Nature | www.nature.com | 1 Article Closed mitosis requires local disassembly of the nuclear envelope Gautam Dey1 ✉, Siân Culley1, Scott Curran2, Uwe Schmidt3,4, Ricardo Henriques1, Wanda Kukulski5 & Buzz Baum1,6 ✉ At the end of mitosis, eukaryotic cells must segregate the two copies of their replicated genome into two new nuclear compartments1. They do this either by first dismantling and later reassembling the nuclear envelope in an ‘open mitosis’ or by reshaping an intact nucleus and then dividing it into two in a ‘closed mitosis’2,3. Mitosis has been studied in a wide variety of eukaryotes for more than a century4, but how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment remains unknown5. Here, using the fission yeast Schizosaccharomyces pombe (a classical model for closed mitosis5), genetics, live-cell imaging and electron tomography, we show that nuclear fission is achieved via local disassembly of nuclear pores within the narrow bridge that links segregating daughter nuclei. In doing so, we identify the protein Les1, which is localized to the inner nuclear envelope and restricts the process of local nuclear envelope breakdown to the bridge midzone to prevent the leakage of material from daughter nuclei. The mechanism of local nuclear envelope breakdown in a closed mitosis therefore closely mirrors nuclear envelope breakdown in open mitosis3, revealing an unexpectedly high conservation of nuclear remodelling mechanisms across diverse eukaryotes. A key event in the process of cell division in eukaryotes is the partition- ing of the nuclear genome into two nuclear compartments. To achieve this, replicated sister chromosomes detach from the inner nuclear envelope3, enabling them to be separated from one another through the work of a microtubule-based spindle6, before being sorted into two new, physically separate nuclei at mitotic exit. Eukaryotic cells have adopted a wide variety of strategies to coordinate nuclear remodel- ling with chromosome segregation2. At one extreme, in open mitosis, cells first disassemble the nuclear lamina and the continuous nuclear envelope at mitotic entry, and then reverse this process by reassembling the structure around separated chromosomes at mitotic exit. At the other extreme, in a closed mitosis, because the nuclear–cytoplasmic compartment barrier remains intact throughout the division process, spindle poles must be inserted into the nuclear envelope7 to form an intranuclear spindle that can drive chromosome segregation. This spindle is then disassembled as the nuclear envelope is divided into two8. These different modes of nuclear division share key features, and despite there being a range of intermediate states9,10, the resolution of a closed mitosis presents a unique topological challenge. How organ- isms undergoing closed mitosis divide the double nuclear envelope without compromising nuclear integrity is not currently understood. To shed light on this process, we chose to study nuclear division in the fission yeast S. pombe, which serves as an experimentally tractable example of an organism that undergoes closed mitosis. Previous stud- ies have shown that the S. pombe nucleus does not tear at mitotic exit11, as it does in the related yeast Schizosaccharomyces japonicus. Instead, the nucleus constricts to form a dumb-bell shape with a thin nuclear bridge around the anaphase spindle5. Whereas the organization and dynamics of the anaphase spindle have been studied in some detail12–14, it is not known how the nuclear envelope is then remodelled to generate two new nuclei at the end of this process without compromising the compartmental barrier between the nucleus and cytoplasm. To investigate this question, we used a synthetic nuclear-localized green fluorescent protein (GFP) construct to characterize the dynam- ics of nuclear fission and to determine the extent to which the nuclear permeability barrier is maintained throughout the process (Fig. 1a, b). Although nuclear GFP levels remained constant throughout the divi- sion process (as expected for a closed mitosis), using an automated analysis pipeline described in Extended Data Fig. 2a and Methods, we observed a gradual loss of GFP from the nuclear bridge before nuclear division (Fig. 1b). Notably, this occurred without the visible leakage of GFP from daughter nuclei (Fig. 1a). Imaging nuclear envelope remodel- ling over the same time period is complicated by the fact that in fission yeast, as in other eukaryotes, the outer face of the nuclear envelope is continuous with the 3D mesh of tubules and sheets constituting the endoplasmic reticulum15 (Fig. 1c, Extended Data Fig. 1a). To bet- ter image this process live, we searched for an effective marker of the inner nuclear envelope. Our search identified the candidate protein SPAC23C4.05c, owing to its homology to the stress-responsive nuclear envelope protein Ish1 (Extended Data Fig. 1c–e) and its strong negative https://doi.org/10.1038/s41586-020-2648-3 Received: 10 September 2019 Accepted: 21 May 2020 Published online: xx xx xxxx Check for updates 1MRC Laboratory for Molecular Cell Biology, London, UK. 2The Francis Crick Institute, London, UK. 3Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. 4Center for Systems Biology Dresden, Dresden, Germany. 5MRC Laboratory of Molecular Biology, Cambridge, UK. 6Institute for the Physics of Living Systems, University College London, London, UK. ✉e-mail: g.dey@ucl.ac.uk; b.baum@ucl.ac.uk ----!@#$NewPage!@#$---- 2 | Nature | www.nature.com Article a b e ������������� c ������� ��� ��� ��� ���� ���� ���� ���� ���� ���� �� � ������ �� ���� ���� ���� ���� ���� ���� ���� ���� �������������������������������� ����������� ���� � ��� ��� ������ ������� �������������������������������� �������������������������������� ������������������ ��� ���� � ��� ��� ��� � �������������������������� ���� ���� ���� �� ����� ���� �� ���� ���� ���� ���� ���� ���� ��� ��� ��� � �� �� ���� ������� �������������������������������� ����� ��� ���� ������� ���� d Fig. 1 | Les1 domains define the site of nuclear division. a, An S. pombe cell (dotted outline) expressing a synthetic NLS–GFP construct undergoing mitosis. Panels are maximum-intensity projections of confocal images acquired every 30 s. Representative of more than 50 cells across 5 biological repeats. Dotted vertical lines represent maximum spindle length (spindle max, t = 0). b, Mean (darker lines) and standard deviations (lighter bands) of averaged single-cell traces (from 11 cells at t = 300 to 19 cells at t = 0, from 2 strains) aligned at spindle max (t = 0) and normalized to daughter nuclear intensity at t = 0. c, Single-plane Airyscan reconstructions of live cells at various stages of the cell cycle expressing a synthetic endoplasmic reticulum-localized mCherry construct (AHDL) and Les1 tagged with mNeonGreen at the endogenous locus. Green arrowheads mark the boundaries of the Les1 stalks, visible in late anaphase. Representative of more than 10 cells across 2 biological and 8 technical repeats. d, Reconstructions using super-resolution radial fluctuations (SRRF) at 28-s intervals on single-plane confocal slices of a cell undergoing anaphase and expressing Atb2–mCherry (tubulin) and Les1–mNeonGreen tagged at the endogenous loci. Green arrowheads mark Les1 stalk boundaries, which first become visible in mid-anaphase. Representative of more than 10 cells across 2 biological and 8 technical repeats. e, Les1 intensity in stalks (1.5 μm from the nuclear periphery) over time: mean (darker line) and standard deviations (lighter band) of between 11 (t = 300) and 36 (t = 0) single-cell traces, aligned at spindle max (t = 0) and normalized to maximum bridge intensity. Scale bars, 2 μm. a c b Nuclear envelope Microtubules Late bridge Inset Nuclear envelope Microtubules Late bridge Inset Early bridge Nuclear envelope Microtubules Les1 Tubulin Late bridge Les1 Tubulin Les1 Tubulin Early bridge Les1 Tubulin Nuclear envelope Microtubules Nuclear envelope Microtubules Fig. 2 | Nuclear division occurs by local nuclear envelope breakdown. a, Representative SRRF reconstructions from single-plane confocal slices of cells in early (left) and late (right) anaphase, expressing Les1–mNeonGreen and Atb2–mCherry (tubulin) tagged at the endogenous loci. Intact spindle microtubules persist in the late anaphase bridge despite midzone clearance of Les1. Representative of more than 10 cells across 2 biological and 8 technical repeats. Scale bar, 2 μm. b, Virtual slice through electron tomogram (left) and 3D segmentation model (right) of early anaphase bridge. Nuclear envelope, green; microtubules, magenta. The outlined region is magnified on the left. Arrowheads indicate nuclear pores. One technical repeat. Scale bars, 100 nm. c, Full and clipped views of 3D segmentation models of late anaphase bridges (nuclear envelope, green; microtubules, magenta), superimposed on virtual slices through electron tomograms, shown without model in second from left. Magnified regions (left) are different virtual slices of the region indicated by white outlines. Magenta arrowheads indicate microtubules, green arrowheads indicate stalk tip and limit of intact nuclear envelope, and blue arrowheads indicate nuclear pores. Two representatives of five technical repeats. Scale bars, 100 nm. ----!