The PA sensor accumulates in NE herniations (supplemental to Fig. 5 ). (A) The NLS-DAG mCherry sensor does not substantially colocalize with NPCs under conditions of herniation formation in nup116Δ cells. Deconvolved fluorescence micrographs of nup116Δ cells expressing NLS-DAG 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, 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-DAG sensor-mCherry from line profiles drawn around the NE of 15 cells; r is the linear correlation coefficient (Pearson’s). (D–F) Examples of correlative light and electron tomography of NLS-PA mCherry-sensor localization in nup116Δ cells. Fluorescence micrographs 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 a 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. Asterisks demark locations of nuclear pores, and arrow points to a spindle pole body. Scale bars, 200 nm.