Limits...
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.

Show MeSH

Related in: MedlinePlus

The dephosphorylation of Spc110 promotes spindle elongation.A. spc11018D91D mutants suppress the binucleate phenotype in swe1Δ cells after CLB2 overexpression. swe1Δ and swe1Δ spc11018D91D 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 30°C, the cells were fixed for DAPI staining. The percentage of binucleate cells is shown after 3 repeats (n>100). B. The dephosphorylation of Spc110 is FEAR dependent. WT, cdc15-2, cdc15-2 spo12Δ, and clb1Δ clb2-VI cells with Spc110-13myc were arrested in G1 phase at 25°C and then released into cell cycle at 37°C. The protein samples were prepared every 20 min and Western blotting was performed to detect Spc110 phosphorylation. The budding index is shown in the left panel; the Spc110 protein levels and band-shift are shown in the right panel. C. spc11018A91A mutants partially suppress the growth defects of spo12Δ mutant cells overexpressing CLB5. Saturated cells with the indicated genotypes were 10-fold diluted and the growth on glucose and galactose plates was examined after 3 day incubation at 30°C. D. spc11018A91A mutants partially suppress the spindle elongation defects in spo12Δ mutants overexpressing CLB5. Cells with the indicated phenotypes were arrested in G1 phase in raffinose medium and then released into 30°C galactose medium. The percentages of large budded cells and cells with an elongated spindle are shown (n>100). E. Overexpression of CLB5 in spo12Δ mutant cells leads to more dramatic Spc110 phosphorylation. SPC110-13Myc and spo12Δ SPC110-13Myc cells with a vector or a PGALCLB5 plasmid were arrested in G1 phase and released into 30°C galactose medium. The protein samples were prepared every 20 min and Western blotting was performed to detect Spc110 phosphorylation. The budding index is shown in the left panel; the Spc110 protein levels and band-shift are shown in the right panel.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3584997&req=5

pgen-1003319-g004: The dephosphorylation of Spc110 promotes spindle elongation.A. spc11018D91D mutants suppress the binucleate phenotype in swe1Δ cells after CLB2 overexpression. swe1Δ and swe1Δ spc11018D91D 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 30°C, the cells were fixed for DAPI staining. The percentage of binucleate cells is shown after 3 repeats (n>100). B. The dephosphorylation of Spc110 is FEAR dependent. WT, cdc15-2, cdc15-2 spo12Δ, and clb1Δ clb2-VI cells with Spc110-13myc were arrested in G1 phase at 25°C and then released into cell cycle at 37°C. The protein samples were prepared every 20 min and Western blotting was performed to detect Spc110 phosphorylation. The budding index is shown in the left panel; the Spc110 protein levels and band-shift are shown in the right panel. C. spc11018A91A mutants partially suppress the growth defects of spo12Δ mutant cells overexpressing CLB5. Saturated cells with the indicated genotypes were 10-fold diluted and the growth on glucose and galactose plates was examined after 3 day incubation at 30°C. D. spc11018A91A mutants partially suppress the spindle elongation defects in spo12Δ mutants overexpressing CLB5. Cells with the indicated phenotypes were arrested in G1 phase in raffinose medium and then released into 30°C galactose medium. The percentages of large budded cells and cells with an elongated spindle are shown (n>100). E. Overexpression of CLB5 in spo12Δ mutant cells leads to more dramatic Spc110 phosphorylation. SPC110-13Myc and spo12Δ SPC110-13Myc cells with a vector or a PGALCLB5 plasmid were arrested in G1 phase and released into 30°C galactose medium. The protein samples were prepared every 20 min and Western blotting was performed to detect Spc110 phosphorylation. The budding index is shown in the left panel; the Spc110 protein levels and band-shift are shown in the right panel.

Mentions: Functional FEAR is required for Clb2-induced premature spindle elongation, and the FEAR promotes Cdc14 release to dephosphorylate Clb5 substrates, such as Ase1 and Fin1 [7], [34], but we found that ase1Δ or fin1Δ mutant did not suppress Clb2-induced premature spindle elongation. A previous study suggests that Clb5-Cdk1-induced phosphorylation of Spc110, one of the SPB proteins, also modulates spindle dynamics [9]. Interestingly, a phospho-mimetic spc11018D91D mutant, in which the CDK phosphorylation sites at Thr18 and Ser91 were mutated to aspartic acid, showed dramatic suppression of the binucleate phenotype in swe1Δ cells overexpressing CLB2 (Figure 4A), indicating that the dephosphorylation of Spc110 might be required for Clb2-induced premature spindle elongation. We further compared the spindle elongation kinetics in WT and spc11018D91D mutant cells and found that the mutant cells did exhibit delayed spindle elongation, although the delay was not pronounced (Figure S8). Nevertheless, the spc11018D91D mutant failed to rescue the sick growth phenotype of swe1Δ cells with PGALCLB2 on galactose medium, suggesting that phosphorylation of other Clb5 substrates can prevent spindle elongation as well. Alternatively, other unidentified defects induced by Clb2 overexpression may also lead to the sick growth.