@#$NewPage!@#$---- Nature | www.nature.com | 3 genetic interactions with ESCRTIII proteins16 (Extended Data Fig. 1b). SPAC23C4.05c localizes exclusively to the nucleoplasmic surface of the inner nuclear envelope throughout the cell cycle (Fig. 1c, Extended Data Fig. 2d, e) without labelling endoplasmic reticulum tubules or cortex. SPAC23C4.05c was also seen concentrated at the stalk of each daughter nucleus during anaphase—a phenotype for which we named the protein Lem-like enriched in stalks (Les1) (see Methods for further details). Using Les1 as a marker, we were able to follow in detail the dynamic changes in nuclear shape that accompany spindle elongation, as the single nucleus divides into two via a characteristic dumb-bell-shaped intermediate (Fig. 1d). The kinetics of spindle elongation are highly reproducible17, allowing us to align single-cell trajectories to the time point at which the spindle reaches its maximum length (Methods). At early stages of bridge formation, Les1 concentrated in stalks originating at the neck of each daughter nucleus (Fig. 1d, e). At maximum spindle elongation, Les1 was visibly depleted from the midzone of the bridge (Fig. 1d), a process that was followed, within a few seconds, by the break- age of the spindle (Fig. 1d, Extended Data Fig. 2b). These observations pointed to the midzone of the bridge as the site where nuclear fission probably occurs. We then used correlative light microscopy and electron tomography of cells labelled with both Les1–mNeonGreen and mCherry–Atb2 (Fig. 2a) to characterize early and late nuclear bridges (Fig. 2b, c, Extended Data Fig. 3a). In early bridges, the nuclear envelope was observed surrounding the spindle (narrowing at the base of the stalks and widening towards the mid- zone) and was studded with nuclear pores (Fig. 2b). At an intermediate stage, the nuclear pores were completely excluded from the stalk and clustered in a central bulge (Extended Data Fig. 3b). By contrast, in late stage bridges, while the nuclear envelope still enveloped the spindle within stalks, there was no evidence of a continuous nuclear envelope in the midzone region of the bridge. Instead, spindle microtubules were seen projecting out of the two newly formed daughter nuclei into the cytoplasm, through stalk membranes that lacked nuclear pores (Fig. 2c). All that appeared to be left of the central part of the nuclear bridge at this stage were fragments of membrane, an observation that explains the loss of nuclear GFP from the midzone of the late anaphase bridge (Fig. 1). We asked, if the nuclear envelope in the central region of the bridge is disassembled to induce nuclear division (as suggested by this unex- pected observation), how this distinct midzone region is specified. A clue came from two observations made using electron tomogra- phy. First, nuclear pores within early-stage bridges were found to be d a e g f b c Tubulin Cut11 0 10 20 30 –10 –20 –30 –40 –50 –60 –70 –80 –90 40 50 Time relative to spindle max (s) Spindle break Transmembrane Nup –200 0 200 0 0.5 1.0 Cut11 Nup60 NLS Time relative to spindle max (s) Normalized bridge intensity Cut11 Cut11 Les1 6 s 2.8 μm Spindle max Cut11 Cut11 Les1 Cut11 Cut11 Les1 Alm1 Cut11 –300 –180 –120 –60 0 –240 60 Time relative to spindle max (s) Les1 Cut11 Δ imp1 Time relative to spindle max (s) Les1 Cut11 Control 5 μM LatA 0 180 360 540 720 900 –180 –360 –540 1,080 1,260 1,440 Expected spindle break Spindle break Expected cytokinesis Cytokinesis Cut11 Alm1 Basket Nup Time relative to spindle max (s) –540 –480 –420 –360 –60 1,140 Δ imp1 Basket Nup Fig. 3 | Les1-restricted stepwise NPC disassembly drives local NEB. a, Schizosaccharomyces pombe expressing Alm1–mNeonGreen and Cut11– mCherry, maximum-intensity projections of confocal images acquired every 60 s. Scale bar, 2 μm. Representative of more than 30 cells across 3 biological repeats. b, iSIM images of cells expressing Cut11–mCherry and Les1– mNeonGreen. Scale bar, 2 μm. Representative of more than 10 cells across 2 biological and 10 technical repeats. c, Kymograph of intensities averaged across a single confocal slice of a dividing cell (white rectangular box in inset) expressing Cut11–mCherry and Les1–mNeonGreen and imaged at three frames per second followed by denoising. Magenta arrows indicate a mobile but corralled NPC cluster. Representative of more than 10 cells across 2 technical repeats. d, Maximum intensity projections of confocal images of cells expressing Cut11–GFP and Atb2–mCherry and acquired at 10-s intervals. Scale bars, 2 μm. Representative of more than 30 cells across 3 biological repeats. e, Averaged normalized-intensity traces for NLS–GFP (between 22 cells at t = −300 and 37 cells at t = 0), Nup60–mNeonGreen (between 11 cells at t = −300 and 34 cells at t = 0) and Cut11–mCherry (between 11 cells at t = −300 and 34 at t = 0), aligned by spindle max (t = 0). The dotted orange line indicates single exponential fit to NLS–GFP average. Data from two strains were combined on the basis of the analysis described in Extended Data Fig. 6b. f, Confocal maximum-intensity projections of imp1∆ cells expressing Les1–mNeonGreen and Cut11–mCherry in the presence (bottom) or absence (top) of 5 μM latrunculin A (LatA). Magenta arrows indicate persistent midzone Cut11 clusters. Scale bar, 2 μm. Representative of more than 20 cells across 2 biological and 4 technical repeats. g, Confocal maximum-intensity projections of imp1∆ cells expressing Alm1–mNeonGreen and Cut11–mCherry. Magenta arrows indicate persistent midzone Cut11 clusters. Representative of more than 20 cells across 2 biological repeats. Scale bar, 5 μm. ----!@#$NewPage!@#$---- 4 | Nature | www.nature.com Article physically too close to spindle microtubules to possess a full nuclear basket, on the basis of steric considerations (Extended Data Figs. 2d, e, 3c). Second, nuclear pores were completely absent from stalks in late-stage bridges (Fig. 2c). Building on these observations, when we tracked various components of the nuclear pore complex (NPC)—a complex of individual proteins referred to as nucleoporins (Nups) (Extended Data Fig. 2d)—through nuclear division by light micros- copy, we discovered that the pores that enter the bridge are completely devoid of the nuclear basket components Alm1 and Nup211 (Fig. 3a, Extended Data Fig. 4a–c). NPCs were also visibly depleted from stalk regions of the bridge where Les1 is concentrated (Fig. 3b, c, Extended Data Fig. 5a). Instead, as previously reported18,19, NPCs were found concentrated in the central bulging section of the bridge, where Les1 levels are low (Fig. 3b, c). Even here, the movement of NPC clusters within the midzone appeared constrained by the stalks on either side (Fig. 3c). Several lines of evidence indicate that Les1 accumulation, and the concomitant corralling of NPCs, requires a close association of the nuclear envelope with spindle microtubules but is not contingent on the formation of a normal bridge midzone. First, occasional ‘stray’ NPC clus- ters located distal to the midzone always correspond to areas of local Les1 depletion and local bridge dilation (Extended Data Fig. 5c–f). Sec- ond, in cells with excess nuclear envelope that generate thicker, floppy bridges, Les1 cannot form stalks and NPCs are no longer restricted to the midzone (Extended Data Fig. 6a). Finally, aberrant tubular projections containing spindle microtubules, induced either by reducing nuclear envelope surface area or by acutely inhibiting Aurora kinase activity before cells enter mitosis (Methods), accumulate Les1 and lack NPCs (Extended Data Fig. 6b, c). Together, these results indicate that the stalks, defined by Les1 accumulation at sites of microtubule–mem- brane contact within the bridge, function to restrict a population of basketless NPCs to the bridge midzone. a c Nup60 NLS–GFP –210 –180 –150 –120 –90 –60 –30 0 30 60 –270 –240 –300 Spindle max –330 Time relative to spindle max (s) Nup60 NLS–GFP –210 –180 –150 –120 –90 –60 –30 0 30 60 –300 –270 –240 Time relative to spindle max (s) –330 Leakage d e Spindle break Relative intensity Time relative to spindle max (s) Les1 stalk NLS TM Nup Local NEB Les1 stalk Loss of nuclear ring Nups Bridge NLS decay begins TM Nup loss Loss of peripheral Nups Bridge NLS decay begins TM Nup loss NLS leakage WT b 0.6 0.7 0.8 0.9 1 Normalized NLS–GFP intensity Leaking (23/71) Leaking (3/66) Nucleus 1 Nucleus 2 Nucleus 1Nucleus 2 –155–35 Time relative to spindle max (s) Time relative to spindle max (s) –155–35 –155–35 –155–35 Spindle max Spindle max Spindle max Spindle max WT f g Nup60 NLS | Pre Nup60 NLS | Cer 0 60 120 180 240 300 360 Time (s) 420 480 Leakage Cmp7 Nup60 Time relative to spindle max (s) 0 –15 –30 –45 –60 –75 –90 15 30 45 60 75 90 105 120 135 les1Δ les1Δ Δ les1 Δ les1 Δ les1 Δ les1 Spindle max Nuclear ring Nup Fig. 4 | Les1 isolates nuclei from local NEB. a, Maximum-intensity projections of spinning-disc confocal images of cells expressing Nup60–mCherry tagged at the endogenous locus and NLS–GFP, either wild type (top) or carrying a Les1 deletion (bottom), imaged at 30-s intervals. Orange arrows indicate sites of NLS–GFP leakage from one or both daughter nuclei. Representative of more than 50 cells across 4 biological repeats. Scale bars, 2 μm. b, Single-cell intensity traces for wild-type (WT) (top) and les1∆ cells (bottom), indicating intensity in each daughter nucleus, aligned at spindle max (dotted line, t = 0) and normalized individually to maximum intensity. Orange bar and text indicate the number of leaky nuclei. c, Virtual slices through electron tomograms of a dividing les1∆ cell. The approximate overlap in field of view of the tomograms is indicated by dashed lines. NPCs are indicated by blue arrowheads. Scale bars, 100 nm. Representative of 2 technical repeats. d, Confocal maximum-intensity projections of les1∆ cells expressing Cmp7–mNeonGreen and Nup60–mCherry. Representative of more than 20 cells across 3 biological repeats. Scale bar, 2 μm. e, les1∆ cells expressing NLS–GFP and Nup60–mCherry, with (bottom) or without (Pre) (top) of 10 μM cerulenin (Cer). Orange arrows indicate sites of NLS–GFP leakage. Representative of more than 40 cells across 3 biological repeats. Scale bar, 5 μm. f, Schematic indicating relative intensity levels of key readouts of the nuclear division process, aligned relative to spindle max. TM, transmembrane. g, Schematic illustrating the process of local NEB and the role of Les1 in structuring the bridge. ----!@#$NewPage!@#$---- Nature | www.nature.com | 5 In an open mitosis, the stepwise removal of NPCs leads to nuclear envelope fenestration and loss of structural integrity during nuclear envelope breakdown10 (NEB). We observed precisely this sequence of events in the S. pombe bridge midzone, in a process that we term ‘local NEB’. Thus, in the few minutes following dumb-bell collapse, as the nuclear-localization signal (NLS)–GFP intensity in the nucleus was seen disappearing from the bridge (Fig. 3e), NPCs were gradually lost from the midzone. This process began with the loss of nuclear ring Nups (Nup60) and was completed with the loss of transmembrane Nups (Cut11) (Fig. 3d, e, Extended Data Fig. 5g–i). Although fast imaging revealed distinct clusters of NPCs within a single bridge disappearing at different times, this order of events was preserved within any given cluster (Extended Data Fig. 5i). Of note, whereas this was independent of Les1 itself (Extended Data Fig. 7d, e), both NPC disassembly and local NEB could be completely arrested by deletion of the importin Imp119 (Fig. 3f, g, Extended Data Fig. 6d). As local NEB precedes spin- dle disassembly (Fig. 2c, 3d, e), this also enables us to reinterpret the previously described Imp1-mutant spindle phenotype19. Our results show that Imp1-dependent removal of bridge NPCs and local nuclear envelope breakdown expose spindle microtubules to the cytoplasm, where cytoplasmic factors trigger their timely disassembly. We considered the function of Les1 in this context. In support of the idea that the site of NEB is restricted to the bridge midzone by Les1-defined stalks, the spatial organization of NPCs in the bridge was completely lost in the les1Δ strain. Light microscopy (Fig. 4a, Extended Data Fig. 7f) and electron tomography (Fig. 4c, Extended Data Fig. 7a, b) of these cells showed that NPC components were uniformly distributed in both early- and late-stage bridges, leading to the onset of local NEB at a random location along the bridge (Extended Data Fig. 7f). Daughter nuclei in the les1Δ strain also suffered transient leakages at the time of maximum spindle elongation, as measured by loss of nuclear NLS–GFP (Fig. 4a, b); this typically occurred in only one of the two daughter-cell nuclei (Fig. 4a, b). These leaks were rapidly repaired in a manner that does not depend on Les1 (Fig. 4a)—an observation that explains the lack of a growth defect in the les1Δ strain. Instead, the repair process was associated with recruitment of the ESCRTIII protein Cmp7 (Fig. 4d) to sites of local NEB20 (Fig. 4d, Extended Data Fig. 8b, c). In line with this, les1Δ is synthetically near-lethal when combined with deletions in cmp7 or lem2 (which encodes Lem2, the binding partner of Cmp7) (Extended Data Fig. 8d, e). These observations are also consistent with the highly conserved role for ESCRTIII proteins in nuclear envelope sealing21–24 and repair25,26 across metazoans and fungi. Together, these data suggest that Les1 stalks functionally isolate daughter nuclei from the process of Imp1-dependent local NEB at the centre of the bridge (Extended Data Fig. 8g). Consistent with this hypothesized role for Les1, deleting Les1 does not strongly affect the kinetics of NPC disassembly (Extended Data Fig. 7d, e) or average inter-NPC spacing (Extended Data Fig. 7b). Instead, Les1 prob- ably acts to create a seal by gathering the inner nuclear envelope tightly around the spindle, leading to the segregation of excess membrane and bulky NPCs into the characteristic bulge at the centre of the bridge in wild-type cells, a structure that is conspicuously absent from les1Δ tomograms (Fig. 4c). Consistent with this model, treating les1Δ cells with cerulenin to increase membrane tension in the bridge (by inhib- iting fatty acid biosynthesis in the endoplasmic reticulum27) forces NPCs back to the centre to rescue the nuclear-leakage phenotype in the proportion of cells that are able to form a bridge (Fig. 4e, Extended Data Fig. 8f). Therefore, Les1 performs a critical role in nuclear division in S. pombe by ensuring that, while nuclei are topologically open to the cytoplasm at this stage of mitosis, the compartment boundary itself remains effectively closed (Fig. 4f, g). In summary, we have identified Les1, a protein that positions the site of nuclear fission during nuclear division in S. pombe. Through the study of Les1 localization and its deletion mutant, we describe a process of local nuclear envelope breakdown that reveals an unexpectedly close similarity between the remodelling of the nuclear envelope in open and closed mitosis (Fig. 4g). In both cases, the new nuclear compart- ment is remodelled as the result of NPC disassembly. Thus, the key difference between mitotic strategies across the eukaryotic tree28 may simply be one of degree, depending on the timing and localization of NPC disassembly. Online content Any methods, additional references, Nature Research reporting sum- maries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author con- tributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-020-2648-3. 1. LaJoie, D. & Ullman, K. S. Coordinated events of nuclear assembly. Curr. Opin. Cell Biol. 46, 39–45 (2017). 2. Sazer, S., Lynch, M. & Needleman, D. Deciphering the evolutionary history of open and closed mitosis. Curr. Biol. 24, R1099–R1103 (2014). 3. Ungricht, R. & Kutay, U. Mechanisms and functions of nuclear envelope remodelling. Nat. Rev. Mol. 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P., Neighbors, B. & McDonald, K. L. Architecture of the microtubule component of mitotic spindles from Dictyostelium discoideum. J. Cell Sci. 75, 93–129 (1985). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. © The Author(s), under exclusive licence to Springer Nature Limited 2020 ----!@#$NewPage!