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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 prevents chromosome alignment and segregation. (A) Mitotic DLD-1 cells stained for tubulin, kinetochores/centromeres (ACA, green), and DNA (red). Bottom panels show examples of abnormal prometaphases frequently observed after treatment with ZM447439 for 1 h. (B) Graph quantitating the number of prophase (P), prometaphase (PM), metaphase (M), and anaphase (A) cells after exposure of DLD-1 cells to ZM447439 for 1 h, showing that chromosome alignment and segregation are inhibited. (C) Graph showing the mitotic index of DLD-1 cultures at the times indicated after treatment with either ZM447439 alone (red diamonds), nocodazole (yellow squares), or ZM447439 plus nocodazole (blue triangles). Values in B and C represent the mean and SEM derived from three independent experiments in which at least 1,000 cells were counted. (D) Electron micrographs of ZM447439-treated HeLa cells showing kinetochores (arrows) and kinetochore fibers.
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fig4: ZM447439 prevents chromosome alignment and segregation. (A) Mitotic DLD-1 cells stained for tubulin, kinetochores/centromeres (ACA, green), and DNA (red). Bottom panels show examples of abnormal prometaphases frequently observed after treatment with ZM447439 for 1 h. (B) Graph quantitating the number of prophase (P), prometaphase (PM), metaphase (M), and anaphase (A) cells after exposure of DLD-1 cells to ZM447439 for 1 h, showing that chromosome alignment and segregation are inhibited. (C) Graph showing the mitotic index of DLD-1 cultures at the times indicated after treatment with either ZM447439 alone (red diamonds), nocodazole (yellow squares), or ZM447439 plus nocodazole (blue triangles). Values in B and C represent the mean and SEM derived from three independent experiments in which at least 1,000 cells were counted. (D) Electron micrographs of ZM447439-treated HeLa cells showing kinetochores (arrows) and kinetochore fibers.

Mentions: The observation that ZM447439-treated cells enter and exit mitosis but fail to divide raises two possibilities: either chromosome segregation takes place normally but cytokinesis fails, or alternatively, chromosome segregation fails, preventing cytokinesis. To determine whether ZM447439 prevents chromosome segregation, we analyzed spindle morphology in ZM447439-treated cultures. In controls, prometaphase rosettes, and metaphase and anaphase spindles were readily apparent (Fig. 4, A and B) . In ZM447439-treated cultures, bipolar spindles were observed and the proportion of cells in prophase appeared normal. However, the proportion of metaphase and anaphase spindles was markedly reduced, indicating that ZM447439 inhibits chromosome segregation (Fig. 4 B). Despite the lack of anaphases, cells treated with ZM447439 alone did not accumulate in mitosis (Fig. 4 C). Cells treated with ZM447439 and nocodazole did however accumulate in mitosis, confirming that ZM447439-treated cells enter and exit mitosis without undergoing chromosome segregation. Chromosome alignment also appeared abnormal in the presence of ZM447439. In particular, the chromosomes either splayed out throughout the cell or, rather than aligning at the spindle equator, lined up along the length of the spindle (Fig. 4 A). In these latter cases, the kinetochores were oriented toward the spindle, suggesting that kinetochore–microtubule interactions were taking place. Indeed, kinetochores and kinetochore fibers were observed in thin sections analyzed by electron microscopy (Fig. 4 D). In 19 control cells, fibers containing multiple microtubules were observed in 12 cases. Eight of these could be traced to kinetochores. In 20 ZM447439-treated cells, fibers containing multiple microtubules were observed in 15 cases, and six were traced to kinetochores. Although we cannot rule out the possibility that ZM447439 effects kinetochore structure and/or microtubule binding capacity, ZM447439 clearly does not prevent kinetochore–microtubule interactions or bipolar spindle formation. However, ZM447439 does inhibit chromosome alignment and segregation. To determine whether ZM447439 prevents sister chromatid separation, we allowed cells to pass through mitosis in the presence of ZM447439, and then analyzed chromosome spreads in the following mitosis. Diplochromosomes were never observed (unpublished data), suggesting that ZM447439 does not prevent loss of sister chromatid cohesion. It is unclear whether ZM447439 directly inhibits cytokinesis or whether cytokinesis is prevented as a secondary consequence of the block to chromosome segregation.


