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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 is a plus end–tracking protein and the yeast orthologue of human CLIP-170. (A) Time-lapse series of Bik1–3GFP. Time is in s. The images are two-dimensional projections of a 0.5-μm Z-focal plane image stack. Arrows indicate Bik1–3GFP spots moving toward the cell periphery. (B) The localization of Bik1, Bik1-NΔ110, Bik1-MTP (which contains four amino acid substitutions in the CAP-Gly MT-binding domain identical to that previously done for CLIP-170; Pierre et al., 1992), and CLIP-170-Bik1. For Bik1, Bik1-MTP, and CLIP-170-Bik1 black-and-white images of the GFP fluorescence are shown. Arrows indicate localization of Bik1 and CLIP-170-Bik1 to the plus ends of astral MTs. For Bik1-NΔ110, a color image is shown of the localization of Bik1-NΔ110-GFP (green) adjacent to Spc42-CFP (red, a SPB marker). All Bik1 constructs are expressed as COOH-terminal fusions to one copy of GFP. (C) Western blot showing the steady-state protein levels of the indicated Bik1 derivatives detected with a polyclonal anti-GFP antibody (50 μg of cell extract were loaded in each lane). Bars, 2 μm.
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fig2: Bik1 is a plus end–tracking protein and the yeast orthologue of human CLIP-170. (A) Time-lapse series of Bik1–3GFP. Time is in s. The images are two-dimensional projections of a 0.5-μm Z-focal plane image stack. Arrows indicate Bik1–3GFP spots moving toward the cell periphery. (B) The localization of Bik1, Bik1-NΔ110, Bik1-MTP (which contains four amino acid substitutions in the CAP-Gly MT-binding domain identical to that previously done for CLIP-170; Pierre et al., 1992), and CLIP-170-Bik1. For Bik1, Bik1-MTP, and CLIP-170-Bik1 black-and-white images of the GFP fluorescence are shown. Arrows indicate localization of Bik1 and CLIP-170-Bik1 to the plus ends of astral MTs. For Bik1-NΔ110, a color image is shown of the localization of Bik1-NΔ110-GFP (green) adjacent to Spc42-CFP (red, a SPB marker). All Bik1 constructs are expressed as COOH-terminal fusions to one copy of GFP. (C) Western blot showing the steady-state protein levels of the indicated Bik1 derivatives detected with a polyclonal anti-GFP antibody (50 μg of cell extract were loaded in each lane). Bars, 2 μm.

Mentions: Human CLIP-170 has the unusual property of associating with the growing plus ends of MTs (Perez et al., 1999). Recently, human EB1 (a Bim1 orthologue), the adenomatous polyposis coli tumor suppressor protein, and components of the dynein motor complex (Xiang et al., 2000; Han et al., 2001) were found to have a similar pattern of association with MT plus ends in vivo (Brunner and Nurse, 2000; Mimori-Kiyosue et al., 2000a,b). We term this behavior “plus end-tracking” (Schuyler and Pellman, 2001a). By time-lapse fluorescence microscopy we found that Bik1–3GFP at the ends of astral MTs moved toward the periphery of the cell as was previously described for CLIP-170 and EB1 (Fig. 2 A). The Bik1–3GFP spots moved at a rate of 3.6 ± 1.5 μm/min in G1 cells (n = 10) and at a rate of 2.9 ± 1.1 μm/min in mitotic cells (n = 20). Although there is some variation in the reported growth rate of astral MTs in budding yeast (measured with different GFP reporters expressed from different promoters), the velocity of the Bik1–3GFP dots was within the range of these values (for review see Adames and Cooper, 2000). Furthermore, we found that in cells where tubulin was labeled with CFP-Tub1, the Bik1–3GFP dots were found at the distal ends of astral MTs, not along the body of the MTs, 96% of the time (n = 170). Together, these data show that Bik1 has the characteristics of a plus end–tracking 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 is a plus end–tracking protein and the yeast orthologue of human CLIP-170. (A) Time-lapse series of Bik1–3GFP. Time is in s. The images are two-dimensional projections of a 0.5-μm Z-focal plane image stack. Arrows indicate Bik1–3GFP spots moving toward the cell periphery. (B) The localization of Bik1, Bik1-NΔ110, Bik1-MTP (which contains four amino acid substitutions in the CAP-Gly MT-binding domain identical to that previously done for CLIP-170; Pierre et al., 1992), and CLIP-170-Bik1. For Bik1, Bik1-MTP, and CLIP-170-Bik1 black-and-white images of the GFP fluorescence are shown. Arrows indicate localization of Bik1 and CLIP-170-Bik1 to the plus ends of astral MTs. For Bik1-NΔ110, a color image is shown of the localization of Bik1-NΔ110-GFP (green) adjacent to Spc42-CFP (red, a SPB marker). All Bik1 constructs are expressed as COOH-terminal fusions to one copy of GFP. (C) Western blot showing the steady-state protein levels of the indicated Bik1 derivatives detected with a polyclonal anti-GFP antibody (50 μg of cell extract were loaded in each lane). Bars, 2 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199317&req=5

