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Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase activity.

Sharp-Baker H, Chen RH - J. Cell Biol. (2001)

Bottom Line: Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro.Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins.Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic spindle. We have isolated a Xenopus homologue of the spindle checkpoint component Bub1, and investigated its role in the spindle checkpoint in Xenopus egg extracts. Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro. Bub1 is essential for the establishment and maintenance of the checkpoint and is localized to kinetochores, similar to the spindle checkpoint complex Mad1-Mad2. However, Bub1 differs from Mad1-Mad2 in that Bub1 remains on kinetochores that have attached to microtubules; the protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins. Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.

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Bub1 and Bub1K872R restore kinetochore binding of Mad1, Mad2, Bub3, and CENP-E in Bub1-depleted extract. Sperm nuclei and nocodazole were incubated with mock-depleted extract or Bub1-depleted extract supplemented with mock, Bub1, or Bub1K872R translation as indicated on the top. Isolated chromosomes were incubated with rabbit antibodies against Mad1, Mad2, Bub3, or CENP-E as indicated on the left, followed by fluorescein-conjugated anti–rabbit antibody. Bub1 was then detected with biotinylated anti-Bub1 antibody and Texas red–conjugated streptavidin. For space conservation, pictures of Bub1 are shown only once for each type of extract. The merge pictures contain all three fluorochromes. Bar, 10 μm.
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Figure 8: Bub1 and Bub1K872R restore kinetochore binding of Mad1, Mad2, Bub3, and CENP-E in Bub1-depleted extract. Sperm nuclei and nocodazole were incubated with mock-depleted extract or Bub1-depleted extract supplemented with mock, Bub1, or Bub1K872R translation as indicated on the top. Isolated chromosomes were incubated with rabbit antibodies against Mad1, Mad2, Bub3, or CENP-E as indicated on the left, followed by fluorescein-conjugated anti–rabbit antibody. Bub1 was then detected with biotinylated anti-Bub1 antibody and Texas red–conjugated streptavidin. For space conservation, pictures of Bub1 are shown only once for each type of extract. The merge pictures contain all three fluorochromes. Bar, 10 μm.

Mentions: For immunofluorescent staining of unreplicated chromosomes, 20 μl of egg extract was incubated with sperm nuclei (1,000/μl of extract) at 23°C for 10 min, followed by addition of nocodazole to 10 μg/ml, and incubation for another 20 min at 23°C to disrupt microtubules. The replicated chromosomes and the in vitro metaphase to anaphase transition were performed as described (Chen et al. 1998). The chromosomes were isolated and processed for immunofluorescent staining as described (Chen et al. 1998). The anti–CENP-E antibody was provided by Dr. T. Yen (Fox Chase Cancer Center, Philadelphia, PA). For results shown in Fig. 8, the coverslips were first incubated with rabbit antibodies against Mad1, Mad2, Bub3, or CENP-E, followed by fluorescein-conjugated anti–rabbit IgG (Jackson ImmunoResearch Laboratories). To detect Bub1 in the same samples, the coverslips were then incubated with biotinylated anti-Bub1 antibody, followed by Texas red–conjugated streptavidin (Jackson ImmunoResearch Laboratories). Images were taken using a charge-coupled device camera (MicroMAX-5MHz; Princeton Instruments, Inc.) attached to a fluorescence microscope (model E800; Nikon). Images were collected and processed with the Metamorph Imaging System (v4.0; Universal Imaging Corp.) and converted to Adobe Photoshop® format. Immunofluorescent staining from samples containing a low nuclear density (1,000/μl of extract) is the same as the high density (9,000/μl of extract) that is required for activation of the spindle checkpoint.


Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase activity.

Sharp-Baker H, Chen RH - J. Cell Biol. (2001)

Bub1 and Bub1K872R restore kinetochore binding of Mad1, Mad2, Bub3, and CENP-E in Bub1-depleted extract. Sperm nuclei and nocodazole were incubated with mock-depleted extract or Bub1-depleted extract supplemented with mock, Bub1, or Bub1K872R translation as indicated on the top. Isolated chromosomes were incubated with rabbit antibodies against Mad1, Mad2, Bub3, or CENP-E as indicated on the left, followed by fluorescein-conjugated anti–rabbit antibody. Bub1 was then detected with biotinylated anti-Bub1 antibody and Texas red–conjugated streptavidin. For space conservation, pictures of Bub1 are shown only once for each type of extract. The merge pictures contain all three fluorochromes. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 8: Bub1 and Bub1K872R restore kinetochore binding of Mad1, Mad2, Bub3, and CENP-E in Bub1-depleted extract. Sperm nuclei and nocodazole were incubated with mock-depleted extract or Bub1-depleted extract supplemented with mock, Bub1, or Bub1K872R translation as indicated on the top. Isolated chromosomes were incubated with rabbit antibodies against Mad1, Mad2, Bub3, or CENP-E as indicated on the left, followed by fluorescein-conjugated anti–rabbit antibody. Bub1 was then detected with biotinylated anti-Bub1 antibody and Texas red–conjugated streptavidin. For space conservation, pictures of Bub1 are shown only once for each type of extract. The merge pictures contain all three fluorochromes. Bar, 10 μm.
Mentions: For immunofluorescent staining of unreplicated chromosomes, 20 μl of egg extract was incubated with sperm nuclei (1,000/μl of extract) at 23°C for 10 min, followed by addition of nocodazole to 10 μg/ml, and incubation for another 20 min at 23°C to disrupt microtubules. The replicated chromosomes and the in vitro metaphase to anaphase transition were performed as described (Chen et al. 1998). The chromosomes were isolated and processed for immunofluorescent staining as described (Chen et al. 1998). The anti–CENP-E antibody was provided by Dr. T. Yen (Fox Chase Cancer Center, Philadelphia, PA). For results shown in Fig. 8, the coverslips were first incubated with rabbit antibodies against Mad1, Mad2, Bub3, or CENP-E, followed by fluorescein-conjugated anti–rabbit IgG (Jackson ImmunoResearch Laboratories). To detect Bub1 in the same samples, the coverslips were then incubated with biotinylated anti-Bub1 antibody, followed by Texas red–conjugated streptavidin (Jackson ImmunoResearch Laboratories). Images were taken using a charge-coupled device camera (MicroMAX-5MHz; Princeton Instruments, Inc.) attached to a fluorescence microscope (model E800; Nikon). Images were collected and processed with the Metamorph Imaging System (v4.0; Universal Imaging Corp.) and converted to Adobe Photoshop® format. Immunofluorescent staining from samples containing a low nuclear density (1,000/μl of extract) is the same as the high density (9,000/μl of extract) that is required for activation of the spindle checkpoint.

Bottom Line: Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro.Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins.Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic spindle. We have isolated a Xenopus homologue of the spindle checkpoint component Bub1, and investigated its role in the spindle checkpoint in Xenopus egg extracts. Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro. Bub1 is essential for the establishment and maintenance of the checkpoint and is localized to kinetochores, similar to the spindle checkpoint complex Mad1-Mad2. However, Bub1 differs from Mad1-Mad2 in that Bub1 remains on kinetochores that have attached to microtubules; the protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins. Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.

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