Figure 6.

Cdc42 and RhoA activities polarize before Rac and Ras activities during de novo polarization and induced chemokinetic movement. (a) Kinetics of cell-averaged GTPase activities after uniform photorelease. Values were normalized by the initial and maximum activity. Green line marks the start of gradient generation by photorelease. Error bars indicate ± s.e.m. of n=30 (Rac), n=35 (Ras), n=31 (Cdc42), and n=67 (RhoA). (b) Left, the contours of the cell edge are overlaid for several color coded timepoints during polarization and movement. Arrow indicates the computationally determined polarization direction. Right, schematics explaining the analysis of the polarization of GTPase activities. The front 20% of cell pixels were determined automatically based on the polarization direction. Scale bar is 10μm. (c) Overlay of timecourses of directed speed for cells with FRET biosensors during de novo polarization in Figure 6d. Directed speed was measured as the rate of movement of the cell centroid in the direction of eventual cell polarization. Each curve is labeled according to the FRET biosensor expressed in the corresponding cells. Green dotted line marks the time of chemoattractant release. Error bars indicate ± s.e.m. of n=26 (Rac), n=28 (Ras), n=33 (Cdc42), and n=26 (RhoA) cells. (d) Timecourse of polarization of GTPase activity (scored by FRET ratio in the front 20% of pixels minus the ratio in the back 80% of pixels) after uniform photorelease of Nv-fMLF. Values were normalized by the maximum (Rac, Ras, and Cdc42) or minimum (RhoA) activity. Green dotted line marks the time of chemoattractant release. Error bars indicate ± s.e.m. of n=26 (Rac), n=28 (Ras), n=33 (Cdc42), and n=26 (RhoA) cells. (e)Time-course of GTPase activities as the cell polarize after photorelease. Color bars indicate the range of biosensor FRET ratios. Scale bar is 10μm.