Localization of gradient-sensing proteins in mating cells. (A–D) Representative time-lapse images. MATa cells expressing reporter genes tagged with GFP in situ were mixed with an equal number of MATα cells and imaged from cytokinesis to zygote formation (fusion). The mating partners are labeled a and α in the DIC images. The fluorescent images show the localization of each reporter as the MATa cells orient toward their mating partners. The blue asterisk indicates reporter localization to the bud neck; dashed arrowheads indicate polarity establishment (PE) at the DS and mark the signal peak; closed arrowheads indicate redistribution and mark the leading peak; filled arrowheads indicate stabilization at the CS and mark the signal peak. The plots show the distribution of each reporter on the PM at the indicated time points (10-point rolling average). The x axes represent distance along the PM; the y axes indicate fluorescence intensity (F.I.). The dashed blue, green, and red lines mark the DS peak, leading peak, and CS peak, respectively. Localization of Ste2-GFP (A), GFP-Gβ (B), Far1-GFP (C), and Sst2-GFP (D). (E and F) PE position. (E) Diagram illustrating how the position of the initial polarity site was determined. δ is the angle formed by rays drawn from the middle of the cell to the center of the cytokinesis site and the center of the polarized crescent (top) or bud (bottom) when they are first detectable. (F) Box scatterplots showing δ values for each reporter. The boxes enclose the middle two quartiles with the horizontal lines indicating the means; the whiskers show the top and bottom quartiles. Mean δ ± SEM in degrees: Bud = 33.7 ± 0.9; Ste2-GFP = 33.9 ± 1.4; GFP-Gβ = 33.2 ± 1.2; Sst2-GFP = 33.6 ± 1.1; Far1-GFP = 33.0 ± 1.2. n ≥ 50 for all strains and measurements.