Figure S3.
Leaky nucleus and ER phenotypes of cdc42-129 mutant are not due to pleiotropy. (A) Still images from epifluorescence movies of cdc42-129 (left) or cdc42-123 (right) cells stained with FM4-64 to visualize the vacuole. 30-min pulse with FM4-64 (10 µg/ml) and chased 1 h. 2-µm maximum-intensity projections at 0.5-µm steps. (B) Confocal spinning-disk images of snf7Δ cells endogenously expressing NLS-GFP (green) and Nup188-mCherry (magenta). White arrowheads indicate SINC. Top panel is a cell with intact nucleo-cytoplasmic partitioning (NLS-GFP nucleus/cytoplasm ratio >3), and bottom panel is a cell with a leaky nucleus (NLS-GFP nucleus/cytoplasm ratio <3). 11-µm maximum-intensity projection at 0.5-µm steps. (C) Quantification of NLS-GFP nucleus/cytoplasm ratios of snf7Δ cells without (black) or with (red) a SINC. Two trials each, mean ± SD. No SINC: 3.13 ± 0.13, n = 51. SINC: 3.13 ± 0.26, n = 23. No significant difference (ns; P = 0.9834) using Student’s t test. nuc, nucleus; cyt, cytoplasm. (D) Montage of still images from epifluorescence movies of snf7Δ cells endogenously expressing Cdc42-mCherryint (magenta) and Nup188-GFP (green). White arrowheads indicate SINC, and yellow arrowheads indicate Cdc42 spot. 2-µm maximum-intensity projections at 0.5-µm steps. (E) Epifluorescence stills of wild-type (top panel) or cdc42-129 mutant cells (bottom panel) endogenously expressing mCherry-Snf7 (magenta) and Vps4-3xHA-GFP (green). 2-µm maximum-intensity projections at 0.5-µm steps. (F) Quantification of Vps4 and Snf7 colocalization in wild-type (wt; black) or cdc42-129 (red) cells. Pearson’s correlation coefficient. Two trials each, mean ± SD. wt: 0.62 ± 0.01, n = 89. cdc42-129: 0.61 ± 0.01, n = 84. No significant difference (P = 0.6429) using Student’s t test. (G) Plot of Snf7 equilibrium binding assay for Cdc42WT (black) and Cdc42V36T (red). Cdc42WT Kd for Snf7 = 40.6 µM; Cdc42V36T Kd for Snf7 = 187.1 µM. Kd = dissociation constant. Three independent trials were conducted for each experiment. Error bars represent standard deviation for three independent measurements. (H) Same quantification shown in Fig. 4 H, but with frequency distribution of Snf7 fluorescence intensity of cdc42-123 mutants at 37°C overlaid in purple. Two trials, n = 40. A.U., arbitrary units. (I) Same quantification of ER area shown in Fig. 4 F for 37°C but with cdc42-123 mutant measurements shown in purple circles. Two trials, n = 40, mean ± SD. Asterisks denote statistical significance compared with control using Student’s t test. *, P ≤ 0.05; ***, P ≤ 0.001. (J) Same micrographs shown in Fig. 4 E, but with cdc42-123 micrograph included. Confocal spinning-disk micrographs of wild-type (left), cdc42-129 (middle), and cdc42-123 (right) cells expressing GFP-HDEL (green) to mark ER grown to early-log phase in minimal media and shifted to 37°C for 6 h. White arrowheads indicate ER tubules emanating from nuclear ER. Single (medial) focal plane. All cells in this figure were grown to log phase in minimal imaging media at 23°–25°C unless otherwise indicated. All scale bars, 5 µm.

Leaky nucleus and ER phenotypes of cdc42-129 mutant are not due to pleiotropy. (A) Still images from epifluorescence movies of cdc42-129 (left) or cdc42-123 (right) cells stained with FM4-64 to visualize the vacuole. 30-min pulse with FM4-64 (10 µg/ml) and chased 1 h. 2-µm maximum-intensity projections at 0.5-µm steps. (B) Confocal spinning-disk images of snf7Δ cells endogenously expressing NLS-GFP (green) and Nup188-mCherry (magenta). White arrowheads indicate SINC. Top panel is a cell with intact nucleo-cytoplasmic partitioning (NLS-GFP nucleus/cytoplasm ratio >3), and bottom panel is a cell with a leaky nucleus (NLS-GFP nucleus/cytoplasm ratio <3). 11-µm maximum-intensity projection at 0.5-µm steps. (C) Quantification of NLS-GFP nucleus/cytoplasm ratios of snf7Δ cells without (black) or with (red) a SINC. Two trials each, mean ± SD. No SINC: 3.13 ± 0.13, n = 51. SINC: 3.13 ± 0.26, n = 23. No significant difference (ns; P = 0.9834) using Student’s t test. nuc, nucleus; cyt, cytoplasm. (D) Montage of still images from epifluorescence movies of snf7Δ cells endogenously expressing Cdc42-mCherryint (magenta) and Nup188-GFP (green). White arrowheads indicate SINC, and yellow arrowheads indicate Cdc42 spot. 2-µm maximum-intensity projections at 0.5-µm steps. (E) Epifluorescence stills of wild-type (top panel) or cdc42-129 mutant cells (bottom panel) endogenously expressing mCherry-Snf7 (magenta) and Vps4-3xHA-GFP (green). 2-µm maximum-intensity projections at 0.5-µm steps. (F) Quantification of Vps4 and Snf7 colocalization in wild-type (wt; black) or cdc42-129 (red) cells. Pearson’s correlation coefficient. Two trials each, mean ± SD. wt: 0.62 ± 0.01, n = 89. cdc42-129: 0.61 ± 0.01, n = 84. No significant difference (P = 0.6429) using Student’s t test. (G) Plot of Snf7 equilibrium binding assay for Cdc42WT (black) and Cdc42V36T (red). Cdc42WT Kd for Snf7 = 40.6 µM; Cdc42V36T Kd for Snf7 = 187.1 µM. Kd = dissociation constant. Three independent trials were conducted for each experiment. Error bars represent standard deviation for three independent measurements. (H) Same quantification shown in Fig. 4 H, but with frequency distribution of Snf7 fluorescence intensity of cdc42-123 mutants at 37°C overlaid in purple. Two trials, n = 40. A.U., arbitrary units. (I) Same quantification of ER area shown in Fig. 4 F for 37°C but with cdc42-123 mutant measurements shown in purple circles. Two trials, n = 40, mean ± SD. Asterisks denote statistical significance compared with control using Student’s t test. *, P ≤ 0.05; ***, P ≤ 0.001. (J) Same micrographs shown in Fig. 4 E, but with cdc42-123 micrograph included. Confocal spinning-disk micrographs of wild-type (left), cdc42-129 (middle), and cdc42-123 (right) cells expressing GFP-HDEL (green) to mark ER grown to early-log phase in minimal media and shifted to 37°C for 6 h. White arrowheads indicate ER tubules emanating from nuclear ER. Single (medial) focal plane. All cells in this figure were grown to log phase in minimal imaging media at 23°–25°C unless otherwise indicated. All scale bars, 5 µm.

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