Fig. 3. pds5 cells are sensitive to delayed progression through mitosis, but do not undergo precocious sister chromatid separation during metaphase arrest. (A) Tenfold serial dilutions of HM123 (pds5⁺), pds5 and pds5-GFP strains were spotted onto YE agar containing 20 µg/ml TBZ or no drug (control). Plates were photographed after 3-5 days incubation at 30°C. (B) HM123 (pds5⁺) and the other strains indicated were streaked in parallel onto YE agar plates and were photographed after 5 days incubation at 23°C or 30°C, as indicated. (C) Micrographs of formaldehyde fixed, DAPI stained nda3-KM311 (pds5⁺) and nda3-KM311 pds5 (pds5) cells before (0 minutes) and 9 minutes after shifting to 36°C to release from a metaphase arrest, imposed previously by holding the cells at 20°C for 8 hours. Examples of anaphase cells with lagging chromosomes are indicated by arrowheads. The percentages of total anaphase cells and anaphase cells with lagging chromosomes 9 minutes after release are indicated graphically on the right. (D) The nda3-KM311 ndc80-GFP pds5 strain was incubated in EMM2 medium at 30°C or shifted to 18°C for 12 hours. Fluorescence micrographs of living cells were acquired after staining with Hoechst 33342, revealing green fluorescence (Ndc80-GFP) and DNA (Hoechst) in the same field, as indicated. Bars, 10 µm.

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