Polyploids require Bik1 for kinetochore-microtubule attachment

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.

Figure 1.

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.

Figure 2.

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.

Figure. 3.

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.

Figure 4.

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.

Figure 5.

BIK1 is essential for viability of polyploids. (A) A series of bik1Δ strains of the indicated ploidy was transformed with a 2μ BIK1 URA3 plasmid and plated on medium containing 5-fluoroorotic acid (5-FOA). 5-fluoroorotic acid selects for the loss of the BIK1 URA3 plasmid revealing the growth defect of triploid and tetraploid bik1Δ strains at 24°C. Control BIK1 strains of the same ploidy do not have a detectable growth defect (Galitski et al., 1999; unpublished data). (B) Growth of BIK1- and bik1-CTΔ40–containing triploids. Serial fivefold dilutions from cultures of the indicated strains at a density of 5 × 10⁷ cells/ml were plated. The indicated strains were spotted onto YPD medium.

Figure 6.

Characterization of the mitotic spindle in triploids bearing bik1-CT Δ40. (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.

Figure 7.

bik1-CTΔ40–containing triploids have a defect in preanaphase kinetochore separation. Kinetochore separation was scored in the indicated strains by coimmunostaining for tubulin (monoclonal antibody YOL1/34) and GFP-marked CEN5 (polyclonal anti-GFP). Unseparated kinetochores are seen as one fluorescent dot, whereas separated kinetochores are seen as two dots. The fraction of cells with separated kinetochores and the average spindle lengths were as follows. BIK1-containing cells: 35/86 and 1.2 μm (haploids); 68/158 and 1.2 μm (diploids); and 68/157 and 1.1 μm (triploids). bik1-CTΔ40–containing cells: 35/88 and 1.3 μm (haploids); 63/158 and 1.2 μm (diploids); and 36/197 and 1.2 μm (triploids).

Figure 8.

Defective kinetochore dynamics in bik1-CT Δ40 –containing triploid cells. Rapid single-focal plane imaging of GFP-tagged kinetochores relative to GFP-tagged SPBs in BIK1- and bik1-CTΔ40–containing triploid cells. Images were acquired approximately every 2 s. >1,300 data points were analyzed for each strain. (A) An example of a BIK1-containing triploid cell where kinetochores remain separated throughout most of the time of imaging. (B) An example of a bik1-CTΔ40–containing triploid cell where kinetochores do not separate. (C) An example of a BIK1-containing triploid cell where kinetochores separate on average for an interval >30 s. (D) An example of a bik1-CTΔ40–containing triploid cell where 9 very short kinetochore separations occur within a 2-min period. (E) Time-lapse images illustrating very short kinetochore separations in a bik1-CTΔ40–containing triploid cell. Arrow indicates separated kinetochores. Bar, 1 μm.