Figure 4.
Cdc42 is at sites of Snf7 disassembly, and its disruption leads to Snf7 aggregates and ER tubule extensions from the nuclear envelope.(A) Still images from epifluorescence movies of cells endogenously expressing Vps4-3xHA-GFP (green) and Sur4-mCherry (magenta) to mark the nuclear envelope. Yellow arrowhead indicates Vps4 spot at base of nuclear filament. (B) Confocal spinning-disk still images of cells endogenously expressing Cdc42-mCherrySW (magenta) and Vps4-3xHA-GFP (green) in unbudded (top panel) or budded cell (lower panel). Plot of normalized fluorescence intensity profiles of Vps4-3xHA-GFP (green) and Cdc42-mCherrySW (magenta) along yellow line shown in inset image. Cells that contain both Cdc42 spot and Vps4 spot, where both are colocalized = 70.6% ± 0.01% (SD; n = 168). (C) Montage of stills from confocal spinning-disk movie of a Cdc42 spot (magenta) and Snf7 (green) punctum. Time is in seconds. (D) GST-fusions of Heh2C-terminal(CT), Heh2N-terminal(NT), Chm7, or Snf7 fragments or full-length protein (Coomassie, lower panel) were immobilized on GST-beads as bait and incubated with purified His6-Cdc42Q61L (constitutively GTP bound) as prey (detected by Western blot, top panel). (E) Confocal spinning-disk micrographs of wild-type (wt; top panel) and cdc42-129 (bottom panel) cells expressing GFP-HDEL (green) to mark ER and mCherry-Snf7 (magenta) grown to early-log phase in minimal media and shifted to 37°C for 6 h. Single (medial) focal plane. (F) Quantification of percentage cell area occupied by ER for same strains shown in E. Open circles indicate wild-type (black) and cdc42-129 mutant (red) strains imaged at the permissive temperature (25°C), and closed circles at the restrictive temperature (37°C). wt 25°C: two trials, n = 40, 37.1 ± 7.8; cdc42-129 25°C: two trials, n = 40, 37.9 ± 7.2; wt 37°C: two trials, n = 40, 28.7 ± 10.2; cdc42-129 37°C: two trials, n = 40, 47.3 ± 14.2. mean ± SD. Asterisks denote statistical significance compared with control using Student’s t test. ***, P ≤ 0.001. (G) Quantification of mCherry-Snf7 punctae of same strain shown in E grown to early-log phase in minimal media at 25°C, as a frequency distribution of the fluorescence intensity of each pixel in a cell normalized to cell area (micrometer squared). Two trials, n = 40. (H) Quantification of mCherry-Snf7 punctae of same strain shown in E grown to early-log phase in minimal media then shifted to 37°C for 6 h. Two trials, n = 40. All scale bars, 5 µm.

Cdc42 is at sites of Snf7 disassembly, and its disruption leads to Snf7 aggregates and ER tubule extensions from the nuclear envelope. (A) Still images from epifluorescence movies of cells endogenously expressing Vps4-3xHA-GFP (green) and Sur4-mCherry (magenta) to mark the nuclear envelope. Yellow arrowhead indicates Vps4 spot at base of nuclear filament. (B) Confocal spinning-disk still images of cells endogenously expressing Cdc42-mCherrySW (magenta) and Vps4-3xHA-GFP (green) in unbudded (top panel) or budded cell (lower panel). Plot of normalized fluorescence intensity profiles of Vps4-3xHA-GFP (green) and Cdc42-mCherrySW (magenta) along yellow line shown in inset image. Cells that contain both Cdc42 spot and Vps4 spot, where both are colocalized = 70.6% ± 0.01% (SD; n = 168). (C) Montage of stills from confocal spinning-disk movie of a Cdc42 spot (magenta) and Snf7 (green) punctum. Time is in seconds. (D) GST-fusions of Heh2C-terminal(CT), Heh2N-terminal(NT), Chm7, or Snf7 fragments or full-length protein (Coomassie, lower panel) were immobilized on GST-beads as bait and incubated with purified His6-Cdc42Q61L (constitutively GTP bound) as prey (detected by Western blot, top panel). (E) Confocal spinning-disk micrographs of wild-type (wt; top panel) and cdc42-129 (bottom panel) cells expressing GFP-HDEL (green) to mark ER and mCherry-Snf7 (magenta) grown to early-log phase in minimal media and shifted to 37°C for 6 h. Single (medial) focal plane. (F) Quantification of percentage cell area occupied by ER for same strains shown in E. Open circles indicate wild-type (black) and cdc42-129 mutant (red) strains imaged at the permissive temperature (25°C), and closed circles at the restrictive temperature (37°C). wt 25°C: two trials, n = 40, 37.1 ± 7.8; cdc42-129 25°C: two trials, n = 40, 37.9 ± 7.2; wt 37°C: two trials, n = 40, 28.7 ± 10.2; cdc42-129 37°C: two trials, n = 40, 47.3 ± 14.2. mean ± SD. Asterisks denote statistical significance compared with control using Student’s t test. ***, P ≤ 0.001. (G) Quantification of mCherry-Snf7 punctae of same strain shown in E grown to early-log phase in minimal media at 25°C, as a frequency distribution of the fluorescence intensity of each pixel in a cell normalized to cell area (micrometer squared). Two trials, n = 40. (H) Quantification of mCherry-Snf7 punctae of same strain shown in E grown to early-log phase in minimal media then shifted to 37°C for 6 h. Two trials, n = 40. All scale bars, 5 µm.

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