Time course analysis of precocious separation of sister centromeres in budding yeast: continuously separated or frequently reassociated?

Background: Sister kinetochores are bioriented toward the spindle poles in eukaryotic metaphase before chromosome segregation. In the budding yeast Sa ccharomyces cerevisiae, sister centromeres/kinetochores are separated in th e early spindle, while the sister arms remain associated. Biorientation is thought to be established in this organism with precocious separation of si ster centromeres in early stages of the cell cycle. It is not, however, set tled whether this pre-anaphase separation is continuous or only transient a nd whether the transient separation has any physiological significance. Results: Time-lapse observation of the behaviour of budding yeast centromer es in living cells was performed using GFP alone or in combination with CFP marking. Sixty-three per cent of the cell population showed permanent sepa ration of centromeres for a long period of time from the small-budded stage to the onset of anaphase in the single-colour GFP-CEN construct. The remai ning cell population (6 of 16) showed brief apparent reassociation of centr omere signals before anaphase, but the frequency of the association was ver y low. In a time-lapse observation of the double-colour marked cells by GFP -CEN and CFP-SPB (the spindle pole body), the continuous separation of sist er centromeres in the short medial spindle was firmly established. Conclusions: In the budding yeast, once sister centromeres separate, they r arely reassociate in pre-anaphase. Sister centromere cohesion at this stage appears to be irrelevant for normal chromosome segregation. Whether abunda nt cohesin in the centromere regions has any role in anaphase remains to be determined.