@#$---- Article Methods Data reporting No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Phylogenetics and protein bioinformatics Secondary structure models for Les1 were generated using the homol- ogy modelling and threading software tool I-TASSER29,30 (https:// zhanglab.ccmb.med.umich.edu/I-TASSER/). All sequence homology searches were carried out using a local installation of the HMMER suite of profile-HMM search tools31 (http://hmmer.org/). Uniprot IDs for sequences analysed in Extended Data Fig. 1 are: O94559 (Les1_Sp), S9RL50 (Les1_So), S9VS84 (Les1_Scr), B6JWA6 (Les1_Sj), Q9Y7X6 (Ish1_Sp), S9PR74 (Ish1_So), S9X3G6 (Ish1_Scr), B6K568 (Ish1_Sj) and Q03104 (MSC1_S.cerevisiae). MAFFT32,33 (https://mafft.cbrc.jp/align- ment/software/) was used to generate sequence alignment with the following command-line options: mafft–maxiterate 1000–localpair < infile.fasta> > . Both trimmed and untrimmed alignments were used to generate phylogenetic trees, though in the case of Les1 this did not significantly affect the resulting trees. Alignments were trimmed, using TrimAl34 (http://trimal.cgenomics.org/trimal) with these command-line options: trimAl/source/trimal -in < infile.align> -gt 0.6 -cons 40 -phylip -out < outfile.trim>. Maximum-likelihood trees were inferred using IQTREE35,36 1.6 (http:// www.iqtree.org/), run using the model test function (for Extended Data Fig. 1d, LG + G4) and 1,000 bootstraps: iqtree-omp-1.5.4-MacOSX/bin/ iqtree-omp -nt 4 -s -m TEST -bb 1000 -redo Conserved motifs were detected in alignments through comparisons with the PFAM database37,38 (https://pfam.xfam.org/). Selected regions of alignments were displayed using ESPript339 (http://espript.ibcp.fr) and default options. Although Ish1 and Les1 are annotated to be type I LEA domain proteins (Uniprot: https://www.uniprot.org/uniprot/ Q9Y7X6), no homology or profile HMM similarity was detected to any LEA domain families. Instead, the easily detected but poorly char- acterized Ish1 motif (PFAM PF10281), present in two copies in both SPAC23C4.05c and Ish1 (as well as Saccharomyces cerevisiae MSC1; Extended Data Fig. 1c–e), bears similarity to the widely conserved HeH/ LEM (PFAM PF12949) and SAP (PFAM PF02037) domains. To reflect this finding, we settled on the name Lem-like enriched in stalks (Les1) for the S. pombe protein SPAC23C4.05c and its best-match orthologues in Schizosaccharomyces species. Schizosaccharomyces pombe culture. Schizosaccharomyces pombe cells were cultured using standard methods40,41 on solid (YES agar) and liquid (YES) rich growth media (ForMedium), at a growth temperature of 32 °C unless stated otherwise. All experiments were performed in exponential growth at 32 °C with at least 48 h of growth (>20 genera- tions) in liquid YES before plating for live imaging. For live imaging, uncoated 35-mm dishes with polymer coverslips (no. 1.5 coverslip, 180 μm thick, Ibidi) were first coated with 1 mg ml−1 soybean lectin (in water, aliquots stored at −80 °C, Sigma-Aldrich) for 15 min. After washing away the excess lectin with fresh YES, cells drawn from exponentially growing liquid cultures were allowed to settle for 30 min in a minimum volume of 500 μl YES. The entire plating volume was replaced with 1 ml of fresh YES prewarmed to 32 °C before transfer to the microscope. For drug treatment experiments, latrunculin A (Sigma-Aldrich), cerulenin (Sigma-Aldrich) and 1NM-PP1 (Calbiochem) were added to YES at 5 μM (10 mM stock in DMSO), 10 μM (10 mM stock in DMSO) and 5 nM (1 mM stock in DMSO), respectively. The maximal amount of DMSO (1/1,000 in YES) added to cells across conditions had no detectable effect on the kinetics of nuclear division—thus, to ease direct kinetic compari- sons across all strains and experiments in the paper, drug-treated cells were compared with untreated controls. For cell culture for electron tomography experiments, see ‘Correlative fluorescence microscopy and electron tomography’. Plasmids and S. pombe strain construction. The full genotypes of all strains used in this study are presented in Extended Data Table 1. Strains generated specifically for this study were constructed using standard methods40,41 for gene editing and crossing. Gene deletions and tagging were performed as previously described42 for PCR-based gene target- ing, using standard primers designed with the Bahler lab web-interface scripts (http://bahlerlab.info/resources/), pFA6a vector templates carrying hygromycin- or kanamycin-resistance cassettes, and transfor- mation using the lithium acetate method43. Antibiotic-resistant clones generated by this method were verified by PCR of the gene locus being targeted as well as fluorescence microscopy, if applicable. The excep- tion to the standard workflow was for the pFA6a-mNeonGreen vectors used in this study, which carry a non-standard linker upstream of the mNeonGreen coding sequence. Instead of the standard 20-mer (CGGAT CCCCGGGTTAATTAA) forward linker, these require a 21-mer forward linker (GATTCTGCTGGATCAGCTGGC). The reverse linker remains unchanged. One new pFA6a vector derivative was generated for this study, replacing the mCherry coding sequence in pFA6a-mCherry:Hph with the coding sequence for the photo-switchable fluorescent protein mEOS3.244 (Addgene) by standard restriction-digestion cloning (using restriction enzymes BamHI and AscI; NEB). This vector is available upon request. Crosses were performed by random spore analysis40,41 followed by marker selection (hygromycin or kanamycin resistance, ura/leu auxotrophy, or sensitivity to 5 μM 1NM-PP1 (Calbiochem) for strains carrying the Ark1-as3 allele, as appropriate) followed by ad- ditional screening for fluorescence, if applicable. The crosses shown in Extended Data Fig. 8d, e were carried out using tetrad dissection45, with each colony grown from a single spore. In brief, cells were mated on low-nitrogen medium (EMM-nitrogen) to produce tetrads of four haploid spores. After 2 days, cells were streaked onto nutrient-rich me- dium (YES) for 2–3 h to degrade the protective membrane surrounding each tetrad. Individual spores were isolated using a Singer MSM300 Tetrad Micro dissecting unit. C-terminal truncation constructs with C-terminal mNeonGreen tags (Extended Data Fig. 8a) were generated at endogenous loci using the standard PCR-based method as described above, but with the following left-hand/forward primers: Les1(1–94), 5′-ATTCTTGGCCTCAACGAAAGCTTGATGACTTTCTCCAAAATCATGG GGTAAAGTCACTGGACGTTCCTCCTATCGAGACTGATTCTGCTGGATC AGCTGGC-3′; Les1(1–204), 5′-CCACCAATGATGAGTTGGAATCCT GGTCAAATAATCT ACTCCTTTCTATGTTGGATCAGAAAAACATTACAGT ACCAATTGATTCTGCTGGATCAGCTGGC-3′; and Les1(1–291) 5′-TTTCTGT TCTTTCACCTCGGGAAACTCTTTTGAAAGAAGCATACGCTAACCGCTT CACACCGCGTGTAATGATTGCCTCCGATTCTGCTGGATCAGCTGGC-3′. These truncation sites were selected to delete both Ish1 motifs and the C2H2 Zn finger, the second Ish1 motif and the Zn finger, or just the Zn finger, with care taken to avoid truncating the protein in the middle of predicted secondary structural elements such as α-helices. The first two constructs, Les1 (1–94) and Les1 (1–204), did not generate any detectable expression by fluorescence microscopy. The localiza- tion pattern of the third construct Les1 (1–291) is shown in Extended Data Fig. 8a. Live-cell fluorescence microscopy All strains were imaged live in regular growth medium (YES) in glass-bottom dishes (see Schizosaccharomyces pombe culture) within stage-top incubation chambers held at 32 °C. No single dish was used for experiments lasting longer than 3 h from the time of plating. Four micro- scopes were used for this study: two spinning-disc confocal systems, a Nikon TiE wide-field system with a VT-iSIM module and a Zeiss LSM880 with an Airyscan module. The first spinning-disc microscope consists of a Nikon TiE inverted stand attached to a Yokogawa CSU-X1 spinning-disc ----!@#$NewPage!@#$---- scan head and a Hamamatsu C9100-13 EMCCD camera. The entire sys- tem was controlled using Volocity software. Cells were imaged using a 100× oil-immersion CFI Plan Apochromat VC objective (1.4 NA, working distance 0.13 mm) with an optional 1.5× additional magnification. The second spinning-disc microscope consists of a Zeiss Axio Observer Z1 inverted stand attached to a Yokogawa CSU-W1 spinning-disc scan head and a Photometrics Prime BSI Scientific CMOS detector. Cells were imaged using 63× oil-immersion Plan Apochromat (1.4 NA, working distance 0.19 mm) and 100× oil-immersion Plan Apochromat (1.4 NA, working distance 0.17 mm) objectives combined with an optional 1.5× additional magnification. The LSM880 is an inverted laser-scanning confocal microscope with an Axio Observer stand. Cells were imaged using a 63× oil-immersion Plan Apochromat objective (1.4 NA, work- ing distance 0.19 mm) and the Airyscan46 detector. Acquisition on the latter systems is controlled via the Zen software (Zeiss). In all cases, samples were illuminated with 488 nm (mNeonGreen or GFP) and 561 nm (mCherry) lasers and appropriate fluorescence filter sets for these fluorophores. Photoconversion of mEOS3.2 was carried out using a 405-nm laser, with the non-converted state imaged using a 488-nm laser and the same filter set as for mNeonGreen/GFP, and the converted state imaged using a 561-nm laser and the same filter set as for mCherry. For regular live imaging on all systems, asynchronous cells were usually imaged using a 4.3 μm Z-stack with 16 slices at 270-nm vertical intervals, and time intervals ranging from 5 s to 120 s, never exceeding 30 min of continuous imaging. For Airyscan imaging, cells were imaged using a larger Z-stack at single time points. For SRRF and Hough fitting, cells were imaged with the system held at a single Z-plane and the imaging rate was increased to yield final