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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 compromises spindle checkpoint function. (A–C) Mitotic HeLa cells were isolated by selective detachment after a 12-h nocodazole block, replated in various drugs combinations, and then harvested at the times indicated in hours and analyzed. (A) DNA content histograms showing that the control cells divide after 1 h. (B) Graph plotting the mitotic index, as determined by MPM-2 reactivity, showing that ZM447439 accelerates mitotic exit. (C) Immunoblots showing that cyclin B1 and phosphohistone H3 levels fall rapidly in ZM447439-treated cells. (D and E) DLD-1 cells were treated with drug combinations and were then fixed and analyzed by fluorescence microscopy. (D) Bar graph plotting the mitotic index after 6 h, showing that ZM447439 compromises paclitaxel- but not nocodazole-induced mitotic arrest. (E) Line graph plotting the mitotic index after 8 h, showing that ZM447439 resolves the effects of nocodazole and paclitaxel with respect to spindle checkpoint function. Values in D and E represent the mean and SEM derived from three independent experiments in which at least 1,000 cells were counted. Drug combinations used were nocodazole alone (Noc, yellow squares); paclitaxel alone (Tax); ZM447439 alone (ZM, red diamonds); nocodazole plus ZM447439 (NZ, blue triangles); paclitaxel plus ZM447439 (TZ, green squares); and no drug (Control, green circles). (F) Mitotic HeLa cells harvested after a 2- or 12-h nocodazole block were replated in nocodazole (black bar) or nocodazole plus ZM447439 (white bars), and the mitotic index was determined by measuring MPM-2 reactivity after 4 h. Bar graph shows that ZM447439 compromises nocodazole-induced checkpoint activation after prolonged mitotic arrest.
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fig5: ZM447439 compromises spindle checkpoint function. (A–C) Mitotic HeLa cells were isolated by selective detachment after a 12-h nocodazole block, replated in various drugs combinations, and then harvested at the times indicated in hours and analyzed. (A) DNA content histograms showing that the control cells divide after 1 h. (B) Graph plotting the mitotic index, as determined by MPM-2 reactivity, showing that ZM447439 accelerates mitotic exit. (C) Immunoblots showing that cyclin B1 and phosphohistone H3 levels fall rapidly in ZM447439-treated cells. (D and E) DLD-1 cells were treated with drug combinations and were then fixed and analyzed by fluorescence microscopy. (D) Bar graph plotting the mitotic index after 6 h, showing that ZM447439 compromises paclitaxel- but not nocodazole-induced mitotic arrest. (E) Line graph plotting the mitotic index after 8 h, showing that ZM447439 resolves the effects of nocodazole and paclitaxel with respect to spindle checkpoint function. Values in D and E represent the mean and SEM derived from three independent experiments in which at least 1,000 cells were counted. Drug combinations used were nocodazole alone (Noc, yellow squares); paclitaxel alone (Tax); ZM447439 alone (ZM, red diamonds); nocodazole plus ZM447439 (NZ, blue triangles); paclitaxel plus ZM447439 (TZ, green squares); and no drug (Control, green circles). (F) Mitotic HeLa cells harvested after a 2- or 12-h nocodazole block were replated in nocodazole (black bar) or nocodazole plus ZM447439 (white bars), and the mitotic index was determined by measuring MPM-2 reactivity after 4 h. Bar graph shows that ZM447439 compromises nocodazole-induced checkpoint activation after prolonged mitotic arrest.

Mentions: The observations described above present a paradox: chromosome alignment defects should activate the spindle checkpoint, and thus cause mitotic arrest. However, ZM447439-treated cells exit mitosis with normal kinetics (Fig. 3 B), suggesting that ZM447439 may compromise checkpoint function. To test this possibility, we analyzed the effect of ZM447439 on cells after release from a nocodazole block. Nocodazole-arrested mitotic cells were isolated by selective detachment, washed to remove the nocodazole, and were then replated in various drug combinations (Fig. 5 , A–C). At various times the cells were harvested to determine DNA content, mitotic index, and cyclin B1 levels. 2 h after release, the majority of the control cells had completed chromosome segregation and divided (Fig. 5 A). In contrast, ZM447439, nocodazole, and ZM447439 plus nocodazole-treated cells failed to divide (unpublished data). The mitotic index of the control cells was initially high, but then fell to 15% 2 h after release. In contrast, in the continued presence of nocodazole the cells remained arrested in mitosis (Fig. 5 B). Consistently, in control cells, cyclin B1 levels fell after 2 h but remained high in the presence of nocodazole (Fig. 5 C). Strikingly, the mitotic index of ZM447439-treated cells fell extremely rapidly: after 1 h, only 4% of the ZM447439-treated cells were still in mitosis, compared with 67% of the controls (Fig. 5 B). Consistently, cyclin B1 was undetectable after 1 h. Thus, ZM447439-treated cells rapidly exit mitosis, consistent with the notion that ZM447439 overrides the spindle checkpoint.


