Limits...
The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint.

Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, Heckel A, van Meel J, Rieder CL, Peters JM - J. Cell Biol. (2003)

Bottom Line: We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation.Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B.Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria.

ABSTRACT
The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling.

Show MeSH

Related in: MedlinePlus

BubR1 localization to kinetochores is abolished in HeLa cells treated with nocodazole and Hesperadin. (A) HeLa cells were arrested in mitosis with 10 μM nocodazole and then additionally treated with 100 nM Hesperadin or the solvent DMSO for 2 h, harvested by mitotic shake off, cytospun on slides, and processed for immunofluorescence with the indicated antibodies (in green). Kinetochores were labeled with CREST serum (in blue). The insets show magnifications of the kinetochore pairs marked by white rectangles. (B) Data, as in A, were quantified. For each cell, the average integrated intensity for the checkpoint protein was related to the average integrated intensity of the CREST signal. Bars show the average of the ratios obtained from 10 cells. The reduction of BubR1 signal intensity was significant in both independent experiments (1 and 2) (P < 0.0001, t test), whereas the reduction of Bub1 signal intensity was only significant in experiment 1 (experiment 1, P < 0.0001; experiment 2, P = 0.059). (C) Average of the two independent experiments shown in B, with the control data set to 100%. (D) Model for Aurora B function.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172906&req=5

fig9: BubR1 localization to kinetochores is abolished in HeLa cells treated with nocodazole and Hesperadin. (A) HeLa cells were arrested in mitosis with 10 μM nocodazole and then additionally treated with 100 nM Hesperadin or the solvent DMSO for 2 h, harvested by mitotic shake off, cytospun on slides, and processed for immunofluorescence with the indicated antibodies (in green). Kinetochores were labeled with CREST serum (in blue). The insets show magnifications of the kinetochore pairs marked by white rectangles. (B) Data, as in A, were quantified. For each cell, the average integrated intensity for the checkpoint protein was related to the average integrated intensity of the CREST signal. Bars show the average of the ratios obtained from 10 cells. The reduction of BubR1 signal intensity was significant in both independent experiments (1 and 2) (P < 0.0001, t test), whereas the reduction of Bub1 signal intensity was only significant in experiment 1 (experiment 1, P < 0.0001; experiment 2, P = 0.059). (C) Average of the two independent experiments shown in B, with the control data set to 100%. (D) Model for Aurora B function.

Mentions: Hesperadin might induce mitotic exit in taxol- or monastrol-treated cells by stabilizing improper microtubule attachments. However, it is possible that Aurora B also has a direct role in the spindle assembly checkpoint, as even under conditions where none of the kinetochores are attached (Fig. 8, nocodazole), cells that are additionally treated with Hesperadin exit mitosis precociously (Fig. 8 D). Recruitment of checkpoint proteins to unattached kinetochores is thought to be necessary to maintain checkpoint signaling (Shah and Cleveland, 2000). We therefore tested whether inhibition of Aurora B function might impair this recruitment. We found that in the presence of nocodazole and Hesperadin, Mad2 and the motor protein CENP-E were still present at kinetochores. In contrast, kinetochore localization of BubR1 was abolished, and the intensity of Bub1 at kinetochores was diminished (Fig. 9 , A–C). Similar results were obtained in logarithmically growing HeLa cells (see Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1), where after addition of Hesperadin, Mad2 and CENP-E could be observed at kinetochores in early prometaphase, whereas BubR1 and Bub1 did not localize to kinetochores at any stage of mitosis. Together, these data suggest that Aurora B function is required for efficient kinetochore recruitment of BubR1 and Bub1, which in turn might be necessary for prolonged checkpoint signaling.


The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint.

Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, Heckel A, van Meel J, Rieder CL, Peters JM - J. Cell Biol. (2003)

BubR1 localization to kinetochores is abolished in HeLa cells treated with nocodazole and Hesperadin. (A) HeLa cells were arrested in mitosis with 10 μM nocodazole and then additionally treated with 100 nM Hesperadin or the solvent DMSO for 2 h, harvested by mitotic shake off, cytospun on slides, and processed for immunofluorescence with the indicated antibodies (in green). Kinetochores were labeled with CREST serum (in blue). The insets show magnifications of the kinetochore pairs marked by white rectangles. (B) Data, as in A, were quantified. For each cell, the average integrated intensity for the checkpoint protein was related to the average integrated intensity of the CREST signal. Bars show the average of the ratios obtained from 10 cells. The reduction of BubR1 signal intensity was significant in both independent experiments (1 and 2) (P < 0.0001, t test), whereas the reduction of Bub1 signal intensity was only significant in experiment 1 (experiment 1, P < 0.0001; experiment 2, P = 0.059). (C) Average of the two independent experiments shown in B, with the control data set to 100%. (D) Model for Aurora B function.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172906&req=5

fig9: BubR1 localization to kinetochores is abolished in HeLa cells treated with nocodazole and Hesperadin. (A) HeLa cells were arrested in mitosis with 10 μM nocodazole and then additionally treated with 100 nM Hesperadin or the solvent DMSO for 2 h, harvested by mitotic shake off, cytospun on slides, and processed for immunofluorescence with the indicated antibodies (in green). Kinetochores were labeled with CREST serum (in blue). The insets show magnifications of the kinetochore pairs marked by white rectangles. (B) Data, as in A, were quantified. For each cell, the average integrated intensity for the checkpoint protein was related to the average integrated intensity of the CREST signal. Bars show the average of the ratios obtained from 10 cells. The reduction of BubR1 signal intensity was significant in both independent experiments (1 and 2) (P < 0.0001, t test), whereas the reduction of Bub1 signal intensity was only significant in experiment 1 (experiment 1, P < 0.0001; experiment 2, P = 0.059). (C) Average of the two independent experiments shown in B, with the control data set to 100%. (D) Model for Aurora B function.
Mentions: Hesperadin might induce mitotic exit in taxol- or monastrol-treated cells by stabilizing improper microtubule attachments. However, it is possible that Aurora B also has a direct role in the spindle assembly checkpoint, as even under conditions where none of the kinetochores are attached (Fig. 8, nocodazole), cells that are additionally treated with Hesperadin exit mitosis precociously (Fig. 8 D). Recruitment of checkpoint proteins to unattached kinetochores is thought to be necessary to maintain checkpoint signaling (Shah and Cleveland, 2000). We therefore tested whether inhibition of Aurora B function might impair this recruitment. We found that in the presence of nocodazole and Hesperadin, Mad2 and the motor protein CENP-E were still present at kinetochores. In contrast, kinetochore localization of BubR1 was abolished, and the intensity of Bub1 at kinetochores was diminished (Fig. 9 , A–C). Similar results were obtained in logarithmically growing HeLa cells (see Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1), where after addition of Hesperadin, Mad2 and CENP-E could be observed at kinetochores in early prometaphase, whereas BubR1 and Bub1 did not localize to kinetochores at any stage of mitosis. Together, these data suggest that Aurora B function is required for efficient kinetochore recruitment of BubR1 and Bub1, which in turn might be necessary for prolonged checkpoint signaling.

Bottom Line: We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation.Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B.Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria.

ABSTRACT
The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling.

Show MeSH
Related in: MedlinePlus