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 prevents chromosome alignment and segregation. (A) Mitotic DLD-1 cells stained for tubulin, kinetochores/centromeres (ACA, green), and DNA (red). Bottom panels show examples of abnormal prometaphases frequently observed after treatment with ZM447439 for 1 h. (B) Graph quantitating the number of prophase (P), prometaphase (PM), metaphase (M), and anaphase (A) cells after exposure of DLD-1 cells to ZM447439 for 1 h, showing that chromosome alignment and segregation are inhibited. (C) Graph showing the mitotic index of DLD-1 cultures at the times indicated after treatment with either ZM447439 alone (red diamonds), nocodazole (yellow squares), or ZM447439 plus nocodazole (blue triangles). Values in B and C represent the mean and SEM derived from three independent experiments in which at least 1,000 cells were counted. (D) Electron micrographs of ZM447439-treated HeLa cells showing kinetochores (arrows) and kinetochore fibers.
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Related In: Results  -  Collection

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fig4: ZM447439 prevents chromosome alignment and segregation. (A) Mitotic DLD-1 cells stained for tubulin, kinetochores/centromeres (ACA, green), and DNA (red). Bottom panels show examples of abnormal prometaphases frequently observed after treatment with ZM447439 for 1 h. (B) Graph quantitating the number of prophase (P), prometaphase (PM), metaphase (M), and anaphase (A) cells after exposure of DLD-1 cells to ZM447439 for 1 h, showing that chromosome alignment and segregation are inhibited. (C) Graph showing the mitotic index of DLD-1 cultures at the times indicated after treatment with either ZM447439 alone (red diamonds), nocodazole (yellow squares), or ZM447439 plus nocodazole (blue triangles). Values in B and C represent the mean and SEM derived from three independent experiments in which at least 1,000 cells were counted. (D) Electron micrographs of ZM447439-treated HeLa cells showing kinetochores (arrows) and kinetochore fibers.
Mentions: The observation that ZM447439-treated cells enter and exit mitosis but fail to divide raises two possibilities: either chromosome segregation takes place normally but cytokinesis fails, or alternatively, chromosome segregation fails, preventing cytokinesis. To determine whether ZM447439 prevents chromosome segregation, we analyzed spindle morphology in ZM447439-treated cultures. In controls, prometaphase rosettes, and metaphase and anaphase spindles were readily apparent (Fig. 4, A and B) . In ZM447439-treated cultures, bipolar spindles were observed and the proportion of cells in prophase appeared normal. However, the proportion of metaphase and anaphase spindles was markedly reduced, indicating that ZM447439 inhibits chromosome segregation (Fig. 4 B). Despite the lack of anaphases, cells treated with ZM447439 alone did not accumulate in mitosis (Fig. 4 C). Cells treated with ZM447439 and nocodazole did however accumulate in mitosis, confirming that ZM447439-treated cells enter and exit mitosis without undergoing chromosome segregation. Chromosome alignment also appeared abnormal in the presence of ZM447439. In particular, the chromosomes either splayed out throughout the cell or, rather than aligning at the spindle equator, lined up along the length of the spindle (Fig. 4 A). In these latter cases, the kinetochores were oriented toward the spindle, suggesting that kinetochore–microtubule interactions were taking place. Indeed, kinetochores and kinetochore fibers were observed in thin sections analyzed by electron microscopy (Fig. 4 D). In 19 control cells, fibers containing multiple microtubules were observed in 12 cases. Eight of these could be traced to kinetochores. In 20 ZM447439-treated cells, fibers containing multiple microtubules were observed in 15 cases, and six were traced to kinetochores. Although we cannot rule out the possibility that ZM447439 effects kinetochore structure and/or microtubule binding capacity, ZM447439 clearly does not prevent kinetochore–microtubule interactions or bipolar spindle formation. However, ZM447439 does inhibit chromosome alignment and segregation. To determine whether ZM447439 prevents sister chromatid separation, we allowed cells to pass through mitosis in the presence of ZM447439, and then analyzed chromosome spreads in the following mitosis. Diplochromosomes were never observed (unpublished data), suggesting that ZM447439 does not prevent loss of sister chromatid cohesion. It is unclear whether ZM447439 directly inhibits cytokinesis or whether cytokinesis is prevented as a secondary consequence of the block to chromosome segregation.

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