Brr6 is required for bipolar spindle formation. Cells were processed for immunofluorescence with anti-tubulin, anti-Sad1, and DAPI as indicated. (A and B) brr6.ds1 cells were grown to early log phase at 32°C in rich YE5S medium before DMSO was added to a final concentration of 4%. Samples were processed for immunofluorescence at discrete time points and scored for the indicated features (B). The images in A are from the 120-min time point. Microtubules (tubulin) extend from adjacent SPBs (Sad1) as chromatin becomes highly condensed, giving it a lumpy appearance (chromatin). (C and D) brr6.ts8 cells were grown to early log phase at 25°C in rich YE5S medium before the temperature of the culture was shifted to 36°C, and samples were processed and scored for the features as indicated in D. The category “no MTs/mitotic” indicates the cells with condensed chromosomes and no microtubule staining. The images in C are from the 30-min time point. (E) brr6.ts8 cells were grown to early log phase at 25°C in rich YE5S medium before the temperature of the culture was shifted to 36°C. 2 h later, a culture of mid-log phase cdc7.A20 spg1.B8 cells (asterisks) that had been cultured at 36°C for 4 h was mixed at a ratio of 1:5 with the brr6.ts8 culture before the mixed culture was processed for immunofluorescence. Although the brr6.ds1 mutation gives rise to monopolar spindles, monopolar spindle formation is transient in the more penetrant brr6.ts8, as prolonged loss of function leads to an inability to nucleate microtubules (arrows). Bars, 5 µm.