Extended Data Figure 8. Partitioning of the NE into core and non-core subdomains depends on the local organization of spindle microtubules.

a-c. 3D-SIM showing exclusion of non-core NE assembly from the region of the lagging chromosome adjacent to CHIMP4B marked microtubules (MTs). a. Scheme summarizing the SIM results after KIF4A knockdown. The ESCRT-III complex is recruited to small membrane holes at the sites where spindle microtubules intersect the reforming NE where it is thought to be required for normal NE sealing²³. Therefore, we used ESCRT-III (CHMP4B) labeling to identify the NE region on the lagging chromosome that is intersected by microtubules for the SIM experiments.

b, Imaris surface three-dimensional renderings from SIM images (from c, below) showing recruitment of Nup133 (white) to the region of a lagging chromosome depleted of CHMP4B (green) and microtubules (red) from a KIF4A-depleted HeLa K cell (representative of 4 lagging chromosomes, from 2 experiments). c, SIM images showing that similar to what occurs on the main chromosome masses during normal NE assembly²³, NPCs (Nup133) are recruited to regions of the lagging chromosome lacking CHMP4-decorated microtubules. Left: A cartoon depicting the entire lagging chromosome visualized from a KIF4A-depleted anaphase/telophase cell (microtubules in red, DNA in blue, z focal planes as indicated). Right: Serial sections from a SIM z-stack of an anaphase/telophase HeLa K cell expressing CHMP4B-GFP (green) labeled for microtubules (red), Nup133 (white) and DNA (blue) after KIF4A depletion. These results are consistent with the idea that microtubules inhibit non-core NE assembly.

d, KIF4A knockdown (Fig. 3b, c, Extended Data Fig. 7b–d) and early anaphase Aurora B inhibition have a similar effect on NPCs/non-core NE assembly to lagging chromosomes. Experiments performed as in Fig. 2a in RPE-1 cells. Left panel images: left column: Representative timelapse images of cells upon ZM447439 or DMSO addition (live, 0 or 6 min post AO); right five columns: Representative images of the same cells after fixation and labeling (12 min post treatment). For the cell exposed to ZM447439 at anaphase onset (middle row), one of the two lagging chromosomes exhibits a small-scale separation of core (white arrowhead, emerin) and non-core (white arrow, Nup133) NE domains. Scale bar, 10 μm. Right: Quantification of the results (mean with 95% CI, n=64, 72, 43, 72, 34, from 2 experiments). **** P < 0.0001, NS: Not significant (P=0.6932), two-tailed Mann-Whitney test.