Extended Data Fig. 1: Defective NE assembly on lagging chromosomes (or chromosome bridges).

a, Cartoon of NE subdomains that form transiently around the main chromosome mass in a telophase cell^8,9 and summarizing results for lagging chromosomes. DNA, light blue. BAF, barrier-to-autointegration factor. The NPC is a complex containing NUPs, including NUP133, NUP107, ELYS, TPR, NUP153, NUP358 and NUP62¹ (see d and Extended Data Fig. 2f). b, Live-cell/fixed cell imaging showing the recruitment of core (emerin) and non-core (NUP133) proteins onto lagging chromosomes (yellow arrows) and the main chromosome mass at the indicated times after anaphase onset (AO, t = 0). Top, experimental scheme. RFP–H2B-expressing RPE-1 cells were released from the nocodazole mitotic block, imaged on gridded coverslips at 2-min intervals, and then fixed and labelled for immunofluorescence (Extended Data Fig. 4e). Bottom, cartoons (left) and images of RPE-1 cells (right) showing DNA (blue), emerin (red) and NUP133 (green). Each image represents 20 cells (from two technical replicates) from the indicated time points. Scale bars, 10 µm. Note there is some variability between cells (~1–2 min) in the times at which different proteins are recruited. During early–mid anaphase (2–4 min post AO), NUP133 is on kinetochores³⁵ (green dots in cartoon). About 4–6 min after AO, NUP133 begins to assemble on the chromosome periphery (right-most column, white arrowheads). About 6–8 min after AO, emerin assembles on lagging chromosome and the main chromosome mass, including the region adjacent to the central spindle (right-most column, orange arrowheads, microtubules not shown). By 8–12 min after AO, emerin becomes concentrated in a recognizable core domain⁸, which is also detected as a gap in NUP133 signal (enlarged image). The peripheral localization of NUP133 (for example, 8 min) marks the non-core domain. About half of the NPCs of the interphase nucleus assemble in this ~8–10-min period of telophase²⁸ (hereafter referred to as late mitotic NPC assembly). Nuclear pore proteins display the most obvious non-core gap, whereas other non-core proteins, such as LBR, more commonly display reduced signal intensity within the core domain (e, top row, RPE-1). Defective NUP133 assembly on lagging chromosomes persists throughout mitotic exit (>15 min after AO). By ~15 min after AO, the core and non-core domains become intermingled on the main nucleus, with fragments of the core domain persisting as pore-free islands that are slowly populated by NPCs during interphase^10,36. c, Similar to RPE-1 cells (b), images of HeLa K cells (representative of 30 cells, from two experiments) showing that the core membrane proteins emerin (top two rows) and LAP2β (bottom two rows) first associate with the chromosome periphery (yellow arrowheads) contemporaneously with the non-core (NPC, mAb414 detects FG-containing nucleoporins) proteins. About 2–4 min later, the core proteins extend into and then concentrate in (emerin, orange arrowheads; LAP2β does not concentrate) the core domain (red arrowheads). Cells were synchronized as in e. In HeLa K cells, lagging chromosomes often exhibit a slight delay (~1–2 min) in the recruitment of core membrane proteins (emerin and LAP2β) as compared to the periphery of the main chromosome mass. Scale bars, 10 µm. It is thought that the NE assembles from a continuous network of mitotic endoplasmic reticulum (ER)¹. It is therefore simplest to propose that the core and non-core subdomains are also in a continuous network, but the core domain is just a region of the continuous ER network that is missing the non-core subgroup of proteins. Supporting this idea, prior work⁸ and our data (b, c) show that the core proteins initially assemble together with the non-core proteins around the chromosome periphery and only later become enriched near the microtubules. This data suggests that the domain partitioning could come solely from NPC precursors and LBR (which requires ELYS for recruitment^37,38) being preferentially retained in fenestrated ER sheets²⁸ that might less readily penetrate bundled spindle microtubules (Fig. 3a). Although we favour this model, we cannot exclude the possibility that core and non-core proteins somehow partition into separate membrane compartments. d, Quantification of defective non-core NE protein recruitment to lagging chromosomes. Synchronization as in Fig. 1a (n = 64, 118, 151, 124, 151, 145, 150, 69, 90, 65, 70, 60, 64, left to right, from three experiments for RPE-1 cells, n = 149, 110, 124, 132, from two experiments for HeLa K cells and n = 44, 76, from two experiments for U2OS cells). e, f, Orthogonal method (1 µM NMS-P715, MPS1i) to generate lagging chromosomes shows a similar non-core NE assembly defect to the nocodazole block-and-release protocol (Fig. 1a). e, Top, experimental scheme. Bottom, representative images of RPE-1 and HeLa K cells. In RPE-1 and U2OS cells there is a near absence of non-core protein on lagging chromosomes, regardless of the method of generation. In HeLa K cells, the effect is less penetrant. About 60% of lagging chromosomes lack detectable non-core protein recruitment, ~15% display strongly reduced levels (scored as labelled but shown in grey bar as reduced, f), and ~25% display a clear signal (NUP133), but often only covering part of the circumference of the lagging chromosome. These differences are probably due to differences in spindle organization between cell lines (Fig. 3; Extended Data Figs. 6–8). f, Quantification of the results (n = 56, 78 for RPE-1 cells, from two experiments; n = 75, 174 for HeLa K cells, from three experiments). Scale bars, 10 µm. g, h, Chromatin bridges (arrows) formed after nocodazole release or partial depletion of SMC2³⁹ show core-only NE protein assembly. g, An RPE-1 cell after release from a nocodazole block (representative of 30 DNA bridges from five experiments). h, Top, experimental scheme for generating chromosome bridges by partial SMC2 depletion. Bottom, an emerin–GFP-expressing RPE-1 cell. Percentages of cells with the indicated staining pattern are shown (n = 30, from two experiments). Scale bars, 10 µm. DNA bridges are uniformly depleted for non-core (LBR) proteins, with no evidence for a gradient as might be expected for the chromosome separation checkpoint hypothesis^15,40. Interphase chromatin bridges have been reported to have an altered NE protein composition, including reduced levels of lamin B1 and NPCs⁴¹, which is consistent with our findings.

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