Automated Analysis of FRET Figure 3. The tracking and analysis of the RhoA activity in different ROIs of a migrating 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 migrating cell. Right: The computed contours and locations of the ROIs in the cell at different time points based on our tracking algorithm. The time-lapse images of the automatically tracked ROIs are also shown in Movie S2. (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.g003 stationary, the location of its centroid was monitored in time. The furthest distance traveled by the centroid from the initial position was divided by the sum of the distances traveled at each time step for about 60 minutes in each experiment. With a ratio greater or equal to 0.5, a cell was considered to be migratory. Otherwise, the cell was considered stationary. The results confirmed that there are clearly two classes of HeLa cells with the distance ratios at 0.71 and 0.1867 (Table 1). The migration direction of each migrating cell can be determined from the trajectory of its centroid by linear fitting. With our method of image registration, the intracellular RhoA activity under growth factor stimulation can be visualized within a uniform reference image frame (Figure 2). The RhoA activity was high at both the front and back edge of migrating cells (Figures 2A– 2B). This confirmed that RhoA contributes to the actin filament elongation and the membrane ruffle at the cell front via mDia whereas regulates actomyosin contractility at the cell rear [2]. In both migratory and stationary cells, spatial patterns of two concentric ring-like subcellular structures with high RhoA activity can be clearly observed (Figure 2 and Movie S1). The outer ring with high RhoA activity may represent the nascent and dynamic integrin activation on RhoA activity [39]. The high RhoA activity in the inner ring may indicate the relatively high stress occurring at the convergent zone where retrograde actin flow of the lamella meets the anterograde actin flow of the cell body [40]. Despite these distinctive subcellular features, the overall RhoA activity of both migratory and non-migratory HeLa cells decreased after the EGF stimulation (Figure 2 and Movie S1). It appears that, upon EGF stimulation, the RhoA activity remained relatively stable at the back of the migratory, but not stationary cells (Figure 2B and Movie S1). Tracking the Regions of Interest To track and quantify the local RhoA activities at subcellular regions in HeLa cells, four ROIs were selected in both the migrating and stationary cells. The four ROIs in the migrating PLoS ONE | www.plosone.org 5 December 2008 | Volume 3 | Issue 12 | e4082