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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 kinase activity increases before spindle disassembly. (A) Ip1lp was immunoprecipitated from a wild-type (SBY3) and a ipl1–321 mutant strain (SBY322) and then incubated with the histone-fold domain of the Cse4 kinetochore protein in a kinase reaction in vitro. The majority of Ipl1p present in the lysates before the immunoprecipitation (pre) was removed (post), and similar amounts of protein were used in the kinase assay (IP). The autoradiogram (right) shows that Cse4p is radiolabeled in the presence of wild-type Ipl1p but not Ipl1–321 mutant protein. (B) pGAL-CDC20 cells expressing Tub1–GFP (SBY952) were synchronized in metaphase by growth in glucose for 3 h. They were then released into galactose medium, and aliquots were taken every 10 min, and kinase assays were performed with the substrate Cse4p in vitro. The autoradiogram shows the phosphate incorporated into Cse4p in one experiment. (C) Microscopy was performed to determine the percent budding (▪) and the percent spindle disassembly (○). The total Ipl1p kinase activity is shown in gray bars (arbitrary units) for three experiments with the standard deviation indicated. Ipl1p kinase activity increases before spindle breakdown.
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fig5: Ipl1p kinase activity increases before spindle disassembly. (A) Ip1lp was immunoprecipitated from a wild-type (SBY3) and a ipl1–321 mutant strain (SBY322) and then incubated with the histone-fold domain of the Cse4 kinetochore protein in a kinase reaction in vitro. The majority of Ipl1p present in the lysates before the immunoprecipitation (pre) was removed (post), and similar amounts of protein were used in the kinase assay (IP). The autoradiogram (right) shows that Cse4p is radiolabeled in the presence of wild-type Ipl1p but not Ipl1–321 mutant protein. (B) pGAL-CDC20 cells expressing Tub1–GFP (SBY952) were synchronized in metaphase by growth in glucose for 3 h. They were then released into galactose medium, and aliquots were taken every 10 min, and kinase assays were performed with the substrate Cse4p in vitro. The autoradiogram shows the phosphate incorporated into Cse4p in one experiment. (C) Microscopy was performed to determine the percent budding (▪) and the percent spindle disassembly (○). The total Ipl1p kinase activity is shown in gray bars (arbitrary units) for three experiments with the standard deviation indicated. Ipl1p kinase activity increases before spindle breakdown.

Mentions: To analyze Ipl1p kinase activity when spindles disassemble, we developed a kinase assay using antibodies generated against a recombinant GST-Ipl1 fusion protein. The affinity-purified antibodies specifically recognize a single major band in yeast lysates that migrates just above 45 kD (Fig. S3 available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1). The antibodies were used to immunoprecipitate wild-type Ipl1p and the Ipl1–321 protein that has reduced kinase activity at high temperatures (Biggins et al., 1999). The majority of Ipl1p present in the yeast lysates (Fig. 5 A, pre) was depleted by the antibody (Fig. 5 A, post). The immunoprecipitates (Fig. 5 A, IP) were then incubated with the histone-fold domain of the kinetochore protein Cse4 in a kinase reaction in vitro. Cse4p was radiolabeled in the presence of wild-type Ipl1p but not the kinase inactive Ipl1–321 protein (Fig. 5 A, right), showing that the assay specifically reflects an Ipl1p-associated kinase activity.


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

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

Ipl1p kinase activity increases before spindle disassembly. (A) Ip1lp was immunoprecipitated from a wild-type (SBY3) and a ipl1–321 mutant strain (SBY322) and then incubated with the histone-fold domain of the Cse4 kinetochore protein in a kinase reaction in vitro. The majority of Ipl1p present in the lysates before the immunoprecipitation (pre) was removed (post), and similar amounts of protein were used in the kinase assay (IP). The autoradiogram (right) shows that Cse4p is radiolabeled in the presence of wild-type Ipl1p but not Ipl1–321 mutant protein. (B) pGAL-CDC20 cells expressing Tub1–GFP (SBY952) were synchronized in metaphase by growth in glucose for 3 h. They were then released into galactose medium, and aliquots were taken every 10 min, and kinase assays were performed with the substrate Cse4p in vitro. The autoradiogram shows the phosphate incorporated into Cse4p in one experiment. (C) Microscopy was performed to determine the percent budding (▪) and the percent spindle disassembly (○). The total Ipl1p kinase activity is shown in gray bars (arbitrary units) for three experiments with the standard deviation indicated. Ipl1p kinase activity increases before spindle breakdown.
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Related In: Results  -  Collection

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fig5: Ipl1p kinase activity increases before spindle disassembly. (A) Ip1lp was immunoprecipitated from a wild-type (SBY3) and a ipl1–321 mutant strain (SBY322) and then incubated with the histone-fold domain of the Cse4 kinetochore protein in a kinase reaction in vitro. The majority of Ipl1p present in the lysates before the immunoprecipitation (pre) was removed (post), and similar amounts of protein were used in the kinase assay (IP). The autoradiogram (right) shows that Cse4p is radiolabeled in the presence of wild-type Ipl1p but not Ipl1–321 mutant protein. (B) pGAL-CDC20 cells expressing Tub1–GFP (SBY952) were synchronized in metaphase by growth in glucose for 3 h. They were then released into galactose medium, and aliquots were taken every 10 min, and kinase assays were performed with the substrate Cse4p in vitro. The autoradiogram shows the phosphate incorporated into Cse4p in one experiment. (C) Microscopy was performed to determine the percent budding (▪) and the percent spindle disassembly (○). The total Ipl1p kinase activity is shown in gray bars (arbitrary units) for three experiments with the standard deviation indicated. Ipl1p kinase activity increases before spindle breakdown.
Mentions: To analyze Ipl1p kinase activity when spindles disassemble, we developed a kinase assay using antibodies generated against a recombinant GST-Ipl1 fusion protein. The affinity-purified antibodies specifically recognize a single major band in yeast lysates that migrates just above 45 kD (Fig. S3 available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1). The antibodies were used to immunoprecipitate wild-type Ipl1p and the Ipl1–321 protein that has reduced kinase activity at high temperatures (Biggins et al., 1999). The majority of Ipl1p present in the yeast lysates (Fig. 5 A, pre) was depleted by the antibody (Fig. 5 A, post). The immunoprecipitates (Fig. 5 A, IP) were then incubated with the histone-fold domain of the kinetochore protein Cse4 in a kinase reaction in vitro. Cse4p was radiolabeled in the presence of wild-type Ipl1p but not the kinase inactive Ipl1–321 protein (Fig. 5 A, right), showing that the assay specifically reflects an Ipl1p-associated kinase activity.

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