Figure 9.

CLEM of Atg39-GFP reveals expanded ONM with INM-derived vesicles in the NE lumen. (A) i: Virtual slice of an electron tomogram of a cell with high expression of Atg39-GFP (fluorescence image overlaying EM in inset). Boxes represent magnified regions shown in the following panels with corresponding colors. ii: 3D model of continuous ONM, with arrowheads pointing to continuities between substructures. iii: 3D model of likely INM-derived vesicles within the ONM compartment, with arrows pointing to constrictions. iv: Example virtual slice of tomogram with ONM and INM traced in blue and teal, respectively. v and vi: Two views of 3D model where ONM and INM are segmented. vii–x: Further magnifications of single tomographic slices (with and without annotation) as defined by surrounding box color with key in i. Arrowheads point to continuity of ONM and arrows to INM constrictions. Scale bars are 200 nm. (B) Quantification of the diameter of INM-derived compartments from cells with high expression (4 cells) and low expression (10 cells) of Atg39 from electron tomography or high expression of Atg39 from FIB-SEM (52 cells). Total number of INM vesicle diameters measured is indicated in the figure. Median and 95% confidence interval are shown. (C) CLEM of low-expressed GFP-Atg39. i: Fluorescence micrograph of GFP-Atg39 with boxed region ultrastructure shown in ii. Arrowhead points to a region of PMN. ii: Virtual slice from electron tomogram acquired at a region indicated by the box in i. iii: Annotation of virtual slice from ii with the ONM in blue and the INM in teal. iv: 3D surface rendering of annotated structures in iii. Scale bars are (i), 2 μm in i and 50 nm in ii–iv. (D) Quantification of indicated morphologies/substructures observed from 26 total GFP-Atg39 NE foci.