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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: 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.The finding that a protein involved in kinetochore-MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics.

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 interacts with MTs but not kinetochores. (A) Cross-linking of Bik1 to CEN DNA requires the cargo-binding domain. The Chip experiment was performed as described in the legend to Figure 3 except that Bik1 and Bik1-CTΔ40 are tagged at the COOH terminus with 13 tandem copies of the myc epitope (Longtine et al., 1998). Bik1 and Bik1-CTΔ40 are expressed at the same levels. Western blot with an anti-myc monoclonal antibody is shown at the bottom (100 μg of cell extract was loaded in each lane). (B) The localization of Bik1-CTΔ40-GFP. Pairs of DIC and fluorescence images are shown. Bar, 2 μm.
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fig4: Bik1-CTΔ40 interacts with MTs but not kinetochores. (A) Cross-linking of Bik1 to CEN DNA requires the cargo-binding domain. The Chip experiment was performed as described in the legend to Figure 3 except that Bik1 and Bik1-CTΔ40 are tagged at the COOH terminus with 13 tandem copies of the myc epitope (Longtine et al., 1998). Bik1 and Bik1-CTΔ40 are expressed at the same levels. Western blot with an anti-myc monoclonal antibody is shown at the bottom (100 μg of cell extract was loaded in each lane). (B) The localization of Bik1-CTΔ40-GFP. Pairs of DIC and fluorescence images are shown. Bar, 2 μm.

Mentions: The COOH-terminal cargo-binding domain of CLIP-170 family proteins is proposed to link MT plus ends to various targets (Pierre et al., 1992). However, the cargo-binding hypothesis has not been directly tested. To test this idea, a bik1 mutant lacking the coding sequence for the cargo-binding domain (bik1-CTΔ40) was generated by homologous recombination at the BIK1 locus (see Materials and methods). Bik1-CTΔ40 is expressed at similar levels to Bik1 (Fig. 4 A) and, when expressed as a COOH-terminal GFP fusion, has a similar pattern of localization as Bik1 (Fig. 4 B). However, in contrast to Bik1, Bik1-CTΔ40 failed to bind to the kinetochore by Chip (Fig. 4 A). These data validate the idea that the COOH-terminal Bik1 domain binds cargo and suggest that Bik1 is indeed a bifunctional linker protein.


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 interacts with MTs but not kinetochores. (A) Cross-linking of Bik1 to CEN DNA requires the cargo-binding domain. The Chip experiment was performed as described in the legend to Figure 3 except that Bik1 and Bik1-CTΔ40 are tagged at the COOH terminus with 13 tandem copies of the myc epitope (Longtine et al., 1998). Bik1 and Bik1-CTΔ40 are expressed at the same levels. Western blot with an anti-myc monoclonal antibody is shown at the bottom (100 μg of cell extract was loaded in each lane). (B) The localization of Bik1-CTΔ40-GFP. Pairs of DIC and fluorescence images are shown. Bar, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Bik1-CTΔ40 interacts with MTs but not kinetochores. (A) Cross-linking of Bik1 to CEN DNA requires the cargo-binding domain. The Chip experiment was performed as described in the legend to Figure 3 except that Bik1 and Bik1-CTΔ40 are tagged at the COOH terminus with 13 tandem copies of the myc epitope (Longtine et al., 1998). Bik1 and Bik1-CTΔ40 are expressed at the same levels. Western blot with an anti-myc monoclonal antibody is shown at the bottom (100 μg of cell extract was loaded in each lane). (B) The localization of Bik1-CTΔ40-GFP. Pairs of DIC and fluorescence images are shown. Bar, 2 μm.
Mentions: The COOH-terminal cargo-binding domain of CLIP-170 family proteins is proposed to link MT plus ends to various targets (Pierre et al., 1992). However, the cargo-binding hypothesis has not been directly tested. To test this idea, a bik1 mutant lacking the coding sequence for the cargo-binding domain (bik1-CTΔ40) was generated by homologous recombination at the BIK1 locus (see Materials and methods). Bik1-CTΔ40 is expressed at similar levels to Bik1 (Fig. 4 A) and, when expressed as a COOH-terminal GFP fusion, has a similar pattern of localization as Bik1 (Fig. 4 B). However, in contrast to Bik1, Bik1-CTΔ40 failed to bind to the kinetochore by Chip (Fig. 4 A). These data validate the idea that the COOH-terminal Bik1 domain binds cargo and suggest that Bik1 is indeed a bifunctional linker protein.

Bottom Line: 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.The finding that a protein involved in kinetochore-MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics.

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