Figure 3.

Topology of AL Insertion

(A–B) NE-openings accompany AL insertions. (A and A′) Transmission electron micrographs (TEM) of two consecutive serial sections of a nucleus with closely aligned AL-NPCs (arrowheads). Note the clear INM-ONM connection of the encircled opening (A), which is replaced by intact NE in (A′), while the boxed opening is seen in both sections. (B) Electron micrograph of a nucleus where the NE is opened (boxed) and continuous with an ER stretch.

(C) Capture of an AL-insertion into the NE by correlative light and electron microscopy (CLEM) and tomography (C′). (C) RFP::Nup107 localizes to the NE and is enriched at proximal AL-NPCs (arrowhead).

(C′) Single slice through a tomogram recorded at the region depicted by the yellow box in (C). Arrowheads point to a membrane connection of the inserting AL to the NE, adjacent to a NE-opening.

(D) Multiple NE openings (arrowheads) are apparent in a volume containing a nucleus that was obtained by focused ion beam-scanning electron microscopy (FIB-SEM; a single slice is shown superimposed with the NE that is isosurface-rendered in blue).

(E) Histogram of NE-opening diameters measured in TEMs as represented in (A, A′, and B). Most NE-openings are in the range of 400–800 nm.

(F–G) FIB-SEM visualizes the continuity of AL membrane sheets with the NE. Slices through a FIB-SEM volume tangential to the nucleus (F–F″) and isosurface rendering of the same region (G). (F–F″) Slices at slightly different angles. Note that cross-sectioned NE with NPCs in side view (yellow arrowheads in F–F″) are perpendicular to an AL membrane sheet that has a branched topology and contains multiple NPC in top view (white arrowheads in F–F″).

See also Figures S5 and S6.