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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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Hesperadin inhibits histone H3 phosphorylation and causes midspindle defects. (A) Samples from the same experiment as in Fig. 1 (C and D) were analyzed by SDS-PAGE and immunoblotting with anti–phospho-histone H3 antibody. (B) HeLa cells were treated with Hesperadin or DMSO (control) for 16 h and immunostained with anti–phospho-histone H3. DNA was stained with DAPI. Chromosomes that have not aligned at the metaphase plate are indicated by arrowheads. (C) HeLa cells were treated as in B. Cells were costained with α-tubulin and either CYK-4 or MKLP-1 antibodies. DNA was stained with DAPI. In Hesperadin-treated cells, chromatin is not segregated properly in anaphase, indicated by arrows. (D) Cdk1 or human Aurora B were immunoprecipitated from mitotic HeLa cell extracts, and in vitro kinase activity was determined using histone H1 or histone H3, respectively, as a substrate. Hesperadin was added to the kinase reactions at the indicated concentrations. As a control, 0.1% DMSO was added (corresponding to the DMSO concentration in the sample containing 5,000 nM Hesperadin). Further controls included a kinase reaction without substrate as well as mock immunoprecipitates by unspecific antibodies.
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fig2: Hesperadin inhibits histone H3 phosphorylation and causes midspindle defects. (A) Samples from the same experiment as in Fig. 1 (C and D) were analyzed by SDS-PAGE and immunoblotting with anti–phospho-histone H3 antibody. (B) HeLa cells were treated with Hesperadin or DMSO (control) for 16 h and immunostained with anti–phospho-histone H3. DNA was stained with DAPI. Chromosomes that have not aligned at the metaphase plate are indicated by arrowheads. (C) HeLa cells were treated as in B. Cells were costained with α-tubulin and either CYK-4 or MKLP-1 antibodies. DNA was stained with DAPI. In Hesperadin-treated cells, chromatin is not segregated properly in anaphase, indicated by arrows. (D) Cdk1 or human Aurora B were immunoprecipitated from mitotic HeLa cell extracts, and in vitro kinase activity was determined using histone H1 or histone H3, respectively, as a substrate. Hesperadin was added to the kinase reactions at the indicated concentrations. As a control, 0.1% DMSO was added (corresponding to the DMSO concentration in the sample containing 5,000 nM Hesperadin). Further controls included a kinase reaction without substrate as well as mock immunoprecipitates by unspecific antibodies.

Mentions: As a mitotic marker, we also analyzed phosphorylation of serine 10 on histone H3 in the same experiment. We found that immunoblotting yielded a phospho-histone H3 signal in mitotic Hesperadin-treated cells that was greatly reduced relative to controls (Fig. 2 A). Immunofluorescence microscopy confirmed this result (Fig. 2 B). Because mitotic H3-Ser10 phosphorylation depends on Aurora B (Adams et al., 2001a), Hesperadin appears to inhibit Aurora B function.


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)

Hesperadin inhibits histone H3 phosphorylation and causes midspindle defects. (A) Samples from the same experiment as in Fig. 1 (C and D) were analyzed by SDS-PAGE and immunoblotting with anti–phospho-histone H3 antibody. (B) HeLa cells were treated with Hesperadin or DMSO (control) for 16 h and immunostained with anti–phospho-histone H3. DNA was stained with DAPI. Chromosomes that have not aligned at the metaphase plate are indicated by arrowheads. (C) HeLa cells were treated as in B. Cells were costained with α-tubulin and either CYK-4 or MKLP-1 antibodies. DNA was stained with DAPI. In Hesperadin-treated cells, chromatin is not segregated properly in anaphase, indicated by arrows. (D) Cdk1 or human Aurora B were immunoprecipitated from mitotic HeLa cell extracts, and in vitro kinase activity was determined using histone H1 or histone H3, respectively, as a substrate. Hesperadin was added to the kinase reactions at the indicated concentrations. As a control, 0.1% DMSO was added (corresponding to the DMSO concentration in the sample containing 5,000 nM Hesperadin). Further controls included a kinase reaction without substrate as well as mock immunoprecipitates by unspecific antibodies.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Hesperadin inhibits histone H3 phosphorylation and causes midspindle defects. (A) Samples from the same experiment as in Fig. 1 (C and D) were analyzed by SDS-PAGE and immunoblotting with anti–phospho-histone H3 antibody. (B) HeLa cells were treated with Hesperadin or DMSO (control) for 16 h and immunostained with anti–phospho-histone H3. DNA was stained with DAPI. Chromosomes that have not aligned at the metaphase plate are indicated by arrowheads. (C) HeLa cells were treated as in B. Cells were costained with α-tubulin and either CYK-4 or MKLP-1 antibodies. DNA was stained with DAPI. In Hesperadin-treated cells, chromatin is not segregated properly in anaphase, indicated by arrows. (D) Cdk1 or human Aurora B were immunoprecipitated from mitotic HeLa cell extracts, and in vitro kinase activity was determined using histone H1 or histone H3, respectively, as a substrate. Hesperadin was added to the kinase reactions at the indicated concentrations. As a control, 0.1% DMSO was added (corresponding to the DMSO concentration in the sample containing 5,000 nM Hesperadin). Further controls included a kinase reaction without substrate as well as mock immunoprecipitates by unspecific antibodies.
Mentions: As a mitotic marker, we also analyzed phosphorylation of serine 10 on histone H3 in the same experiment. We found that immunoblotting yielded a phospho-histone H3 signal in mitotic Hesperadin-treated cells that was greatly reduced relative to controls (Fig. 2 A). Immunofluorescence microscopy confirmed this result (Fig. 2 B). Because mitotic H3-Ser10 phosphorylation depends on Aurora B (Adams et al., 2001a), Hesperadin appears to inhibit Aurora B function.

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