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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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ZM447439-treated cells enter mitosis, but fail to divide. HeLa cells were released from G1/S into various drug combinations, harvested at the times indicated in hours, and then analyzed by flow cytometry and immunoblotting. The data shown are representative of three independent experiments. The data in A and B are from the same experiment where the mitotic index peaked 10 h after release from G1/S. The data in C are from an independent experiment where the mitotic index peaked 12 h after release from G1/S. (A) DNA content histograms showing that ZM447439-treated cells fail to divide, but re-replicate their genomes with similar kinetics to the control cells. (B) Graph plotting mitotic index, as determined by MPM-2 reactivity, against time showing that ZM447439-treated cells enter and exit mitosis with similar kinetics to the controls. (C) Immunoblots showing that cyclin B1 levels rise and fall in ZM447439-treated cells with similar kinetics to the controls. Tubulin is used as a loading control.
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fig3: ZM447439-treated cells enter mitosis, but fail to divide. HeLa cells were released from G1/S into various drug combinations, harvested at the times indicated in hours, and then analyzed by flow cytometry and immunoblotting. The data shown are representative of three independent experiments. The data in A and B are from the same experiment where the mitotic index peaked 10 h after release from G1/S. The data in C are from an independent experiment where the mitotic index peaked 12 h after release from G1/S. (A) DNA content histograms showing that ZM447439-treated cells fail to divide, but re-replicate their genomes with similar kinetics to the control cells. (B) Graph plotting mitotic index, as determined by MPM-2 reactivity, against time showing that ZM447439-treated cells enter and exit mitosis with similar kinetics to the controls. (C) Immunoblots showing that cyclin B1 levels rise and fall in ZM447439-treated cells with similar kinetics to the controls. Tubulin is used as a loading control.

Mentions: The observation that ZM447439-treated cells can enter additional S phases without dividing raises two possibilities: either the cells re-replicate their genomes without entering mitosis, or alternatively, the cells enter and exit mitosis without dividing and then enter a second S phase. To distinguish between these two possibilities, we analyzed HeLa cells after release from a G1/S block into fresh medium or media supplemented with either ZM447439 or nocodazole, a spindle toxin that prevents microtubule polymerization (Fig. 3) . At various times after G1/S, the cells were harvested to determine DNA content, mitotic index, and cyclin B1 levels. DNA content histograms show that the vast majority of control cells divided by 12 h then entered a second S phase such that by 18 h, the majority had DNA contents greater than 2N (Fig. 3 A). Consistently, the mitotic index peaked at 10 h and cyclin B1 levels decreased as the cells completed mitosis (Fig. 3, B and C). Nocodazole-treated cells entered mitosis, and then remained arrested with high cyclin B1 levels for the remainder of the experiment. Cells released into ZM447439 progressed through S phase, failed to divide, degraded cyclin B1 normally, and then entered a second S phase (Fig. 3, A and C). Significantly, the kinetics with which the mitotic index increased and decreased was very similar to the control culture (Fig. 3 B). Thus, in the presence of ZM447439, HeLa cells enter and exit mitosis normally, but fail to divide. A549 and HME cells exhibited a similar response (unpublished data).


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)

ZM447439-treated cells enter mitosis, but fail to divide. HeLa cells were released from G1/S into various drug combinations, harvested at the times indicated in hours, and then analyzed by flow cytometry and immunoblotting. The data shown are representative of three independent experiments. The data in A and B are from the same experiment where the mitotic index peaked 10 h after release from G1/S. The data in C are from an independent experiment where the mitotic index peaked 12 h after release from G1/S. (A) DNA content histograms showing that ZM447439-treated cells fail to divide, but re-replicate their genomes with similar kinetics to the control cells. (B) Graph plotting mitotic index, as determined by MPM-2 reactivity, against time showing that ZM447439-treated cells enter and exit mitosis with similar kinetics to the controls. (C) Immunoblots showing that cyclin B1 levels rise and fall in ZM447439-treated cells with similar kinetics to the controls. Tubulin is used as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172902&req=5

fig3: ZM447439-treated cells enter mitosis, but fail to divide. HeLa cells were released from G1/S into various drug combinations, harvested at the times indicated in hours, and then analyzed by flow cytometry and immunoblotting. The data shown are representative of three independent experiments. The data in A and B are from the same experiment where the mitotic index peaked 10 h after release from G1/S. The data in C are from an independent experiment where the mitotic index peaked 12 h after release from G1/S. (A) DNA content histograms showing that ZM447439-treated cells fail to divide, but re-replicate their genomes with similar kinetics to the control cells. (B) Graph plotting mitotic index, as determined by MPM-2 reactivity, against time showing that ZM447439-treated cells enter and exit mitosis with similar kinetics to the controls. (C) Immunoblots showing that cyclin B1 levels rise and fall in ZM447439-treated cells with similar kinetics to the controls. Tubulin is used as a loading control.
Mentions: The observation that ZM447439-treated cells can enter additional S phases without dividing raises two possibilities: either the cells re-replicate their genomes without entering mitosis, or alternatively, the cells enter and exit mitosis without dividing and then enter a second S phase. To distinguish between these two possibilities, we analyzed HeLa cells after release from a G1/S block into fresh medium or media supplemented with either ZM447439 or nocodazole, a spindle toxin that prevents microtubule polymerization (Fig. 3) . At various times after G1/S, the cells were harvested to determine DNA content, mitotic index, and cyclin B1 levels. DNA content histograms show that the vast majority of control cells divided by 12 h then entered a second S phase such that by 18 h, the majority had DNA contents greater than 2N (Fig. 3 A). Consistently, the mitotic index peaked at 10 h and cyclin B1 levels decreased as the cells completed mitosis (Fig. 3, B and C). Nocodazole-treated cells entered mitosis, and then remained arrested with high cyclin B1 levels for the remainder of the experiment. Cells released into ZM447439 progressed through S phase, failed to divide, degraded cyclin B1 normally, and then entered a second S phase (Fig. 3, A and C). Significantly, the kinetics with which the mitotic index increased and decreased was very similar to the control culture (Fig. 3 B). Thus, in the presence of ZM447439, HeLa cells enter and exit mitosis normally, but fail to divide. A549 and HME cells exhibited a similar response (unpublished data).

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