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Mutations in the essential spindle checkpoint gene bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila.

Basu J, Bousbaa H, Logarinho E, Li Z, Williams BC, Lopes C, Sunkel CE, Goldberg ML - J. Cell Biol. (1999)

Bottom Line: We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase.Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry.Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies.

View Article: PubMed Central - PubMed

Affiliation: Section of Genetics and Development, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
We have characterized the Drosophila mitotic checkpoint control protein Bub1 and obtained mutations in the bub1 gene. Drosophila Bub1 localizes strongly to the centromere/kinetochore of mitotic and meiotic chromosomes that have not yet reached the metaphase plate. Animals homozygous for P-element-induced, near- mutations of bub1 die during late larval/pupal stages due to severe mitotic abnormalities indicative of a bypass of checkpoint function. These abnormalities include accelerated exit from metaphase and chromosome missegregation and fragmentation. Chromosome fragmentation possibly leads to the significantly elevated levels of apoptosis seen in mutants. We have also investigated the relationship between Bub1 and other kinetochore components. We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase. Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry. Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies.

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Phenotype of bub1 mutants in Drosophila neuroblasts. (A–C) Orcein-stained mitotic figures from third instar larval brains treated with colchicine and hypotonic solution to perturb spindle assembly. Wild-type neuroblasts arrest in a prometaphase like configuration with attached sister chromatids as in A. In bub1 mutant neuroblasts treated in the same fashion (B and C), sister chromatids separate, and some evidence for aneuploidy or chromosome fragmentation is observed (diploid cells should contain 12 large chromatids and four dot-like 4th chromosome chromatids). (D–I) Orcein-stained anaphase figures from untreated brains. In contrast with a wild-type (D), bub1 mutant anaphases show several abnormalities including extensive chromatin bridging (E and F), chromosomes that lag in the vicinity of the metaphase plate (G), and apparent widespread chromosome fragmentation (H and I). Bar, 5 μm.
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Figure 4: Phenotype of bub1 mutants in Drosophila neuroblasts. (A–C) Orcein-stained mitotic figures from third instar larval brains treated with colchicine and hypotonic solution to perturb spindle assembly. Wild-type neuroblasts arrest in a prometaphase like configuration with attached sister chromatids as in A. In bub1 mutant neuroblasts treated in the same fashion (B and C), sister chromatids separate, and some evidence for aneuploidy or chromosome fragmentation is observed (diploid cells should contain 12 large chromatids and four dot-like 4th chromosome chromatids). (D–I) Orcein-stained anaphase figures from untreated brains. In contrast with a wild-type (D), bub1 mutant anaphases show several abnormalities including extensive chromatin bridging (E and F), chromosomes that lag in the vicinity of the metaphase plate (G), and apparent widespread chromosome fragmentation (H and I). Bar, 5 μm.

Mentions: We initially observed that bub1 mutants possess the hallmark of a defect in the spindle checkpoint: that is, a failure to maintain sister chromatid cohesion when the spindle is disrupted. In wild-type brains incubated with colchicine for 1 h, sister chromatids remain attached in 98% of all mitotic figures (Fig. 4 A and Table ), revealing activity of the spindle checkpoint. Similar values have been obtained in previous experiments by our laboratory (Williams et al. 1992) and by others (Gonzales et al. 1991). Under identical conditions, sister chromatids remain attached in only 32% of mitotic figures in bub1 brains, indicating that the spindle checkpoint has often been bypassed (Fig. 4B and Fig. C, and Table ). In fact, the frequency of neuroblasts with separated sister chromatids in bub1 mutant brains is essentially unaffected by colchicine treatment, in stark contrast with wild-type.


Mutations in the essential spindle checkpoint gene bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila.

Basu J, Bousbaa H, Logarinho E, Li Z, Williams BC, Lopes C, Sunkel CE, Goldberg ML - J. Cell Biol. (1999)

Phenotype of bub1 mutants in Drosophila neuroblasts. (A–C) Orcein-stained mitotic figures from third instar larval brains treated with colchicine and hypotonic solution to perturb spindle assembly. Wild-type neuroblasts arrest in a prometaphase like configuration with attached sister chromatids as in A. In bub1 mutant neuroblasts treated in the same fashion (B and C), sister chromatids separate, and some evidence for aneuploidy or chromosome fragmentation is observed (diploid cells should contain 12 large chromatids and four dot-like 4th chromosome chromatids). (D–I) Orcein-stained anaphase figures from untreated brains. In contrast with a wild-type (D), bub1 mutant anaphases show several abnormalities including extensive chromatin bridging (E and F), chromosomes that lag in the vicinity of the metaphase plate (G), and apparent widespread chromosome fragmentation (H and I). Bar, 5 μm.
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Figure 4: Phenotype of bub1 mutants in Drosophila neuroblasts. (A–C) Orcein-stained mitotic figures from third instar larval brains treated with colchicine and hypotonic solution to perturb spindle assembly. Wild-type neuroblasts arrest in a prometaphase like configuration with attached sister chromatids as in A. In bub1 mutant neuroblasts treated in the same fashion (B and C), sister chromatids separate, and some evidence for aneuploidy or chromosome fragmentation is observed (diploid cells should contain 12 large chromatids and four dot-like 4th chromosome chromatids). (D–I) Orcein-stained anaphase figures from untreated brains. In contrast with a wild-type (D), bub1 mutant anaphases show several abnormalities including extensive chromatin bridging (E and F), chromosomes that lag in the vicinity of the metaphase plate (G), and apparent widespread chromosome fragmentation (H and I). Bar, 5 μm.
Mentions: We initially observed that bub1 mutants possess the hallmark of a defect in the spindle checkpoint: that is, a failure to maintain sister chromatid cohesion when the spindle is disrupted. In wild-type brains incubated with colchicine for 1 h, sister chromatids remain attached in 98% of all mitotic figures (Fig. 4 A and Table ), revealing activity of the spindle checkpoint. Similar values have been obtained in previous experiments by our laboratory (Williams et al. 1992) and by others (Gonzales et al. 1991). Under identical conditions, sister chromatids remain attached in only 32% of mitotic figures in bub1 brains, indicating that the spindle checkpoint has often been bypassed (Fig. 4B and Fig. C, and Table ). In fact, the frequency of neuroblasts with separated sister chromatids in bub1 mutant brains is essentially unaffected by colchicine treatment, in stark contrast with wild-type.

Bottom Line: We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase.Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry.Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies.

View Article: PubMed Central - PubMed

Affiliation: Section of Genetics and Development, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
We have characterized the Drosophila mitotic checkpoint control protein Bub1 and obtained mutations in the bub1 gene. Drosophila Bub1 localizes strongly to the centromere/kinetochore of mitotic and meiotic chromosomes that have not yet reached the metaphase plate. Animals homozygous for P-element-induced, near- mutations of bub1 die during late larval/pupal stages due to severe mitotic abnormalities indicative of a bypass of checkpoint function. These abnormalities include accelerated exit from metaphase and chromosome missegregation and fragmentation. Chromosome fragmentation possibly leads to the significantly elevated levels of apoptosis seen in mutants. We have also investigated the relationship between Bub1 and other kinetochore components. We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase. Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry. Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies.

Show MeSH
Related in: MedlinePlus