Turnover-defective GTP-locked Cdc42p causes multiple growth sites in a fMAPK-dependent manner. (A) Wild-type cells (WT, PC538), and the ste11Δ (PC3862), tec1Δ (PC6102), bni1Δ (PC7086), gic1Δ gic2Δ (PC7044), rsr1Δ (PC4256), and bem4Δ (PC3551) mutants expressing GFP-Cdc42p^Q61L+TD (PC7654, Q61L+TD) were examined by fluorescence microscopy. Blue arrows indicate multiple growth projections. Scale bar, 5 µm. (B) Multiple growth sites expressed as percentage for the strains described in A. Error bars indicate SD from two biological replicates (n = 2); 300 cells were analyzed in each replicate. Data were analyzed by one-way ANOVA, and the P value (asterisk, <0.01, all samples were compared to WT) was calculated using Tukey’s multiple comparison test. (C) Wild-type cells (PC538) expressing GFP-Cdc42p (Cdc42, PC6454), GFP-Cdc42p^Q61L (Q61L, PC7458), or GFP-Cdc42p^Q61L+TD (Q61L+TD, PC7654). Cells were stained with Phalloidin-Atto 532 (F-actin, magenta) and fluorescent brightener #28 (CFW, calcofluor white, yellow, cell wall). Magenta arrows refer to multiple sites of actin polymerization. Scale bar, 5 µm. (D) Fluorescence microscopy of Cdc3p-mCherry cells (PC7365) expressing same plasmids as in C. Cdc3p-mCherry (red) is a septin ring marker. Red arrows refer to septin rings. Scale bar, 5 µm. (E) The cdc42Δ mutant (PC6684) harboring the pGFP-Cdc42 (URA3, PC6454) and pGFP-Cdc42 (LEU2-based plasmid, PC6457) or pGFP-Cdc42^TD (TD, K5,94,96R; LEU2-based plasmid, PC7698) were examined for growth on SD-URA-LEU (top) and 5-FOA (middle) media. Bottom: Morphology of the cdc42Δ mutant harboring the pRS316-GFP-Cdc42 (URA3) and pRS315-GFP-Cdc42 (LEU2) or pRS315-GFP-Cdc42^TD (TD, K5,94,96R; LEU2) cells after 2 d on 5-FOA media. Blue arrows indicate multiple buds. Scale bar, 5 µm.