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LEM2 phase separation promotes ESCRT-mediated nuclear envelope reformation

Abstract

During cell division, remodelling of the nuclear envelope enables chromosome segregation by the mitotic spindle1. The reformation of sealed nuclei requires ESCRTs (endosomal sorting complexes required for transport) and LEM2, a transmembrane ESCRT adaptor2,3,4. Here we show how the ability of LEM2 to condense on microtubules governs the activation of ESCRTs and coordinated spindle disassembly. The LEM motif of LEM2 binds BAF, conferring on LEM2 an affinity for chromatin5,6, while an adjacent low-complexity domain (LCD) promotes LEM2 phase separation. A proline–arginine-rich sequence within the LCD binds to microtubules and targets condensation of LEM2 to spindle microtubules that traverse the nascent nuclear envelope. Furthermore, the winged-helix domain of LEM2 activates the ESCRT-II/ESCRT-III hybrid protein CHMP7 to form co-oligomeric rings. Disruption of these events in human cells prevented the recruitment of downstream ESCRTs, compromised spindle disassembly, and led to defects in nuclear integrity and DNA damage. We propose that during nuclear reassembly LEM2 condenses into a liquid-like phase and coassembles with CHMP7 to form a macromolecular O-ring seal at the confluence between membranes, chromatin and the spindle. The properties of LEM2 described here, and the homologous architectures of related inner nuclear membrane proteins7,8, suggest that phase separation may contribute to other critical envelope functions, including interphase repair8,9,10,11,12,13 and chromatin organization14,15,16,17.

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Fig. 1: Targeting of LEM2 to the nuclear envelope core at anaphase chromatin discs depends on BAF binding and a LCD that can form liquid-like droplets.
Fig. 2: LEM2 concentrates around spindle microtubules and its LCD forms a liquid-like coating around microtubules.
Fig. 3: LEM2 coassembles with CHMP7 to form an O-ring that facilitates early nuclear sealing.

Data availablility

Raw data and peaklists from the quantitative crosslinking mass spectrometry analysis can be accessed with MassIVE: ftp://massive.ucsd.edu/MSV000084837/. Crosslinked peptide spectral assignments are accessible using accession: rgf6hriush at http://msviewer.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer. The uncropped blots and gels are provided in Supplementary Fig. 1. Source Data for Figs. 13 and Extended Data Figs. 14, 5, 7, 8 are provided online.

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Acknowledgements

We thank W. I. Sundquist and S. Redding for critically reading the manuscript. For reagents, technical advice, and discussions we thank the Redding, Narlikar, Ullman, and Frost laboratories, as well as the Nikon Imaging Center at UCSF. We thank L. Williams for helping to score cell phenotypes and M. Mendoza and J. Rosenblatt for sharing microscope and other resources. We also thank the UCSF Center for Advanced cryoEM, including A. Myasnikov, D. Bulkley and M. Braunfeld. Our research was supported by NIH grants P50 GM082545, 1DP2-GM110772 (A.F.), 1R01-GM131052 (K.S.U.), and the Huntsman Cancer Foundation (K.S.U.). Shared resources used at the University of Utah were funded in part by the Huntsman Cancer Institute Cancer Center Support Grant NIH P30CA042014. Mass spectrometry analysis at the UCSF Mass Spectrometry Resource was supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and a shared instrument grant (NIH S10OD016229). A.v.A. was funded by EMBO (ALTF 455-2016) and the German Research Foundation (DFG AP 298/1-1). I.E.J. was funded by the NSF Graduate Research Fellowship (1000232072) and a Mortiz-Heyman Discovery Fellowship. A.F. is a Chan Zuckerberg Biohub investigator and an HHMI faculty scholar.

