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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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Characterization of the mitotic spindle in triploids bearing bik1-CTΔ40. (A) Triploid cells containing bik1-CTΔ40 have normal spindle morphology. The left panel shows spindles in triploid cells containing BIK1. The right panel shows spindles in triploid cells containing bik1-CTΔ40. MTs are labeled in both strains by GFP-Tub-1. (B) FRAP in BIK1- and bik1-CTΔ40–containing triploid cells. MTs are labeled by GFP-Tub1. The graph shows intensity measurements of a 5 × 5 pixel area from preanahase spindles in triploid cells bearing either BIK1 (Δ, bleached; ⋄, unbleached) or bik1-CTΔ40 (•, bleached; ▪, unbleached). The predicted exponential curves for FRAP are shown as the plotted lines. Curves I and III represent the bleached and unbleached regions of triploid cell bearing bik1-CTΔ40, respectively. Curves II and IV represent the bleached and unbleached regions of cells bearing BIK1, respectively. These theoretical curves were derived from the first-order rate constant, calculated as described in the Materials and methods (also see Table III). The intensity of the unbleached region at the 0 time point was assumed to be 100% and the intensity of bleached region at the 0 time point was assumed to be 0%. (C) Example of a FRAP experiment with a preanaphase triploid cell bearing bik1-CTΔ40. Time is in seconds. Bar, 1 μm. (D) The kinetics of anaphase spindle elongation are not altered in bik1-CTΔ40–containing triploids relative to BIK1-containing triploids. Time-lapse images were acquired from GFP-Tub1–containing strains at ∼90-s intervals.
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fig6: Characterization of the mitotic spindle in triploids bearing bik1-CTΔ40. (A) Triploid cells containing bik1-CTΔ40 have normal spindle morphology. The left panel shows spindles in triploid cells containing BIK1. The right panel shows spindles in triploid cells containing bik1-CTΔ40. MTs are labeled in both strains by GFP-Tub-1. (B) FRAP in BIK1- and bik1-CTΔ40–containing triploid cells. MTs are labeled by GFP-Tub1. The graph shows intensity measurements of a 5 × 5 pixel area from preanahase spindles in triploid cells bearing either BIK1 (Δ, bleached; ⋄, unbleached) or bik1-CTΔ40 (•, bleached; ▪, unbleached). The predicted exponential curves for FRAP are shown as the plotted lines. Curves I and III represent the bleached and unbleached regions of triploid cell bearing bik1-CTΔ40, respectively. Curves II and IV represent the bleached and unbleached regions of cells bearing BIK1, respectively. These theoretical curves were derived from the first-order rate constant, calculated as described in the Materials and methods (also see Table III). The intensity of the unbleached region at the 0 time point was assumed to be 100% and the intensity of bleached region at the 0 time point was assumed to be 0%. (C) Example of a FRAP experiment with a preanaphase triploid cell bearing bik1-CTΔ40. Time is in seconds. Bar, 1 μm. (D) The kinetics of anaphase spindle elongation are not altered in bik1-CTΔ40–containing triploids relative to BIK1-containing triploids. Time-lapse images were acquired from GFP-Tub1–containing strains at ∼90-s intervals.

