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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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Distribution of 3F3/2 phosphoepitopes in bub1 mutant neuroblasts. DNA is in blue, and 3F3/2 phosphoepitopes are in green. In all panels, the two strongest sites of 3F3/2 staining are the centrosomes. 3F3/2 distribution in prophase (A) and metaphase (B) neuroblasts from bub1 mutants. 3F3/2 epitopes at the centrosomes and kinetochores are strongly recognized, demonstrating that the Bub1 kinase is not a significant source of 3F3/2 phosphorylation activity in vivo. (C) 3F3/2 epitopes are completely dephosphorylated during anaphase in wild-type neuroblasts (see Bousbaa et al. 1997 for a detailed description of 3F3/2 distribution in wild-type Drosophila neuroblasts). (D) 3F3/2 distribution in an anaphase figure from a bub1 mutant neuroblast. 3F3/2 epitopes continue to remain phosphorylated in bub1 anaphases, though at reduced levels relative to those seen during prophase/prometaphase. Thus, total dephosphorylation of 3F3/2 phosphoepitopes cannot be a prerequisite for entry into anaphase. Bar, 5 μm.
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Figure 7: Distribution of 3F3/2 phosphoepitopes in bub1 mutant neuroblasts. DNA is in blue, and 3F3/2 phosphoepitopes are in green. In all panels, the two strongest sites of 3F3/2 staining are the centrosomes. 3F3/2 distribution in prophase (A) and metaphase (B) neuroblasts from bub1 mutants. 3F3/2 epitopes at the centrosomes and kinetochores are strongly recognized, demonstrating that the Bub1 kinase is not a significant source of 3F3/2 phosphorylation activity in vivo. (C) 3F3/2 epitopes are completely dephosphorylated during anaphase in wild-type neuroblasts (see Bousbaa et al. 1997 for a detailed description of 3F3/2 distribution in wild-type Drosophila neuroblasts). (D) 3F3/2 distribution in an anaphase figure from a bub1 mutant neuroblast. 3F3/2 epitopes continue to remain phosphorylated in bub1 anaphases, though at reduced levels relative to those seen during prophase/prometaphase. Thus, total dephosphorylation of 3F3/2 phosphoepitopes cannot be a prerequisite for entry into anaphase. Bar, 5 μm.

Mentions: Dephosphorylation of 3F3/2 epitopes is associated with the metaphase–anaphase transition (Campbell and Gorbsky 1995). Microinjection of anti-3F3/2 antibodies into cultured cells blocks 3F3/2 dephosphorylation and delays anaphase onset, implying that dephosphorylation of 3F3/2 epitopes may be a prerequisite for entry into anaphase (Campbell and Gorbsky 1995). The Bub1 kinase has been suggested as a candidate 3F3/2 kinase, both because of its function in the spindle checkpoint and because its intracellular distribution shows similarities with that of 3F3/2 epitopes (Chan et al. 1998). In order to examine these questions in more detail, we asked whether bub1 mutations would affect the distribution of 3F3/2 epitopes. As shown in Fig. 7A and Fig. B, Fig. 3F3/2 signals are present at the kinetochores in bub1 prophase/prometaphase and metaphase figures at levels comparable to that of wild-type brains (see Bousbaa et al. 1997 for a description of 3F3/2 staining in wild-type Drosophila neuroblasts). This result demonstrates that Bub1 kinase does not contribute significantly to 3F3/2 kinase activity in vivo.


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)

Distribution of 3F3/2 phosphoepitopes in bub1 mutant neuroblasts. DNA is in blue, and 3F3/2 phosphoepitopes are in green. In all panels, the two strongest sites of 3F3/2 staining are the centrosomes. 3F3/2 distribution in prophase (A) and metaphase (B) neuroblasts from bub1 mutants. 3F3/2 epitopes at the centrosomes and kinetochores are strongly recognized, demonstrating that the Bub1 kinase is not a significant source of 3F3/2 phosphorylation activity in vivo. (C) 3F3/2 epitopes are completely dephosphorylated during anaphase in wild-type neuroblasts (see Bousbaa et al. 1997 for a detailed description of 3F3/2 distribution in wild-type Drosophila neuroblasts). (D) 3F3/2 distribution in an anaphase figure from a bub1 mutant neuroblast. 3F3/2 epitopes continue to remain phosphorylated in bub1 anaphases, though at reduced levels relative to those seen during prophase/prometaphase. Thus, total dephosphorylation of 3F3/2 phosphoepitopes cannot be a prerequisite for entry into anaphase. Bar, 5 μm.
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Related In: Results  -  Collection

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Figure 7: Distribution of 3F3/2 phosphoepitopes in bub1 mutant neuroblasts. DNA is in blue, and 3F3/2 phosphoepitopes are in green. In all panels, the two strongest sites of 3F3/2 staining are the centrosomes. 3F3/2 distribution in prophase (A) and metaphase (B) neuroblasts from bub1 mutants. 3F3/2 epitopes at the centrosomes and kinetochores are strongly recognized, demonstrating that the Bub1 kinase is not a significant source of 3F3/2 phosphorylation activity in vivo. (C) 3F3/2 epitopes are completely dephosphorylated during anaphase in wild-type neuroblasts (see Bousbaa et al. 1997 for a detailed description of 3F3/2 distribution in wild-type Drosophila neuroblasts). (D) 3F3/2 distribution in an anaphase figure from a bub1 mutant neuroblast. 3F3/2 epitopes continue to remain phosphorylated in bub1 anaphases, though at reduced levels relative to those seen during prophase/prometaphase. Thus, total dephosphorylation of 3F3/2 phosphoepitopes cannot be a prerequisite for entry into anaphase. Bar, 5 μm.
Mentions: Dephosphorylation of 3F3/2 epitopes is associated with the metaphase–anaphase transition (Campbell and Gorbsky 1995). Microinjection of anti-3F3/2 antibodies into cultured cells blocks 3F3/2 dephosphorylation and delays anaphase onset, implying that dephosphorylation of 3F3/2 epitopes may be a prerequisite for entry into anaphase (Campbell and Gorbsky 1995). The Bub1 kinase has been suggested as a candidate 3F3/2 kinase, both because of its function in the spindle checkpoint and because its intracellular distribution shows similarities with that of 3F3/2 epitopes (Chan et al. 1998). In order to examine these questions in more detail, we asked whether bub1 mutations would affect the distribution of 3F3/2 epitopes. As shown in Fig. 7A and Fig. B, Fig. 3F3/2 signals are present at the kinetochores in bub1 prophase/prometaphase and metaphase figures at levels comparable to that of wild-type brains (see Bousbaa et al. 1997 for a description of 3F3/2 staining in wild-type Drosophila neuroblasts). This result demonstrates that Bub1 kinase does not contribute significantly to 3F3/2 kinase activity in vivo.

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