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Authors

Contributions

A.v.A., D.L., I.E.J., M.J.T., A.L.B., K.S.U., and A.F. designed research; A.v.A. and I.E.J. purified proteins and performed in vitro fluorescent and negative stain electron microscopy imaging, FRAP, and analysis. A.v.A. performed protein droplet imaging. A.v.A. performed 2D averaging and homology modeling. D.L. performed mammalian cell experiments and analysis. D.L. prepared fixed, immunostained samples for STED and A.v.A performed STED imaging and analysis. I.E.J. performed and analysed turbidimetry and analytical SEC experiments. A.v.A. performed XL-MS sample preparation and analysis. M.J.T. performed mass spectrometry and analysis. A.v.A. performed and analysed polymer pelleting experiments. S.M.P. purified CHMP7ESCRT-III. A.v.A., D.L., I.E.J., M.T., K.S.U., and A.F. analysed data. A.v.A., D.L., I.E.J., K.S.U., and A.F. wrote the manuscript.

Corresponding authors

Correspondence to Katharine S. Ullman or Adam Frost.

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Peer review information Nature thanks Stephen Michnick and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 The N terminus of LEM2 possesses a canonical BAF-binding LEM domain and an LCD.

a, Live-cell imaging of GFP–lamin B2 and LEM2–mChr; DNA is stained with NucBlue and tubulin detected with SiR–tubulin. Time 0 refers to complete CFI. Representative of ten or more cells imaged across at least three biological replicates. Scale bar, 2 μm. b, Multiple sequence alignment of LEM domains across LEM family proteins, highlighting a conserved four-amino-acid sequence that, when mutated in emerin (EMDm24), disrupts BAF binding20. The position of an analogous mutation in LEM2 (LEM2m21) is indicated. c, HeLa cells stably expressing LEM2–mCherry and EGFP–BAF live-imaged during anaphase. Representative of ten or more cells imaged across at least three biological replicates for both fixed and live-cell imaging. Scale bar, 10 μm. d, Top, a homology model for the LEM21–72 –BAF–DNA complex51, based on Protein Data Bank code (PDB): 2BZF and PDB: 2ODG32,51. Middle, absorbance at 280 nm as a function of retention volume (ml) from analytical size exclusion chromatography. Retention volumes for major peaks (arrowheads) and predicted molecular weights for protein or protein–DNA complexes are listed. Bottom, SDS–PAGE of major peak for LEM21–72 + BAF + DNA sample. Representative of three technical replicates. For gel source data, see Supplementary Fig. 1. e, Percentage amino acid composition for the LEM2 LCD, and the compositions of two subregions, compared to an average amino acid composition. f, Schematic of LEMNTD with amino acid substitutions (S, T, or Y to D) relative to SY-rich (yellow) and PR-rich (rust) regions, in LEM2NTD Mim1 and Mim2 constructs. LEM2 immunoblot assessing the migration pattern of full-length (also shown in Fig. 1e) and mutant LEM2–mChr constructs following separation by Phos-tag SDS–PAGE. Cell lysates were prepared from G1/S- and prometaphase- arrested cells expressing the indicated exogenous LEM2; lysates treated with λ-PP are indicated. Representative data from two biological replicates, with one and three technical replicates per biological replicate. For immunoblot source data, see Supplementary Fig. 1. g, Top, amino acid sequences of the peptides corresponding to the SY-rich and PR-rich regions of LEM2. Bottom, concentration-dependent droplet formation by the LEM2SY peptide, juxtaposed with similar data collected for full LEM2NTD as in Fig. 1d. Representative of three technical replicates. Scale bar, 2 μm. h, Fluorescence microscopy of purified LEM2NTD with indicated molecular anions. Image representative of two technical replicates. Scale bar, 2 μm. Source Data

Extended Data Fig. 2 LEM2 wets the surface of microtubules with a liquid-like coat.

