Cdc42p forms a focus at the center of the ZCF. (A) Live imaging of GFP–Cdc42p during mating. GFP–Cdc42 cells (MY15747) were mated to WT (MY8093) and imaged at 3-min intervals. Bar, 2 µm. (B and C) Images and quantification of a representative prezygote and zygote containing GFP–Cdc42p. (B) GFP–CDC42 cells (MY15747) were mated to a fus1Δfus2Δ strain (JY429) for 2.5 h at 30°, fixed with 2% formaldehyde, and imaged. Bar, 2 μm. (C) The fluorescence intensity was measured from the top to the bottom of the ZCF, as denoted by the arrows on the reference image, and plotted as a function of the distance across the ZCF. (D and E) Cdc42p focus is not observed in shmoos. (D) GFP–CDC42 cells (MY15747) were imaged after incubation with pheromone for 1.5 h. Bar, 2 μm. (E) The fluorescence intensity was measured around the shmoo tip from the top arrow to the bottom arrow, as denoted on the reference image, and plotted as a function of the distance. (F) Quantification of WT Cdc42p ZCF localization for all prezygotes imaged. Fluorescence intensities across the ZCF were measured for all prezygotes, interpolated to account for differences in ZCF length, and plotted as a ratio of the center values to the mean of the two edges. Data in F were pooled from three independent experiments. Error bars denote the SEM of all cells imaged. n > 120 prezygotes.