Fig. 2.

RhoC is activated in areas behind the leading edge of the cell during EGF-stimulated protrusions. (A) Representative image of an MTLn3 cell expressing the RhoC biosensor and stimulated globally with EGF. White arrows point to the areas at the protrusion where RhoC activity is high. (B) Quantification of RhoC activity in areas of 1 µm depth and 10 µm length, starting at the cell edge of the MTLn3 cell stimulated globally with EGF for 1 minute. Values are normalized to the 0–1 µm values. Number of cells = 19. (C) RhoC activity and staining of cofilin in MTLn3 cells expressing RhoC biosensor before and 1 minute after stimulation with an EGF-filled micropipette. Asterisks indicate where the pipette was placed. RhoC activity and cofilin radial sweep of stimulated cell is shown (this sweep is the outermost 4 µm strip of the cell). Vertical white lines in the radial sweep indicate the positions where the front and the back of the cells are located with respect to the pipette. Spatial distances and horizontal line in the radial sweep images highlight the first and the second micrometers (D) RhoC activity and cofilin intensity profiles along the indicated dotted line in the radial-sweep images. Grey rectangles highlight the first and the second micrometer behind the cell edge. (E) Quantification of RhoC activity in an area of 1 µm width, 1 µm behind the cell edge in MTLn3 cells expressing RhoC biosensor and non-stimulated or stimulated for 1 minute with an EGF-filled pipette. Areas were analyzed in the regions facing the pipette and in the mirror back (see Materials and Methods for detailed description and supplementary material Fig. S2d). Scale bars: 10 µm (main image); 1 µm (insert). **P<0.01, ***P<0.001. The pseudocolor scale shows ratio limits of black to red for RhoC activity. Values are normalized to the first micrometer values. Error bars indicate the s.e.m.