Brr6 is not required for spindle formation after SPB insertion into the nuclear envelope. (A) wee1.50 brr6.ts8 cells were grown to early log phase in rich YE5S medium at 25°C before the temperature of the culture was shifted to 36°C. Cells were processed for immunofluorescence with anti-tubulin, anti-Sad1, and DAPI, and scored as indicated. (B and C) brr6.ts8 nda3.KM311 (B) or brr6.ts8 nda3.KM311 cdc7.HA cells (C) were grown to early log phase at 30°C before the temperature of the culture was dropped to 20°C. 8 h later, the temperature of each culture was shifted to 36°C and samples were processed at the indicated times (min) for immunofluorescence with anti-tubulin (B) or 12CA5 antibodies (C), then stained with DAPI. (B) From top to bottom, microtubules, chromatin, and combined chromatin and differential interference contrast (DIC). (C) Combined staining of Cdc7.HA (red) and chromatin (green). Arrows indicate Cdc7.HA at new SPBs. Bars: (B) 10 µm; (C) 5 µm.