JCS031781 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. The figure shows the fluorescence intensity at the membrane relative to that of the cytosol for different depletion of the fluorescence in the cytosol. The result was calculated with equation 7 using different values of a, the fractions of pixels of a cell present at the boundary. The dashed line indicates at which condition the fluorescence intensity at the boundary becomes higher than in the cytosol. We estimated that for the present images a=0.25, indicating that the fluorescence intensity at the boundary pixels will be higher than the fluorescence intensity of the interior pixels only at relatively large stimulations inducing more than 12% depletion in the cytosol.

• Supplemental Table S1 -

  Table S1. Chemotaxis index and speed of wild-type cells and pi3k-null cells with and without PtdIns(3,4,5)P₃ signaling.

• Movie 1 -

  Movie S1. Enhanced visualization of PH_CRAC-GFP by subtraction of cytosol signal. Cells expressing both PH_CRAC-GFP and cytosolic mRFP chemotaxing towards a cAMP-filled microtip (tip is outside field of view towards upper-left corner) were recorded with a confocal laser-scanning microscope (frame interval is 8 seconds). GFP- and mRFP-signals were recorded separately. The cell of interest is marked with a white arrow in the first frame. Panel A contains the original PH_CRAC-GFP signal. After background subtraction, the red signal was normalized to the mean cytosolic green fluorescence of the cell of interest; panel B shows the normalized mRFP signal. This normalized signal was subtracted from the green channel, yielding the cytosol-corrected PH_CRAC signal (panel C). Note that the cytosol correction must be performed separately for each individual cell. The movie was used for quantification presented in Fig. 4.