Cdc42-mCherry^SW localizes to a single spot at sites of ER remodeling in a subpopulation of cells. (A) Montage of wide-field epifluorescence time lapse imaging of cells endogenously expressing Cdc42-mCherry^SW during polarized cell growth. Cells were imaged in early-log phase in minimal imaging media at 23°C–25°C. 3-µm maximum-intensity projection at 0.5-µm steps. (B) Field of view of same strain as in A showing two examples of the 23% ± 6.9% of cells with Cdc42 spots (±SD; n = 1,527; four trials). White arrowhead indicates bud containing Cdc42 spot, and yellow arrowhead indicates a budding mother containing Cdc42 spot. (C) Quantification of frequency of Cdc42 or Chm7 spot colocalized with the vacuole or ER. Two trials for each, mean ± SD. Cdc42 and vacuole: 100% ± 0%; n = 27. Cdc42 and ER: 84.6% ± 6.9%; n = 170. Chm7 and ER: 98.2% ± 0.72%; n = 165. (D) Frequency of Cdc42 localization to the nuclear envelope during unbudded, budding, and cytokinesis stages of the cell cycle. Unbudded: 82%, n = 34; Budding: 50%, n = 38; Cytokinesis: 90%, n = 20. Also depicts percentage of cells undergoing nuclear fission within cytokinetic cells (n = 18) with Cdc42 spot at bottleneck region of nuclear filament undergoing nuclear fission (68%). (E) Montage of confocal spinning-disk movie stills of the same cells as in A undergoing nuclear fission. 3-µm maximum-intensity projections at 0.5-µm steps. (F) Montage of confocal spinning-disk movie stills of cells endogenously expressing Cdc42-mCherry^SW (magenta) and GFP-HDEL (green) undergoing fission of ER tubule emanating from the nucleus. White arrowhead in first frame indicates ER tubule, and yellow arrowhead in second frame indicates site of severed ER tubule on the nuclear ER. 2-µm maximum-intensity projections at 0.5-µm steps. All scale bars, 5 µm.