Movie 2.

mNG-Rac1 cells were serum starved, placed onto microscope stage into environmental chamber, and 3D live-cell images were acquired through 488-nm (green) and 561-nm (red) channels from living cells at 37^°C for 60 min. EGF-Rh (final concentration 4 ng/ml) was perfused 5 min after the start of time-lapse imaging. Images were deconvolved. Imaging parameters for acquiring EGF-Rh images were used to avoid saturating signals at later time points, which resulted in the lack of visible EGF-Rh fluorescence at early time points. MIP images of 4 consecutive confocal planes are presented. Scale bar, 10 μm.