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The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly.

Buvelot S, Tatsutani SY, Vermaak D, Biggins S - J. Cell Biol. (2003)

Bottom Line: As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules.Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly.We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

View Article: PubMed Central - PubMed

Affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1-GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

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Ipl1p's role in spindle disassembly is independent from its role in chromosome segregation. (A) pGAL-CDC20 (SBY952) and pGAL-CDC20 ipl1–321 (SBY943) cells containing Tub1–GFP were shifted to glucose to arrest cells in metaphase and then shifted to 37°C to inactivate Ipl1–321p. Cells were then released into galactose medium at 37°C to restore Cdc20 protein synthesis in the presence of α-factor to arrest cells in the following G1. The percentage of cells with a pole to pole distance ≥9 μm were monitored for the presence or absence of a spindle. Spindle disassembly occurred in 68% of pGAL-CDC20 cells compared with 36% of pGAL-CDC20 ipl1–321 mutant cells released from metaphase. (B) Wild-type (SBY130) and ipl1–321 (SBY97) cells containing Tub1–GFP were released from α-factor (T = 0) into the restrictive temperature (37°C). Time points were taken at 60, 70, and 80 min after release and monitored for the presence or absence of a spindle as in A. In wild-type cells, 78% of the spindles have depolymerized compared with only 40% of ip1l-321 mutant spindles. The bars represent the 95% confidence interval. (C) Cells from A were taken every 5 min, and Clb2p and Tub1p (loading control) protein levels were monitored by immunoblotting. Clb2p levels decline with similar kinetics in both strains, indicating that ipl1–321 mutant cells exit mitosis normally. (D) ipl1–321 mutants have hyperstable microtubules. cdc26Δ mcd1–1 (SBY965) and cdc26Δ mcd1–1 ipl1–321 (SBY2066) cells were arrested in G1 using α-factor then released into the cell cycle at the restrictive temperature (37°C). Cells were monitored for budding index, spindle formation, and spindle breakdown. 93% of cdc26Δ mcd1–1 mutant cells underwent spindle disassembly by 150 min compared with only 48% of cdc26Δ mcd1–1 ipl1–321 cells.
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fig4: Ipl1p's role in spindle disassembly is independent from its role in chromosome segregation. (A) pGAL-CDC20 (SBY952) and pGAL-CDC20 ipl1–321 (SBY943) cells containing Tub1–GFP were shifted to glucose to arrest cells in metaphase and then shifted to 37°C to inactivate Ipl1–321p. Cells were then released into galactose medium at 37°C to restore Cdc20 protein synthesis in the presence of α-factor to arrest cells in the following G1. The percentage of cells with a pole to pole distance ≥9 μm were monitored for the presence or absence of a spindle. Spindle disassembly occurred in 68% of pGAL-CDC20 cells compared with 36% of pGAL-CDC20 ipl1–321 mutant cells released from metaphase. (B) Wild-type (SBY130) and ipl1–321 (SBY97) cells containing Tub1–GFP were released from α-factor (T = 0) into the restrictive temperature (37°C). Time points were taken at 60, 70, and 80 min after release and monitored for the presence or absence of a spindle as in A. In wild-type cells, 78% of the spindles have depolymerized compared with only 40% of ip1l-321 mutant spindles. The bars represent the 95% confidence interval. (C) Cells from A were taken every 5 min, and Clb2p and Tub1p (loading control) protein levels were monitored by immunoblotting. Clb2p levels decline with similar kinetics in both strains, indicating that ipl1–321 mutant cells exit mitosis normally. (D) ipl1–321 mutants have hyperstable microtubules. cdc26Δ mcd1–1 (SBY965) and cdc26Δ mcd1–1 ipl1–321 (SBY2066) cells were arrested in G1 using α-factor then released into the cell cycle at the restrictive temperature (37°C). Cells were monitored for budding index, spindle formation, and spindle breakdown. 93% of cdc26Δ mcd1–1 mutant cells underwent spindle disassembly by 150 min compared with only 48% of cdc26Δ mcd1–1 ipl1–321 cells.

Mentions: Since Ipl1p is required for chromosome segregation and the spindle checkpoint, we tested whether the spindle disassembly delay was a consequence of defects in these functions. We showed previously that if bipolar spindle assembly occurs before Ipl1p inactivation, chromosome segregation is normal (Biggins and Murray, 2001). Therefore, we arrested cells containing Tub1–GFP in metaphase by depleting the Cdc20 protein, shifted the cells to the restrictive temperature to inactivate Ipl1p, and then released them into the cell cycle. Aliquots were taken every 5 min, and cells with a pole to pole distance corresponding to a late anaphase cell (equal or greater than 9 μm) were analyzed for the presence or absence of a spindle. Since tubulin is always at the SPB, the pole to pole distance can be measured regardless of whether a spindle is present. Spindle disassembly occurred in 68% of Cdc20-depleted cells compared with only 36% of the Cdc20-depleted ipl1–321 double mutant cells (Fig. 4 A). In addition, there was no defect in spindle elongation in either strain. This result suggests that the spindle breakdown defect in ipl1–321 mutants is independent from Ipl1p's role in chromosome segregation.


The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly.

