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. 2019 Aug 19;8:e47646. doi: 10.7554/eLife.47646

Figure 9. Visualization of a CyclinB1-Cdk1 activity gradient in the vicinity of the midzone during anaphase.

Figure 9.

(a) The chromatin targeted reporter exhibits a longer lifetime and lower FRET efficiency in interphase versus prometaphase. The sensor on lagging chromosomes in the vicinity of the midzone MTs, demarcated by the dashed lines, exhibits higher FRET efficiency and shorter donor lifetime than the reporter on the segregated DNA. The activity gradient is no longer evident in the presence of 10 μM RO-3306. (b) Quantification of donor lifetimes for segregated versus lagging chromatin in untreated and RO-3306-treated cells (N = 14 cells for each condition). (c) Quantification of FRET efficiencies for segregated versus lagging chromatin in untreated and RO-3306-treated cells (N = 14 cells for each condition). Error bars are SEM. Scale bars are 5 μm. Color wedges indicate 0.6 ns (blue) – 3.8 ns (red) for donor lifetimes and 0% (blue) to 55% (red) for FRET E. Two-tailed P-values from a Student’s t-test are reported. n.s. is not significant (p>0.05).

Figure 9—source data 1. Mean lifetimes, percentage of fluorophores exhibiting short and long lifetimes, and the short lifetimes for the chromatin-targeted Cyclin B1-Cdk1 activity reporter.
Mitotic cells included prophase, prometaphase, and metaphase, but excluded anaphase and telophase/cytokinesis. * indicates p<0.05 relative to the control, non-phosphorylatable FRET reporter in interphase. # indicates p<0.05 relative to the phosphorylatable FRET reporter in interphase. Two-tailed P-values from a Student’s t-test are reported.
elife-47646-fig9-data1.xlsx (8.9KB, xlsx)
DOI: 10.7554/eLife.47646.037
Figure 9—source data 2. Mean FRET efficiency statistics of chromatin-targeted Cyclin B1-Cdk1 FRET sensors.
Analysis of mitotic cells includes prophase, prometaphase, and metaphase, but excludes anaphase and telophase/cytokinesis. The active sensor reported increased FRET in mitosis relative to the non-phosphorylatable control in interphase (p<0.001). P-values calculated using the PlotsOfDifferences web app (Goedhart, 2019). N-values reported in the table apply to Figure 9—source data 1.
elife-47646-fig9-data2.xlsx (8.8KB, xlsx)
DOI: 10.7554/eLife.47646.038