Figure 3.
Cdc42 functions in the same NE sealing pathway as ESCRT and LEM proteins. (A) Stills from epifluorescence movies of cells endogenously expressing Chm7-GFP (green) and Sur4-mCherry (magenta) to mark the nuclear envelope. Yellow arrowhead indicates Chm7 spot at postfission nuclear membrane. (B) Stills from epifluorescence movies of cells endogenously expressing mCherry-Snf7 (magenta) and GFP-HDEL (green) to mark the ER. Yellow arrowhead indicates Snf7 punctum at base of ER tubule neck undergoing nuclear fission. (C) Confocal spinning-disk still of a cell endogenously expressing Cdc42-mCherrySW (red), GFP-HDEL (green), and mtagBFP2-Snf7 (blue) undergoing nuclear fission. White arrowhead indicates Snf7 and Cdc42 structure on ER membrane. (D) Confocal spinning-disk micrographs of indicated mutant strains, with wild-type (wt) cells (marked with yellow asterisks) in the same field of view. (E) Quantification of each mutant’s mean nuclear:cytosolic GFP fluorescence intensity normalized to that of wild-type cells in the same field of view. Colored dots report means from individual trials. Asterisks denote statistical significance compared with control after one-way ANOVA (F = 10.74), followed by Sidak’s multiple comparison test to determine P values between bracketed strains. Three trials were conducted for each mutant. n, mean ± SD are as follows: control n = 434 (wt n = 332), 1.02 ± 0.05; snf7Δn = 286 (wt n = 224), 0.57 ± 0.09; cdc42-129n = 115 (wt n = 266), 0.60 ± 0.05; cdc42-129 snf7Δn = 293 (wt n = 633), 0.47 ± 0.13; chm7Δn = 422 (wt n = 443), 0.66 ± 0.13; cdc42-129 chm7Δn = 233 (wt n = 443), 0.53 ± 0.04; heh1Δheh2Δn = 366 (wt n = 169), 0.63 ± 0.10; cdc42-129 heh1Δheh2Δn = 229 (wt n = 192), 0.64 ± 0.02; cdc42-129 vps4Δn = 92 (wt n = 137), 0.60 ± 0.08; cdc42-129 apq12Δn = 181 (wt n = 446), 0.60 ± 0.08; cdc42-123n = 95 (wt n = 141), 0.93 ± 0.05. (F) Quantification of wild-type, chm7Δ, snf7Δ, or heh1Δheh2Δ mutant cells with a Cdc42 spot. wt: 23% ± 6.9%, four trials, n = 1,527. chm7Δ: 25% ± 0.6%, two trials, n = 1,120. snf7Δ: 42% ± 3.8%, two trials, n = 1,136. heh1Δheh2Δ 11% ± 0.9%, n = 752. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (A–C) Shown are 2-µm maximum-intensity projections at 0.5-µm steps. (D) Shown is 11-µm maximum-intensity projection at 0.5-µm steps. All cells in this figure were grown to log phase in minimal imaging media at 23°C–25°C. All scale bars, 5 µm. ns, not significant. Cyt, cytosol; Nuc, nucleus.

Cdc42 functions in the same NE sealing pathway as ESCRT and LEM proteins. (A) Stills from epifluorescence movies of cells endogenously expressing Chm7-GFP (green) and Sur4-mCherry (magenta) to mark the nuclear envelope. Yellow arrowhead indicates Chm7 spot at postfission nuclear membrane. (B) Stills from epifluorescence movies of cells endogenously expressing mCherry-Snf7 (magenta) and GFP-HDEL (green) to mark the ER. Yellow arrowhead indicates Snf7 punctum at base of ER tubule neck undergoing nuclear fission. (C) Confocal spinning-disk still of a cell endogenously expressing Cdc42-mCherrySW (red), GFP-HDEL (green), and mtagBFP2-Snf7 (blue) undergoing nuclear fission. White arrowhead indicates Snf7 and Cdc42 structure on ER membrane. (D) Confocal spinning-disk micrographs of indicated mutant strains, with wild-type (wt) cells (marked with yellow asterisks) in the same field of view. (E) Quantification of each mutant’s mean nuclear:cytosolic GFP fluorescence intensity normalized to that of wild-type cells in the same field of view. Colored dots report means from individual trials. Asterisks denote statistical significance compared with control after one-way ANOVA (F = 10.74), followed by Sidak’s multiple comparison test to determine P values between bracketed strains. Three trials were conducted for each mutant. n, mean ± SD are as follows: control n = 434 (wt n = 332), 1.02 ± 0.05; snf7Δn = 286 (wt n = 224), 0.57 ± 0.09; cdc42-129n = 115 (wt n = 266), 0.60 ± 0.05; cdc42-129 snf7Δn = 293 (wt n = 633), 0.47 ± 0.13; chm7Δn = 422 (wt n = 443), 0.66 ± 0.13; cdc42-129 chm7Δn = 233 (wt n = 443), 0.53 ± 0.04; heh1Δheh2Δn = 366 (wt n = 169), 0.63 ± 0.10; cdc42-129 heh1Δheh2Δn = 229 (wt n = 192), 0.64 ± 0.02; cdc42-129 vps4Δn = 92 (wt n = 137), 0.60 ± 0.08; cdc42-129 apq12Δn = 181 (wt n = 446), 0.60 ± 0.08; cdc42-123n = 95 (wt n = 141), 0.93 ± 0.05. (F) Quantification of wild-type, chm7Δ, snf7Δ, or heh1Δheh2Δ mutant cells with a Cdc42 spot. wt: 23% ± 6.9%, four trials, n = 1,527. chm7Δ: 25% ± 0.6%, two trials, n = 1,120. snf7Δ: 42% ± 3.8%, two trials, n = 1,136. heh1Δheh2Δ 11% ± 0.9%, n = 752. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (A–C) Shown are 2-µm maximum-intensity projections at 0.5-µm steps. (D) Shown is 11-µm maximum-intensity projection at 0.5-µm steps. All cells in this figure were grown to log phase in minimal imaging media at 23°C–25°C. All scale bars, 5 µm. ns, not significant. Cyt, cytosol; Nuc, nucleus.

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