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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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Bub1 distribution in cycling Drosophila S2 tissue culture cells. DNA is shown in blue and Bub1 is in red. (A) Prophase. Bub1 is strongly associated with the kinetochores of the condensing chromosomes. (B) Strong kinetochore staining continues to be observed into prometaphase. (C–E) As cells approach metaphase, chromosomes that are aligned along the metaphase plate show only weak Bub1 staining, while chromosomes that have not yet reached the metaphase plate continue to show intense Bub1 staining at one or both kinetochores. Occasionally, as in D, the two kinetochores of the lagging chromosome stain show different intensities of Bub1 signals. (F) At metaphase, all the chromosomes show weak Bub1 staining at the kinetochores, which continues to be detectable into early anaphase (G). (H) Later in anaphase, kinetochore staining is no longer detectable, although some staining of the spindle midzone is visible. In addition to the mitotic figures, interphase nuclei are also visible in G and H. (I) During telophase, no specific Bub1 staining pattern is observed. Bar, 5 μm.
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Figure 3: Bub1 distribution in cycling Drosophila S2 tissue culture cells. DNA is shown in blue and Bub1 is in red. (A) Prophase. Bub1 is strongly associated with the kinetochores of the condensing chromosomes. (B) Strong kinetochore staining continues to be observed into prometaphase. (C–E) As cells approach metaphase, chromosomes that are aligned along the metaphase plate show only weak Bub1 staining, while chromosomes that have not yet reached the metaphase plate continue to show intense Bub1 staining at one or both kinetochores. Occasionally, as in D, the two kinetochores of the lagging chromosome stain show different intensities of Bub1 signals. (F) At metaphase, all the chromosomes show weak Bub1 staining at the kinetochores, which continues to be detectable into early anaphase (G). (H) Later in anaphase, kinetochore staining is no longer detectable, although some staining of the spindle midzone is visible. In addition to the mitotic figures, interphase nuclei are also visible in G and H. (I) During telophase, no specific Bub1 staining pattern is observed. Bar, 5 μm.

Mentions: Next we used our affinity-purified anti-Bub1 antibodies to examine in detail the distribution of Bub1 during mitosis in cycling Drosophila S2 cells. Interphase cells show a generalized, diffuse nucleoplasmic staining pattern (not shown). At prophase (Fig. 3 A), Bub1 associates strongly with the kinetochore regions of the condensed chromosomes; as shown in Fig. 2D–F, Bub1 indeed substantially colocalizes with the kinetochore marker ZW10 (Williams et al. 1992, 1994, Williams et al. 1996). Kinetochore staining becomes weaker at prometaphase (Fig. 3 B). At metaphase, the Bub1 signal weakens specifically for those chromosomes that have migrated to the metaphase plate (Fig. 3, C–F). Chromosomes in the same cells that have not yet reached the metaphase plate continue to show strong Bub1 staining at their kinetochores (Fig. 3, C–E). Depending on the orientation of the chromosome with respect to the spindle, one kinetochore may stain more strongly for Bub1 than the other (Fig. 3 D). Very weak kinetochore signals continue to be visible into anaphase (Fig. 3 G), but are not observed during late anaphase (Fig. 3 H) or telophase (Fig. 3 I). Some staining of the spindle midzone is detectable at late anaphase (Fig. 3 H).


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)

Bub1 distribution in cycling Drosophila S2 tissue culture cells. DNA is shown in blue and Bub1 is in red. (A) Prophase. Bub1 is strongly associated with the kinetochores of the condensing chromosomes. (B) Strong kinetochore staining continues to be observed into prometaphase. (C–E) As cells approach metaphase, chromosomes that are aligned along the metaphase plate show only weak Bub1 staining, while chromosomes that have not yet reached the metaphase plate continue to show intense Bub1 staining at one or both kinetochores. Occasionally, as in D, the two kinetochores of the lagging chromosome stain show different intensities of Bub1 signals. (F) At metaphase, all the chromosomes show weak Bub1 staining at the kinetochores, which continues to be detectable into early anaphase (G). (H) Later in anaphase, kinetochore staining is no longer detectable, although some staining of the spindle midzone is visible. In addition to the mitotic figures, interphase nuclei are also visible in G and H. (I) During telophase, no specific Bub1 staining pattern is observed. Bar, 5 μm.
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Figure 3: Bub1 distribution in cycling Drosophila S2 tissue culture cells. DNA is shown in blue and Bub1 is in red. (A) Prophase. Bub1 is strongly associated with the kinetochores of the condensing chromosomes. (B) Strong kinetochore staining continues to be observed into prometaphase. (C–E) As cells approach metaphase, chromosomes that are aligned along the metaphase plate show only weak Bub1 staining, while chromosomes that have not yet reached the metaphase plate continue to show intense Bub1 staining at one or both kinetochores. Occasionally, as in D, the two kinetochores of the lagging chromosome stain show different intensities of Bub1 signals. (F) At metaphase, all the chromosomes show weak Bub1 staining at the kinetochores, which continues to be detectable into early anaphase (G). (H) Later in anaphase, kinetochore staining is no longer detectable, although some staining of the spindle midzone is visible. In addition to the mitotic figures, interphase nuclei are also visible in G and H. (I) During telophase, no specific Bub1 staining pattern is observed. Bar, 5 μm.
Mentions: Next we used our affinity-purified anti-Bub1 antibodies to examine in detail the distribution of Bub1 during mitosis in cycling Drosophila S2 cells. Interphase cells show a generalized, diffuse nucleoplasmic staining pattern (not shown). At prophase (Fig. 3 A), Bub1 associates strongly with the kinetochore regions of the condensed chromosomes; as shown in Fig. 2D–F, Bub1 indeed substantially colocalizes with the kinetochore marker ZW10 (Williams et al. 1992, 1994, Williams et al. 1996). Kinetochore staining becomes weaker at prometaphase (Fig. 3 B). At metaphase, the Bub1 signal weakens specifically for those chromosomes that have migrated to the metaphase plate (Fig. 3, C–F). Chromosomes in the same cells that have not yet reached the metaphase plate continue to show strong Bub1 staining at their kinetochores (Fig. 3, C–E). Depending on the orientation of the chromosome with respect to the spindle, one kinetochore may stain more strongly for Bub1 than the other (Fig. 3 D). Very weak kinetochore signals continue to be visible into anaphase (Fig. 3 G), but are not observed during late anaphase (Fig. 3 H) or telophase (Fig. 3 I). Some staining of the spindle midzone is detectable at late anaphase (Fig. 3 H).

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