Fig. 1. Formation of PH_CRAC-GFP patches in steep cAMP gradients. Cells expressing PH_CRAC-GFP were stimulated with a micropipette filled with 10^–4 M cAMP. (A) The confocal image reveals PH_CRAC-GFP patch formation at the leading edge of cells close to the pipette, but more uniform cytosolic localization of PH_CRAC-GFP in cells further away from the pipette. Two cells are shown at higher magnification. The asterisk indicates the position of the pipette. Scales bars: 50 µm (middle) and 10 µm (left and right). (B) The fraction of cells containing a PH_CRAC-GFP patch at the leading edge and the chemotaxis index were determined at different distances from the pipette. Half-maximal PH_CRAC-GFP patch formation occurs at a distance of about 50 µm from the pipette, and significant chemotaxis is observed up to 1000 µm from the pipette. (C) The chemotaxis index and speed of cells at 40-60 µm from the pipette was determined for 35 cells without and 22 cells with a PH_CRAC-GFP patch. Values are averages ± s.d.