Figure 1.

apc15Δ Mutants Are Checkpoint Defective

(A) Checkpoint arrest. nda3 strains were grown to log phase and then shifted to 18°C to de-polymerize microtubules and thereby activate the spindle checkpoint. At the time points indicated, cells were fixed in methanol and the mitotic index was scored by analyzing the levels and localization of Cdc13-GFP (cyclin B). Cdc13-GFP localizes to the spindle pole bodies in early mitosis. The apc14Δ mutant arrests proficiently, but apc15Δ and mad2Δ mutants do not. This experiment was repeated three times (with at least 100 cells scored per strain at each time point), and the data are plotted as the mean ± SD.

(B) Mad2 and Mph1 overexpression. Cultures containing plasmids expressing Mad2 from the nmt1 promoter or Mph1 from the nmt41 promoter were induced (−thiamine) for 18 hr and the mitotic index was scored by immunostaining of microtubules and spindle length. This experiment was repeated twice (with at least 100 cells scored per strain at each time point), and the data are plotted as the mean ± SD.

(C) MCC assembly is not affected. cdc25-22 cdc20-FLAG cultures were synchronized at G2/M by cdc25 block and release, cell samples were taken at 15-min intervals, and Cdc20 was subjected to immunoprecipitation (IP) and analyzed for associated checkpoint proteins (Mad3 and Mad2). This experiment was repeated three times, and a representative example is shown here.

See also Figure S1.