Overexpression of <i>CLB2</i> in <i>pds1</i>Δ mutants causes uncoupled sister-chromatid separation and spindle elongation.

<p>A. Saturated cells with the indicated genotypes were examined for the growth on glucose and galactose plates after 4 day incubation at 25°C. B. Cells with the indicated genotypes were grown to mid-log phase in raffinose medium and then shifted to galactose medium. Cells were collected over time and the viability was examined using platting efficiency (n>300). C. WT, <i>pds1</i>Δ, and <i>pds1</i>Δ <i>spo12</i>Δ cells in <i>CEN4</i>-<i>GFP TUB1-mCherry</i> background with a vector or a <i>P_GALCLB2</i> plasmid were arrested in G₁-phase and then released into 25°C galactose medium to induce <i>CLB2</i> overexpression. Cells were collected at 160 and 180 min and fixed to examine chromosome segregation. The spindle morphology and <i>CEN4-</i>GFP segregation in some representative cells at 180 min are shown in the left panel. The arrow indicates a cell with an elongated spindle and mis-segregated <i>CEN4-</i>GFP. The average percentage of cells with mis-segregated <i>CEN4-</i>GFP at 160 and 180 min from three experiments is shown in the right panel (n>100). Scale bar, 5 µm. D. WT and <i>pds1</i>Δ cells in <i>CEN4</i>-<i>GFP TUB1-mCherry</i> background with a vector or <i>P_GALCLB2</i> plasmid were grown to mid-log phase in raffinose medium and then shifted to galactose medium. The cells were collected after 4 hr incubation at 25°C and fixed for DAPI staining. The segregation of <i>CEN4-</i>GFP as well as the spindle and nuclear morphology in some representative cells is shown. The scale bar is 5 µm. E. WT, <i>pds1</i>Δ, and <i>pds1</i>Δ <i>spo12</i>Δ cells in <i>TEL5-GFP NUF2-mCherry</i> background were treated as described in C. The experiment was repeated for 3 times. The segregation of telomere of chromosome V (<i>TEL5-</i>GFP) in cells at 180 min is shown in the left panel. The arrow indicates a cell with unseparated <i>TEL5</i>. The average percentage of cells with two Nuf2 foci and one <i>TEL5-</i>GFP dot at 160 and 180 min is shown in the right panel after three repeats (n>100). Scale bar, 5 µm.