reconstructed SRRF images at >3 frames per second. Instant SIM (iSIM) images were acquired with a Visitech VT-iSIM module and Hamamatsu Flash4.0 v.3 scientific CMOS cam- era, attached to an inverted Nikon TiE microscope stand with Perfect Focus System and motorized stage (100× oil-immersion 1.45 NA Plan Apo λ objective). Image processing All basic image processing (cropping, viewing stacks, scaling for visual presentation and producing maximum-intensity projections) was carried out in Fiji47 (ImageJ48). All time-lapse images subjected to SRRF analysis were processed with NanoJ-LiveSRRF, the newest implemen- tation of NanoJ-SRRF49. NanoJ-LiveSRRF is available on request and is expected to be available for download soon. NanoJ-SRRF is already released and available as open-source software for Fiji and ImageJ. Airyscan processing and iSIM 3D deconvolution were carried out using proprietary Zen (Zeiss) and Elements (Nikon) software, respectively. Image denoising for fast-acquired data Denoising (Fig. 3c, Extended Data Fig. 4b) was performed using the Noise2Noise image restoration technique50 as implemented using the CSBDeep/CARE framework51. Three separate Noise2Noise models were trained independently for Alm1–mNeonGreen images, Les1– mNeonGreen images and Cut11–mCherry images. Training data com- prised 2,000 pairs of intensity-normalized 64 × 64-pixel patches in two adjacent frames, randomly selected from across all acquired datasets for each model. Validation data comprised an additional 200 pairs of 64 × 64-pixel patches randomly selected and normalized via the same method. Each model was trained with the following network param- eters: training loss = mean-squared error, UNet kernel size = 3 × 3, train- ing steps per epoch = 200, training epochs = 100. Jupyter notebooks for network training and prediction and trained Noise2Noise models are available at https://github.com/superresolusian/local-NEB. Statistics and reproducibility Through this paper, the basic independent biological unit of com- parison is the single cell (undergoing nuclear division). For experi- ments using light microscopy, biological repeats refer to cells drawn from two different cultures and plated separately; technical repeats refer to cells drawn from the same culture but plated separately, usu- ally imaged on the same day. For high-resolution (in time or space) imaging of strains already analysed by conventional confocal imag- ing, we focused on collecting data from additional technical repeats. The number of cells indicated in the legends that accompany repre- sentative images is a conservative estimate of the number of cells, across biological and technical repeats, at the same cell cycle stage. Special considerations apply for electron microscopy (see ‘Correla- tive fluorescence microscopy and electron tomography’). As cells for all experiments were cultured at 32 °C in rich YES growth medium, we were able to pool cells across technical and biological repeats, as well as different clones carrying the same deletion or fluorescent protein tag, for population-level analyses (see ‘Analysis framework for single-cell trajectories’ and ‘Population-level analyses’). Analysis framework for single-cell trajectories Analysis of single-cell trajectories was carried out using custom soft- ware written for the open-source platform Fiji47. This is available from https://github.com/superresolusian/local-NEB. Regions of interest selection. Regions of interest (ROIs) containing dividing cells were manually selected in time-series data. Only divi- sion events that completed were selected for analysis (Extended Data Fig. 2a, ‘Manually select ROIs’, ‘Extracted ROI’). A ROI devoid of nuclei over the whole timelapse was also selected for background subtraction of measured intensities. Detecting divisions. Each ROI was maximum-intensity projected and this projection was then blurred, binarized, hole-filled and skeletonized using ImageJ1 ‘binary’ functions in Fiji47. The longest path of the skeleton structure was assumed to correspond to the dividing nucleus, and the angle formed by this line-like path measured to be the division angle θ (Extended Data Fig. 2a, ‘Determine division angle’). Circle detection. Within ROIs, nuclei were identified for each frame and their radii determined using a custom-written Fiji plugin imple- menting the circular Hough transform52 (Extended Data Fig. 2a, ‘Circle detection’; each coloured circle denotes a separate detected nucleus). For two-colour images, the mNeonGreen channel was used to identify nuclei owing to their superior signal-to-noise ratio, and these nuclei centroid coordinates were assumed to be the same across both chan- nels. For NLS images, a Sobel filter was applied to extract the perimeters of the nuclei before performing the circular Hough transform. Identification of dividing nucleus pairs. For all possible pairs of detected circles in the ROI, the angle between the circle centres was calculated. Circle pairs oriented at angles different from the division angle θ by more than 30° were rejected. For proteins distributed along the whole length of the bridge between the two daughter nuclei (for example, Les1 as shown in Fig. 1d), all candidate bridge paths in each ROI frame were identified by blurring, binarizing, hole-filling and skel- etonizing the images (skeleton paths shown in Extended Data Fig. 2a, ‘Path segmentation’). Paths of length < 3 pixels were rejected as these corresponded to isolated nuclei (for example, Extended Data Fig. 2a, ‘Path segmentation’, blue paths). The end points of the remaining paths were then checked against the coordinates of the remaining circle pairs. Paths without anchoring circle detections were rejected (for example, Extended Data Fig. 2a, ‘Path segmentation’, pink paths), as were any detected circles lacking an associated path (for example, Extended Data Fig. 2a, ‘Circle detection’, red circle). The final result for each frame was either that no division events were successfully detected, or two nuclei and joining path were detected (Extended Data Fig. 2a, ‘Filter circles and paths satisfying selection criteria’). Following breakage of the bridge, there is no longer a continuous path between the daughter ----!@#$NewPage!@#$---- Article nuclei. In this case, for a detected circle pair at an appropriate angle we joined two paths, each with one endpoint anchored at one of the circles, with a straight line between the ‘free’ endpoints (Extended Data Fig. 2a, ‘Filter circles and paths satisfying selection criteria’, yellow dotted line). For proteins not continuously present along the whole length of the bridge (for example, NLS as shown in Fig. 1a), the path corresponding to the bridge location was defined as a straight line between the two daughter nucleus circle centres. Definition of bridge and time points. The bridge was defined as the connection between the two daughter nuclei excluding any pixels within the nuclei perimeters. The initiation of division was defined as the first frame in which two distinct nuclei were successfully detected, and dumb-bell appearance was defined as the first frame where a bridge could be discerned (that is, the first time point where all path pixels were not contained by daughter nuclei). Nucleus separation distance was defined as the Euclidean distance between the centroids of the daughter nuclei. Measurement of nuclear intensities. Nuclear intensities were the background-subtracted average intensities within the detected circles (Fig. 1b, ‘Nucleus’, Fig. 4b). Measurement of bridge intensities. Total bridge intensity was the background-subtracted average intensity along the bridge path (Fig. 1b, ‘Bridge’, Fig. 3e, Extended Data Figs. 4a, 5h, 7d, 8f). In all cases, the path line width was set to 5 (that is, 2 pixels perpendicularly either side of the path) to account for the full thickness of the bridges. Measurement of vertical displacement of nuclear envelope pro- teins. Circle detection and analysis was again performed using the circular Hough transform, but this time on whole frames (that is, no manually selected ROIs) so that all nuclei within a single time frame were detected. All data analysed had Cut11–mCherry as a reference protein in one channel and another nuclear envelope protein of inter- est in the second channel. For each nucleus, the difference in radius between the two channels was calculated (Extended Data Fig. 2c, e). SRRF processing was performed on images before radius measure- ment to increase resolution. As a control, Nup60 was labelled with a green-to-red photoconvertable fluorescent protein (mEOS3.2) and im- ages were taken before and after photoconversion. These two channels were then analysed using the same pipeline as for the Cut11 two-colour strains to check that there were no systematic errors in radius measure- ment