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Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores.

Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T, Mortlock A, Keen N, Taylor SS - J. Cell Biol. (2003)

Bottom Line: Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension.Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment.Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Rd., Manchester M13 9PT, UK.

ABSTRACT
The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

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Repression of Aurora B prevents kinetochore localization of BubR1. HeLa and DLD-1 cells were transfected with siRNA duplexes to repress either Aurora A or B. (A) Immunoblots of HeLa cell lysates showing repression of Aurora A and B. (B) Transfected DLD-1 cells were exposed to spindle toxins, and the mitotic index was measured over time. Values represent the mean and SEM from three independent experiments in which at least 1,000 cells were counted. (C) Projected deconvolved image stacks of nocodazole-treated mitotic DLD-1 cells after transfection of siRNAs targeting Aurora A (top) and Aurora B (bottom). (D) Examples of spindle morphology in control and Aurora B RNAi cultures. (E) Plot of interkinetochore distance in control and Aurora B–repressed cells.
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fig8: Repression of Aurora B prevents kinetochore localization of BubR1. HeLa and DLD-1 cells were transfected with siRNA duplexes to repress either Aurora A or B. (A) Immunoblots of HeLa cell lysates showing repression of Aurora A and B. (B) Transfected DLD-1 cells were exposed to spindle toxins, and the mitotic index was measured over time. Values represent the mean and SEM from three independent experiments in which at least 1,000 cells were counted. (C) Projected deconvolved image stacks of nocodazole-treated mitotic DLD-1 cells after transfection of siRNAs targeting Aurora A (top) and Aurora B (bottom). (D) Examples of spindle morphology in control and Aurora B RNAi cultures. (E) Plot of interkinetochore distance in control and Aurora B–repressed cells.

Mentions: The ZM447439 data suggest that Aurora kinase activity is required for chromosome alignment and spindle checkpoint function. To determine which Aurora is required for these functions, and to rule out the possibility that the ZM447439 phenotypes might be due to inhibition of another kinase, we repressed Aurora A and B by RNAi (Fig. 8 A). Although control and Aurora A RNAi cultures had robust spindle checkpoints, repression of Aurora B reduced the accumulation of mitotic cells after spindle damage (Fig. 8 B). Repression of Aurora B (but not Aurora A) inhibited kinetochore localization of BubR1, Cenp-E, and Mad2 (Fig. 8 C and Fig. S3). Thus, these observations indicate that the ZM447439-induced phenotypes described above are due to inhibition of Aurora B, not Aurora A or some other kinase.


Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores.

Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T, Mortlock A, Keen N, Taylor SS - J. Cell Biol. (2003)

Repression of Aurora B prevents kinetochore localization of BubR1. HeLa and DLD-1 cells were transfected with siRNA duplexes to repress either Aurora A or B. (A) Immunoblots of HeLa cell lysates showing repression of Aurora A and B. (B) Transfected DLD-1 cells were exposed to spindle toxins, and the mitotic index was measured over time. Values represent the mean and SEM from three independent experiments in which at least 1,000 cells were counted. (C) Projected deconvolved image stacks of nocodazole-treated mitotic DLD-1 cells after transfection of siRNAs targeting Aurora A (top) and Aurora B (bottom). (D) Examples of spindle morphology in control and Aurora B RNAi cultures. (E) Plot of interkinetochore distance in control and Aurora B–repressed cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172902&req=5

fig8: Repression of Aurora B prevents kinetochore localization of BubR1. HeLa and DLD-1 cells were transfected with siRNA duplexes to repress either Aurora A or B. (A) Immunoblots of HeLa cell lysates showing repression of Aurora A and B. (B) Transfected DLD-1 cells were exposed to spindle toxins, and the mitotic index was measured over time. Values represent the mean and SEM from three independent experiments in which at least 1,000 cells were counted. (C) Projected deconvolved image stacks of nocodazole-treated mitotic DLD-1 cells after transfection of siRNAs targeting Aurora A (top) and Aurora B (bottom). (D) Examples of spindle morphology in control and Aurora B RNAi cultures. (E) Plot of interkinetochore distance in control and Aurora B–repressed cells.
Mentions: The ZM447439 data suggest that Aurora kinase activity is required for chromosome alignment and spindle checkpoint function. To determine which Aurora is required for these functions, and to rule out the possibility that the ZM447439 phenotypes might be due to inhibition of another kinase, we repressed Aurora A and B by RNAi (Fig. 8 A). Although control and Aurora A RNAi cultures had robust spindle checkpoints, repression of Aurora B reduced the accumulation of mitotic cells after spindle damage (Fig. 8 B). Repression of Aurora B (but not Aurora A) inhibited kinetochore localization of BubR1, Cenp-E, and Mad2 (Fig. 8 C and Fig. S3). Thus, these observations indicate that the ZM447439-induced phenotypes described above are due to inhibition of Aurora B, not Aurora A or some other kinase.

Bottom Line: Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension.Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment.Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Rd., Manchester M13 9PT, UK.

ABSTRACT
The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

Show MeSH
Related in: MedlinePlus