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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 BubR1 prevents chromosome alignment. DLD-1 cells were transfected with siRNA duplexes to repress BubR1 and were then stained to detect Bub1 (green), tubulin (red), and DNA (blue). (A) Examples of abnormal prometaphase cells with chromosomes aligned along the length of the spindle rather than along the metaphase plate. Bar graph shows that ∼40% of prometaphases appear abnormal with chromosomes aligned along the length of the spindle. (B) Plot of interkinetochore distances measured using Bub1 foci to determine kinetochore position. (C) Proportion of mitotic cells exhibiting either prometaphase or metaphase chromosome alignment after a 1-h exposure to MG132. Values represent the mean and SEM derived from three independent experiments in which at least 100 mitotic cells were counted.
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fig9: Repression of BubR1 prevents chromosome alignment. DLD-1 cells were transfected with siRNA duplexes to repress BubR1 and were then stained to detect Bub1 (green), tubulin (red), and DNA (blue). (A) Examples of abnormal prometaphase cells with chromosomes aligned along the length of the spindle rather than along the metaphase plate. Bar graph shows that ∼40% of prometaphases appear abnormal with chromosomes aligned along the length of the spindle. (B) Plot of interkinetochore distances measured using Bub1 foci to determine kinetochore position. (C) Proportion of mitotic cells exhibiting either prometaphase or metaphase chromosome alignment after a 1-h exposure to MG132. Values represent the mean and SEM derived from three independent experiments in which at least 100 mitotic cells were counted.

Mentions: Our observations are consistent with the notion that Aurora B kinase activity regulates the spindle checkpoint, at least in part, by targeting BubR1 to kinetochores. Because BubR1 binds Cenp-E (Chan et al., 1999; Yao et al., 2000), we reasoned that the requirement for Aurora B in promoting correct chromosome alignment might also be mediated, at least in part, via its affect on BubR1. To test this, we used RNAi to determine whether repression of BubR1 inhibited chromosome alignment. Consistent with antibody injection experiments (Chan et al., 1999), repression of BubR1 compromised spindle checkpoint function. In particular, in asynchronous cultures, the number of metaphases was reduced and the anaphases frequently displayed lagging chromosomes (Fig. S4). Furthermore, BubR1 RNAi cultures did not accumulate mitotic cells on exposure to spindle toxins (unpublished data). Strikingly, prometaphase cells in BubR1 RNAi cultures often appeared abnormal with the chromosomes aligned along the length of the spindle rather than at the metaphase plate (Fig. 9 A). Although these chromosomes appear to be attached to the spindle, the mean interkinetochore distance was reduced compared with control cells (Fig. 9 B), consistent with a reduction in pulling forces.


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 BubR1 prevents chromosome alignment. DLD-1 cells were transfected with siRNA duplexes to repress BubR1 and were then stained to detect Bub1 (green), tubulin (red), and DNA (blue). (A) Examples of abnormal prometaphase cells with chromosomes aligned along the length of the spindle rather than along the metaphase plate. Bar graph shows that ∼40% of prometaphases appear abnormal with chromosomes aligned along the length of the spindle. (B) Plot of interkinetochore distances measured using Bub1 foci to determine kinetochore position. (C) Proportion of mitotic cells exhibiting either prometaphase or metaphase chromosome alignment after a 1-h exposure to MG132. Values represent the mean and SEM derived from three independent experiments in which at least 100 mitotic cells were counted.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172902&req=5

fig9: Repression of BubR1 prevents chromosome alignment. DLD-1 cells were transfected with siRNA duplexes to repress BubR1 and were then stained to detect Bub1 (green), tubulin (red), and DNA (blue). (A) Examples of abnormal prometaphase cells with chromosomes aligned along the length of the spindle rather than along the metaphase plate. Bar graph shows that ∼40% of prometaphases appear abnormal with chromosomes aligned along the length of the spindle. (B) Plot of interkinetochore distances measured using Bub1 foci to determine kinetochore position. (C) Proportion of mitotic cells exhibiting either prometaphase or metaphase chromosome alignment after a 1-h exposure to MG132. Values represent the mean and SEM derived from three independent experiments in which at least 100 mitotic cells were counted.
Mentions: Our observations are consistent with the notion that Aurora B kinase activity regulates the spindle checkpoint, at least in part, by targeting BubR1 to kinetochores. Because BubR1 binds Cenp-E (Chan et al., 1999; Yao et al., 2000), we reasoned that the requirement for Aurora B in promoting correct chromosome alignment might also be mediated, at least in part, via its affect on BubR1. To test this, we used RNAi to determine whether repression of BubR1 inhibited chromosome alignment. Consistent with antibody injection experiments (Chan et al., 1999), repression of BubR1 compromised spindle checkpoint function. In particular, in asynchronous cultures, the number of metaphases was reduced and the anaphases frequently displayed lagging chromosomes (Fig. S4). Furthermore, BubR1 RNAi cultures did not accumulate mitotic cells on exposure to spindle toxins (unpublished data). Strikingly, prometaphase cells in BubR1 RNAi cultures often appeared abnormal with the chromosomes aligned along the length of the spindle rather than at the metaphase plate (Fig. 9 A). Although these chromosomes appear to be attached to the spindle, the mean interkinetochore distance was reduced compared with control cells (Fig. 9 B), consistent with a reduction in pulling forces.

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