a, Additional example of STED imaging of endogenous LEM2 localization during late anaphase. Scale bars, 150 nm. Representative of data from four cells. b, Fluorescence imaging of indicated combinations of LEM2FL–Alexa488 (magenta), tubulin–Alexa647 (green), and lipids labelled with PE-rhodamine (cyan). Scale bar, 2 μm. Representative of two technical replicates. c, Light scattering at 340 nm (turbidity) of microtubule bundling by indicated LEM2 constructs. Half maximal concentration of LEM2NTD is 1.303 ± 0.1 μM. Mean ± s.e.m. for n = 3 independent samples. d, Negative stain electron microscopy of microtubules alone or microtubules with indicated concentrations of LEM2NTD, corresponding to the concentrations measured by light scattering. Images representative of at least six; scale bars, 25 nm. e, Fluorescence microscopy of LEM2NTD–Alexa488 in combination with tubulin–Alexa647, LEM2WH–Alexa555, and GTP/MgCl2, as indicated. Scale bar, 10 μm. Images are representative of at least three. f, Example images for FRAP of LEM2FL- and LEM2NTD-coated microtubule bundles, representative of five independent samples (LEM2FL) or 17 independent samples (LEM2NTD). Scale bar, 2 μm. g, Top, kymograph across bleached region. Bottom, FRAP of LEM2NTD-coated microtubule bundle. Images show fluorescent LEM2NTD channel. Scale bar, 2 μm. Representative of eight technical replicates. Source Data

Extended Data Fig. 3 LEM2–LCD bundles microtubules in vitro through a regulated microtubule-binding domain.

a, Amino-acid sequences of six LEM2 peptides tiling the LCD. LEM2145–165 is LEM2-PRA, and LEM2188–212 is LEM2-PRB. b, Light scattering at 340 nm (turbidity) of microtubule bundling by indicated LEM2 peptides. Half maximal concentration of LEM2188–212 (LEM2-PRB) is 85.11 μM. For LEM2188–212, data plotted are mean ± s.e.m. for n = 3 technical replicates. LEM240–60, LEM261–81, LEM2123–144 and LEM2166–187, did not bundle. c, Negative stain electron microscopy of microtubules alone or microtubules with indicated concentrations of LEM2188–212, corresponding to turbidity reactions. Scale bars, 25 nm. For each condition, images are representatives of seven. d, Live-cell imaging of indicated LEM2∆SY–mChr and LEM2∆PR–mChr deletion constructs (magenta) and GFP–tubulin (green). Time 0 refers to time of complete CFI. Scale bar, 2 μm. Images are representative of three independent experiments. e, Negative stain electron microscopy of microtubules co-incubated with the indicated phosphomimetic proteins. Scale bar, 25 nm. Images are representative of two technical replicates. f, Light scattering at 340 nm (turbidity) of microtubule bundling by phosphomimetic LEM2 constructs. Vertical lines are half maximum, shaded regions show s.e.; LEM2NTD (green) half maximum 1.303 μM (1.144–1.493 μM), LEM2Mim1 (red) half maximum 2.824 μM (2.283–3.397 μM), LEM2Mim2 (blue) half maximum 8.442 μM, (7.511–10.06 μM). Data plotted are mean ± s.e.m. from three technical replicates. Source Data

Extended Data Fig. 4 The LEM2 WH domain is required for recruitment of IST1 to the nascent nuclear envelope and mediates polymer formation with CHMP7.

a, Top, quantification of robust IST1 recruitment to chromatin discs in late anaphase, as assessed by blind scoring. Mean ± s.e.m. determined from three independent experiments (siControl parental: n = 80, 58, 106; siLEM2-2/parental: n = 78, 50, 58; siControl/LEM2–mChr: n = 46, 42, 62; siLEM2-2/LEM2–mChr: n = 78, 51, 62; siControl/LEM2∆WH–mChr: n = 138, 52, 42; siLEM2-2/LEM2∆WH–mChr: n = 112, 48, 51). Two-tailed unpaired t-test, no multiple comparisons. Bottom, representative images by widefield showing localization of endogenous IST1 in late anaphase cells depleted of endogenous LEM2 and expressing the indicated siRNA-resistant LEM2–mChr constructs. Scale bar, 2 μm. b, Top, quantification of the percent of early anaphase discs with robust IST1 recruitment, as assessed by blind scoring. Mean ± s.e.m. determined from three independent experiments (siControl/parental: n = 38, 16, 20; siLEM2-2/parental: n = 24, 10, 25; siControl/LEM2–mChr: n = 50, 20, 24; siLEM2-2/LEM2–mChr: n = 38, 14, 8; siControl/LEM2∆WH–mChr: n = 44, 16, 20; siLEM2-2/LEM2∆WH–mChr: n = 20, 4, 14). Two-tailed unpaired t-test, no multiple comparisons. Bottom, representative images by widefield showing localization of endogenous IST1 in early anaphase cells depleted of endogenous LEM2 and expressing the indicated siRNA-resistant LEM2–mChr constructs. Scale bar, 2 μm. c, Negative stain electron microscopy corresponding to the CHMP7 polymerization assay showing no polymerization for the control condition CHMP7 + LEM2NTD. Representative of two technical replicates. Source Data

