Fig. 1
Distribution of distinct pools of nascent CENP-A is equivalent between sister centromeres and between chromosomes. a Outline for using quench-pulse-chase experiments to detect distinct nascent pools of SNAP-tagged CENP-A over several cell cycles. After quench-pulse-chase experiments, cells were arrested in metaphase, and total CENP-A was detected by immunostaining with CENP-A antibodies. Quantitative measurements of sister centromeres of individual chromosomes revealed how nascent CENP-A had been distributed in the most recent or previous cell cycles. b Frequency distribution of the difference in the total CENP-A intensity (amount of CENP-A) between sister centromeres (n > 13,000). c Results of multi-labeling of distinct SNAP-CENP-A pools to follow temporal dynamics of CENP-A distribution. Nascent CENP-A from consecutive (Cycle 1 vs Cycle 2; Cycle 2 vs Cycle 3) and alternating cell cycles (Cycle 1 vs Cycle 3) was detected. Scale bars 15 μm. d Quantitation of CENP-A fluorescence (arbitrary fluorescence units, AFU) at single sister centromeres in two different cell cycles. Chromosome insets highlight nascent CENP-A at single sister centromeres and detected in different cell cycles (red, green arrows). e A subset of the data from d showing proportionate CENP-A distribution in Cycle 1 followed by disproportionate distribution in Cycle 2. f, g Quantitation of SNAP-CENP-A distribution between sister centromeres over different cell cycles, showing the extreme edges (upper 80 % and lower 20 %) of the data set and illustrating disproportionate CENP-A distribution in Cycle 1 followed by proportionate distribution in Cycle 2