Mentions: Triploid cells bearing bik1-CTΔ40 had morphologically normal mitotic spindles (Fig. 6 A). In addition, the average length of preanaphase spindles in triploids bearing bik1-CTΔ40 was not significantly different from BIK1-containing triploids (1.14 ± 0.3 μm [n = 197] and 1.10 ± 0.3 μm [n = 157], respectively, p > 0.05). We calculated the approximate volume of preanaphase spindles by measuring the width and length of the GFP-Tub1–labeled preanaphase spindles (Tub1 is the major α-tubulin isoform), assuming that they are cylinders. These values were also not significantly different between bik1-CTΔ40–containing triploids and the control (0.9 ± 0.3 μm3 and 1.0 ± 0.4 μm3, respectively, p > 0.05). By contrast, the spindle volume was smaller in isogenic wild-type haploids (0.4 ± 0.2 μm3, p < 0.001). Thus, bik1-CTΔ40 does not alter the size or shape of preanaphase spindles in triploid cells. The preanaphase spindles in triploids are similar in length to the preanaphase spindles in haploids, but they are somewhat thicker. The relatively short length of preanaphase spindles in triploids might reflect increased tension on the spindle poles from the additional bioriented kinetochores (Goshima 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)

Characterization of the mitotic spindle in triploids bearing bik1-CTΔ40. (A) Triploid cells containing bik1-CTΔ40 have normal spindle morphology. The left panel shows spindles in triploid cells containing BIK1. The right panel shows spindles in triploid cells containing bik1-CTΔ40. MTs are labeled in both strains by GFP-Tub-1. (B) FRAP in BIK1- and bik1-CTΔ40–containing triploid cells. MTs are labeled by GFP-Tub1. The graph shows intensity measurements of a 5 × 5 pixel area from preanahase spindles in triploid cells bearing either BIK1 (Δ, bleached; ⋄, unbleached) or bik1-CTΔ40 (•, bleached; ▪, unbleached). The predicted exponential curves for FRAP are shown as the plotted lines. Curves I and III represent the bleached and unbleached regions of triploid cell bearing bik1-CTΔ40, respectively. Curves II and IV represent the bleached and unbleached regions of cells bearing BIK1, respectively. These theoretical curves were derived from the first-order rate constant, calculated as described in the Materials and methods (also see Table III). The intensity of the unbleached region at the 0 time point was assumed to be 100% and the intensity of bleached region at the 0 time point was assumed to be 0%. (C) Example of a FRAP experiment with a preanaphase triploid cell bearing bik1-CTΔ40. Time is in seconds. Bar, 1 μm. (D) The kinetics of anaphase spindle elongation are not altered in bik1-CTΔ40–containing triploids relative to BIK1-containing triploids. Time-lapse images were acquired from GFP-Tub1–containing strains at ∼90-s intervals.
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fig6: Characterization of the mitotic spindle in triploids bearing bik1-CTΔ40. (A) Triploid cells containing bik1-CTΔ40 have normal spindle morphology. The left panel shows spindles in triploid cells containing BIK1. The right panel shows spindles in triploid cells containing bik1-CTΔ40. MTs are labeled in both strains by GFP-Tub-1. (B) FRAP in BIK1- and bik1-CTΔ40–containing triploid cells. MTs are labeled by GFP-Tub1. The graph shows intensity measurements of a 5 × 5 pixel area from preanahase spindles in triploid cells bearing either BIK1 (Δ, bleached; ⋄, unbleached) or bik1-CTΔ40 (•, bleached; ▪, unbleached). The predicted exponential curves for FRAP are shown as the plotted lines. Curves I and III represent the bleached and unbleached regions of triploid cell bearing bik1-CTΔ40, respectively. Curves II and IV represent the bleached and unbleached regions of cells bearing BIK1, respectively. These theoretical curves were derived from the first-order rate constant, calculated as described in the Materials and methods (also see Table III). The intensity of the unbleached region at the 0 time point was assumed to be 100% and the intensity of bleached region at the 0 time point was assumed to be 0%. (C) Example of a FRAP experiment with a preanaphase triploid cell bearing bik1-CTΔ40. Time is in seconds. Bar, 1 μm. (D) The kinetics of anaphase spindle elongation are not altered in bik1-CTΔ40–containing triploids relative to BIK1-containing triploids. Time-lapse images were acquired from GFP-Tub1–containing strains at ∼90-s intervals.
Mentions: Triploid cells bearing bik1-CTΔ40 had morphologically normal mitotic spindles (Fig. 6 A). In addition, the average length of preanaphase spindles in triploids bearing bik1-CTΔ40 was not significantly different from BIK1-containing triploids (1.14 ± 0.3 μm [n = 197] and 1.10 ± 0.3 μm [n = 157], respectively, p > 0.05). We calculated the approximate volume of preanaphase spindles by measuring the width and length of the GFP-Tub1–labeled preanaphase spindles (Tub1 is the major α-tubulin isoform), assuming that they are cylinders. These values were also not significantly different between bik1-CTΔ40–containing triploids and the control (0.9 ± 0.3 μm3 and 1.0 ± 0.4 μm3, respectively, p > 0.05). By contrast, the spindle volume was smaller in isogenic wild-type haploids (0.4 ± 0.2 μm3, p < 0.001). Thus, bik1-CTΔ40 does not alter the size or shape of preanaphase spindles in triploid cells. The preanaphase spindles in triploids are similar in length to the preanaphase spindles in haploids, but they are somewhat thicker. The relatively short length of preanaphase spindles in triploids might reflect increased tension on the spindle poles from the additional bioriented kinetochores (Goshima 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