Fig. 4: Directional control of cAMP increase and decrease upon localized stimulation.

Giant AX2 cells expressing Flamindo2-RFP were generated by adding 10 μM blebbistatin and culturing under shaking conditions. Eight hours after starvation with DB, time-lapse fluorescence imaging was initiated using a confocal microscope. A micromanipulator-mounted glass needle was used to locally deliver 10 μM cAMP. Imaging conditions: 40× objective, 5 s intervals. a Ratiometric images of the entire field of view were generated by dividing the RFP signal by the Flamindo2 signal. Differential interference contrast (DIC) images of the glass needle were overlaid. Time elapsed from the start of observation is shown in the upper left corner. Scale bars: 50 μm. The red circle indicates a representative giant cell within the field. b Cropped images of a representative giant cell in (a). The white circles at 115 s indicate regions of cAMP increase; the circle at 160 s indicates a region of cAMP signal decrease. Time is indicated in the upper right corner. Scale bars: 50 μm. c Cell contour and centroid trajectory of the giant cell in (b). An arrow indicates the direction of cell movement. d Same as (a), with the micromanipulator repositioned. Scale bar: 50 μm. e Cell contour and centroid trajectory after repositioning of the micromanipulator. An arrow indicates the direction of cell movement.