A functional internal fluorescent protein fusion of Cdc42 reveals a novel subcellular localization. (A) Cell growth of indicated yeast strains was compared by spotting serial dilutions of liquid cultures on plates at 25°C, 30°C, 37°C, or 39°C. (B) Micrographs of three separate cells expressing endogenous Cdc42-mCherry^SW showing Cdc42 spot in the mother, the bud, or the mother and bud. (C) Montage of confocal spinning-disk movie stills of cells endogenously expressing Cdc42-mCherry^SW showing Cdc42 spot segregating from mother cell into bud. Time is in minutes. (D) Quantification of maximum intensity of Cdc42 spot in wild-type (wt) cells (black) or cells overexpressing VPS21 (red) imaged at the restrictive temperature (37°C). Two trials each, mean ± SD. wt: 226 ± 3.5, n = 44. VPS21 overexpression: 220 ± 2.4, n = 78. No significant difference (ns; P = 0.1420) using Student’s t test. (E) Illustration depicting the organization of membranes and lumen spaces of the nuclear envelope and ER. The perinuclear space is continuous with the ER lumen (dark blue) because the outer nuclear membrane is continuous with the ER membrane. Nuclear fission events introduce a break in the neck of the dividing nucleus to expose the nucleoplasm (light blue) to the cytosolic space. (F) Still images from epifluorescence movies of cells endogenously expressing Cdc42-mCherry^SW (magenta) and GFP-HDEL (green) to mark the ER and show the Cdc42 spot on nuclear envelope after nuclear fission in both mother cells and buds. All cells in micrographs of this figure were imaged during log phase in minimal imaging media at 23°C–25°C. 2-µm maximum-intensity projections at 0.5-µm steps. All scale bars, 5 µm.