fig2: Bik1 is a plus end–tracking protein and the yeast orthologue of human CLIP-170. (A) Time-lapse series of Bik1–3GFP. Time is in s. The images are two-dimensional projections of a 0.5-μm Z-focal plane image stack. Arrows indicate Bik1–3GFP spots moving toward the cell periphery. (B) The localization of Bik1, Bik1-NΔ110, Bik1-MTP (which contains four amino acid substitutions in the CAP-Gly MT-binding domain identical to that previously done for CLIP-170; Pierre et al., 1992), and CLIP-170-Bik1. For Bik1, Bik1-MTP, and CLIP-170-Bik1 black-and-white images of the GFP fluorescence are shown. Arrows indicate localization of Bik1 and CLIP-170-Bik1 to the plus ends of astral MTs. For Bik1-NΔ110, a color image is shown of the localization of Bik1-NΔ110-GFP (green) adjacent to Spc42-CFP (red, a SPB marker). All Bik1 constructs are expressed as COOH-terminal fusions to one copy of GFP. (C) Western blot showing the steady-state protein levels of the indicated Bik1 derivatives detected with a polyclonal anti-GFP antibody (50 μg of cell extract were loaded in each lane). Bars, 2 μm.
Mentions: Human CLIP-170 has the unusual property of associating with the growing plus ends of MTs (Perez et al., 1999). Recently, human EB1 (a Bim1 orthologue), the adenomatous polyposis coli tumor suppressor protein, and components of the dynein motor complex (Xiang et al., 2000; Han et al., 2001) were found to have a similar pattern of association with MT plus ends in vivo (Brunner and Nurse, 2000; Mimori-Kiyosue et al., 2000a,b). We term this behavior “plus end-tracking” (Schuyler and Pellman, 2001a). By time-lapse fluorescence microscopy we found that Bik1–3GFP at the ends of astral MTs moved toward the periphery of the cell as was previously described for CLIP-170 and EB1 (Fig. 2 A). The Bik1–3GFP spots moved at a rate of 3.6 ± 1.5 μm/min in G1 cells (n = 10) and at a rate of 2.9 ± 1.1 μm/min in mitotic cells (n = 20). Although there is some variation in the reported growth rate of astral MTs in budding yeast (measured with different GFP reporters expressed from different promoters), the velocity of the Bik1–3GFP dots was within the range of these values (for review see Adames and Cooper, 2000). Furthermore, we found that in cells where tubulin was labeled with CFP-Tub1, the Bik1–3GFP dots were found at the distal ends of astral MTs, not along the body of the MTs, 96% of the time (n = 170). Together, these data show that Bik1 has the characteristics of a plus end–tracking protein.

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