Figure 3: Dynamics of damage repair and PLK1 activity from single-cell measurements. | Nature Communications

Figure 3: Dynamics of damage repair and PLK1 activity from single-cell measurements.

From: Homeostatic control of polo-like kinase-1 engenders non-genetic heterogeneity in G2 checkpoint fidelity and timing

Figure 3

(a) Serial imaging of U2OS cells expressing the PLK1 sensor and MDC1-mCherry. U2OS cells synchronized in G2 were pulsed with etoposide as before and tracked by live cell imaging until just before NEBD at mitotic entry as described in Fig. 2b. The images were analysed using custom-written MATLAB software to measure the nuclear PLK1 FRET index and MDC1 foci number at different time points. Scale bar, 5 μm. (b) Changes in the checkpoint activation signal (MDC1 foci number) in single cells (left) or on average (right) from the time of damage treatment until just before NEBD at mitotic entry (n=20). The coloured lines represent the trajectories of individual cells, and the single black lines represent the population average, with the standard deviation for each data point (bars). (c) The relative PLK1 activity at each time point, measured using the FRET index as described in Methods, was tracked by live cell imaging under various experimental conditions. Plots marked ‘damage’ shows results on U2OS cells, treated as described in Fig. 2a, followed from the time of damage treatment until mitotic entry (n=20); ‘asynchronous’, shows results on asynchronous U2OS cells followed from initial PLK1 activation until mitotic entry (n=19); ‘synchronized’, shows results on U2OS cells synchronized in G2 after thymidine release, followed until mitotic entry (n=20); ‘damaged+caffeine’, shows results from a similar experiment on U2OS cells synchronized in G2, pulsed with 10 μM etoposide for 8 min, and then left in caffeine during live cell imaging (n=25). The coloured lines represent the trajectories of individual cells. (d) Traces from individual cells exposed to different treatments described in c were synchronized at mitotic entry and averaged within the same treatment groups. The mean and standard error of the average trajectories are shown. ‘Terminal slopes’ depicted in the top-left corner display the linear regression of the last 3 h of the traces, with the very last time point excluded. Analysis of covariance shows that the slopes in the different treatment groups are significantly different from one another. (****P<0.0001).

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