Molecular Imaging Reveals a Novel Yeast Centromere Structural Transition in Anaphase

We use in vivo fluorescence correlation spectroscopy (FCS) and high sensitivity quantitative confocal imaging to demonstrate a dramatic structural rearrangement of the yeast centromere in early anaphase. using FCS, we show that the yeast centromeric histone, Cse4, is present in the early kinetochore at a stoichiometry of one protein per chromosome and doubles to two copies during the structural change. This transition reconciles the controversy between previous results observing both forms in vitro. We have confirmed this result by quantifying nuclear pore complex stoichiometries which are well known. Fluorescence recovery after photobleaching (FRAP) experiments further confirm our result, demonstrating centromeric protein addition specifically during anaphase. Fluorescence resonance energy transfer (FRET) experiments also confirm the result, demonstrating homotypic interactions only in late anaphase. The yeast centromere is an important model complex whose functions are central to many important biological processes. These results have revolutionized our understanding of these basic processes and provide fertile ground for future studies of the kinetochore. Our method demonstrates a unique modality for FCS and quantitative imaging and will likely provide much needed insight into biologically important structures in the future.