Overexpression of mitotic cyclin <i>CLB2</i> results in premature spindle elongation in <i>swe1Δ</i> mutants.

<p>A. Overexpression of <i>CLB2</i> is toxic to <i>swe1Δ</i> mutants. WT and <i>swe1Δ</i> mutant cells with a control vector or <i>P_GALCLB1</i>, <i>P_GALCLB2</i>, <i>P_GALCLB3</i>, and <i>P_GALCLB5</i> plasmids were grown to saturation in glucose medium, 10-fold diluted, and spotted onto glucose or galactose plates. The plates were scanned after incubation at 30°C for 3 days. B. Overexpression of <i>CLB2</i> in <i>swe1</i>Δ mutant cells leads to premature spindle elongation. G₁-arrested <i>PDS1-Myc TUB1-GFP</i> and <i>swe1Δ PDS1-Myc TUB1-GFP</i> cells with a vector or a <i>P_GALCLB2</i> plasmid were released into 30°C galactose medium to induce <i>CLB2</i> overexpression. Cells were collected over time and fixed to examine GFP signal and for DAPI staining. Spindles longer than 3 µm were counted as elongated. The percentage of cells with an elongated spindle is shown in the left panel (n>100). The percentage of binucleate cells is shown in the middle panel and the spindle morphology in cells at 120 min time point is shown in the right panel. The arrow indicates a binucleate cell with premature spindle elongation. Scale bar, 5 µm. The budding index, FACS analysis and Pds1 protein level are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003319#pgen.1003319.s002" target="_blank">Figure S2</a>. C. Live-cell imaging shows the premature spindle elongation in <i>swe1</i>Δ mutants overexpressing <i>CLB2</i>. <i>swe1</i>Δ <i>TUB1-GFP</i> cells with a vector or a <i>P_GALCLB2</i> plasmid were arrested in G₁-phase in raffinose medium. After released into galactose medium for 1.5 hr, the cells were transferred to an agarose pad on a microscope slide to perform live-cell imaging at 25°C. Scale bar, 5 µm.