Fig. 2. Polo boxes are required for interference of bipolar spindle formation, but not for induction of septation upon overexpression of Plo1. Wild-type cells carrying plo1 mutants in pREP1 under the control of the thiamine repressible nmt1 promoter were grown in the absence of thiamine. The cells were fixed and stained with DAPI, which highlights DNA, cell outlines and septa. No defects were apparent with any of the plasmids in the presence of thiamine. Bar, 10 µm. (A) Empty vector, pREP1 has no effect on cell growth. (B) Overexpression of wildtype plo1 (pREP1plo1⁺) leads to mitotic arrest and cells with extra septa. (C,D) Overexpression of mutants in the polo boxes (plo1W497F,plo1.1-583 are shown here as examples) resulted in uncontrolled formation of septa but no mitotically arrested cells. (E) Overexpression of a catalytic inactive plo1 (plo1K69R) results only in mitotically arrested cells with highly overcondensed chromosomes with no cells forming extra septa. (F) Overexpression of a catalytically inactive polo-box mutant plo1K69R YQL508AAA. No abnormalities can be seen. (G-I) Monopolar spindles were associated with overcondensed chromosomes in cells overexpressing plo1K69R. The SPB component -tubulin (G), Sad1 (H) and DNA (I). (J-L) Quantification of cytological defects seen upon overproduction of wild-type Plo1 (J), Plo1 with a polo box mutation (Plo1DHK625AAA; K) or kinase inactive Plo1 (Plo1K69R; L) in wild-type or mad2 deletion strains. Cells were observed by DAPI staining after the removal of thiamine to induce expression from the nmt1 promoter. Mitotic arrest caused by overproduction of inactive Plo1 is abolished by a mutation in the spindle checkpoint, mad2.

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