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)

The dephosphorylation of Spc110 promotes spindle elongation.A. spc11018D91D mutants suppress the binucleate phenotype in swe1Δ cells after CLB2 overexpression. swe1Δ and swe1Δ spc11018D91D 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 30°C, the cells were fixed for DAPI staining. The percentage of binucleate cells is shown after 3 repeats (n>100). B. The dephosphorylation of Spc110 is FEAR dependent. WT, cdc15-2, cdc15-2 spo12Δ, and clb1Δ clb2-VI cells with Spc110-13myc were arrested in G1 phase at 25°C and then released into cell cycle at 37°C. The protein samples were prepared every 20 min and Western blotting was performed to detect Spc110 phosphorylation. The budding index is shown in the left panel; the Spc110 protein levels and band-shift are shown in the right panel. C. spc11018A91A mutants partially suppress the growth defects of spo12Δ mutant cells overexpressing CLB5. Saturated cells with the indicated genotypes were 10-fold diluted and the growth on glucose and galactose plates was examined after 3 day incubation at 30°C. D. spc11018A91A mutants partially suppress the spindle elongation defects in spo12Δ mutants overexpressing CLB5. Cells with the indicated phenotypes were arrested in G1 phase in raffinose medium and then released into 30°C galactose medium. The percentages of large budded cells and cells with an elongated spindle are shown (n>100). E. Overexpression of CLB5 in spo12Δ mutant cells leads to more dramatic Spc110 phosphorylation. SPC110-13Myc and spo12Δ SPC110-13Myc cells with a vector or a PGALCLB5 plasmid were arrested in G1 phase and released into 30°C galactose medium. The protein samples were prepared every 20 min and Western blotting was performed to detect Spc110 phosphorylation. The budding index is shown in the left panel; the Spc110 protein levels and band-shift are shown in the right panel.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003319-g004: The dephosphorylation of Spc110 promotes spindle elongation.A. spc11018D91D mutants suppress the binucleate phenotype in swe1Δ cells after CLB2 overexpression. swe1Δ and swe1Δ spc11018D91D 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 30°C, the cells were fixed for DAPI staining. The percentage of binucleate cells is shown after 3 repeats (n>100). B. The dephosphorylation of Spc110 is FEAR dependent. WT, cdc15-2, cdc15-2 spo12Δ, and clb1Δ clb2-VI cells with Spc110-13myc were arrested in G1 phase at 25°C and then released into cell cycle at 37°C. The protein samples were prepared every 20 min and Western blotting was performed to detect Spc110 phosphorylation. The budding index is shown in the left panel; the Spc110 protein levels and band-shift are shown in the right panel. C. spc11018A91A mutants partially suppress the growth defects of spo12Δ mutant cells overexpressing CLB5. Saturated cells with the indicated genotypes were 10-fold diluted and the growth on glucose and galactose plates was examined after 3 day incubation at 30°C. D. spc11018A91A mutants partially suppress the spindle elongation defects in spo12Δ mutants overexpressing CLB5. Cells with the indicated phenotypes were arrested in G1 phase in raffinose medium and then released into 30°C galactose medium. The percentages of large budded cells and cells with an elongated spindle are shown (n>100). E. Overexpression of CLB5 in spo12Δ mutant cells leads to more dramatic Spc110 phosphorylation. SPC110-13Myc and spo12Δ SPC110-13Myc cells with a vector or a PGALCLB5 plasmid were arrested in G1 phase and released into 30°C galactose medium. The protein samples were prepared every 20 min and Western blotting was performed to detect Spc110 phosphorylation. The budding index is shown in the left panel; the Spc110 protein levels and band-shift are shown in the right panel.
Mentions: Functional FEAR is required for Clb2-induced premature spindle elongation, and the FEAR promotes Cdc14 release to dephosphorylate Clb5 substrates, such as Ase1 and Fin1 [7], [34], but we found that ase1Δ or fin1Δ mutant did not suppress Clb2-induced premature spindle elongation. A previous study suggests that Clb5-Cdk1-induced phosphorylation of Spc110, one of the SPB proteins, also modulates spindle dynamics [9]. Interestingly, a phospho-mimetic spc11018D91D mutant, in which the CDK phosphorylation sites at Thr18 and Ser91 were mutated to aspartic acid, showed dramatic suppression of the binucleate phenotype in swe1Δ cells overexpressing CLB2 (Figure 4A), indicating that the dephosphorylation of Spc110 might be required for Clb2-induced premature spindle elongation. We further compared the spindle elongation kinetics in WT and spc11018D91D mutant cells and found that the mutant cells did exhibit delayed spindle elongation, although the delay was not pronounced (Figure S8). Nevertheless, the spc11018D91D mutant failed to rescue the sick growth phenotype of swe1Δ cells with PGALCLB2 on galactose medium, suggesting that phosphorylation of other Clb5 substrates can prevent spindle elongation as well. Alternatively, other unidentified defects induced by Clb2 overexpression may also lead to the sick growth.

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