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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 RNAi in human cells induces a similar phenotype as Hesperadin. (A) HeLa cells were transfected with Aurora B–targeting siRNA duplex (RNAi AurB) or with H2O replacing the siRNA (control). After 50 h, cells were lysed in sample buffer. Samples were resolved by SDS-PAGE and immunoblotted. (B) Cells treated as in A were processed for immunofluorescence 48 h after transfection. Cells were costained for Aurora B (in green) and phospho-histone H3 (in red). DNA was stained with DAPI (left panel, right panel in blue, also in C and D). Chromosomes that have not aligned to the metaphase plate are indicated by arrowheads. (C and D) Cells treated as in A were processed for immunofluorescence 72 h after transfection. Cells were stained for α-tubulin (in green) and Survivin (C, in red). Cells treated with Aurora B siRNA duplex did not form a proper midspindle and showed segregation defects, indicated by an arrow.
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fig3: Aurora B RNAi in human cells induces a similar phenotype as Hesperadin. (A) HeLa cells were transfected with Aurora B–targeting siRNA duplex (RNAi AurB) or with H2O replacing the siRNA (control). After 50 h, cells were lysed in sample buffer. Samples were resolved by SDS-PAGE and immunoblotted. (B) Cells treated as in A were processed for immunofluorescence 48 h after transfection. Cells were costained for Aurora B (in green) and phospho-histone H3 (in red). DNA was stained with DAPI (left panel, right panel in blue, also in C and D). Chromosomes that have not aligned to the metaphase plate are indicated by arrowheads. (C and D) Cells treated as in A were processed for immunofluorescence 72 h after transfection. Cells were stained for α-tubulin (in green) and Survivin (C, in red). Cells treated with Aurora B siRNA duplex did not form a proper midspindle and showed segregation defects, indicated by an arrow.

Mentions: To further test if the phenotype induced by Hesperadin is due to the inhibition of Aurora B function, we used RNA interference (RNAi). In HeLa cells treated with small inhibitory RNAs (siRNAs) directed against Aurora B, the level of Aurora B protein was substantially reduced, as judged by both immunoblotting (Fig. 3 A) and immunofluorescence (Fig. 3 B), whereas Aurora A levels were affected only to a minor extent (Fig. 3 A). Immunofluorescence microscopy revealed that mitotic cells without detectable Aurora B staining frequently contained chromosomes that were not aligned on the spindle equator (Fig. 3 B) and phospho-histone H3 staining was often reduced or absent (Fig. 3 B). We also observed elongated cells, presumably undergoing anaphase, with some chromosomes that were located at opposite poles but others that were lagging (Fig. 3 C). α-Tubulin staining revealed that these cells did not form a midspindle structure (Fig. 3 C). Instead of being highly localized, as in control anaphase cells, Survivin was diffusely localized in the region between the poles (Fig. 3 C). siRNA cultures also contained enlarged cells with multiple nuclei and micronuclei (Fig. 3 D) that were likely the consequence of cytokinesis defects. Together, these observations reveal that human Aurora B is required for mitotic phosphorylation of histone H3, chromosome alignment, chromosome segregation, formation of a midspindle, and cytokinesis, consistent with earlier observations in C. elegans embryos and Drosophila melanogaster (for review see Adams et al., 2001a). Hesperadin-treated cells exhibit a very similar phenotype (Figs. 1 and 2), which is further evidence that Hesperadin inhibits Aurora B function in vivo. We therefore named our inhibitor Hesperadin in reference to the antique goddess of dusk, Hespera, who is the counterpart of Aurora, the goddess of dawn.


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 RNAi in human cells induces a similar phenotype as Hesperadin. (A) HeLa cells were transfected with Aurora B–targeting siRNA duplex (RNAi AurB) or with H2O replacing the siRNA (control). After 50 h, cells were lysed in sample buffer. Samples were resolved by SDS-PAGE and immunoblotted. (B) Cells treated as in A were processed for immunofluorescence 48 h after transfection. Cells were costained for Aurora B (in green) and phospho-histone H3 (in red). DNA was stained with DAPI (left panel, right panel in blue, also in C and D). Chromosomes that have not aligned to the metaphase plate are indicated by arrowheads. (C and D) Cells treated as in A were processed for immunofluorescence 72 h after transfection. Cells were stained for α-tubulin (in green) and Survivin (C, in red). Cells treated with Aurora B siRNA duplex did not form a proper midspindle and showed segregation defects, indicated by an arrow.
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Related In: Results  -  Collection

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fig3: Aurora B RNAi in human cells induces a similar phenotype as Hesperadin. (A) HeLa cells were transfected with Aurora B–targeting siRNA duplex (RNAi AurB) or with H2O replacing the siRNA (control). After 50 h, cells were lysed in sample buffer. Samples were resolved by SDS-PAGE and immunoblotted. (B) Cells treated as in A were processed for immunofluorescence 48 h after transfection. Cells were costained for Aurora B (in green) and phospho-histone H3 (in red). DNA was stained with DAPI (left panel, right panel in blue, also in C and D). Chromosomes that have not aligned to the metaphase plate are indicated by arrowheads. (C and D) Cells treated as in A were processed for immunofluorescence 72 h after transfection. Cells were stained for α-tubulin (in green) and Survivin (C, in red). Cells treated with Aurora B siRNA duplex did not form a proper midspindle and showed segregation defects, indicated by an arrow.
Mentions: To further test if the phenotype induced by Hesperadin is due to the inhibition of Aurora B function, we used RNA interference (RNAi). In HeLa cells treated with small inhibitory RNAs (siRNAs) directed against Aurora B, the level of Aurora B protein was substantially reduced, as judged by both immunoblotting (Fig. 3 A) and immunofluorescence (Fig. 3 B), whereas Aurora A levels were affected only to a minor extent (Fig. 3 A). Immunofluorescence microscopy revealed that mitotic cells without detectable Aurora B staining frequently contained chromosomes that were not aligned on the spindle equator (Fig. 3 B) and phospho-histone H3 staining was often reduced or absent (Fig. 3 B). We also observed elongated cells, presumably undergoing anaphase, with some chromosomes that were located at opposite poles but others that were lagging (Fig. 3 C). α-Tubulin staining revealed that these cells did not form a midspindle structure (Fig. 3 C). Instead of being highly localized, as in control anaphase cells, Survivin was diffusely localized in the region between the poles (Fig. 3 C). siRNA cultures also contained enlarged cells with multiple nuclei and micronuclei (Fig. 3 D) that were likely the consequence of cytokinesis defects. Together, these observations reveal that human Aurora B is required for mitotic phosphorylation of histone H3, chromosome alignment, chromosome segregation, formation of a midspindle, and cytokinesis, consistent with earlier observations in C. elegans embryos and Drosophila melanogaster (for review see Adams et al., 2001a). Hesperadin-treated cells exhibit a very similar phenotype (Figs. 1 and 2), which is further evidence that Hesperadin inhibits Aurora B function in vivo. We therefore named our inhibitor Hesperadin in reference to the antique goddess of dusk, Hespera, who is the counterpart of Aurora, the goddess of dawn.

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