Figure 4.
Chm7 hyperactivation and NPC misassembly lead to local PA sensor accumulation at the INM. (A) Diagram of the PA and DAG sensors used to visualize the localization of PA and DAG in cells. (B) chm7OPEN is a dominant “activated” form of Chm7 that accumulates at the INM (Thaller et al., 2019). Schematic of Chm7 and chm7OPEN with predicted winged helices (WHs), hydrophobic stretch (H), α helices (α), MIT-interacting motif type 1 (MIM1), and NESs. Numbers denote amino acids. (C) The PA sensor specifically accumulates at sites of Chm7 hyperactivation. Deconvolved fluorescence micrographs of cells with wild-type CHM7 (no GFP) or chm7OPEN-GFP expressed with an NLS-PA sensor-mCherry. GFP (inverted), mCherry (inverted), and merged images are shown. Numbers are the mean percentage and SD of cells with colocalization (co-loc) of chm7OPEN-GFP with either lipid sensor from three independent replicates of 100 cells per replicate. (D) As in C, but with the NLS-DAG sensor-mCherry. Inset is representative of the few cells where there is colocalization between NLS-DAG sensor-mCherry and chm7OPEN-GFP. Note that as DAG sensor is constitutively at the NE, we only scored cells where there was a discernable enrichment of the DAG sensor at the chm7OPEN-GFP site. Red asterisks demark the nucleus. (E) The PA sensor specifically accumulates at the NE in strains with NPC misassembly-associated herniations. Deconvolved inverted fluorescence images of NLS-PA sensor-mCherry (maximum-intensity projections from a z-series) in an nup116Δ strain at 23°C or after 3 h at 37°C, which triggers herniation formation. Numbers at bottom are the mean percentage and SD of cells with NLS-PA sensor-mCherry at foci at the NE where 300 total cells were counted from three independent experiments. (F) As in E, but with the NLS-DAG sensor-mCherry and only a middle focal section shown. Asterisks mark the nucleus. (G) Deconvolved fluorescence micrographs of NLS-PA sensor-mCherry in the indicated strains after 3 h at 30°C (upper panels) or 37°C (lower panels). (H) Plot of the mean and SD of the percentage of cells (from three independent experiments) where the NLS-PA sensor-mCherry accumulates at foci at the NE. >50 cells were quantified per experiment. P values were calculated from a one-way ANOVA with Tukey’s post hoc test where ns (not significant) is P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. Scale bars, 5 µm.

Chm7 hyperactivation and NPC misassembly lead to local PA sensor accumulation at the INM. (A) Diagram of the PA and DAG sensors used to visualize the localization of PA and DAG in cells. (B) chm7OPEN is a dominant “activated” form of Chm7 that accumulates at the INM (Thaller et al., 2019). Schematic of Chm7 and chm7OPEN with predicted winged helices (WHs), hydrophobic stretch (H), α helices (α), MIT-interacting motif type 1 (MIM1), and NESs. Numbers denote amino acids. (C) The PA sensor specifically accumulates at sites of Chm7 hyperactivation. Deconvolved fluorescence micrographs of cells with wild-type CHM7 (no GFP) or chm7OPEN-GFP expressed with an NLS-PA sensor-mCherry. GFP (inverted), mCherry (inverted), and merged images are shown. Numbers are the mean percentage and SD of cells with colocalization (co-loc) of chm7OPEN-GFP with either lipid sensor from three independent replicates of 100 cells per replicate. (D) As in C, but with the NLS-DAG sensor-mCherry. Inset is representative of the few cells where there is colocalization between NLS-DAG sensor-mCherry and chm7OPEN-GFP. Note that as DAG sensor is constitutively at the NE, we only scored cells where there was a discernable enrichment of the DAG sensor at the chm7OPEN-GFP site. Red asterisks demark the nucleus. (E) The PA sensor specifically accumulates at the NE in strains with NPC misassembly-associated herniations. Deconvolved inverted fluorescence images of NLS-PA sensor-mCherry (maximum-intensity projections from a z-series) in an nup116Δ strain at 23°C or after 3 h at 37°C, which triggers herniation formation. Numbers at bottom are the mean percentage and SD of cells with NLS-PA sensor-mCherry at foci at the NE where 300 total cells were counted from three independent experiments. (F) As in E, but with the NLS-DAG sensor-mCherry and only a middle focal section shown. Asterisks mark the nucleus. (G) Deconvolved fluorescence micrographs of NLS-PA sensor-mCherry in the indicated strains after 3 h at 30°C (upper panels) or 37°C (lower panels). (H) Plot of the mean and SD of the percentage of cells (from three independent experiments) where the NLS-PA sensor-mCherry accumulates at foci at the NE. >50 cells were quantified per experiment. P values were calculated from a one-way ANOVA with Tukey’s post hoc test where ns (not significant) is P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. Scale bars, 5 µm.

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