FIG. 2.
bir1-17 mutant cells show mono-oriented attachment of sister centromeres to microtubules during metaphase. Wild-type BIR1 (VMY33) and bir1-17 (VMY34) cells containing CEN3-(tetO)336, tetR-GFP, YFP-TUB1, and pMET3-CDC20 were arrested in G1 with α-factor at 26°C and then released to a metaphase block in rich medium (containing 2 mM methionine to deplete Cdc20) at 37°C for 2.5 h. (A) Representative time-lapse images taken from live cells during 4 min. Bioriented chromosomes show the dynamic splitting and reassociation of sister CEN3 centromeres, whereas mono-oriented chromosomes show continuously unresolved sister CEN3 centromeres localized to one end of the spindle. Green, CEN3 labeled with tetR-GFP; red, YFP-tubulin. (B) Quantification of chromosome biorientation in metaphase-arrested cells from multiple time-lapse fields (n is the number of cells scored in each category). Examples of wild-type and bir1-17 time-lapse movies can be found in the supplemental material.