between red and green channels. The values obtained for various Nups provide a good match with a recent electron microscopy analysis of the S. pombe NPC53, which also provided the estimate of the width of the lumen between inner and outer nuclear envelopes. Spindle detection and breakpoint measurement. Spindles were seg- mented from ROIs in images containing fluorescently labelled tubulin (mCherry–Atb2) by blurring, binarization, hole-filling and skeletoniza- tion. The longest path of the skeleton was determined for each frame; the frame in which spindle breakage occurred was determined as the first frame where the maximum skeleton path length decreased by ≥ 25% compared to the maximum skeleton path length measured across all previous frames. Quantifying early bridge intensities. Normalized bridge intensity for early bridges (Extended Data Fig. 7c) was calculated as I I f I f I f I f = ( ) − ( ) ( ) − ( ) bridge bridge bg nuc bg in which f is the first frame in a nuclear division sequence in which a bridge appears that has 1 μm ≤ bridge length ≤ 2μm; Ibridge (f) is the average intensity along the bridge length in frame f (with a linewidth of 3 pixels for averaging adjacent to the bridge axis); Ibg (f) is the average intensity of the image background in frame f; and Inuc (f) is the average intensity of interphase nuclei in frame f. Bridge numbers were as follows: Cut11, n = 19; Cut11-les1Δ, n = 27 (P = 0.0179, unpaired two-tailed t-test); Nup60, n = 44; Nup60- les1Δ, n = 102 (P = 0.0722, unpaired two-tailed t-test). Data curation. Every detected nucleus division was manually checked to ensure that the correct nuclei had been identified, and ROIs contain- ing false detections were excluded from analysis. Manual quantification. The breakpoint analysis in Extended Data Fig. 7f was scored manually owing to the high error rates of identifying the precise site of breakage from automatically extracted time-series data. The timings in Extended Data Fig. 6e–g (with reference to the first appearance of the nuclear bridge) were manually quantified. This was due to the presence of a second green label (Cdc15–mNeon- Green) in addition to Cut11–GFP; while this allowed the visualization of the cytokinetic ring, it also prevented the automated analysis of maximum-intensity projections. Population-level analyses Unless otherwise specified, single-cell trajectories (see ‘Manual quan- tification’ for specific exceptions) were aligned to the time of maxi- mal spindle elongation (if a tubulin label was not present, measured indirectly using the maximal separation between daughter nuclei as a proxy for spindle length)—making use of the fact that spindle elonga- tion kinetics are highly reproducible from cell to cell. When combining trajectories from different strains (for example, Fig. 3e), we made use of the observation that mitotic timing tends to be consistent even when spindles reach different maximum lengths (Extended Data Fig. 5b). This is probably owing to a Klp9-dependent adjustment in spindle elonga- tion rates between strains (strains with longer spindles elongate their spindles faster)17. Statistical analyses were carried out using GraphPad (mean and standard deviation of averaged traces), MATLAB (ANOVA; mean and standard deviation of averaged traces) and the Estimation Stats platform54 (two-sided Mann–Whitney). Graphs and heat maps were generated using either GraphPad or MATLAB. Correlative fluorescence microscopy and electron tomography Correlative microscopy was done as described before for resin-embedded yeast cells55,56, with minor modifications. In brief, Les1–mNeonGreen/mCherry–Atb2-expressing cells and Cut11–GFP/ mCherry–AHDL-expressing les1Δ cells were grown in YES at 32 °C to mid-log phase, pelleted by vacuum-filtration and high-pressure fro- zen in the 100 μm recess of aluminium carriers (Wohlwend) using an HPM100 (Leica Microsystems). Samples were freeze-substituted and embedded in Lowicryl HM20 (Polysciences) according to the published protocol55, except with 0.03% uranyl acetate in the freeze-substitution solution, and for the les1Δ experiment, samples were shaken on dry ice for 2 h during freeze-substitution. Resin blocks were sectioned at 320-nm nominal thickness, picked up onto carbon-coated copper grids (AGS160, Agar Scientific) and imaged on the same day on a Nikon TE2000 or Ti2 microscope using a 100× TIRF objective, a NEO sCMOS DC-152Q-C00-FI camera (Andor), and a Niji LED light source. Based on the fluorescence images, cells with profiles in which an elongated bridge was visible within the section plane were selected for electron tomography. Protein A-coated gold 15-nm beads (EMS) were adhered to the grids before Reynolds’ lead citrate staining. Dual-axis electron tomographic tilt series were acquired approximately from +60° to −60° on a TF20 electron microscope (FEI) operated in STEM mode, using a 50 μm C2 aperture, at 1° increment and 1.1-nm pixel size on an axial bright field detector57, using SerialEM58. Both wild-type and les1Δ data are each from one high-pressure freezing and freeze-substitution ----!@#$NewPage!@#$---- experiment. Tomograms were reconstructed using IMOD59. Segmenta- tion models were generated using Amira (Thermo Fisher Scientific) by manual tracing of membranes and microtubules, followed by extensive simplification and smoothening of the generated surfaces. Therefore, segmentation models are for purely illustrative purposes. Segmenta- tion models are inverted in z or rotated around x or y relative to the origi- nal tomograms, thus the corresponding electron tomographic slices in figure panels Fig. 2b, c, Extended Data Fig. 3c are shown flipped relative to the original tomograms. Some of the electron tomographic slices shown in figures have been mildly gauss-filtered to improve visibility. NPC nearest neighbour distances were measured in IMOD59. An IMOD model file was manually generated of the centres of all visible nuclear pores, and the programme imod-dist was used to obtain distances of each pore to all other pores. For each nuclear pore, the shortest distance was determined to be the distance to its nearest neighbour. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this paper. Data availability Bulk microscopy time-series data, comprising more than 50 files with an average size over 1 GB, are available upon request. 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R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996). Acknowledgements We thank M. Balasubramanian, S. Oliferenko, S. Hauf, K. Gould, J. Bahler, P. Nurse and their laboratories for sharing S. pombe strains, plasmids, expertise and S. pombe protocols; J. O. Patterson for the gift of pFA6a-mNeonGreen plasmids; the LMB electron microscopy facility for electron microscopy support; T.-O. Buchholz for advice on the Noise2Noise implementation.; and D. Albrecht, I. Raote, A. Chaigne, P. Pereira, C. Jacobs and members of the Baum laboratory, in particular G. Paci, G. Cazzagon and H. Matthews, for their feedback on this manuscript. G.D. was funded by a European Union Marie Sklodowska-Curie Individual Fellowship (704281-CCDSA) and the Wellcome Trust (203276/Z/16/Z). S. Culley and R.H. were supported by the UK Biotechnology and Biological Sciences Research Council (BB/R021805/1; BB/S507532/1), the UK Medical Research Council (MR/K015826/1) and the Wellcome Trust (203276/Z/16/Z). S. Curran was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001121), the UK Medical Research Council (FC001121) and the Wellcome Trust (FC001121). W.K. was funded by the Medical Research Council (MC_UP_1201/8). B.B. was supported by UCL’s Institute for the Physics of Living Systems, the MRC-LMCB, the Wellcome Trust (203276/Z/16/Z) and Cancer Research UK (C1529/A28276). Author contributions G.D. co-conceived the project, designed and implemented all the experiments, generated strains and reagents, acquired, analysed and interpreted the data (with the exception of the electron microscopy data shown in parts of Figs. 2, 4, Extended Data Figs. 3, 7), and led the drafting of the paper. S. Culley and U.S. created new software used in the work, and analysed and assisted in the interpretation of data. S. Curran advised on experimental design, helped to generate strains and reagents, and provided protocols and training. R.H. advised on the creation of new software used for the project. W.K. performed all electron tomography and analysis of electron tomograms (parts of Figs 2, 4, Extended Data Figs. 3, 7). B.B. co-conceived and supervised the project, provided advice on experimental design, implementation and analysis and co-drafted the manuscript. All authors provided input during the manuscript drafting stage. Competing interests The authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020- 2648-3. Correspondence and requests for materials should be addressed to G.D. or B.B. Peer review information Nature thanks Brian Burke and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Reprints and permissions information is available at http://www.nature.com/reprints. ----!