Loss of Scd1 localization in CK-666-treated cells is mediated by Pak1. (A) Scd1-mNG localization in pak1+ and pak1-ts mutant cells. Asterisks depict bipolar Scd1-mNG localization. (B) Quantification of Scd1-mNG intensities at the cell ends in pak1+ and pak1-ts cells treated with DMSO and CK-666. Colors represent each experimental replicate. Large circles are the means of each experimental replicate (N = 3, n ≥ 12 cells). (C) Modeling simulation of Scd1 and Cdc42-GTP localization in pak1-ts cells in Model 2 shows only monopolar localization of Scd1 and Cdc42-GTP in the absence of pak1. (D i) Model 3 incorporates feedback loops involving Cdc42-GTP, Cdc42-GDP, GEF (Scd1), Pak1, and endocytosis, and an additional negative signaling pathway inhibiting Cdc42-GTP. The asterisk highlights the additional new arrow to Model 3. (D ii) The left panel shows Model 3 simulations and the right panel shows simulations with a decreased detachment rate of Pak1. (E) Model 3 simulations with an additional negative signaling pathway simulated via decreased detachment rate of Cdc42 and Scd1. Scale bar, 10 µm. n.s., not significant, P value, *<0.05, ***<0.0003, one-way ANOVA, followed by Tukey’s multiple comparison test.