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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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Localization of Bik1 to MT plus ends and to the kinetochore. (A) Colocalization of Bik1–3GFP (green) with CFP-Tub1 (red) at different stages of the cell cycle. Pairs of DIC (left) and merged fluorescence (right) images are shown. From top to bottom: G1 cell; preanaphase cell; anaphase cell; and telophase cell. Bar, 2 μm. (B) Bik1–3GFP localization at the kinetochore during mitosis in cells expressing Bik1–3GFP (green) and Spc42-CFP (red), a spindle pole body marker. Bar, 2 μm.
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fig1: Localization of Bik1 to MT plus ends and to the kinetochore. (A) Colocalization of Bik1–3GFP (green) with CFP-Tub1 (red) at different stages of the cell cycle. Pairs of DIC (left) and merged fluorescence (right) images are shown. From top to bottom: G1 cell; preanaphase cell; anaphase cell; and telophase cell. Bar, 2 μm. (B) Bik1–3GFP localization at the kinetochore during mitosis in cells expressing Bik1–3GFP (green) and Spc42-CFP (red), a spindle pole body marker. Bar, 2 μm.

Mentions: Bik1 has sequence homology to the MT plus end–associated protein CLIP-170 (Pierre et al., 1992), but previous localization studies on cells overexpressing Bik1 indicated that, unlike CLIP-170, the protein was present along the length of all MT structures (Berlin et al., 1990). To determine whether this distribution was a consequence of overexpression, we reexamined Bik1 localization in cells expressing the protein at native levels. Bik1 was fused to three tandem copies of GFP (Bik1–3GFP). This chimera was functional as assayed by complementation of bik1Δ cells (see Materials and methods). Bik1–3GFP gave bright and strikingly discontinuous labeling of MTs, with discrete dots located at the plus ends of MTs (Fig. 1 A). During mitosis, Bik1–3GFP was also present in a bilobed distribution on spindle MTs where the kinetochores are known to localize. This bilobed distribution of Bik1–3GFP near the spindle poles persisted throughout all stages of mitosis (Fig. 1 B). This contrasts with a previous study that found that CLIP-170 is only transiently associated with kinetochores at prometaphase in tissue culture cells (Dujardin et al., 1998). Overall, these data show that the localization of Bik1 is similar to that of human CLIP-170 and to that of the other budding yeast MT plus end–binding protein Bim1 (Pierre et al., 1992; Perez et al., 1999; Tirnauer et al., 1999).


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)

Localization of Bik1 to MT plus ends and to the kinetochore. (A) Colocalization of Bik1–3GFP (green) with CFP-Tub1 (red) at different stages of the cell cycle. Pairs of DIC (left) and merged fluorescence (right) images are shown. From top to bottom: G1 cell; preanaphase cell; anaphase cell; and telophase cell. Bar, 2 μm. (B) Bik1–3GFP localization at the kinetochore during mitosis in cells expressing Bik1–3GFP (green) and Spc42-CFP (red), a spindle pole body marker. Bar, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Localization of Bik1 to MT plus ends and to the kinetochore. (A) Colocalization of Bik1–3GFP (green) with CFP-Tub1 (red) at different stages of the cell cycle. Pairs of DIC (left) and merged fluorescence (right) images are shown. From top to bottom: G1 cell; preanaphase cell; anaphase cell; and telophase cell. Bar, 2 μm. (B) Bik1–3GFP localization at the kinetochore during mitosis in cells expressing Bik1–3GFP (green) and Spc42-CFP (red), a spindle pole body marker. Bar, 2 μm.
Mentions: Bik1 has sequence homology to the MT plus end–associated protein CLIP-170 (Pierre et al., 1992), but previous localization studies on cells overexpressing Bik1 indicated that, unlike CLIP-170, the protein was present along the length of all MT structures (Berlin et al., 1990). To determine whether this distribution was a consequence of overexpression, we reexamined Bik1 localization in cells expressing the protein at native levels. Bik1 was fused to three tandem copies of GFP (Bik1–3GFP). This chimera was functional as assayed by complementation of bik1Δ cells (see Materials and methods). Bik1–3GFP gave bright and strikingly discontinuous labeling of MTs, with discrete dots located at the plus ends of MTs (Fig. 1 A). During mitosis, Bik1–3GFP was also present in a bilobed distribution on spindle MTs where the kinetochores are known to localize. This bilobed distribution of Bik1–3GFP near the spindle poles persisted throughout all stages of mitosis (Fig. 1 B). This contrasts with a previous study that found that CLIP-170 is only transiently associated with kinetochores at prometaphase in tissue culture cells (Dujardin et al., 1998). Overall, these data show that the localization of Bik1 is similar to that of human CLIP-170 and to that of the other budding yeast MT plus end–binding protein Bim1 (Pierre et al., 1992; Perez et al., 1999; Tirnauer et al., 1999).

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