Fig. 5. alp4 mutants become irreversibly lethal before entry into mitosis. (A) The timing of lethality. Nitrogen-starved, G₁-synchronized alp4 mutants at 26°C were shifted to 36°C upon addition of nitrogen. Samples were taken every 30 min, and viability loss (shaded boxes) and percentage of septated cells (closed circles) were measured. At each time point, aliquots were shifted down to 26°C and the percentage of cells that progressed through an abnormal mitosis (hatched) was counted. (B) DNA content upon nitrogen addition. Samples used in (A) were fixed and processed for FACS analysis. (C) Appearance of monopolar spindles under permissive conditions. alp4 mutant cells grown at 26°C were shifted to 36°C and HU (10 mM) was added simultaneously. After 4 h incubation, HU was washed out, and the cultures were returned to 26°C. Samples were then taken at different time points for immunofluorescence microscopy with anti-tubulin antibody (red) and Cut12–GFP (green, an integral SPB protein; Bridge et al., 1998). The figure (confocal microscopy) shows representative abnormal mitotic cells that have monopolar spindles (2 h at 26°C). The bar indicates 5 µm.