The Cdc42 spot is localized to vacuolar and ER membranes and vertices of fusing vacuoles. (A) Epifluorescence still images of cells endogenously expressing Cdc42-mCherry^SW (magenta) and Vph1-GFP (green) in unbudded (top panel) or budded cell (lower panel). Plot of normalized fluorescence intensity profiles of Vph1-GFP (green) and Cdc42-mCherry^SW (magenta) along yellow line shown in inset image. (B) Epifluorescence still images of cells endogenously expressing Cdc42-mCherry^SW (magenta) and GFP-HDEL (green) in unbudded (top panel) or budded cell (lower panel). Plot of normalized fluorescence intensity profiles of GFP-HDEL (green) and Cdc42-mCherry^SW (magenta) along yellow line shown in inset image. All images are 2-µm maximum-intensity projections at 0.5-µm steps. All scale bars, 5 µm. (C) Montage of stills from epifluorescence movies of cells expressing endogenous Cdc42-mCherry^SW (magenta) and Vph1-GFP (green). All cells in micrographs of this figure were imaged in log phase growth in minimal imaging media at 23°–25°C. Scale bar, 5 µm. 2-µm maximum-intensity projections at 0.5-µm steps. (D) Two-color versions of the micrographs in Fig. 3 D showing wild-type cells expressing Vph1-mCherry (marked with white asterisks in micrographs) in the same field of view as the mutant indicated by genotype above. Mutant and wild-type cells both express NLS-GFP. All scale bars, 5 µm.