Figure 4.

Time-lapse Imaging Reveals a Negative Correlation between ERK Activity and Cell Motility

(A) Histogram of the FRET/CFP ratio of thymocytes in the thymic cortex in the sliced thymic lobes. Thymocytes with high ERK activity (top 3% of activity; the 20 cells shown in red), intermediate (int.) ERK activity (10 cells, green), and low ERK activity (bottom 3% of activity, the 20 cells in blue) were categorized into three groups and tracked for the analysis. Data represent the analysis of samples from three individual mice. See also Table S1.

(B) Representative time-lapse FRET/CFP ratio images of thymocytes in intensity-modulated display (IMD) mode. (Left) The white lines indicate the cell tracks for the duration of the movie, with the yellow spot indicating the location of the cell at 0 min. (Right) Time-lapse FRET/CFP ratio images in IMD mode. The movie was obtained for 5 min. Scale bar, 15 μm.

(C) Time-lapse analysis of the speed of two representative cells shown in (B). To smooth a dataset, the averaged value of five successive time frames was adopted.

(D) The relationship between the average FRET/CFP ratio and the average speed (μm/min) of thymocytes. Each dot indicates an individual cell track and is color coded as in panel (B): n = 20 tracks (High); n = 10 tracks (Int.); n = 20 tracks (Low). Data represent the analysis of samples from three individual mice. The relationship between pairs of variables was analyzed using the Pearson correlation analysis.