@#$NewPage!@#$---- Article Extended Data Fig. 1 | ER topology, Les1 structure and phylogeny. a, Airyscan reconstructions of cells expressing the mCherry-AHDL synthetic construct. Orange arrows indicate tubules linking the outer nuclear envelope to the cortical ER. Purple arrows indicate the displacement of the cortical ER by the division ring (not shown). The blue bar indicates the central ER plate formed during late anaphase. Representative of more than 20 cells across 3 technical repeats. Scale bar. 2 μm. b, Genome-wide genetic interaction scores (E-MAP) with Les1/SPAC23C4.05c. Data from ref. 16. c, Schematic of Les1 sequence with positions of conserved motifs indicated. d, Phylogenetic tree of Les1 homologues across Schizosaccharomyces species with single S. cerevisiae homologue MSC1 grouping separately, indicating a duplication in the lineage leading up to Schizosaccharomyces. Bootstrap values indicated at nodes. See Methods for details of tree construction. e, Alignment of Les1 homologues with key conserved motifs highlighted. Colour-coded by % similarity. See Methods for details. ----!@#$NewPage!@#$---- Extended Data Fig. 2 | See next page for caption. ----!@#$NewPage!@#$---- Article Extended Data Fig. 2 | Image analysis workflow and its application to NE protein localization and spindle breakdown timings. a, Schematic demonstrating pipeline for detecting and measuring nuclei and bridges in time- lapse data. Individual steps are described fully in the Methods. Representative images shown here are of Les1-mNeonGreen and Atb2-mCherry. b, The delay between reaching maximum spindle length (spindle max.) and spindle breakage, in seconds. Spindle breakage always follows maximal extension. Sample size (n) = 39 individual cells drawn from 3 biological repeats of the entire experiment. Central line represents the mean and the error bars represent the standard deviation of the population. c, Images of two-colour strains used for relative localization of nuclear envelope and NPC components and nucleus radii (r) as measured using the circular Hough transform (Methods), representative of more than 130 individual cells each drawn from 2 biological repeats. d, e, The vertical displacement (relative to the plane of the nuclear envelope) of various nuclear pore complex components (e) (schematized in d; Nup60, in the nuclear ring; Alm1 and Nup211 in the basket; transmembrane Nup Pom34) and nuclear envelope (NE) membrane proteins (Cta4) relative to Cut11. These measurements correspond to the differences in r as shown in c. Nup60-mEOS was used as an internal control, with the values representing the displacement of photo- converted Nup60-mEOS (red channel) relative to un-converted Nup60-mEOS (green channel). The dotted lines represent an estimate of the thickness of the nuclear envelope (see Methods for details). n = 393 (Cta4), 455 (Pom34), 266 (Les1), 185 (Nup60), 280 (Nup211), 218 (Alm1), and 135 (Nup60-mEOS) individual cells, each drawn from across 2 biological repeats of the entire experiment. The measure of centre (central line) shows the mean of each population and the error bars represent the standard deviation. ----!@#$NewPage!@#$---- Extended Data Fig. 3 | See next page for caption. ----!@#$NewPage!@#$---- Article Extended Data Fig. 3 | Electron tomography of mid- and early-stage bridges with correlative fluorescence images. a, Fluorescence images of resin sections through cells expressing Les1-mNeonGreen (green) and Atb2- mCherry (magenta), corresponding to cells that were imaged by electron tomography and are shown in Fig. 2b (left image), Fig. 2c top panel (middle image) and Fig. 2c lower panel (right image). Images have been rotated to match approximately the orientation of electron tomograms. Scale bars, 1 μm. Representative of 1 (Fig. 2b) and 5 technical repeats (Fig. 2c), respectively. b, Fluorescence image of resin section through a cell expressing Les1- mNeonGreen (green) and Atb2-mCherry (magenta), corresponding to cell imaged by electron tomography shown to the right, indicated by white region. Scale bar, 1 μm. The right two panels are virtual slices through electron tomograms of the cell shown to the left. The approximate overlap in field of view of the tomograms is indicated by dashed lines. No NPCs are visible in the stalk part shown in top image. NPCs (indicated by blue arrowheads) are constrained to the midzone (bottom image). Scale bars, 100 nm. Representative of 2 technical repeats. c, Early stage bridge of a dividing cell; same electron tomogram and cell as shown in Fig. 2b. Top panel is the segmentation model; white area corresponds to the panels below. Inset slice 1 is a virtual slice through the electron tomogram showing the centres of microtubules, indicated by white arrowheads. Inset slice 2 is 18 nm apart in z and shows a nuclear pore, indicated by blue arrowheads. The NPC thus projects less than 18 nm into the nucleoplasm before encountering the microtubules. 1 technical repeat. Scale bars, 50 nm. ----!@#$NewPage!@#$---- Extended Data Fig. 4 | See next page for caption. ----!@#$NewPage!@#$---- Article Extended Data Fig. 4 | Dynamics of individual nucleoporins. a, Averaged normalized intensity traces for Alm1-mNeonGreen (between 9 cells at t = -300 to 26 cells at t = 0) aligned by spindle max (t = 0). The central line represents the mean of the population; the shaded area depicts the standard deviation. Images on right are confocal maximum intensity projections of dividing cells expressing Alm1-mNeonGreen and Cut11-mCherry, representative of more than 20 cells across 3 biological repeats. Scale bar, 2 μm. b, Kymograph of intensities averaged across a single confocal plane of a dividing cell expressing Cut11-mCherry and Alm1-mNeonGreen and imaged at 3 frames per second followed by denoising (Methods). Representative of more than 10 cells across 2 technical repeats. c, Confocal maximum intensity projections of dividing cells expressing Nup211-mNeonGreen and Cut11-mCherry. Representative of more than 30 cells across 3 biological repeats. Scale bar, 2 μm. d, Schematic indicating effect of deleting Nem1 on nuclear envelope surface area and tension. e, Confocal maximum intensity projections of nem1∆ cells expressing Nup211-mNeonGreen and Cut11-mCherry. Representative of more than 30 cells across 2 biological repeats. Scale bar, 2 μm. The basket Nup is able to enter the nuclear bridge in this condition. f, Confocal maximum intensity projections of dividing cells expressing various NPC subcomplex components tagged with mNeonGreen at their C termini, co-expressed in each case with Cut11-mCherry. Each set of panels is representative of >20 cells across 2 biological repeats. Scale bars, 2 μm. ----!@#$NewPage!@#$---- Extended Data Fig. 5 | See next page for caption. ----!@#$NewPage!@#$---- Article Extended Data Fig. 5 | Relative localization of Les1 and nucleoporins. a, Averaged line traces (darker lines = mean, lighter bands = standard deviation) of Les1 and Cut11 intensities along the bridge for 16 cells aligned at bridge length 3 μm. b, Time from bridge formation to maximum spindle length measured for strains pooled to generate Fig. 3e (GD173, GD250) and Extended Data 7d (GD176, GD253). Numbers in brackets (n) indicate number of cells in each population, with bars representing mean and standard deviation. The ANOVA F statistic and P value are listed above each plot. The line and pairwise P value within each plot refer to the comparison between pooled strains. c, Single Airyscan reconstructions of cells expressing Cut11 tagged at the endogenous locus with mNeonGreen and a synthetic mCherry-AHDL construct. Magenta arrow highlights stray nuclear pore cluster accompanied by widening of the nuclear envelope. Representative of more than 10 cells across 2 technical replicates. Scale bar, 2 μm. d, Maximum intensity projections of confocal images acquired every 60 s of cells expressing Les1-mNeonGreen and Cut11-mCherry. Bridge formation is at t = 0. Green arrows mark boundaries of Les1 stalks, magenta arrow indicates stray pore cluster. Representative of more than 50 cells across 6 biological repeats. Scale bar, 2 μm. e, SRRF reconstruction from single confocal planes of a cell expressing Les1-mNeonGreen and Cut11-mCherry. Magenta arrow indicates stray nuclear pore cluster, with examples marked upon the line scans along the bridges of 3 illustrative cells in f, Representative of more than 20 cells across 4 biological repeats. g, Confocal maximum intensity projections of cells expressing Cut11-GFP and Atb2-mCherry and acquired at 10 s intervals. Scale bars, 2 μm. Representative of more than 20 cells across 2 biological repeats. h, Relative NPC decay rates, calculated for a strain expressing both Nup60 and Cut11 (GD173; between 11 cells at t = -300 and 34 cells at t = 0), showing similar relative rates to a cross-strain comparison, as in b or Fig. 3e. Line shows mean of the population. i, Kymograph generated using 10 fps single plane imaging of a strain expressing Nup60 and Cut11 tagged at the endogenous loci with mNeonGreen and mCherry respectively. Blue (Nup60) and magenta (Cut11) arrows represent the staggered decay of individual clusters of nuclear pores. Representative of more than 10 cells drawn from 2 technical replicates. ----!