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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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MT-independent binding of Bik1 to the kinetochore. (A) Bik1 can be cross-linked to CEN DNA in the absence of MTs. Chip with strains bearing the indicated Bik1 derivatives. Each construct is expressed from a CEN plasmid in a bik1Δ strain and contains the coding sequence for 3 tandem copies of the myc epitope at the 3′ end of the BIK1 sequence. CEN3 and CEN16 are centromere DNA sequences. HMRa, PGK1, LEU2, and MET16 are control flanking sequences. The nocodazole-treated cells were incubated in medium containing 15 μg/ml nocodazole for 1 h. Complete MT depolymerization was confirmed in a parallel culture expressing GFP-Tub1. (B) Cross-linking of Bik1 to CEN DNA requires a functional kinetochore. Chip assay of Bik1–3GFP in an ndc10–1 strain after incubation at the indicated temperature for 3 h. (C) Coimmunoprecipitation of Bik1 with Stu2. Cell extracts from strains expressing either Bik1–3GFP or untagged Bik1 were immunoprecipitated with a polyclonal anti-GFP antibody. Stu2-HA was detected by Western blotting with an anti-HA monoclonal antibody.
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fig3: MT-independent binding of Bik1 to the kinetochore. (A) Bik1 can be cross-linked to CEN DNA in the absence of MTs. Chip with strains bearing the indicated Bik1 derivatives. Each construct is expressed from a CEN plasmid in a bik1Δ strain and contains the coding sequence for 3 tandem copies of the myc epitope at the 3′ end of the BIK1 sequence. CEN3 and CEN16 are centromere DNA sequences. HMRa, PGK1, LEU2, and MET16 are control flanking sequences. The nocodazole-treated cells were incubated in medium containing 15 μg/ml nocodazole for 1 h. Complete MT depolymerization was confirmed in a parallel culture expressing GFP-Tub1. (B) Cross-linking of Bik1 to CEN DNA requires a functional kinetochore. Chip assay of Bik1–3GFP in an ndc10–1 strain after incubation at the indicated temperature for 3 h. (C) Coimmunoprecipitation of Bik1 with Stu2. Cell extracts from strains expressing either Bik1–3GFP or untagged Bik1 were immunoprecipitated with a polyclonal anti-GFP antibody. Stu2-HA was detected by Western blotting with an anti-HA monoclonal antibody.

Mentions: Because of the prominent Bik1 signal at kinetochores, chromatin immunoprecipitation (Chip) was used to determine whether Bik1 bound to the kinetochore (Meluh and Koshland, 1997). Epitope-tagged Bik1 was specifically cross-linked to centromeric DNA but not to flanking chromosome arm sequences (Fig. 3 A). Further, the interaction of Bik1 with centromeric DNA was completely dependent upon Ndc10, a core component of the CDEIII DNA–binding subcomplex of the kinetochore (Fig. 3 B). Finally, we found that Bik1 specifically coimmunoprecipitated with another candidate kinetochore protein, Stu2 (Fig. 3 C). Stu2 is the budding yeast member of the XMAP215 family of MT-binding proteins (Gard and Kirschner, 1987; Wang and Huffaker, 1997; Chen et al., 1998; He et al., 2001; Nakaseko et al., 2001). Stu2 was previously shown to interact with Bik1 by two-hybrid analysis (Chen et al., 1998). Both Stu2 and its S. pombe orthologue dis1 were recently shown to bind to the kinetochore by Chip (He et al., 2001; Nakaseko et al., 2001). These data strongly suggest that Bik1 is a component of the yeast kinetochore.


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)

MT-independent binding of Bik1 to the kinetochore. (A) Bik1 can be cross-linked to CEN DNA in the absence of MTs. Chip with strains bearing the indicated Bik1 derivatives. Each construct is expressed from a CEN plasmid in a bik1Δ strain and contains the coding sequence for 3 tandem copies of the myc epitope at the 3′ end of the BIK1 sequence. CEN3 and CEN16 are centromere DNA sequences. HMRa, PGK1, LEU2, and MET16 are control flanking sequences. The nocodazole-treated cells were incubated in medium containing 15 μg/ml nocodazole for 1 h. Complete MT depolymerization was confirmed in a parallel culture expressing GFP-Tub1. (B) Cross-linking of Bik1 to CEN DNA requires a functional kinetochore. Chip assay of Bik1–3GFP in an ndc10–1 strain after incubation at the indicated temperature for 3 h. (C) Coimmunoprecipitation of Bik1 with Stu2. Cell extracts from strains expressing either Bik1–3GFP or untagged Bik1 were immunoprecipitated with a polyclonal anti-GFP antibody. Stu2-HA was detected by Western blotting with an anti-HA monoclonal antibody.
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Related In: Results  -  Collection

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fig3: MT-independent binding of Bik1 to the kinetochore. (A) Bik1 can be cross-linked to CEN DNA in the absence of MTs. Chip with strains bearing the indicated Bik1 derivatives. Each construct is expressed from a CEN plasmid in a bik1Δ strain and contains the coding sequence for 3 tandem copies of the myc epitope at the 3′ end of the BIK1 sequence. CEN3 and CEN16 are centromere DNA sequences. HMRa, PGK1, LEU2, and MET16 are control flanking sequences. The nocodazole-treated cells were incubated in medium containing 15 μg/ml nocodazole for 1 h. Complete MT depolymerization was confirmed in a parallel culture expressing GFP-Tub1. (B) Cross-linking of Bik1 to CEN DNA requires a functional kinetochore. Chip assay of Bik1–3GFP in an ndc10–1 strain after incubation at the indicated temperature for 3 h. (C) Coimmunoprecipitation of Bik1 with Stu2. Cell extracts from strains expressing either Bik1–3GFP or untagged Bik1 were immunoprecipitated with a polyclonal anti-GFP antibody. Stu2-HA was detected by Western blotting with an anti-HA monoclonal antibody.
Mentions: Because of the prominent Bik1 signal at kinetochores, chromatin immunoprecipitation (Chip) was used to determine whether Bik1 bound to the kinetochore (Meluh and Koshland, 1997). Epitope-tagged Bik1 was specifically cross-linked to centromeric DNA but not to flanking chromosome arm sequences (Fig. 3 A). Further, the interaction of Bik1 with centromeric DNA was completely dependent upon Ndc10, a core component of the CDEIII DNA–binding subcomplex of the kinetochore (Fig. 3 B). Finally, we found that Bik1 specifically coimmunoprecipitated with another candidate kinetochore protein, Stu2 (Fig. 3 C). Stu2 is the budding yeast member of the XMAP215 family of MT-binding proteins (Gard and Kirschner, 1987; Wang and Huffaker, 1997; Chen et al., 1998; He et al., 2001; Nakaseko et al., 2001). Stu2 was previously shown to interact with Bik1 by two-hybrid analysis (Chen et al., 1998). Both Stu2 and its S. pombe orthologue dis1 were recently shown to bind to the kinetochore by Chip (He et al., 2001; Nakaseko et al., 2001). These data strongly suggest that Bik1 is a component of the yeast kinetochore.

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