FIG. 3.

Bir1 is not required for maintenance of biorientation. (A) Wild-type BIR1 (VMY33), bir1-17 (VMY34), and ask1-3 (T2997) cells with CEN3-(tetO)₃₃₆, tetR-GFP, YFP-TUB1, and pMET3-CDC20 were arrested in G₁ with α-factor, incubated for 2.5 h at 26°C in rich medium containing 2 mM methionine (to deplete Cdc20), and then shifted to 37°C in the same medium (defined as time zero). Samples were fixed with paraformaldehyde at the indicated time points. The graphs show the percentage of cells with separated and unseparated sister CEN3 centromeres on the bipolar spindle, plotting the mean values from three separate experiments (error bars indicate standard errors of the means). Sister CEN3 centromere separation was not scored after 45 min in ask1-3 cells, as they exhibited a collapsed spindle at later times. Note that while essentially all separated sister CEN3 centromeres should represent instances where chromosome III is bioriented, the unseparated sister CEN3 category will contain both mono-oriented chromosomes and bioriented chromosomes where the sister CEN centromeres had transiently reassociated at the point of fixation. (B) Metaphase-arrested cells at 37°C from panel A were released at 37°C in methionine-free medium, and the distribution of sister CEN3 centromeres was scored on elongated, anaphase spindles observed within the first 30 min. Cumulative data from two experiments are shown. (C) Representative images of cells to indicate the phenotypes scored in panel B. Red, tubulin (YFP-TUB1); green, CEN3 (tetR-GFP).