@#$NewPage!@#$---- Extended Data Fig. 6 | See next page for caption. ----!@#$NewPage!@#$---- Article Extended Data Fig. 6 | Response of nuclear division dynamics to acute perturbations. a, Confocal maximum intensity projections of nem1∆ cells expressing Les1-mNeonGreen and Cut11-mCherry. Note the absence of Les1 stalk formation. Schematic indicates opposing effects of nem1∆ and Cerulenin treatment. Representative of more than 30 cells drawn from 2 biological repeats. Scale bar, 2 μm. b, Two examples of nuclei attempting to divide in the presence of 10 μM Cerulenin. Note Les1 stalk formation along aberrant bridge- like projections. Magenta arrows indicate NPC clusters. Representative of more than 30 cells drawn from 3 biological repeats. c, Dividing cells expressing the Ark1-as3 analogue-sensitive allele, either co-expressing Les1-mNeonGreen and mCherry-Atb2 or Les1-mNeonGreen and Cut11-mCherry and treated with 5 nM 1NM-PP1 and 5 μM latrunculin A. In both cases, representative of more than 30 cells across 2 biological repeats. Scale bars, 2 μm. d, iSIM image of imp1∆ cells expressing Les1-mNeonGreen and Cut11-mCherry. Arrows highlight isolated NPC clusters in Les1-depleted regions. Representative of more than 40 cells across 2 biological replicates. Scale bar, 2 μm. e, Time-lapse confocal images of cells expressing Cut11-GFP, Cdc15-mNeonGreen, and mCherry-AHDL acquired at 60 s intervals with frames displayed at 3-min intervals. Representative of more than 50 cells across 3 biological replicates. Scale bars,  4 μm. f, Treatment with latrunculin A depolymerizes the actin ring (marked by Cdc15) but has a minimal effect on the time of nuclear division, as marked by the time from bridge formation to complete NPC signal loss. Numbers above and below the horizontal bar represent the difference in means with 95% confidence interval and the two-sided Mann–Whitney P value. n = 180 (untreated) and 146 (Lat A) individual cells in each population, in each case pooled from 3 biological replicates. Overlaid on individual data points, the upper and lower extent of the boxes span the inter-quartile range and the central bar denotes the median. g, Cytokinetic ring constriction only begins after nuclear division completes, and it takes almost 30 min for the ring to constrict and septation to complete. n = 222 (NPC loss to constriction start), 103 (constriction start to end) and 111 (constriction end to septation) individual cells in each population, in each case pooled from 3 biological replicates. Overlaid on individual data points, the upper and lower extent of the boxes span the inter-quartile range and the central bar denotes the median. ----!@#$NewPage!@#$---- Extended Data Fig. 7 | See next page for caption. ----!@#$NewPage!@#$---- Article Extended Data Fig. 7 | Nucleoporin localization and dynamics in les1Δ cells. a, Fluorescence image of resin section through a les1Δ cell expressing Cut11- GFP (green) and a synthetic mCherry-AHDL construct (magenta), corresponding to the cell that was imaged by electron tomography shown in Fig. 4c. Representative of 2 technical repeats. Scale bar, 1 μm. b, Inter-pore distances for tomograms shown in Extended Data Fig. 2b (WT Early stage), 3b (WT Mid-stage), and tomogram not shown (les1∆ Mid-stage). See Methods for details on measurement. n = 26 (WT mid-stage), 18 (WT early stage) and 18 (les1∆ mid-stage) individual nuclear pores in each dataset. The central lines represent the mean of the population, with the error bars representing standard deviation. c, Bridge intensity at bridge formation as an indirect readout of nucleoporin copy number in individual cells, for wild-type (WT) and les1∆ cells. n = 19 (WT Cut11), 27 (les1∆ Cut11), 44 (WT Nup60) and 102 (les1∆ Nup60) individual cells pooled from a minimum of 2 biological replicates. Bars overlaid on top of individual data points represent the mean (central line) and standard deviation (error bars). P values derive from a two-tailed unpaired t-test. See Methods for details. d, Decay curves for NLS-GFP (orange, from 28 cells at t = -300 to 39 cells at t = 0), Nup60 (blue, from 10 cells at t = -300 to 15 cells at t = 0) and Cut11 (magenta, from 10 cells at t = -300 to 15 cells at t = 0) in a les1∆ background, drawn from a minimum of 2 biological replicates. Each trace was normalized by division by maximum bridge signal for that cell before averaging. The plot on the left represents the population averages for each marker in a les1∆ background, and the dotted orange line indicates exponential fit to the NLS-GFP average. The 3 subsequent plots show the averages (darker line) and standard deviation (shaded area) for each marker (NLS-GFP, orange; Nup60, blue; Cut11, magenta) in a les1∆ background, overlaid on the wild-type equivalents in grey. e, les1∆ cells expressing Alm1-mNeonGreen and Cut11- mCherry. Arrow indicates a small cluster of Alm1 that enters the bridge but then disappears. Representative of more than 20 cells from 2 biological repeats. Scale bar, 2 μm. f. SRRF reconstructions of confocal slices at 28 s intervals of les1∆ cells expressing Nup60-mNeonGreen and Cut11-mCherry tagged at the endogenous loci, aligned relative to spindle max (t = 0). Representative of more than 10 cells across 2 technical replicates. Magenta arrow indicates breakpoint. Scale bar, 2 μm. On the right, the cumulative distribution (42 cells from 2 strains across 3 biological replicates) of breakpoint locations relative to the midzone in les1∆ cells. The shaded grey area represents the mean +/− standard deviation of breakpoint locations in wild-type cells (31 cells from 2 biological replicates), with the cumulative distribution as a grey line. ----!@#$NewPage!@#$---- Extended Data Fig. 8 | Les1 truncations and genetic interactions. a, Schematic indicating Les1 truncation constructs, with numbers representing amino acid positions starting from 1 at the N terminus. Lower panels, cells expressing Les1 (1-291)-mNeonGreen, replacing Les1 at the endogenous locus, as well as Cut11-mCherry. See Methods for details on the truncation constructs (all at endogenous locus, replacing endogenous copy). Note the absence of detectable stalks and the delocalization of Cut11 in the bridge. Representative of more than 10 cells across 2 biological repeats. Scale bar, 5 μm. b, Confocal maximum intensity projections of cells expressing Cmp7-mNeonGreen and Les1-mScarlet. Arrows indicate Cmp7 foci at the tips of retracting stalks. Representative of more than 20 cells across 4 biological repeats. Scale bar, 2 μm. c, Single iSIM slice of a dividing cell expressing Cmp7-mNeonGreen and Les1-mScarlet, representative of more than 5 cells drawn from 2 technical repeats. Scale bar, 2 μm. d, Tetrad dissection assay for les1∆ crossed with either lem2∆ or cmp7∆, showing colonies grown from individual spores. See Methods for details. e, Single image of double deletion strains, derived from the tetrad assay clones shown in d, expressing a synthetic NLS-GFP construct. Scale bar, 10 μm. Representative of more than 200 cells drawn from 2 biological repeats. f, Averaged line traces (darker lines = mean, lighter bands = standard deviation) of Nup60-mCherry intensities in les1∆ cells (grey: control; blue: treated with 10 μM Cerulenin) at bridge length 3 μm (n = 45 cells for cerulenin-treated; n = 27 cells for control). g, Confocal maximum intensity projections of les1∆ lem2∆ cells expressing Cut11-mCherry, either in the presence (lower panel) or absence (upper panel) of 5 μM latrunculin A. Representative of more than 20 cells across 3 technical repeats. Scale bar, 2 μm. ----!@#$NewPage!@#$---- Article Extended Data Table 1 | Complete list of S. pombe strains used in this study List of all the S. pombe strains used in this study along with their full genotypes and any relevant strain construction notes. ----!@#$NewPage!@#$---- �       !"#$"%&'()* +, -& ('!#&(#./&'()* +, 0!($"%1'22&/ 3&('0 &4)5$ ) ($2!6()!#'4$.$7$(/8()59()&(5!'.7$ ):;)$ 82!6$# ('4('84" $ ("4/&"#(&" !&"4/ $"!($"%:<8'()$"82&($""3&('0 &4)!7$4$ = >'() ?08 &"#()@#$($&7A7$4/)497$ (: 1(&($ ($4 <&77 (&($ ($4&7&"&7/  =4"8$2()&(()8775$"%$(2 &! 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