Biosensor measurements of Cdc24 GEF activity level in various strains. (A) Schematic representation of the FRET-based biosensor for Cdc42 activation. Active Cdc42^GTP binds the CRIB domain, bringing the flanking GFP and mCherry into proximity and allowing FRET. (B) A representative wild-type cell expressing the biosensor containing wild-type Cdc42. Leftmost image shows the sum of the time series for GFP. The center image shows the same image with cortex mask applied (see Materials and methods). The right image shows the FRET efficiency at each pixel within the cell as indicated by the heat bar. Bar, 5 µm. (C) Mean FRET efficiency (orange curve) was measured and plotted in 10° increments along the cortex in the masked image (see B, center image). Mean normalized GFP intensity at the cortex (blue curve) was also measured and plotted. Each plot shows means from >25 cells, and error bars show SEMs. (D) FRET efficiency in the cap region in indicated strains. The left two bars were from wild-type cells expressing positive and negative control sensors. Mean FRET efficiency was measured within the yellow region highlighted in C (see Materials and methods for details). Each histogram shows means from >25 cells, and error bars show SEMs. *, P < 0.01; **, P < 0.001; ***, P < 10⁻⁸.