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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 responds to tension. DNA is shown in blue and Bub1 is in red. (A) Prometaphase I spermatocyte from a X^Y; C(4)RM stock, showing strong kinetochore staining of comparable intensity on both bivalents and univalents. (B–D) Once the bivalents align at the metaphase plate, the intensity of kinetochore staining is sharply decreased. Univalents continue to show strong kinetochore association with Bub1, indicating that Bub1 can discriminate between the presence and absence of bipolar tension at the kinetochores. Bar, 5 μm.
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Figure 10: Bub1 responds to tension. DNA is shown in blue and Bub1 is in red. (A) Prometaphase I spermatocyte from a X^Y; C(4)RM stock, showing strong kinetochore staining of comparable intensity on both bivalents and univalents. (B–D) Once the bivalents align at the metaphase plate, the intensity of kinetochore staining is sharply decreased. Univalents continue to show strong kinetochore association with Bub1, indicating that Bub1 can discriminate between the presence and absence of bipolar tension at the kinetochores. Bar, 5 μm.

Mentions: Is the association of Bub1 with the kinetochores during male meiosis in Drosophila responsive to tension? To answer this question, we analyzed the distribution of Bub1 in primary spermatocytes containing univalents: the attached XY (X^Y) and the compound 4th [C(4)RM], which are never subject to bipolar tension as they can attach only to a single pole (Ault and Lin 1984; Ault and Nicklas 1989). Fig. 10 A shows that the intensity of Bub1 staining is comparable between univalents and bivalents at prometaphase I. However, once the bivalents align at the metaphase plate, the intensity of Bub1 staining on their kinetochores decreases drastically, while the univalents in the same cell retain strong Bub1 signals (Fig. 10, B–D). Thus, the spindle checkpoint component Bub1 is not only properly localized during male meiosis, but it is also capable of discriminating between the presence or absence of bipolar tension at kinetochores. Tension has also been recently implicated in regulating the localization of Mad2 at the kinetochores in maize spermatocytes (Yu et al. 1999).


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 responds to tension. DNA is shown in blue and Bub1 is in red. (A) Prometaphase I spermatocyte from a X^Y; C(4)RM stock, showing strong kinetochore staining of comparable intensity on both bivalents and univalents. (B–D) Once the bivalents align at the metaphase plate, the intensity of kinetochore staining is sharply decreased. Univalents continue to show strong kinetochore association with Bub1, indicating that Bub1 can discriminate between the presence and absence of bipolar tension at the kinetochores. Bar, 5 μm.
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Related In: Results  -  Collection

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Figure 10: Bub1 responds to tension. DNA is shown in blue and Bub1 is in red. (A) Prometaphase I spermatocyte from a X^Y; C(4)RM stock, showing strong kinetochore staining of comparable intensity on both bivalents and univalents. (B–D) Once the bivalents align at the metaphase plate, the intensity of kinetochore staining is sharply decreased. Univalents continue to show strong kinetochore association with Bub1, indicating that Bub1 can discriminate between the presence and absence of bipolar tension at the kinetochores. Bar, 5 μm.
Mentions: Is the association of Bub1 with the kinetochores during male meiosis in Drosophila responsive to tension? To answer this question, we analyzed the distribution of Bub1 in primary spermatocytes containing univalents: the attached XY (X^Y) and the compound 4th [C(4)RM], which are never subject to bipolar tension as they can attach only to a single pole (Ault and Lin 1984; Ault and Nicklas 1989). Fig. 10 A shows that the intensity of Bub1 staining is comparable between univalents and bivalents at prometaphase I. However, once the bivalents align at the metaphase plate, the intensity of Bub1 staining on their kinetochores decreases drastically, while the univalents in the same cell retain strong Bub1 signals (Fig. 10, B–D). Thus, the spindle checkpoint component Bub1 is not only properly localized during male meiosis, but it is also capable of discriminating between the presence or absence of bipolar tension at kinetochores. Tension has also been recently implicated in regulating the localization of Mad2 at the kinetochores in maize spermatocytes (Yu et al. 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.

Show MeSH
Related in: MedlinePlus