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Aurora B kinase and protein phosphatase 1 have opposing roles in modulating kinetochore assembly.

Emanuele MJ, Lan W, Jwa M, Miller SA, Chan CS, Stukenberg PT - J. Cell Biol. (2008)

Bottom Line: PP1 is necessary for kinetochores to disassemble at the exit from M phase, and purified enzyme is sufficient to cause disassembly on isolated mitotic nuclei.These data demonstrate that Aurora B activity is required for kinetochore maintenance and that PP1 is necessary and sufficient to disassemble kinetochores.We suggest that Aurora B and PP1 coordinate cell cycle-dependent changes in kinetochore assembly though phosphorylation of kinetochore substrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT
The outer kinetochore binds microtubules to control chromosome movement. Outer kinetochore assembly is restricted to mitosis, whereas the inner kinetochore remains tethered to centromeres throughout the cell cycle. The cues that regulate this transient assembly are unknown. We find that inhibition of Aurora B kinase significantly reduces outer kinetochore assembly in Xenopus laevis and human tissue culture cells, frog egg extracts, and budding yeast. In X. leavis M phase extracts, preassembled kinetochores disassemble after inhibiting Aurora B activity with either drugs or antibodies. Kinetochore disassembly, induced by Aurora B inhibition, is rescued by restraining protein phosphatase 1 (PP1) activity. PP1 is necessary for kinetochores to disassemble at the exit from M phase, and purified enzyme is sufficient to cause disassembly on isolated mitotic nuclei. These data demonstrate that Aurora B activity is required for kinetochore maintenance and that PP1 is necessary and sufficient to disassemble kinetochores. We suggest that Aurora B and PP1 coordinate cell cycle-dependent changes in kinetochore assembly though phosphorylation of kinetochore substrates.

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Aurora B inhibition compromises kinetochore assembly in X. laevis and human tissue culture cells and in budding yeast. (A) Asynchronous X. laevis S3 cells were treated with hesperadin and fixed for immunofluorescence. Cells were immunostained with antibodies to Ndc80 and phosphorylated serine 10 on histone H3 (pH3S10). The graph shows levels of Ndc80 staining at kinetochores in control and hesperadin-treated cells (n = 60 kinetochores in three different cells per condition). (B) A549 human lung epithelial cells and primary human foreskin fibroblasts were treated with either DMSO or hesperadin and ZM447439 (Hesp + ZM) together. Cells were fixed and processed for immunofluorescence with ACA and Ndc80 antibodies. The amount of Ndc80 staining at kinetochores relative to ACA staining on the same kinetochore was determined and is graphed (n = 60 kinetochores in three cells per condition). Bars, = 5 μm. (C) Wild type or sli15-3 yeast cells were grown at a restrictive temperature of 37° for 3 h and kinetochore assembly was assessed by ChIP for Ndc80, Mtw1, Kip1, Bim1, Ipl1, Dam1, Cse4, and Ctf19. The outer kinetochore proteins are displaced from kinetochores in sli15-3 cells. IN and + indicate PCR performed on either total input or immunoprecipitated DNA, respectively. Centromeres on chromosome 16 (Cen16) were amplified. Graphs show mean plus standard deviation.
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fig1: Aurora B inhibition compromises kinetochore assembly in X. laevis and human tissue culture cells and in budding yeast. (A) Asynchronous X. laevis S3 cells were treated with hesperadin and fixed for immunofluorescence. Cells were immunostained with antibodies to Ndc80 and phosphorylated serine 10 on histone H3 (pH3S10). The graph shows levels of Ndc80 staining at kinetochores in control and hesperadin-treated cells (n = 60 kinetochores in three different cells per condition). (B) A549 human lung epithelial cells and primary human foreskin fibroblasts were treated with either DMSO or hesperadin and ZM447439 (Hesp + ZM) together. Cells were fixed and processed for immunofluorescence with ACA and Ndc80 antibodies. The amount of Ndc80 staining at kinetochores relative to ACA staining on the same kinetochore was determined and is graphed (n = 60 kinetochores in three cells per condition). Bars, = 5 μm. (C) Wild type or sli15-3 yeast cells were grown at a restrictive temperature of 37° for 3 h and kinetochore assembly was assessed by ChIP for Ndc80, Mtw1, Kip1, Bim1, Ipl1, Dam1, Cse4, and Ctf19. The outer kinetochore proteins are displaced from kinetochores in sli15-3 cells. IN and + indicate PCR performed on either total input or immunoprecipitated DNA, respectively. Centromeres on chromosome 16 (Cen16) were amplified. Graphs show mean plus standard deviation.

