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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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Drosophila Bub1 is recruited to kinetochores. DNA is shown in blue and Bub1 is in red. (A) A chromosome isolated from a Drosophila S2 cell arrested with colchicine, showing strong Bub1 staining at the kinetochores. (B) Bub1 is localized to the kinetochores in a wild-type (Oregon-R) neuroblast arrested with colchicine. (C) A neuroblast from a bub1 mutant (l(2)K06109/l(2)K06109) brain arrested with colchicine and stained with affinity-purified anti-Bub1 antibodies under identical conditions to those used in B. Note the complete absence of Bub1 staining in this figure; complete lack of Bub1 staining is also observed in all other bub1 allelic and deficiency combinations (not shown). D–F show that Bub1 (red) colocalizes with the kinetochore marker ZW10 (green) in wild-type neuroblasts, although the levels of staining of individual kinetochores with the two reagents are not always in concert. Bars, 5 μm. A–C are at the same magnification, as are D–F.
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Figure 2: Drosophila Bub1 is recruited to kinetochores. DNA is shown in blue and Bub1 is in red. (A) A chromosome isolated from a Drosophila S2 cell arrested with colchicine, showing strong Bub1 staining at the kinetochores. (B) Bub1 is localized to the kinetochores in a wild-type (Oregon-R) neuroblast arrested with colchicine. (C) A neuroblast from a bub1 mutant (l(2)K06109/l(2)K06109) brain arrested with colchicine and stained with affinity-purified anti-Bub1 antibodies under identical conditions to those used in B. Note the complete absence of Bub1 staining in this figure; complete lack of Bub1 staining is also observed in all other bub1 allelic and deficiency combinations (not shown). D–F show that Bub1 (red) colocalizes with the kinetochore marker ZW10 (green) in wild-type neuroblasts, although the levels of staining of individual kinetochores with the two reagents are not always in concert. Bars, 5 μm. A–C are at the same magnification, as are D–F.

Mentions: To identify third instar larvae homozygous for bub1 mutations, chromosomes bearing both the l(2)K06109 and l(2)K03113 P-element insertions were rebalanced over T(2;3)SM6a-TM6B, a translocation between the second chromosome balancer SM6a and the third chromosome balancer TM6B synthesized in the laboratory of A. Garcia-Bellido (Universidad Autonoma de Madrid, Madrid, Spain). T(2;3)SM6a-TM6B includes the dominant larval/pupal marker Tubby, so the desired mutant animals were chosen on the basis of their non-Tubby phenotype. Orcein stained preparations of neuroblasts from the brains of third instar larvae were obtained as described by Gatti and Goldberg 1991. Cytological preparation and immunolocalization studies of these larval neuroblasts were performed as described by Williams and Goldberg 1994. Living testes from third instar larvae were observed by the techniques of Cenci et al. 1994, while fixed larval testes were analyzed by immunofluorescence as described by Williams et al. 1996. Preparation and immunolocalization analysis of Drosophila S2 tissue culture cells, and of metaphase-arrested chromosomes isolated from these cells, was as described by Bousbaa et al. 1997; the same reference describes experimental protocols involving the 3F3/2 antibody. Secondary antibodies used for immunofluorescence localization of Bub1 were TRITC or FITC conjugated Affinipure donkey anti–chicken IgY (Jackson ImmunoResearch Labs), both at dilutions of 1:200. In all figures requiring comparisons of Bub1 or 3F3/2 staining between panels (Fig. 2B and Fig. C; 3, A–I; 7, A–D; 9, A–F; and 10, A–D), the gain on the digital camera was held constant, and all images were digitally processed in the same fashion.


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)

Drosophila Bub1 is recruited to kinetochores. DNA is shown in blue and Bub1 is in red. (A) A chromosome isolated from a Drosophila S2 cell arrested with colchicine, showing strong Bub1 staining at the kinetochores. (B) Bub1 is localized to the kinetochores in a wild-type (Oregon-R) neuroblast arrested with colchicine. (C) A neuroblast from a bub1 mutant (l(2)K06109/l(2)K06109) brain arrested with colchicine and stained with affinity-purified anti-Bub1 antibodies under identical conditions to those used in B. Note the complete absence of Bub1 staining in this figure; complete lack of Bub1 staining is also observed in all other bub1 allelic and deficiency combinations (not shown). D–F show that Bub1 (red) colocalizes with the kinetochore marker ZW10 (green) in wild-type neuroblasts, although the levels of staining of individual kinetochores with the two reagents are not always in concert. Bars, 5 μm. A–C are at the same magnification, as are D–F.
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Related In: Results  -  Collection

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

Figure 2: Drosophila Bub1 is recruited to kinetochores. DNA is shown in blue and Bub1 is in red. (A) A chromosome isolated from a Drosophila S2 cell arrested with colchicine, showing strong Bub1 staining at the kinetochores. (B) Bub1 is localized to the kinetochores in a wild-type (Oregon-R) neuroblast arrested with colchicine. (C) A neuroblast from a bub1 mutant (l(2)K06109/l(2)K06109) brain arrested with colchicine and stained with affinity-purified anti-Bub1 antibodies under identical conditions to those used in B. Note the complete absence of Bub1 staining in this figure; complete lack of Bub1 staining is also observed in all other bub1 allelic and deficiency combinations (not shown). D–F show that Bub1 (red) colocalizes with the kinetochore marker ZW10 (green) in wild-type neuroblasts, although the levels of staining of individual kinetochores with the two reagents are not always in concert. Bars, 5 μm. A–C are at the same magnification, as are D–F.
Mentions: To identify third instar larvae homozygous for bub1 mutations, chromosomes bearing both the l(2)K06109 and l(2)K03113 P-element insertions were rebalanced over T(2;3)SM6a-TM6B, a translocation between the second chromosome balancer SM6a and the third chromosome balancer TM6B synthesized in the laboratory of A. Garcia-Bellido (Universidad Autonoma de Madrid, Madrid, Spain). T(2;3)SM6a-TM6B includes the dominant larval/pupal marker Tubby, so the desired mutant animals were chosen on the basis of their non-Tubby phenotype. Orcein stained preparations of neuroblasts from the brains of third instar larvae were obtained as described by Gatti and Goldberg 1991. Cytological preparation and immunolocalization studies of these larval neuroblasts were performed as described by Williams and Goldberg 1994. Living testes from third instar larvae were observed by the techniques of Cenci et al. 1994, while fixed larval testes were analyzed by immunofluorescence as described by Williams et al. 1996. Preparation and immunolocalization analysis of Drosophila S2 tissue culture cells, and of metaphase-arrested chromosomes isolated from these cells, was as described by Bousbaa et al. 1997; the same reference describes experimental protocols involving the 3F3/2 antibody. Secondary antibodies used for immunofluorescence localization of Bub1 were TRITC or FITC conjugated Affinipure donkey anti–chicken IgY (Jackson ImmunoResearch Labs), both at dilutions of 1:200. In all figures requiring comparisons of Bub1 or 3F3/2 staining between panels (Fig. 2B and Fig. C; 3, A–I; 7, A–D; 9, A–F; and 10, A–D), the gain on the digital camera was held constant, and all images were digitally processed in the same fashion.

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