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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 activity is required for kinetochore maintenance in CSF extracts. (A) Kinetochore maintenance was assessed after hesperadin treatment in extracts containing nuclei with preassembled kinetochores. The relative intensity of Ndc80 to Cenp-A immunostaining on the same kinetochore was determined and graphed (bottom; n = 60 individual kinetochores on four nuclei per condition). Graphs show mean plus standard deviation. (B) The addition of anti–Aurora B antibodies caused preassembled kinetochores to disassemble in an identical manner. (C) A panel of antibodies was used to assess the sensitivity of outer kinetochore proteins to hesperadin treatment in extracts with preassembled kinetochores. The outer kinetochore proteins Dsn1, Knl1, Ndc80, and Zwint and the checkpoint proteins BubR1, Mad1, and Mad2 are displaced after hesperadin treatment. The inner kinetochore proteins Cenp-A and Cenp-C are unaffected. Bars, 5 μm.
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fig3: Aurora B activity is required for kinetochore maintenance in CSF extracts. (A) Kinetochore maintenance was assessed after hesperadin treatment in extracts containing nuclei with preassembled kinetochores. The relative intensity of Ndc80 to Cenp-A immunostaining on the same kinetochore was determined and graphed (bottom; n = 60 individual kinetochores on four nuclei per condition). Graphs show mean plus standard deviation. (B) The addition of anti–Aurora B antibodies caused preassembled kinetochores to disassemble in an identical manner. (C) A panel of antibodies was used to assess the sensitivity of outer kinetochore proteins to hesperadin treatment in extracts with preassembled kinetochores. The outer kinetochore proteins Dsn1, Knl1, Ndc80, and Zwint and the checkpoint proteins BubR1, Mad1, and Mad2 are displaced after hesperadin treatment. The inner kinetochore proteins Cenp-A and Cenp-C are unaffected. Bars, 5 μm.

Mentions: We next tested if continuous Aurora B activity is required to maintain assembly of the kinetochore. In vitro egg extracts are well suited for this because they allow us to temporally control Aurora B activity. We preassembled kinetochores on sperm chromatin in extracts for 30 min and then added hesperadin (Fig. 3 A, top). Reactions were fixed at 5-min intervals and immunostained with Ndc80 antibodies (Fig. 3 A). The ratio of Ndc80 to Cenp-A staining on the same centromeres was calculated as described previously (Ditchfield et al., 2003). Ndc80 staining was reduced >90% after 5 min and >95% after 10 min (Fig. 3 A). Identical effects were seen after adding anti–Aurora B (Fig. 3 B) or anti-INCENP (not depicted) antibodies instead of hesperadin. Therefore, Aurora B is required to both establish and maintain the Ndc80 protein at centromeres.


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 activity is required for kinetochore maintenance in CSF extracts. (A) Kinetochore maintenance was assessed after hesperadin treatment in extracts containing nuclei with preassembled kinetochores. The relative intensity of Ndc80 to Cenp-A immunostaining on the same kinetochore was determined and graphed (bottom; n = 60 individual kinetochores on four nuclei per condition). Graphs show mean plus standard deviation. (B) The addition of anti–Aurora B antibodies caused preassembled kinetochores to disassemble in an identical manner. (C) A panel of antibodies was used to assess the sensitivity of outer kinetochore proteins to hesperadin treatment in extracts with preassembled kinetochores. The outer kinetochore proteins Dsn1, Knl1, Ndc80, and Zwint and the checkpoint proteins BubR1, Mad1, and Mad2 are displaced after hesperadin treatment. The inner kinetochore proteins Cenp-A and Cenp-C are unaffected. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Aurora B activity is required for kinetochore maintenance in CSF extracts. (A) Kinetochore maintenance was assessed after hesperadin treatment in extracts containing nuclei with preassembled kinetochores. The relative intensity of Ndc80 to Cenp-A immunostaining on the same kinetochore was determined and graphed (bottom; n = 60 individual kinetochores on four nuclei per condition). Graphs show mean plus standard deviation. (B) The addition of anti–Aurora B antibodies caused preassembled kinetochores to disassemble in an identical manner. (C) A panel of antibodies was used to assess the sensitivity of outer kinetochore proteins to hesperadin treatment in extracts with preassembled kinetochores. The outer kinetochore proteins Dsn1, Knl1, Ndc80, and Zwint and the checkpoint proteins BubR1, Mad1, and Mad2 are displaced after hesperadin treatment. The inner kinetochore proteins Cenp-A and Cenp-C are unaffected. Bars, 5 μm.
Mentions: We next tested if continuous Aurora B activity is required to maintain assembly of the kinetochore. In vitro egg extracts are well suited for this because they allow us to temporally control Aurora B activity. We preassembled kinetochores on sperm chromatin in extracts for 30 min and then added hesperadin (Fig. 3 A, top). Reactions were fixed at 5-min intervals and immunostained with Ndc80 antibodies (Fig. 3 A). The ratio of Ndc80 to Cenp-A staining on the same centromeres was calculated as described previously (Ditchfield et al., 2003). Ndc80 staining was reduced >90% after 5 min and >95% after 10 min (Fig. 3 A). Identical effects were seen after adding anti–Aurora B (Fig. 3 B) or anti-INCENP (not depicted) antibodies instead of hesperadin. Therefore, Aurora B is required to both establish and maintain the Ndc80 protein at centromeres.

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