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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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Relationship of Bub1 to other kinetochore components. In all panels, DNA is in blue. Neuroblasts were treated with colchicine in all panels except D. (A) Bub1 (red) in a zw10S1/Y mutant neuroblast. Note the Bub1 staining at the kinetochores of separated sister chromatids. (B) ZW10 (red) stains the kinetochores in bub1 mutant neuroblasts. (C) In neuroblasts homozygous for the mutation polo1, Bub1 associates with the kinetochores. (D) In bub1 mutant neuroblasts (here shown at anaphase), Polo protein is bound to the kinetochores of the separating chromosomes, as in wild-type. (E) Null mutations in rough deal (rod) do not prevent the association of Bub1 with kinetochores. (F) Bub1 also binds to kinetochores in the neuroblasts of animals homozygous for the mutation fizzy6. Bar, 5 μm.
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Figure 8: Relationship of Bub1 to other kinetochore components. In all panels, DNA is in blue. Neuroblasts were treated with colchicine in all panels except D. (A) Bub1 (red) in a zw10S1/Y mutant neuroblast. Note the Bub1 staining at the kinetochores of separated sister chromatids. (B) ZW10 (red) stains the kinetochores in bub1 mutant neuroblasts. (C) In neuroblasts homozygous for the mutation polo1, Bub1 associates with the kinetochores. (D) In bub1 mutant neuroblasts (here shown at anaphase), Polo protein is bound to the kinetochores of the separating chromosomes, as in wild-type. (E) Null mutations in rough deal (rod) do not prevent the association of Bub1 with kinetochores. (F) Bub1 also binds to kinetochores in the neuroblasts of animals homozygous for the mutation fizzy6. Bar, 5 μm.

Mentions: Mutations in zw10 and rough deal disrupt the segregation of chromosomes during anaphase of mitosis and meiosis. Intriguingly, mutations in both genes cause precocious sister chromatid separation in colchicine treated larval neuroblasts, indicating a bypass of the spindle checkpoint (Smith et al. 1985; Karess and Glover 1989; Williams et al. 1992). Both the ZW10 and Rod proteins are associated with the kinetochore during prophase/prometaphase of mitosis and both meiotic divisions (Williams et al. 1992; Williams and Goldberg 1994; Scaerou, F., and R. Karess, personal communication). We found that mutations in zw10 or rod do not affect the localization of Bub1 to the kinetochore (Fig. 8A and Fig. E). Interestingly, in these mutant cells Bub1 continues to be associated with the kinetochores of precociously separated sister chromatids (Fig. 8 A), indicating that sister chromatid separation does not require the loss of Bub1 from the kinetochore. Similar results were observed when precocious sister chromatid separation was induced in wild-type colchicine-arrested neuroblasts subjected to prolonged hypotonic shock (data not shown). Conversely, bub1 mutations do not block the association of ZW10 with the kinetochore (Fig. 8 B).


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)

Relationship of Bub1 to other kinetochore components. In all panels, DNA is in blue. Neuroblasts were treated with colchicine in all panels except D. (A) Bub1 (red) in a zw10S1/Y mutant neuroblast. Note the Bub1 staining at the kinetochores of separated sister chromatids. (B) ZW10 (red) stains the kinetochores in bub1 mutant neuroblasts. (C) In neuroblasts homozygous for the mutation polo1, Bub1 associates with the kinetochores. (D) In bub1 mutant neuroblasts (here shown at anaphase), Polo protein is bound to the kinetochores of the separating chromosomes, as in wild-type. (E) Null mutations in rough deal (rod) do not prevent the association of Bub1 with kinetochores. (F) Bub1 also binds to kinetochores in the neuroblasts of animals homozygous for the mutation fizzy6. Bar, 5 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199734&req=5

Figure 8: Relationship of Bub1 to other kinetochore components. In all panels, DNA is in blue. Neuroblasts were treated with colchicine in all panels except D. (A) Bub1 (red) in a zw10S1/Y mutant neuroblast. Note the Bub1 staining at the kinetochores of separated sister chromatids. (B) ZW10 (red) stains the kinetochores in bub1 mutant neuroblasts. (C) In neuroblasts homozygous for the mutation polo1, Bub1 associates with the kinetochores. (D) In bub1 mutant neuroblasts (here shown at anaphase), Polo protein is bound to the kinetochores of the separating chromosomes, as in wild-type. (E) Null mutations in rough deal (rod) do not prevent the association of Bub1 with kinetochores. (F) Bub1 also binds to kinetochores in the neuroblasts of animals homozygous for the mutation fizzy6. Bar, 5 μm.
Mentions: Mutations in zw10 and rough deal disrupt the segregation of chromosomes during anaphase of mitosis and meiosis. Intriguingly, mutations in both genes cause precocious sister chromatid separation in colchicine treated larval neuroblasts, indicating a bypass of the spindle checkpoint (Smith et al. 1985; Karess and Glover 1989; Williams et al. 1992). Both the ZW10 and Rod proteins are associated with the kinetochore during prophase/prometaphase of mitosis and both meiotic divisions (Williams et al. 1992; Williams and Goldberg 1994; Scaerou, F., and R. Karess, personal communication). We found that mutations in zw10 or rod do not affect the localization of Bub1 to the kinetochore (Fig. 8A and Fig. E). Interestingly, in these mutant cells Bub1 continues to be associated with the kinetochores of precociously separated sister chromatids (Fig. 8 A), indicating that sister chromatid separation does not require the loss of Bub1 from the kinetochore. Similar results were observed when precocious sister chromatid separation was induced in wild-type colchicine-arrested neuroblasts subjected to prolonged hypotonic shock (data not shown). Conversely, bub1 mutations do not block the association of ZW10 with the kinetochore (Fig. 8 B).

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