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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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Larval brains of bub1 mutants contain many apoptotic nuclei. bub1 mutant (A–C) and wild-type (Oregon-R; D–F) brains were labeled by a TUNEL-based assay (A and D) for the presence of apoptotic nuclei, and stained with propidium iodide for DNA (B and E); a merged view with DNA in red and the TUNEL signal in green is shown in C and F. Many apoptotic nuclei are seen in bub1 mutant brains but not in wild-type; the majority of these TUNEL-positive nuclei are also pycnotic as seen by the abnormally condensed DNA signal. G–I show bub1 mutant larval brains stained with annexin V to reveal phosphatidylserine on the outside of the cell membrane (G), propidium iodide (H), and a merged view (I) with DNA staining in red and the annexin V signal in green. No annexin V staining is observed within wild-type brains (not shown). Bars: (A) 10 μm; (G) 5 μm. A–F are at the same magnification, as are G–I.
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Figure 5: Larval brains of bub1 mutants contain many apoptotic nuclei. bub1 mutant (A–C) and wild-type (Oregon-R; D–F) brains were labeled by a TUNEL-based assay (A and D) for the presence of apoptotic nuclei, and stained with propidium iodide for DNA (B and E); a merged view with DNA in red and the TUNEL signal in green is shown in C and F. Many apoptotic nuclei are seen in bub1 mutant brains but not in wild-type; the majority of these TUNEL-positive nuclei are also pycnotic as seen by the abnormally condensed DNA signal. G–I show bub1 mutant larval brains stained with annexin V to reveal phosphatidylserine on the outside of the cell membrane (G), propidium iodide (H), and a merged view (I) with DNA staining in red and the annexin V signal in green. No annexin V staining is observed within wild-type brains (not shown). Bars: (A) 10 μm; (G) 5 μm. A–F are at the same magnification, as are G–I.

Mentions: A striking feature of bub1 mutant brains examined with DNA staining is the occurrence of extremely high frequencies of pycnotic nuclei with highly condensed chromatin. These nuclei are strongly positive when labeled by Tdt-mediated dUTP-biotin nick end labeling (TUNEL)-based techniques (Fig. 5, A–F; see Materials and Methods). Because the TUNEL procedure detects chromosome damage (normally induced in the pathway for apoptosis), the TUNEL signals could reflect either the occurrence of bona fide programmed cell death, or alternatively simply the chromosome fragmentation that occurs during anaphase in bub1 mutant cells. To discriminate between these possibilities, we asked whether mutant nuclei showed elevated expression of two apoptotic events independent of chromosome breakage. The first of these markers was the redistribution of phosphatidylserine, which early in apoptosis rapidly moves from the internal face of the plasma membrane to the outside of the membrane; this redistribution was detected by use of FITC-conjugated Annexin V, a protein with very strong affinity for the serine in phosphatidylserine (Martin et al. 1995; see Materials and Methods). The second marker was a β-galactosidase reporter for reaper, a gene whose expression is needed to activate programmed cell death in Drosophila (White et al. 1994). Use of both markers verifies that mitotic cells in bub1 mutants undergo vastly elevated levels of apoptosis (Fig. 5, G–I, and 6). Levels of apoptotic nuclei are similar in l(2)K06109 or l(2)K03113 homozygotes as well as in trans-heterozygotes for either of the two alleles with deletions of the region (not shown).


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)

Larval brains of bub1 mutants contain many apoptotic nuclei. bub1 mutant (A–C) and wild-type (Oregon-R; D–F) brains were labeled by a TUNEL-based assay (A and D) for the presence of apoptotic nuclei, and stained with propidium iodide for DNA (B and E); a merged view with DNA in red and the TUNEL signal in green is shown in C and F. Many apoptotic nuclei are seen in bub1 mutant brains but not in wild-type; the majority of these TUNEL-positive nuclei are also pycnotic as seen by the abnormally condensed DNA signal. G–I show bub1 mutant larval brains stained with annexin V to reveal phosphatidylserine on the outside of the cell membrane (G), propidium iodide (H), and a merged view (I) with DNA staining in red and the annexin V signal in green. No annexin V staining is observed within wild-type brains (not shown). Bars: (A) 10 μm; (G) 5 μm. A–F are at the same magnification, as are G–I.
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Related In: Results  -  Collection

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Figure 5: Larval brains of bub1 mutants contain many apoptotic nuclei. bub1 mutant (A–C) and wild-type (Oregon-R; D–F) brains were labeled by a TUNEL-based assay (A and D) for the presence of apoptotic nuclei, and stained with propidium iodide for DNA (B and E); a merged view with DNA in red and the TUNEL signal in green is shown in C and F. Many apoptotic nuclei are seen in bub1 mutant brains but not in wild-type; the majority of these TUNEL-positive nuclei are also pycnotic as seen by the abnormally condensed DNA signal. G–I show bub1 mutant larval brains stained with annexin V to reveal phosphatidylserine on the outside of the cell membrane (G), propidium iodide (H), and a merged view (I) with DNA staining in red and the annexin V signal in green. No annexin V staining is observed within wild-type brains (not shown). Bars: (A) 10 μm; (G) 5 μm. A–F are at the same magnification, as are G–I.
Mentions: A striking feature of bub1 mutant brains examined with DNA staining is the occurrence of extremely high frequencies of pycnotic nuclei with highly condensed chromatin. These nuclei are strongly positive when labeled by Tdt-mediated dUTP-biotin nick end labeling (TUNEL)-based techniques (Fig. 5, A–F; see Materials and Methods). Because the TUNEL procedure detects chromosome damage (normally induced in the pathway for apoptosis), the TUNEL signals could reflect either the occurrence of bona fide programmed cell death, or alternatively simply the chromosome fragmentation that occurs during anaphase in bub1 mutant cells. To discriminate between these possibilities, we asked whether mutant nuclei showed elevated expression of two apoptotic events independent of chromosome breakage. The first of these markers was the redistribution of phosphatidylserine, which early in apoptosis rapidly moves from the internal face of the plasma membrane to the outside of the membrane; this redistribution was detected by use of FITC-conjugated Annexin V, a protein with very strong affinity for the serine in phosphatidylserine (Martin et al. 1995; see Materials and Methods). The second marker was a β-galactosidase reporter for reaper, a gene whose expression is needed to activate programmed cell death in Drosophila (White et al. 1994). Use of both markers verifies that mitotic cells in bub1 mutants undergo vastly elevated levels of apoptosis (Fig. 5, G–I, and 6). Levels of apoptotic nuclei are similar in l(2)K06109 or l(2)K03113 homozygotes as well as in trans-heterozygotes for either of the two alleles with deletions of the region (not shown).

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