Automated Analysis of FRET Figure 4. The tracking and analysis of the RhoA activity in different ROIs of a stationary HeLa cell. (A) Left: Four ROIs were chosen in the first frame to monitor the RhoA activity at the front, back, and two sides of a stationary cell. Right: The computed contours and locations of the ROIs in the cell at different time points based on our tracking algorithm. (B) The time courses of FRET ratios of the Raichu-RhoA biosensor averaged on the four ROIs shown in (A). doi:10.1371/journal.pone.0004082.g004 cells span the front, back and two sides of the cell (Figure 3A and Movie S2). Since a ROI may move out of the cell body (Figure 3A, Region 1), a threshold value of the YFP image of the cell was used to judge and calculate the FRET signal within the part of the ROI inside the cell body. Therefore, the average FRET ratios within each ROI of the migrating cells at different time points were calculated to monitor the dynamic RhoA activity at subcellular locations (Figure 3B). It appeared that the RhoA activity in all four regions immediately dropped and slightly recovered after EGF stimulation. The local RhoA activity then decreased continuously in regions at the front and both sides. However, in the region near the trailing edge of the migrating cell, the RhoA activity remained relatively constant without significant down-regulation after EGF stimulation (Figure 3B). This is a representative feature among different migrating cells analyzed. As a result, the difference between the activity of the ROIs near the front and the back of the cell displayed a significant increase for the migrating cells. This finding corroborates the previous study that high RhoA activities are important in mediating the tail contraction of migrating cells [2]. In the stationary cells, because the cells were not moving significantly, the positions of ROIs remained relatively unchanged in time (Figure 4A). The RhoA activity in all four regions immediately dropped and slightly recovered before continuing decrease after EGF stimulation. In contrast to the sub-cellular differences of RhoA activity observed in the migrating cells, the RhoA activity in all four ROIs of the stationary cells appeared to decrease with similar kinetics upon EGF stimulation (Figure 4). Due to the relatively stable RhoA activity at the tail of the migrating cells upon EGF stimulation, the global activity of RhoA in migrating cells was expected to decrease slower than that of the stationary cells. Hence we quantified the global activity of the RhoA biosensor in the migrating and stationary cells by taking the average of RhoA activities in all four ROIs and normalized according to their pre-EGF ratios. The results confirmed that the RhoA activity decreased in both the migrating and the stationary cells upon EGF, at a slower rate in migrating cells than that in stationary cells (Figure 5). The difference between the normalized ratios of the RhoA biosensor in migrating and stationary cells became statistically significant at 13–26 minutes after EGF stimulation (Figure 5), consistent with the time when the RhoA activity at the tail of the migrating cells started to show difference from other subcellular regions (,25 minutes after EGF in PLoS ONE | www.plosone.org 6 December 2008 | Volume 3 | Issue 12 | e4082