Mentions: To examine the role of Aurora B kinase in kinetochore assembly, X. laevis–derived S3 tissue culture cells were treated with the Aurora kinase inhibitor hesperadin and prepared for immunofluorescence. Because many proteins are removed from kinetochores after microtubule attachment, we added nocodazole 15 min before fixing cells. Hesperadin treatment depleted pH3S10 staining on chromatin, indicating the effectiveness of the drug treatment (Fig. 1 A). In DMSO controls, 90% of mitotic cells that had progressed beyond prophase had Ndc80 kinetochore staining on chromatin. After treatment with 250 nM hesperadin, 49% of cells had visibly undetectable levels of Ndc80 and 47% showed strongly reduced Ndc80 staining. Quantification of immunofluorescent intensity at centromeres shows that Ndc80 kinetochore staining was reduced 76% in cells treated with 250 nM hesperadin (Fig. 1 A, right). Immunostaining with antibodies to Zwilch and the dynactin subunit p150Glued produced similar results (unpublished data). These experiments also revealed that Zwilch and Ndc80 appear at kinetochores after Aurora B has been activated, which is indicated by costaining for pH3S10 (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200710019/DC1). This suggests that Aurora B kinase activity is required for the localization of Ndc80, Zwilch, and dynactin at kinetochores in X. laevis tissue culture cells.


Aurora B kinase and protein phosphatase 1 have opposing roles in modulating kinetochore assembly.

Emanuele MJ, Lan W, Jwa M, Miller SA, Chan CS, Stukenberg PT - J. Cell Biol. (2008)

Aurora B inhibition compromises kinetochore assembly in X. laevis and human tissue culture cells and in budding yeast. (A) Asynchronous X. laevis S3 cells were treated with hesperadin and fixed for immunofluorescence. Cells were immunostained with antibodies to Ndc80 and phosphorylated serine 10 on histone H3 (pH3S10). The graph shows levels of Ndc80 staining at kinetochores in control and hesperadin-treated cells (n = 60 kinetochores in three different cells per condition). (B) A549 human lung epithelial cells and primary human foreskin fibroblasts were treated with either DMSO or hesperadin and ZM447439 (Hesp + ZM) together. Cells were fixed and processed for immunofluorescence with ACA and Ndc80 antibodies. The amount of Ndc80 staining at kinetochores relative to ACA staining on the same kinetochore was determined and is graphed (n = 60 kinetochores in three cells per condition). Bars, = 5 μm. (C) Wild type or sli15-3 yeast cells were grown at a restrictive temperature of 37° for 3 h and kinetochore assembly was assessed by ChIP for Ndc80, Mtw1, Kip1, Bim1, Ipl1, Dam1, Cse4, and Ctf19. The outer kinetochore proteins are displaced from kinetochores in sli15-3 cells. IN and + indicate PCR performed on either total input or immunoprecipitated DNA, respectively. Centromeres on chromosome 16 (Cen16) were amplified. Graphs show mean plus standard deviation.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2315672&req=5

