Figure 5.
A PA sensor accumulates at NE herniations associated with NPC assembly defects. (A) The PA sensor colocalizes with NPCs under conditions of NE herniation formation. Deconvolved fluorescence micrographs of nup116Δ cells expressing NLS-PA sensor-mCherry and GFP-Nup49 at the indicated temperatures. In the GFP and mCherry images, the fluorescence is inverted, whereas the merge shows the GFP signal in green and mCherry in magenta. Scale bar is 5 µm. (B) Line profiles of the fluorescence intensities (in a.u.) along the nuclear rim of the boxed cells from corresponding top and bottom panels in A. (C) Correlation of the fluorescence intensity (in a.u.) of GFP-Nup49 and NLS-PA sensor-mCherry from line profiles drawn around the NE of 15 cells; r is the linear correlation coefficient (Pearson’s). Top and bottom panels correspond to top and bottom panels in A. (D) NLS-PA sensor-mCherry accumulates at NE herniations. Correlative light and electron microscopy of an nup116Δ strain grown at 37°C for 3 h before processing for EM. Fluorescence micrograph of NLS-PA sensor-mCherry alone (i) or overlaid on the corresponding low-magnification electron micrograph (ii) after correlation. Blue arrowheads point out focal accumulation of NLS-PA sensor-mCherry that are magnified in iv. Scale bars, 1 µm. (iii) Virtual slices from electron tomograms of the region indicated by the box in ii with 200-nm scale bar. (iv) Annotation of virtual slices from iii, with the ONM drawn in blue and the INM in teal and with the location of mCherry signal denoted with blue arrowheads (see Video 1). Scale bars, 200 nm. (E) Plot of the number of NE structures correlated with a total of 12 NLS-PA sensor-mCherry foci acquired from six unique cells/tomograms (other examples in Fig. S3). Area of colored rectangles is proportional to the total number of groups of herniations (31 individual herniations) and NPCs (1) identified. (F) Cartoon model incorporating PA into an NE/NPC assembly surveillance mechanism. From left to right: NPC assembly proceeds through the evagination of the INM and ultimate fusion with the ONM. Local PA (shown as red lipids) or DAG (not shown) might contribute to these events, likely through regulation of Pah1 (not shown). In contexts in which the NPC cannot be fully assembled (e.g., nup116Δ), Chm7 is recruited to the INM–ONM fusion site by a local accumulation of PA likely driven by its natural affinity for highly curved membranes. This local PA and membrane curvature would contribute to Chm7 recruitment. Chm7 activation is triggered by Heh1. The resulting local polymerization of Chm7 and other ESCRT-III subunits (teal spiral) would lead to membrane sealing. It is possible that the high local concentration of Chm7 during polymerization might also locally recruit PA and help to destabilize the nuclear membranes to promote fusion. Under conditions (like in nup116Δ) where NPCs cannot assemble properly, PA would continually build up, which might lead to the expansion of the herniations and likely instability in the herniation itself, leading to additional rupture (and repair) events. At right is a magnification of the boxed regions.

A PA sensor accumulates at NE herniations associated with NPC assembly defects. (A) The PA sensor colocalizes with NPCs under conditions of NE herniation formation. Deconvolved fluorescence micrographs of nup116Δ cells expressing NLS-PA sensor-mCherry and GFP-Nup49 at the indicated temperatures. In the GFP and mCherry images, the fluorescence is inverted, whereas the merge shows the GFP signal in green and mCherry in magenta. Scale bar is 5 µm. (B) Line profiles of the fluorescence intensities (in a.u.) along the nuclear rim of the boxed cells from corresponding top and bottom panels in A. (C) Correlation of the fluorescence intensity (in a.u.) of GFP-Nup49 and NLS-PA sensor-mCherry from line profiles drawn around the NE of 15 cells; r is the linear correlation coefficient (Pearson’s). Top and bottom panels correspond to top and bottom panels in A. (D) NLS-PA sensor-mCherry accumulates at NE herniations. Correlative light and electron microscopy of an nup116Δ strain grown at 37°C for 3 h before processing for EM. Fluorescence micrograph of NLS-PA sensor-mCherry alone (i) or overlaid on the corresponding low-magnification electron micrograph (ii) after correlation. Blue arrowheads point out focal accumulation of NLS-PA sensor-mCherry that are magnified in iv. Scale bars, 1 µm. (iii) Virtual slices from electron tomograms of the region indicated by the box in ii with 200-nm scale bar. (iv) Annotation of virtual slices from iii, with the ONM drawn in blue and the INM in teal and with the location of mCherry signal denoted with blue arrowheads (see Video 1). Scale bars, 200 nm. (E) Plot of the number of NE structures correlated with a total of 12 NLS-PA sensor-mCherry foci acquired from six unique cells/tomograms (other examples in Fig. S3). Area of colored rectangles is proportional to the total number of groups of herniations (31 individual herniations) and NPCs (1) identified. (F) Cartoon model incorporating PA into an NE/NPC assembly surveillance mechanism. From left to right: NPC assembly proceeds through the evagination of the INM and ultimate fusion with the ONM. Local PA (shown as red lipids) or DAG (not shown) might contribute to these events, likely through regulation of Pah1 (not shown). In contexts in which the NPC cannot be fully assembled (e.g., nup116Δ), Chm7 is recruited to the INM–ONM fusion site by a local accumulation of PA likely driven by its natural affinity for highly curved membranes. This local PA and membrane curvature would contribute to Chm7 recruitment. Chm7 activation is triggered by Heh1. The resulting local polymerization of Chm7 and other ESCRT-III subunits (teal spiral) would lead to membrane sealing. It is possible that the high local concentration of Chm7 during polymerization might also locally recruit PA and help to destabilize the nuclear membranes to promote fusion. Under conditions (like in nup116Δ) where NPCs cannot assemble properly, PA would continually build up, which might lead to the expansion of the herniations and likely instability in the herniation itself, leading to additional rupture (and repair) events. At right is a magnification of the boxed regions.

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