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 compromises spindle checkpoint function. (A–C) Mitotic HeLa cells were isolated by selective detachment after a 12-h nocodazole block, replated in various drugs combinations, and then harvested at the times indicated in hours and analyzed. (A) DNA content histograms showing that the control cells divide after 1 h. (B) Graph plotting the mitotic index, as determined by MPM-2 reactivity, showing that ZM447439 accelerates mitotic exit. (C) Immunoblots showing that cyclin B1 and phosphohistone H3 levels fall rapidly in ZM447439-treated cells. (D and E) DLD-1 cells were treated with drug combinations and were then fixed and analyzed by fluorescence microscopy. (D) Bar graph plotting the mitotic index after 6 h, showing that ZM447439 compromises paclitaxel- but not nocodazole-induced mitotic arrest. (E) Line graph plotting the mitotic index after 8 h, showing that ZM447439 resolves the effects of nocodazole and paclitaxel with respect to spindle checkpoint function. Values in D and E represent the mean and SEM derived from three independent experiments in which at least 1,000 cells were counted. Drug combinations used were nocodazole alone (Noc, yellow squares); paclitaxel alone (Tax); ZM447439 alone (ZM, red diamonds); nocodazole plus ZM447439 (NZ, blue triangles); paclitaxel plus ZM447439 (TZ, green squares); and no drug (Control, green circles). (F) Mitotic HeLa cells harvested after a 2- or 12-h nocodazole block were replated in nocodazole (black bar) or nocodazole plus ZM447439 (white bars), and the mitotic index was determined by measuring MPM-2 reactivity after 4 h. Bar graph shows that ZM447439 compromises nocodazole-induced checkpoint activation after prolonged mitotic arrest.
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Related In: Results  -  Collection

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fig5: ZM447439 compromises spindle checkpoint function. (A–C) Mitotic HeLa cells were isolated by selective detachment after a 12-h nocodazole block, replated in various drugs combinations, and then harvested at the times indicated in hours and analyzed. (A) DNA content histograms showing that the control cells divide after 1 h. (B) Graph plotting the mitotic index, as determined by MPM-2 reactivity, showing that ZM447439 accelerates mitotic exit. (C) Immunoblots showing that cyclin B1 and phosphohistone H3 levels fall rapidly in ZM447439-treated cells. (D and E) DLD-1 cells were treated with drug combinations and were then fixed and analyzed by fluorescence microscopy. (D) Bar graph plotting the mitotic index after 6 h, showing that ZM447439 compromises paclitaxel- but not nocodazole-induced mitotic arrest. (E) Line graph plotting the mitotic index after 8 h, showing that ZM447439 resolves the effects of nocodazole and paclitaxel with respect to spindle checkpoint function. Values in D and E represent the mean and SEM derived from three independent experiments in which at least 1,000 cells were counted. Drug combinations used were nocodazole alone (Noc, yellow squares); paclitaxel alone (Tax); ZM447439 alone (ZM, red diamonds); nocodazole plus ZM447439 (NZ, blue triangles); paclitaxel plus ZM447439 (TZ, green squares); and no drug (Control, green circles). (F) Mitotic HeLa cells harvested after a 2- or 12-h nocodazole block were replated in nocodazole (black bar) or nocodazole plus ZM447439 (white bars), and the mitotic index was determined by measuring MPM-2 reactivity after 4 h. Bar graph shows that ZM447439 compromises nocodazole-induced checkpoint activation after prolonged mitotic arrest.
Mentions: The observations described above present a paradox: chromosome alignment defects should activate the spindle checkpoint, and thus cause mitotic arrest. However, ZM447439-treated cells exit mitosis with normal kinetics (Fig. 3 B), suggesting that ZM447439 may compromise checkpoint function. To test this possibility, we analyzed the effect of ZM447439 on cells after release from a nocodazole block. Nocodazole-arrested mitotic cells were isolated by selective detachment, washed to remove the nocodazole, and were then replated in various drug combinations (Fig. 5 , A–C). At various times the cells were harvested to determine DNA content, mitotic index, and cyclin B1 levels. 2 h after release, the majority of the control cells had completed chromosome segregation and divided (Fig. 5 A). In contrast, ZM447439, nocodazole, and ZM447439 plus nocodazole-treated cells failed to divide (unpublished data). The mitotic index of the control cells was initially high, but then fell to 15% 2 h after release. In contrast, in the continued presence of nocodazole the cells remained arrested in mitosis (Fig. 5 B). Consistently, in control cells, cyclin B1 levels fell after 2 h but remained high in the presence of nocodazole (Fig. 5 C). Strikingly, the mitotic index of ZM447439-treated cells fell extremely rapidly: after 1 h, only 4% of the ZM447439-treated cells were still in mitosis, compared with 67% of the controls (Fig. 5 B). Consistently, cyclin B1 was undetectable after 1 h. Thus, ZM447439-treated cells rapidly exit mitosis, consistent with the notion that ZM447439 overrides the spindle checkpoint.

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