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Polyploids require Bik1 for kinetochore-microtubule attachment.

Lin H, de Carvalho P, Kho D, Tai CY, Pierre P, Fink GR, Pellman D - J. Cell Biol. (2001)

Bottom Line: Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface.Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids.The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, The Dana-Farber Cancer Institute, The Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The attachment of kinetochores to spindle microtubules (MTs) is essential for maintaining constant ploidy in eukaryotic cells. Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface. Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids. The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton. In polyploid cells, Bik1 is required before anaphase to maintain kinetochore separation and therefore contributes to the force that opposes the elastic recoil of attached sister chromatids. The role of Bik1 in kinetochore separation appears to be independent of the role of Bik1 in regulating MT dynamics. The finding that a protein involved in kinetochore-MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics.

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bik1-CTΔ40–containing triploids have a defect in preanaphase kinetochore separation. Kinetochore separation was scored in the indicated strains by coimmunostaining for tubulin (monoclonal antibody YOL1/34) and GFP-marked CEN5 (polyclonal anti-GFP). Unseparated kinetochores are seen as one fluorescent dot, whereas separated kinetochores are seen as two dots. The fraction of cells with separated kinetochores and the average spindle lengths were as follows. BIK1-containing cells: 35/86 and 1.2 μm (haploids); 68/158 and 1.2 μm (diploids); and 68/157 and 1.1 μm (triploids). bik1-CTΔ40–containing cells: 35/88 and 1.3 μm (haploids); 63/158 and 1.2 μm (diploids); and 36/197 and 1.2 μm (triploids).
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fig7: bik1-CTΔ40–containing triploids have a defect in preanaphase kinetochore separation. Kinetochore separation was scored in the indicated strains by coimmunostaining for tubulin (monoclonal antibody YOL1/34) and GFP-marked CEN5 (polyclonal anti-GFP). Unseparated kinetochores are seen as one fluorescent dot, whereas separated kinetochores are seen as two dots. The fraction of cells with separated kinetochores and the average spindle lengths were as follows. BIK1-containing cells: 35/86 and 1.2 μm (haploids); 68/158 and 1.2 μm (diploids); and 68/157 and 1.1 μm (triploids). bik1-CTΔ40–containing cells: 35/88 and 1.3 μm (haploids); 63/158 and 1.2 μm (diploids); and 36/197 and 1.2 μm (triploids).

Mentions: We next determined if the strain containing bik1-CTΔ40 had a defect in preanaphase kinetochore separation. A GFP-based system was used to visualize kinetochores in bik1-CTΔ40 and BIK1 cells (Straight et al., 1996). Tandem arrays of the Tet operator sequence (224 copies) were introduced 1.9 Kb from the chromosome V centromere (CEN5; He et al., 2000), allowing the position of CEN5 to be detected by a Tet repressor–GFP fusion. In fixed populations of wild-type haploids kinetochores were separated in the majority of preanaphase cells (Goshima and Yanagida, 2000). Preanaphase kinetochore separation was also observed in our wild-type strains. In haploid and diploid cells bearing the bik1-CTΔ40 allele, there was no defect in kinetochore separation relative to control strain (p > 0.05, Fig. 7). By contrast, kinetochore separation was reduced from 43% in the control triploid strain to 18% in the bik1-CTΔ40 triploid strain (p < 0.001). Thus, Bik1, and specifically its cargo-binding domain, was required for preanaphase kinetochore separation in triploid cells.


Polyploids require Bik1 for kinetochore-microtubule attachment.

Lin H, de Carvalho P, Kho D, Tai CY, Pierre P, Fink GR, Pellman D - J. Cell Biol. (2001)

bik1-CTΔ40–containing triploids have a defect in preanaphase kinetochore separation. Kinetochore separation was scored in the indicated strains by coimmunostaining for tubulin (monoclonal antibody YOL1/34) and GFP-marked CEN5 (polyclonal anti-GFP). Unseparated kinetochores are seen as one fluorescent dot, whereas separated kinetochores are seen as two dots. The fraction of cells with separated kinetochores and the average spindle lengths were as follows. BIK1-containing cells: 35/86 and 1.2 μm (haploids); 68/158 and 1.2 μm (diploids); and 68/157 and 1.1 μm (triploids). bik1-CTΔ40–containing cells: 35/88 and 1.3 μm (haploids); 63/158 and 1.2 μm (diploids); and 36/197 and 1.2 μm (triploids).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199317&req=5

fig7: bik1-CTΔ40–containing triploids have a defect in preanaphase kinetochore separation. Kinetochore separation was scored in the indicated strains by coimmunostaining for tubulin (monoclonal antibody YOL1/34) and GFP-marked CEN5 (polyclonal anti-GFP). Unseparated kinetochores are seen as one fluorescent dot, whereas separated kinetochores are seen as two dots. The fraction of cells with separated kinetochores and the average spindle lengths were as follows. BIK1-containing cells: 35/86 and 1.2 μm (haploids); 68/158 and 1.2 μm (diploids); and 68/157 and 1.1 μm (triploids). bik1-CTΔ40–containing cells: 35/88 and 1.3 μm (haploids); 63/158 and 1.2 μm (diploids); and 36/197 and 1.2 μm (triploids).
Mentions: We next determined if the strain containing bik1-CTΔ40 had a defect in preanaphase kinetochore separation. A GFP-based system was used to visualize kinetochores in bik1-CTΔ40 and BIK1 cells (Straight et al., 1996). Tandem arrays of the Tet operator sequence (224 copies) were introduced 1.9 Kb from the chromosome V centromere (CEN5; He et al., 2000), allowing the position of CEN5 to be detected by a Tet repressor–GFP fusion. In fixed populations of wild-type haploids kinetochores were separated in the majority of preanaphase cells (Goshima and Yanagida, 2000). Preanaphase kinetochore separation was also observed in our wild-type strains. In haploid and diploid cells bearing the bik1-CTΔ40 allele, there was no defect in kinetochore separation relative to control strain (p > 0.05, Fig. 7). By contrast, kinetochore separation was reduced from 43% in the control triploid strain to 18% in the bik1-CTΔ40 triploid strain (p < 0.001). Thus, Bik1, and specifically its cargo-binding domain, was required for preanaphase kinetochore separation in triploid cells.

Bottom Line: Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface.Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids.The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, The Dana-Farber Cancer Institute, The Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The attachment of kinetochores to spindle microtubules (MTs) is essential for maintaining constant ploidy in eukaryotic cells. Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface. Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids. The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton. In polyploid cells, Bik1 is required before anaphase to maintain kinetochore separation and therefore contributes to the force that opposes the elastic recoil of attached sister chromatids. The role of Bik1 in kinetochore separation appears to be independent of the role of Bik1 in regulating MT dynamics. The finding that a protein involved in kinetochore-MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics.

Show MeSH
Related in: MedlinePlus