fig1: Aurora B inhibition compromises kinetochore assembly in X. laevis and human tissue culture cells and in budding yeast. (A) Asynchronous X. laevis S3 cells were treated with hesperadin and fixed for immunofluorescence. Cells were immunostained with antibodies to Ndc80 and phosphorylated serine 10 on histone H3 (pH3S10). The graph shows levels of Ndc80 staining at kinetochores in control and hesperadin-treated cells (n = 60 kinetochores in three different cells per condition). (B) A549 human lung epithelial cells and primary human foreskin fibroblasts were treated with either DMSO or hesperadin and ZM447439 (Hesp + ZM) together. Cells were fixed and processed for immunofluorescence with ACA and Ndc80 antibodies. The amount of Ndc80 staining at kinetochores relative to ACA staining on the same kinetochore was determined and is graphed (n = 60 kinetochores in three cells per condition). Bars, = 5 μm. (C) Wild type or sli15-3 yeast cells were grown at a restrictive temperature of 37° for 3 h and kinetochore assembly was assessed by ChIP for Ndc80, Mtw1, Kip1, Bim1, Ipl1, Dam1, Cse4, and Ctf19. The outer kinetochore proteins are displaced from kinetochores in sli15-3 cells. IN and + indicate PCR performed on either total input or immunoprecipitated DNA, respectively. Centromeres on chromosome 16 (Cen16) were amplified. Graphs show mean plus standard deviation.
Mentions: To examine the role of Aurora B kinase in kinetochore assembly, X. laevis–derived S3 tissue culture cells were treated with the Aurora kinase inhibitor hesperadin and prepared for immunofluorescence. Because many proteins are removed from kinetochores after microtubule attachment, we added nocodazole 15 min before fixing cells. Hesperadin treatment depleted pH3S10 staining on chromatin, indicating the effectiveness of the drug treatment (Fig. 1 A). In DMSO controls, 90% of mitotic cells that had progressed beyond prophase had Ndc80 kinetochore staining on chromatin. After treatment with 250 nM hesperadin, 49% of cells had visibly undetectable levels of Ndc80 and 47% showed strongly reduced Ndc80 staining. Quantification of immunofluorescent intensity at centromeres shows that Ndc80 kinetochore staining was reduced 76% in cells treated with 250 nM hesperadin (Fig. 1 A, right). Immunostaining with antibodies to Zwilch and the dynactin subunit p150Glued produced similar results (unpublished data). These experiments also revealed that Zwilch and Ndc80 appear at kinetochores after Aurora B has been activated, which is indicated by costaining for pH3S10 (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200710019/DC1). This suggests that Aurora B kinase activity is required for the localization of Ndc80, Zwilch, and dynactin at kinetochores in X. laevis tissue culture cells.

Bottom Line: PP1 is necessary for kinetochores to disassemble at the exit from M phase, and purified enzyme is sufficient to cause disassembly on isolated mitotic nuclei.These data demonstrate that Aurora B activity is required for kinetochore maintenance and that PP1 is necessary and sufficient to disassemble kinetochores.We suggest that Aurora B and PP1 coordinate cell cycle-dependent changes in kinetochore assembly though phosphorylation of kinetochore substrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT
The outer kinetochore binds microtubules to control chromosome movement. Outer kinetochore assembly is restricted to mitosis, whereas the inner kinetochore remains tethered to centromeres throughout the cell cycle. The cues that regulate this transient assembly are unknown. We find that inhibition of Aurora B kinase significantly reduces outer kinetochore assembly in Xenopus laevis and human tissue culture cells, frog egg extracts, and budding yeast. In X. leavis M phase extracts, preassembled kinetochores disassemble after inhibiting Aurora B activity with either drugs or antibodies. Kinetochore disassembly, induced by Aurora B inhibition, is rescued by restraining protein phosphatase 1 (PP1) activity. PP1 is necessary for kinetochores to disassemble at the exit from M phase, and purified enzyme is sufficient to cause disassembly on isolated mitotic nuclei. These data demonstrate that Aurora B activity is required for kinetochore maintenance and that PP1 is necessary and sufficient to disassemble kinetochores. We suggest that Aurora B and PP1 coordinate cell cycle-dependent changes in kinetochore assembly though phosphorylation of kinetochore substrates.

Show MeSH
Related in: MedlinePlus