Figure 3. Sun2 contains a membrane... | Rockefeller University Press

Figure 3.

$Sun2 contains a membrane binding amphipathic helix that is sensitive to the INM lipid environment. (A) Schematic of N-terminal region of Sun2 highlighting predicted amphipathic helix (AH; residues 155–180) and mutation of bulky hydrophobic residues. Helical wheel projections generated by HeliQuest with hydrophobic moment <µH>, net charge z, and the discriminant factor D. (B and C) Representative image of coomassie stained gel of indicated in vitro purified proteins after liposome-cosedimentation assays with indicated lipid compositions. Arrowhead: GST-AH; Asterisk: purification byproduct. In C, percent of protein in pellet as a ratio of soluble fraction is shown. n = 3 or 4 experimental replicates. P values: ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. (D) Spinning disk confocal images of live U2OS cells expressing indicated constructs. (E) Spinning disk confocal images of live U2OS cells expressing AH-mNG-NLS treated as indicated. Box plot: NE enrichment scores pooled from two independent experiments. n indicates the number of cells quantified. (F) Spinning disk confocal image of live CTDNEP1 KO cells expressing AH-mNG-NLS. (G) Plot of ER:NE fluorescence ratio of AH-mNG-NLS in indicated cells pooled from two or three independent experiments. n indicates the number of cells quantified. Bars indicate median. (H) Spinning disk confocal images of live U2OS cells expressing indicated constructs. Plot (left): Fluorescence intensity of mNG normalized to maximum fluorescence along line scan. Plot (right): ER:NE fluorescence ratio. P values, Kruskal–Wallis test followed by Dunn’s multiple comparisons test. n indicates the number of cells quantified. Bars indicate median. (I–K) Immunoblot of whole cell lysates from cells treated with cycloheximide for indicated hours expressing indicated constructs. In J, plot shows mNG level at 0 h (untreated cells). Each dot indicates an independent experiment. In K, plot shows n = 2 (2 h) or 3 (4 h) independent experiments. Mean (and SD where n = 3) shown. All scale bars: 10 µm. Source data are available for this figure: SourceData F3.$

Sun2 contains a membrane binding amphipathic helix that is sensitive to the INM lipid environment. (A) Schematic of N-terminal region of Sun2 highlighting predicted amphipathic helix (AH; residues 155–180) and mutation of bulky hydrophobic residues. Helical wheel projections generated by HeliQuest with hydrophobic moment <µH>, net charge z, and the discriminant factor D. (B and C) Representative image of coomassie stained gel of indicated in vitro purified proteins after liposome-cosedimentation assays with indicated lipid compositions. Arrowhead: GST-AH; Asterisk: purification byproduct. In C, percent of protein in pellet as a ratio of soluble fraction is shown. n = 3 or 4 experimental replicates. P values: ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. (D) Spinning disk confocal images of live U2OS cells expressing indicated constructs. (E) Spinning disk confocal images of live U2OS cells expressing AH-mNG-NLS treated as indicated. Box plot: NE enrichment scores pooled from two independent experiments. n indicates the number of cells quantified. (F) Spinning disk confocal image of live CTDNEP1 KO cells expressing AH-mNG-NLS. (G) Plot of ER:NE fluorescence ratio of AH-mNG-NLS in indicated cells pooled from two or three independent experiments. n indicates the number of cells quantified. Bars indicate median. (H) Spinning disk confocal images of live U2OS cells expressing indicated constructs. Plot (left): Fluorescence intensity of mNG normalized to maximum fluorescence along line scan. Plot (right): ER:NE fluorescence ratio. P values, Kruskal–Wallis test followed by Dunn’s multiple comparisons test. n indicates the number of cells quantified. Bars indicate median. (I–K) Immunoblot of whole cell lysates from cells treated with cycloheximide for indicated hours expressing indicated constructs. In J, plot shows mNG level at 0 h (untreated cells). Each dot indicates an independent experiment. In K, plot shows n = 2 (2 h) or 3 (4 h) independent experiments. Mean (and SD where n = 3) shown. All scale bars: 10 µm. Source data are available for this figure: SourceData F3.

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