Panel A shows a schematic diagram illustrating cohesin cleavage and subsequent sister chromatid separation, highlighting cohesin complexes and the progression of chromosome segregation events. Panel B shows a schematic of fluorescent chromosome labeling near centromeres, indicating positions of tetO and lacO repeats relative to centromeric regions. Panel C shows a line graph depicting sister centromere distance over time, alongside a kymograph visualizing dynamic separation behavior in a single observed cell. Panel D shows a line graph with kymograph explaining how the time difference between separation of two centromere markers is measured and quantified precisely. Panel E shows a histogram displaying frequency distribution of time differences between separation of markers located on the same chromosome with Gaussian fit overlay. Panel F shows histograms comparing time differences between centromere marker separations across chromosomes one and two and chromosomes two and three with statistical analysis. Panel G shows a schematic of fluorescent labeling at inner centromeric regions, illustrating positioning of markers across chromosomes using lacO/LacI-GFP and tetO/TetR-tdTomato systems. Panel H shows histograms with Gaussian fits comparing time differences between central centromere marker separations, contrasted with outer centromere marker distributions for statistical comparison.
Centromeres segregate within a narrow time window but not with perfect synchrony. (A) Schematic depicting cohesin cleavage and subsequent sister chromatid separation; cohesin complexes in orange. (B) Fluorescent labeling of chromosomes close to their centromeric regions. The schematic depicts the localization of the tandem tetO or lacO repeats relative to the centromere, which comprises central core (cnt), innermost repeats (imr), and different numbers of dg/dh repeat pairs. Chromosome II was marked with lacO/LacI-GFP (Yamamoto and Hiraoka, 2003); chromosome I and III were marked with tetO/TetR-tdTomato (Sakuno et al., 2009 and this study). The lengths of the centromere regions (distance between furthest dh/dg repeats) are shown in kbp. (C and D) Sister centromere distance and corresponding kymograph for a strain with cen2-GFP and cen3-tdTomato markers. The same cell in C and D. (D) shows how the time difference between two markers (Δt) is scored. (E and F) Frequency distributions and Gaussian fit (continuous lines) of the time difference (Δt) between the separation of two markers, either on the same chromosome (E) or on chromosomes I and II or chromosomes II and III (F). Mean ± SD of the fit and number of cells are shown; P value from the one-sample t test against zero. (G) Fluorescent labeling of chromosomes at the inner centromeric regions. Chromosome II was marked with tetO/tetR-tdTomato (Sakuno et al., 2009); chromosomes I and III were marked with lacO/LacI-GFP (Sakuno et al., 2009 and this study). (H) Cyan: Frequency distributions and Gaussian fit (continuous lines) of the time difference (Δt) between the separation of central centromere markers on chromosomes I and II or chromosomes II and III. The fitted Gaussian distributions for cells using outer centromere markers (F) are shown in black for comparison. Mean ± SD of the fit; n = number of cells; P values from a two-sample Kolmogorov–Smirnov test.