Extended Data Fig. 5 Homology modelling and XL-MS consistent with a WH domain-swap mechanism for activation of CHMP7 by LEM2.

a, Workflow of lysine–lysine hybrid peptide mapping using XL-MS. BS3 cross-links surface-accessible lysine residues with Cα–Cα distances below about 3 nm. b, Homology models for the WH domains of CHMP7 and LEM2 from reference structures described in Supplementary Table 2. WH1 of CHMP7 contains a membrane-binding region indicated as a loop4. c, Homology models of the CHMP7 ESCRT-III-fold in open and closed conformations. Green and orange, α-helices 1–2 and 3–4, respectively. d, Distance restraints identified from XL-MS analysis of the CHMP7 monomer were mapped to open and closed homology models. Cα–Cα distances over 3 nm are considered violations. Blue, satisfied restraints; red, violated restraints. The open ESCRT-III conformation was rejected. e, Top. a crystallographic interface between VPS25 and an ESCRT-III helix28 serves as a template for the XL-MS-informed homology model of the CHMP7 WH2 interaction with the CHMP7 ESCRT-III domain. For details on template structures see Supplementary Table 2. Middle, bottom, all cross-links are satisfied when mapped to the closed CHMP7-ESCRT-III model and agree with domain connectivity. A subset of cross-links was not satisfied when mapping WH1 instead to the same interface (data not shown). f, Left, distance restraints identified from XL-MS analysis of CHMP7 monomer mapped to conformation of polymerized CHMP7 consistent with 2D class averages. Violated restraints suggest a hinge region between CHMP7 WH1 and WH2 that allows its WH1 to move closer to the ESCRT-III core (black arrow). Right, violated restraints to WH2.

Extended Data Fig. 6 Activated CHMP7 forms polymeric rings via its ESCRT-III domain, exposing its tandem WH domains.

a, CHMP7 point mutations are indicated in an open ESCRT-III fold, representing polymerized CHMP7. b, Negative stain electron microscopy of His6-SUMO–LEM2WH co-incubated with CHMP7 mutants. Scale bars, 50 nm. Images representative of three technical replicates. c, SDS–PAGE of pellet (P) or supernatant (S) following centrifugation of LEM2WH incubated with wild-type or mutant CHMP7. For gel source data, see Supplementary Fig. 1. d, Quantification of pelleted protein after SDS–PAGE and Coomassie blue staining for mutant versus wild-type proteins. Mean ± s.d. quantified from n = 3 independent experiments. e, SDS–PAGE-based relative quantification of polymerized and pelleted CHMP7 with different ratios of LEM2WH present. Red dashed lines indicate expected fraction of CHMP7 in the pelleted polymer, assuming 1:1 stoichiometric polymer. Mean ± s.d. quantified from n = 3 independent experiments. For gel source data, see Supplementary Fig. 1. f, Negative stain electron microscopy of CHMP7 polymers on a liposome. Scale bar, 100 nm. g, Top, negative stain electron microscopy of membrane-induced CHMP7 polymers used for 2D averaging. Scale bar, 20 nm. Bottom, representative 2D class averages from manually picked particles from polymers shown at top. f, g, Representative of five technical replicates. h, Negative stain electron microscopy of CHMP7ESCRT-III (AA 229–453). Representative of three technical replicates. Scale bar, 20 nm.