Buvelot S, Tatsutani SY, Vermaak D, Biggins S - J. Cell Biol. (2003)

Ipl1p's role in spindle disassembly is independent from its role in chromosome segregation. (A) pGAL-CDC20 (SBY952) and pGAL-CDC20 ipl1–321 (SBY943) cells containing Tub1–GFP were shifted to glucose to arrest cells in metaphase and then shifted to 37°C to inactivate Ipl1–321p. Cells were then released into galactose medium at 37°C to restore Cdc20 protein synthesis in the presence of α-factor to arrest cells in the following G1. The percentage of cells with a pole to pole distance ≥9 μm were monitored for the presence or absence of a spindle. Spindle disassembly occurred in 68% of pGAL-CDC20 cells compared with 36% of pGAL-CDC20 ipl1–321 mutant cells released from metaphase. (B) Wild-type (SBY130) and ipl1–321 (SBY97) cells containing Tub1–GFP were released from α-factor (T = 0) into the restrictive temperature (37°C). Time points were taken at 60, 70, and 80 min after release and monitored for the presence or absence of a spindle as in A. In wild-type cells, 78% of the spindles have depolymerized compared with only 40% of ip1l-321 mutant spindles. The bars represent the 95% confidence interval. (C) Cells from A were taken every 5 min, and Clb2p and Tub1p (loading control) protein levels were monitored by immunoblotting. Clb2p levels decline with similar kinetics in both strains, indicating that ipl1–321 mutant cells exit mitosis normally. (D) ipl1–321 mutants have hyperstable microtubules. cdc26Δ mcd1–1 (SBY965) and cdc26Δ mcd1–1 ipl1–321 (SBY2066) cells were arrested in G1 using α-factor then released into the cell cycle at the restrictive temperature (37°C). Cells were monitored for budding index, spindle formation, and spindle breakdown. 93% of cdc26Δ mcd1–1 mutant cells underwent spindle disassembly by 150 min compared with only 48% of cdc26Δ mcd1–1 ipl1–321 cells.
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Related In: Results  -  Collection

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fig4: Ipl1p's role in spindle disassembly is independent from its role in chromosome segregation. (A) pGAL-CDC20 (SBY952) and pGAL-CDC20 ipl1–321 (SBY943) cells containing Tub1–GFP were shifted to glucose to arrest cells in metaphase and then shifted to 37°C to inactivate Ipl1–321p. Cells were then released into galactose medium at 37°C to restore Cdc20 protein synthesis in the presence of α-factor to arrest cells in the following G1. The percentage of cells with a pole to pole distance ≥9 μm were monitored for the presence or absence of a spindle. Spindle disassembly occurred in 68% of pGAL-CDC20 cells compared with 36% of pGAL-CDC20 ipl1–321 mutant cells released from metaphase. (B) Wild-type (SBY130) and ipl1–321 (SBY97) cells containing Tub1–GFP were released from α-factor (T = 0) into the restrictive temperature (37°C). Time points were taken at 60, 70, and 80 min after release and monitored for the presence or absence of a spindle as in A. In wild-type cells, 78% of the spindles have depolymerized compared with only 40% of ip1l-321 mutant spindles. The bars represent the 95% confidence interval. (C) Cells from A were taken every 5 min, and Clb2p and Tub1p (loading control) protein levels were monitored by immunoblotting. Clb2p levels decline with similar kinetics in both strains, indicating that ipl1–321 mutant cells exit mitosis normally. (D) ipl1–321 mutants have hyperstable microtubules. cdc26Δ mcd1–1 (SBY965) and cdc26Δ mcd1–1 ipl1–321 (SBY2066) cells were arrested in G1 using α-factor then released into the cell cycle at the restrictive temperature (37°C). Cells were monitored for budding index, spindle formation, and spindle breakdown. 93% of cdc26Δ mcd1–1 mutant cells underwent spindle disassembly by 150 min compared with only 48% of cdc26Δ mcd1–1 ipl1–321 cells.
Mentions: Since Ipl1p is required for chromosome segregation and the spindle checkpoint, we tested whether the spindle disassembly delay was a consequence of defects in these functions. We showed previously that if bipolar spindle assembly occurs before Ipl1p inactivation, chromosome segregation is normal (Biggins and Murray, 2001). Therefore, we arrested cells containing Tub1–GFP in metaphase by depleting the Cdc20 protein, shifted the cells to the restrictive temperature to inactivate Ipl1p, and then released them into the cell cycle. Aliquots were taken every 5 min, and cells with a pole to pole distance corresponding to a late anaphase cell (equal or greater than 9 μm) were analyzed for the presence or absence of a spindle. Since tubulin is always at the SPB, the pole to pole distance can be measured regardless of whether a spindle is present. Spindle disassembly occurred in 68% of Cdc20-depleted cells compared with only 36% of the Cdc20-depleted ipl1–321 double mutant cells (Fig. 4 A). In addition, there was no defect in spindle elongation in either strain. This result suggests that the spindle breakdown defect in ipl1–321 mutants is independent from Ipl1p's role in chromosome segregation.

Bottom Line: As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules.Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly.We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

View Article: PubMed Central - PubMed

Affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1-GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

Show MeSH
Related in: MedlinePlus