Fig. 4. Accumulation of PH_CRAC-GFP at the leading edge of cells stimulated with a gradient of cAMP. Cells expressing PH_CRAC-GFP and cytosolic mRFP were stimulated with a micropipette filled with 100 µM cAMP (see supplementary material Movie 1). (A) A typical cell at a distance of about 100 µm from the pipette is shown. The cell in frame 41 exhibits a faint PH_CRAC-GFP patch that is just visible in the confocal image of the GFP signal. The normalized mRFP signal of each pixel was subtracted from the GFP signal in that pixel to correct for the difference in cytosolic content of boundary pixel, yielding ccPH_CRAC-GFP. Scale bar: 10 µm. (B) The fluorescence intensity of PH_CRAC-GFP and ccPH_CRAC-GFP at the boundary of the cell for all frames is presented. For the PH_CRAC-GFP signal, a patch is visible around frames 3 and 40, but otherwise not many details can be recognized. In the panel showing ccPH_CRAC-GFP, not only these patches can be seen but nearly all frames show an increased fluorescence at the leading edge. (C) The fluorescence intensity at the boundary of the cell is quantified for the PH_CRAC-GFP and ccPH_CRAC-GFP signal. Data for a visible PH_CRAC-GFP patch are obtained from frames 3 or 4 and 39-42, and data for no visible PH_CRAC-GFP patch are obtained from the remaining frames. The data show the means ± s.d. (D) The fluorescence intensity at the boundary in the front (f, black symbols) or back (b, white symbols) of the cell for the PH_CRAC-GFP (triangles) and ccPH_CRAC-GFP (circles) signal is shown. The left panel in D is the same cell as is shown in A-C; the gray area indicates the strong patches. The right panel in D was of the same batch of cells after incubation with 50 µM LY294002.