The old pole nucleates microtubules in cut12.1 and cut11.1 defective mitoses. (A–C) cut12.1 pcp1.RFP nmt81.atb2GFP (A) or cut11.1 pcp1.RFP nmt81.atb2GFP (B and C) cells were grown to mid-log phase at 25°C and starved by the removal of a nitrogen source for 14 h and 8 h, respectively, before being filtered and resuspended in nitrogen-containing minimal medium at a density of 1.5 × 106 cells/ml. 4 h after refeeding, both mutants were shifted to 36°C for 3 h, and samples were taken either for live cell imaging (A and C) or anti-Sad1/anti–α-tubulin immunofluorescence (IF) microscopy (B). In this case, DAPI was used to stain the chromatin. All combinations of two signals are shown using false colors. Although microtubules emerge from only one of two dots of Sad1 staining in cut11.1 cells in B, microtubules emerge from the single (old SPB) staining in A and C. Note the asymmetry of Sad1 staining in cut11.1 immunofluorescence (B, left). The more intense signal correlates with the nonactive SPB. Bars: (A and C) 5 µm; (B) 10 µm.