Extended Data Fig. 7 LEM2 promotes early nuclear compartmentalization via cooperation between its LCD and WH domains.

a, Example images of cells treated with siLEM2 and expressing NLS–3GFP in combination with siRNA-resistant LEM2 constructs corresponding to the quantification graphs shown in b and Fig. 3f. DNA labelled using NucBlue. Time 0 refers to the time of complete CFI. Scale bar, 2 μm. b, Mean + s.d. nuclear/cytoplasmic ratio of NLS–3GFP fluorescence over time in cells treated with the indicated siRNAs and expressing the indicated siRNA-resistant constructs. Cells imaged at 15-s intervals though 1-min increments are plotted. Data were collected across at least three biological replicates (siCon/LEM2–mChr; n = 26; siLEM2-2/LEM2–mChr: n = 44; siCon/LEM2∆SY–mChr: n = 20; siLEM2-2/LEM2∆SY–mChr: n = 24; siCon/LEM2∆PR–mChr: n = 20; siLEM2-2/LEM2∆PR–mChr: n = 20; siCon/LEM2∆WH–mChr: n = 24; siLEM2-2/LEM2∆WH–mChr: n = 16). Two-tailed unpaired t-test was used to determine P values comparing deletion mutant lines to the full-length LEM2 line under endogenous LEM2 depletion conditions at each time point. No multiple comparisons. *P < 0.05, **P < 0.005; exact P values below. c, Quantification of nuclear/cytoplasmic ratio of NLS–3GFP approximately 30 min after complete CFI in parental HeLa cells treated with the indicated siRNAs. Data were collected across three biological replicates and plotted as mean ± s.d. (siControl: n = 11, 12, 6; siLEM2-2: n = 18, 18, 14; siCHMP7: n = 11, 6, 14). Two-tailed unpaired t-test, no multiple comparisons. Source Data

Extended Data Fig. 8 Loss of LEM2 function leads to DNA damage and abnormal nuclear morphologies.

a, Live-cell imaging of GFP–tubulin and H2B–mChr in siRNA-treated cells. Images representative of two biological replicates, quantified in b. Time 0 refers to the time of complete CFI. Scale bar, 2 μm. b, Left, orthogonal view of the tubulin phenotype following LEM2 depletion co-stained for the nuclear envelope protein SUN2. Images representative of three biological replicates. Scale bar, 5 μm. Right, mean ± s.e.m. percentage of telophase cells with nuclear tubulin defects, lined with inner nuclear membrane (as assessed by immunofluorescence of lamin B2). Three biological replicates (siControl: n = 75, 35, 37; siLEM2-1: n = 72, 35, 37; siLEM2-2: n = 45, 34, 34). Two-tailed unpaired t-test, no multiple comparisons. c, Left, example images of 53BP1 localization by immunofluorescence in telophase U2OS cells following siRNA treatment. Scale bar, 5 μm. Right, mean ± s.e.m. percentage of telophase cells with five or more 53BP1 nuclear foci. Three biological replicates (siControl: n = 56, 52, 44; siLEM2-1: n = 64, 56, 26; siLEM2-2: n = 74, 50, 40). Two-tailed unpaired t-test, no multiple comparisons. Bottom, immunoblot confirming depletion of endogenous LEM2 in U2OS cells, using siRNA oligos previously validated in other human cell lines, including HeLa4,19 (immunoblot source data shown in Supplementary Fig. 1). d, Negative stain electron microscopy of indicated combinations of microtubules, LEM2NTD-linker-WH, and CHMP7FL. Scale bars, 25 nm. Images representative of two technical replicates. e, Example images of cells expressing the indicated siRNA-resistant constructs and treated with the indicated siRNAs. Cells were arrested in S-phase and then allowed to progress through one round of division, resulting in an interphase population of cells that had just exited mitosis. We observed an increased number of highly irregular nuclei in cells expressing either LEM2∆PR–mChr or LEM2∆WH–mChr compared to cells expressing full-length LEM2 or even those depleted of LEM2. Notably, deformed nuclei were commonly associated with microtubule disorganization and aberrant accumulation of LEM2∆PR–mChr and LEM2∆WH–mChr. Representative nuclear, tubulin, and LEM2 phenotypes and the correspondence to nuclear circularity score are shown. Nuclear borders and circularity scores annotated in tubulin channel. Scale bar, 5 μm. These findings suggest that interfering with cooperation between the microtubule-interacting and ESCRT-binding domains of LEM2 alters nuclear morphology, indicating that both activities are necessary, but neither is sufficient for nuclear envelope reformation. Moreover, the presence of one activity without the other is detrimental to nuclear morphology. f, Quantification of nuclear circularity in interphase parental HeLa cells and cells expressing the indicated siRNA-resistant LEM2 constructs, treated with the indicated siRNAs. Mean ± s.e.m. from three biological replicates (siControl/parental: n = 105, 46, 80; siLEM2-2/parental: n = 102, 116, 59; siControl/LEM2–mChr: n = 153, 53, 122; siLEM2-2/LEM2–mChr: n = 84, 81, 105; siControl/LEM2∆SY–mChr: n = 123, 68, 144; siLEM2-2/LEM2∆SY–mChr: n = 93, 95, 105; siControl/LEM2∆PR–mChr: n = 149, 123, 58; siLEM2-2/LEM2∆PR–mChr: n = 49, 31, 42; siControl/LEM2∆WH–mChr: n = 116, 96, 94; siLEM2-2/LEM2∆WH–mChr: n = 85, 32, 68). Two-tailed unpaired t-test comparing circularity scores less than 0.6 (indicated by blue) between the indicated treatments; no multiple comparisons. g, Immunoblot showing relative levels of the siRNA-resistant constructs fused with mCherry in parallel with endogenous LEM2. Representative of two technical replicates. For immunoblot source data, see Supplementary Fig. 1. Source Data

