FIGURE 1:

Ras-GTP patches in unpolarized Dictyostelium cells. Movies were made of cells in buffer expressing RBD-Raf-GFP and cytosolic (cyt) RFP (Supplemental Movies S1–S3). (A) Images of frame 67 in the green channel, the red channel, and the calculated GFP minus RFP signal Ψ, which is proportional to Ras-GTP (see Materials and Methods); scale bar, 5 μm. (B) Values of Ψ recorded in a line scan at a boundary 3 pixels wide (0.6 μm), starting at the arrow indicated in A. The dashed line at Ψ = 0.5 indicates that details below this line are not visible in the GFP channel. (C) Kymograph of the values of Ψ  for the entire movie. This cell formed ∼53 Ras-GTP patches and extended 16 protrusions in the period of the movie. (D) Cumulative probability distribution of RAS-GTP patches in unpolarized cells with increasing intensity. The data represent 63 patches in buffer and 55 in LatA. Dashed line at Ψ = 0.5 indicates that only 10% of the patches in buffer and 5% of those in LatA are detectable in the GFP channel.