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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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Role of Bub1 during Drosophila spermatogenesis. DNA is shown in blue and Bub1 is in red. (A–C) Localization of Bub1 during the first meiotic division. (A) Bub1 is strongly associated with the kinetochores at prometaphase I. (B) Kinetochore staining is significantly reduced by metaphase I and lost completely by anaphase I (C). (D–F) Localization of Bub1 during the second meiotic division parallels the behaviour of Bub1 during the first meiotic division. (G and H) Living spermatids from third instar larval testes viewed by phase contrast optics. A field of wild-type “onion stage” spermatids is shown in (G). Note that each spermatid contains a single phase light nucleus and a single phase dark Nebenkern (mitochondrial derivative), and that the volume of all nuclei are the same, indicating that chromosome segregation has occurred correctly. In contrast, a field of spermatids from a bub1 mutant testis (H) displays evidence of chromosome missegregation, in the form of spermatids with abnormal numbers of nuclei (arrows) or with micronuclei (arrowheads). Bars, 5 μm. A–F appear at the same magnification, as do G and H.
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Figure 9: Role of Bub1 during Drosophila spermatogenesis. DNA is shown in blue and Bub1 is in red. (A–C) Localization of Bub1 during the first meiotic division. (A) Bub1 is strongly associated with the kinetochores at prometaphase I. (B) Kinetochore staining is significantly reduced by metaphase I and lost completely by anaphase I (C). (D–F) Localization of Bub1 during the second meiotic division parallels the behaviour of Bub1 during the first meiotic division. (G and H) Living spermatids from third instar larval testes viewed by phase contrast optics. A field of wild-type “onion stage” spermatids is shown in (G). Note that each spermatid contains a single phase light nucleus and a single phase dark Nebenkern (mitochondrial derivative), and that the volume of all nuclei are the same, indicating that chromosome segregation has occurred correctly. In contrast, a field of spermatids from a bub1 mutant testis (H) displays evidence of chromosome missegregation, in the form of spermatids with abnormal numbers of nuclei (arrows) or with micronuclei (arrowheads). Bars, 5 μm. A–F appear at the same magnification, as do G and H.

Mentions: To explore the apparent absence or weakness of the spindle checkpoint in meiotic Drosophila spermatocytes, we examined the distribution of Bub1 during spermatogenesis using techniques we have previously developed (Williams et al. 1996). Bub1 localizes to the kinetochores of bivalents in primary spermatocytes during prometaphase I, as shown in Fig. 9 A. The kinetochore association of Bub1 decreases significantly as the bivalents align at the metaphase plate (Fig. 9 B) and becomes undetectable at anaphase (Fig. 9 C), although some nuclear and spindle staining above background is visible during these cell cycle stages. This dynamic localization pattern is repeated during the second meiotic division (Fig. 9, D–F). Thus, the pattern of Bub1 distribution during both meiotic divisions parallels that seen during mitosis.


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)

Role of Bub1 during Drosophila spermatogenesis. DNA is shown in blue and Bub1 is in red. (A–C) Localization of Bub1 during the first meiotic division. (A) Bub1 is strongly associated with the kinetochores at prometaphase I. (B) Kinetochore staining is significantly reduced by metaphase I and lost completely by anaphase I (C). (D–F) Localization of Bub1 during the second meiotic division parallels the behaviour of Bub1 during the first meiotic division. (G and H) Living spermatids from third instar larval testes viewed by phase contrast optics. A field of wild-type “onion stage” spermatids is shown in (G). Note that each spermatid contains a single phase light nucleus and a single phase dark Nebenkern (mitochondrial derivative), and that the volume of all nuclei are the same, indicating that chromosome segregation has occurred correctly. In contrast, a field of spermatids from a bub1 mutant testis (H) displays evidence of chromosome missegregation, in the form of spermatids with abnormal numbers of nuclei (arrows) or with micronuclei (arrowheads). Bars, 5 μm. A–F appear at the same magnification, as do G and H.
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Related In: Results  -  Collection

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

Figure 9: Role of Bub1 during Drosophila spermatogenesis. DNA is shown in blue and Bub1 is in red. (A–C) Localization of Bub1 during the first meiotic division. (A) Bub1 is strongly associated with the kinetochores at prometaphase I. (B) Kinetochore staining is significantly reduced by metaphase I and lost completely by anaphase I (C). (D–F) Localization of Bub1 during the second meiotic division parallels the behaviour of Bub1 during the first meiotic division. (G and H) Living spermatids from third instar larval testes viewed by phase contrast optics. A field of wild-type “onion stage” spermatids is shown in (G). Note that each spermatid contains a single phase light nucleus and a single phase dark Nebenkern (mitochondrial derivative), and that the volume of all nuclei are the same, indicating that chromosome segregation has occurred correctly. In contrast, a field of spermatids from a bub1 mutant testis (H) displays evidence of chromosome missegregation, in the form of spermatids with abnormal numbers of nuclei (arrows) or with micronuclei (arrowheads). Bars, 5 μm. A–F appear at the same magnification, as do G and H.
Mentions: To explore the apparent absence or weakness of the spindle checkpoint in meiotic Drosophila spermatocytes, we examined the distribution of Bub1 during spermatogenesis using techniques we have previously developed (Williams et al. 1996). Bub1 localizes to the kinetochores of bivalents in primary spermatocytes during prometaphase I, as shown in Fig. 9 A. The kinetochore association of Bub1 decreases significantly as the bivalents align at the metaphase plate (Fig. 9 B) and becomes undetectable at anaphase (Fig. 9 C), although some nuclear and spindle staining above background is visible during these cell cycle stages. This dynamic localization pattern is repeated during the second meiotic division (Fig. 9, D–F). Thus, the pattern of Bub1 distribution during both meiotic divisions parallels that seen during mitosis.

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