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion and Supplementary References.

Reporting Summary

Supplementary Figure 1

This file contains the uncropped blots.

Supplementary Table 1

Quantified Cross-links from comparative mass spectrometry analysis between monomeric CHMP7 and CHMP7 polymerized by LEM2WH. Summed peak areas for identified cross-links are listed for Heavy (polymeric CHMP7) and Light (Monomeric CHMP7). In case the identified cross-link was absent in Light sample, the value was set to 40000 for analysis purposes and the log2 ratio is shown as n/a. Cross-links with log2 ratios between 2 and -2 as well as cross-links falling into the region of the His6-SUMO-tag present in LEM2WH were filtered and not considered in the analysis.

Supplementary Table 2

Template Structures Used for Homology Modeling

Supplementary Table 3

Plasmids and cell lines used in this study. Cell lines stably expressing exogenous, fluorescently labeled proteins were generated by transfecting parental HeLa cells with the described plasmids using Lipofectamine LTX Plus (Thermo Fisher) for 24 h before selection with the specified antibiotics.

Video 1: Droplet fusion

Droplet fusion of LEM2NTD droplets on PEGylated glass 30 min after induction. Droplet formation of 32 μM LEM2NTD was induced by reducing the salt concentration from 500 mM to 150 mM KCl in presence of 0.5 μM Alexa488 labeled His6-SUMO- LEM2NTD. Images were recorded every 2 sec. Representative of 2 technical replicates.

Video 2: FRAP of LEM2NTD droplets fused to MTs

24 uM LEM2NTD (magenta) and 1 uM polymerized MTs (green) were mixed at 150 mM KCl in presence of 0.5 μM Alexa488 labeled His6-SUMO- LEM2NTD. Images were recorded every 2 sec. Representative of 2 technical replicates.

Video 3: A series of Z-stack images illustrative of the LEM2-depletion phenotype in telophase cells

HeLa cells stably expressing H2B-mCherry and GFP-Tubulin were treated with control or LEM2-targeting siRNA and live imaged by spinning disk confocal microscopy. Representative of two biological replicates.

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von Appen, A., LaJoie, D., Johnson, I.E. et al. LEM2 phase separation promotes ESCRT-mediated nuclear envelope reformation. Nature 582, 115–118 (2020). https://doi.org/10.1038/s41586-020-2232-x

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