Fig. 7. Bub2/Bfa1 act during metaphase arrest caused by means other than spindle damage. (A) Bfa1 shows the same pattern of phosphorylation during apc2-8 temperature arrest as after nocodazole-induced arrest. Three strains expressing 3HA-Bfa1, a wild type, SLY116 (apc2-8) and JB5A1-51C (apc2-8 cdc5) were grown to mid-log at 25°C and transferred to 37°C for 3 hours in the presence or absence of nocodazole and an immunoblot prepared. (B) Bub2 restrains actin ring formation in metaphase arrest caused by apc2-8 temperature shift or overexpression of indestructible Pds1. Mid-log cultures of the apc2-8 strains were transferred to 37°C for 3 hours. For overexpression of indesructible Pds1, the strains were grown to mid-log phase in YEP sucrose, arrested with 
-factor and released into fresh YEP sucrose and galactose added to 2% when all cells had budded. Once large-budded cells had accumulated, samples were removed for microscopy. Cells were stained with DAPI and rhodamine-phalloidin for actin distribution (Frenz et al., 2000). The cells shown above (B) are representative examples of the large-budded cells examined. (C, D) Bub2 restrains mitotic exit in apc2-8-arrested cells. Strains containing TUB1.GFP in an apc2-8 or apc2-8 bub2
background were grown to mid-log phase at 23°C, shifted to 37°C and sampled at 2 hour intervals. Rebudding was assessed microscopically following sonication (C) and the state of the spindles and SPBs examined by fluorescent microscopy (D).
