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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.

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Aurora B function is required at the time of microtubule attachment to kinetochores. (A and C) HeLa cells were arrested in nocodazole. Hesperadin or the solvent DMSO were added shortly before release from nocodazole (outlined in A). Chromosome spreads were performed with the arrested cells after addition of Hesperadin or DMSO (noc-arrest), directly after washing out nocodazole (0'), and at different time points after release from nocodazole (15–150'). Representative images are shown in C. For a quantification, see Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1). (B and D) HeLa cells were arrested in nocodazole in the presence of Hesperadin or the solvent DMSO. Hesperadin and DMSO were washed out before releasing cells from nocodazole (outlined in B). Chromosome spreads were performed with the arrested cells after Hesperadin or DMSO washout (noc-arrest), directly after washing out nocodazole (0'), and at different time points after release from nocodazole (15–150'). Representative images are shown in D. For a quantification, see Fig. S2.
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fig5: Aurora B function is required at the time of microtubule attachment to kinetochores. (A and C) HeLa cells were arrested in nocodazole. Hesperadin or the solvent DMSO were added shortly before release from nocodazole (outlined in A). Chromosome spreads were performed with the arrested cells after addition of Hesperadin or DMSO (noc-arrest), directly after washing out nocodazole (0'), and at different time points after release from nocodazole (15–150'). Representative images are shown in C. For a quantification, see Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1). (B and D) HeLa cells were arrested in nocodazole in the presence of Hesperadin or the solvent DMSO. Hesperadin and DMSO were washed out before releasing cells from nocodazole (outlined in B). Chromosome spreads were performed with the arrested cells after Hesperadin or DMSO washout (noc-arrest), directly after washing out nocodazole (0'), and at different time points after release from nocodazole (15–150'). Representative images are shown in D. For a quantification, see Fig. S2.

Mentions: To test if Hesperadin's inhibitory effect on sister chromatid resolution in prometaphase could be responsible for the observed chromosome alignment defect, e.g., by preventing complete resolution of sister kinetochores, we analyzed when in mitosis Hesperadin addition causes chromosome segregation defects. Cells were arrested by nocodazole, and Hesperadin was only added shortly before releasing cells from nocodazole so that cells progressed through prometaphase in the absence of Hesperadin (Fig. 5 A). In this case, the resolution of sister chromatids was not affected, but the cells nevertheless exited mitosis with the same chromosome alignment and segregation defects as described above (Fig. 5 C; see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1). In contrast, cells exited mitosis without abnormalities in anaphase or telophase (Fig. 5 D) and with similar kinetics as controls (Fig. S2) when Hesperadin was washed out before the cells were released from nocodazole (Fig. 5 B), i.e., when spindles assembled in the absence of Hesperadin. These observations indicate that the defect in chromosome segregation induced by Hesperadin can be specifically ascribed to the inhibition of Aurora B function during the process of chromosome attachment.


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)

Aurora B function is required at the time of microtubule attachment to kinetochores. (A and C) HeLa cells were arrested in nocodazole. Hesperadin or the solvent DMSO were added shortly before release from nocodazole (outlined in A). Chromosome spreads were performed with the arrested cells after addition of Hesperadin or DMSO (noc-arrest), directly after washing out nocodazole (0'), and at different time points after release from nocodazole (15–150'). Representative images are shown in C. For a quantification, see Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1). (B and D) HeLa cells were arrested in nocodazole in the presence of Hesperadin or the solvent DMSO. Hesperadin and DMSO were washed out before releasing cells from nocodazole (outlined in B). Chromosome spreads were performed with the arrested cells after Hesperadin or DMSO washout (noc-arrest), directly after washing out nocodazole (0'), and at different time points after release from nocodazole (15–150'). Representative images are shown in D. For a quantification, see Fig. S2.
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Related In: Results  -  Collection

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fig5: Aurora B function is required at the time of microtubule attachment to kinetochores. (A and C) HeLa cells were arrested in nocodazole. Hesperadin or the solvent DMSO were added shortly before release from nocodazole (outlined in A). Chromosome spreads were performed with the arrested cells after addition of Hesperadin or DMSO (noc-arrest), directly after washing out nocodazole (0'), and at different time points after release from nocodazole (15–150'). Representative images are shown in C. For a quantification, see Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1). (B and D) HeLa cells were arrested in nocodazole in the presence of Hesperadin or the solvent DMSO. Hesperadin and DMSO were washed out before releasing cells from nocodazole (outlined in B). Chromosome spreads were performed with the arrested cells after Hesperadin or DMSO washout (noc-arrest), directly after washing out nocodazole (0'), and at different time points after release from nocodazole (15–150'). Representative images are shown in D. For a quantification, see Fig. S2.
Mentions: To test if Hesperadin's inhibitory effect on sister chromatid resolution in prometaphase could be responsible for the observed chromosome alignment defect, e.g., by preventing complete resolution of sister kinetochores, we analyzed when in mitosis Hesperadin addition causes chromosome segregation defects. Cells were arrested by nocodazole, and Hesperadin was only added shortly before releasing cells from nocodazole so that cells progressed through prometaphase in the absence of Hesperadin (Fig. 5 A). In this case, the resolution of sister chromatids was not affected, but the cells nevertheless exited mitosis with the same chromosome alignment and segregation defects as described above (Fig. 5 C; see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1). In contrast, cells exited mitosis without abnormalities in anaphase or telophase (Fig. 5 D) and with similar kinetics as controls (Fig. S2) when Hesperadin was washed out before the cells were released from nocodazole (Fig. 5 B), i.e., when spindles assembled in the absence of Hesperadin. These observations indicate that the defect in chromosome segregation induced by Hesperadin can be specifically ascribed to the inhibition of Aurora B function during the process of chromosome attachment.

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