 6B). A FEAR mutant spo12Δ failed to suppress the growth defect of pds1Δ (PGALCLB2) cells on galactose plates, but it partially suppressed the lethality of pds1Δ (PGALCLB2) cells after incubation in liquid galactose media (Figure 6B). Therefore, we conclude that high level of Clb2 proteins causes the lethality in pds1Δ mutant cells.


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 CLB2 in pds1Δ mutants causes uncoupled sister-chromatid separation and spindle elongation.A. Saturated cells with the indicated genotypes were examined for the growth on glucose and galactose plates after 4 day incubation at 25°C. B. Cells with the indicated genotypes were grown to mid-log phase in raffinose medium and then shifted to galactose medium. Cells were collected over time and the viability was examined using platting efficiency (n>300). C. WT, pds1Δ, and pds1Δ spo12Δ cells in CEN4-GFP TUB1-mCherry background with a vector or a PGALCLB2 plasmid were arrested in G1-phase and then released into 25°C galactose medium to induce CLB2 overexpression. Cells were collected at 160 and 180 min and fixed to examine chromosome segregation. The spindle morphology and CEN4-GFP segregation in some representative cells at 180 min are shown in the left panel. The arrow indicates a cell with an elongated spindle and mis-segregated CEN4-GFP. The average percentage of cells with mis-segregated CEN4-GFP at 160 and 180 min from three experiments is shown in the right panel (n>100). Scale bar, 5 µm. D. WT and pds1Δ cells in CEN4-GFP TUB1-mCherry background with a vector or PGALCLB2 plasmid were grown to mid-log phase in raffinose medium and then shifted to galactose medium. The cells were collected after 4 hr incubation at 25°C and fixed for DAPI staining. The segregation of CEN4-GFP as well as the spindle and nuclear morphology in some representative cells is shown. The scale bar is 5 µm. E. WT, pds1Δ, and pds1Δ spo12Δ cells in TEL5-GFP NUF2-mCherry background were treated as described in C. The experiment was repeated for 3 times. The segregation of telomere of chromosome V (TEL5-GFP) in cells at 180 min is shown in the left panel. The arrow indicates a cell with unseparated TEL5. The average percentage of cells with two Nuf2 foci and one TEL5-GFP dot at 160 and 180 min is shown in the right panel after three repeats (n>100). Scale bar, 5 µm.
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pgen-1003319-g006: Overexpression of CLB2 in pds1Δ mutants causes uncoupled sister-chromatid separation and spindle elongation.A. Saturated cells with the indicated genotypes were examined for the growth on glucose and galactose plates after 4 day incubation at 25°C. B. Cells with the indicated genotypes were grown to mid-log phase in raffinose medium and then shifted to galactose medium. Cells were collected over time and the viability was examined using platting efficiency (n>300). C. WT, pds1Δ, and pds1Δ spo12Δ cells in CEN4-GFP TUB1-mCherry background with a vector or a PGALCLB2 plasmid were arrested in G1-phase and then released into 25°C galactose medium to induce CLB2 overexpression. Cells were collected at 160 and 180 min and fixed to examine chromosome segregation. The spindle morphology and CEN4-GFP segregation in some representative cells at 180 min are shown in the left panel. The arrow indicates a cell with an elongated spindle and mis-segregated CEN4-GFP. The average percentage of cells with mis-segregated CEN4-GFP at 160 and 180 min from three experiments is shown in the right panel (n>100). Scale bar, 5 µm. D. WT and pds1Δ cells in CEN4-GFP TUB1-mCherry background with a vector or PGALCLB2 plasmid were grown to mid-log phase in raffinose medium and then shifted to galactose medium. The cells were collected after 4 hr incubation at 25°C and fixed for DAPI staining. The segregation of CEN4-GFP as well as the spindle and nuclear morphology in some representative cells is shown. The scale bar is 5 µm. E. WT, pds1Δ, and pds1Δ spo12Δ cells in TEL5-GFP NUF2-mCherry background were treated as described in C. The experiment was repeated for 3 times. The segregation of telomere of chromosome V (TEL5-GFP) in cells at 180 min is shown in the left panel. The arrow indicates a cell with unseparated TEL5. The average percentage of cells with two Nuf2 foci and one TEL5-GFP dot at 160 and 180 min is shown in the right panel after three repeats (n>100). Scale bar, 5 µm.
Mentions: Securin Pds1 binds to and inhibits separase Esp1, whose activity is required for the cleavage of cohesion Scc1/Mcd1 and the subsequent anaphase onset [2]. Thus, cell cycle-regulated Pds1 protein levels control the timing of cohesion cleavage and anaphase onset. A previous study showed that deletion of a nonessential gene CDH1 caused lethality in pds1Δ cells and expression of an extra copy of SWE1 suppressed this lethality [39]. Because CDH1 encodes an APC activator required for Clb2 degradation, it is possible that the high Clb2 levels contribute to the synthetic lethality of cdh1 pds1 mutants. Therefore, we examined the growth of pds1Δ cells overexpressing CLB2 and found that overexpression of CLB2 was very toxic to pds1Δ cells (Figure 6A). One possibility is that high levels of mitotic CDK cause chromosome biorientation defects [40], which require the intact spindle assembly checkpoint for survival. However, overexpression of CLB2 was not toxic to checkpoint mutant mad2Δ (Figure S9), excluding the possibility that the checkpoint defect in pds1Δ contributes to the lethality. After CLB2 expression for 4 hrs, 68% of pds1Δ cells lost viability (Figure 6B). A FEAR mutant spo12Δ failed to suppress the growth defect of pds1Δ (PGALCLB2) cells on galactose plates, but it partially suppressed the lethality of pds1Δ (PGALCLB2) cells after incubation in liquid galactose media (Figure 6B). Therefore, we conclude that high level of Clb2 proteins causes the lethality in pds1Δ mutant cells.

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