Real-time in vivo imaging of SNAP-macroH2A incorporation timing. (A) Examples of in vivo imaging of macroH2A dynamics in individual cells. Preexisting macroH2A in nonsynchronized cells was labeled with SNAP–Oregon Green and blocked with SNAP-Block. Newly incorporated macroH2A was detected with JF646. Live-cell images were acquired every 20 min for 18–20 h. Signal intensity transitions were measured as shown in the left panels (Fucci, upper: S/G2, red; G1, blue; SNAP-tagging; lower: preexisting macroH2A, green; newly incorporated macroH2A, magenta). Representative images from four hourly intervals are shown to the right (with the component of the cell cycle captured shown with a gray box). The upper cell is captured entering G1 and shows a substantial accumulation of new macroH2A, whereas the lower cell is captured during S/G2 and accumulates new macroH2A to a much lesser extent. (B) The summarized data from imaging of 22 cells. The cells were aligned temporally using the Fucci cell cycle images. The lines in lower panel represent the detection phase in each cell. The rate of change of signal of newly incorporated macroH2A was calculated as the difference of intensity of newly incorporated macroH2A normalized to the signal in first time point of preexisting macroH2A between two consecutive time points [Delta (X_t − X_{(t − 1)})/2]. These delta values were then plotted through the cell cycle. Signal saturation at metaphase distorts the data, but the right panel allows comparison of temporal changes compared with the average delta (0.17) during S–G2 phase. The period of sustained accumulation of macroH2A is between hours ∼13 and 17 (red box). Error bars represent standard error of the mean from 22 cells.