During telophase, the nuclear envelope (NE) reforms around daughter nuclei to ensure proper segregation of nuclear and cytoplasmic contents1,2,3,4. NE reformation requires the coating of chromatin by membrane derived from the endoplasmic reticulum, and a subsequent annular fusion step to ensure that the formed envelope is sealed1,2,4,5. How annular fusion is accomplished is unknown, but it is thought to involve the p97 AAA-ATPase complex and bears a topological equivalence to the membrane fusion event that occurs during the abscission phase of cytokinesis1,6. Here we show that the endosomal sorting complex required for transport-III (ESCRT-III) machinery localizes to sites of annular fusion in the forming NE in human cells, and is necessary for proper post-mitotic nucleo-cytoplasmic compartmentalization. The ESCRT-III component charged multivesicular body protein 2A (CHMP2A) is directed to the forming NE through binding to CHMP4B, and provides an activity essential for NE reformation. Localization also requires the p97 complex member ubiquitin fusion and degradation 1 (UFD1). Our results describe a novel role for the ESCRT machinery in cell division and demonstrate a conservation of the machineries involved in topologically equivalent mitotic membrane remodelling events.
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J.G.C. is a Wellcome Trust Research Career Development Fellow. We acknowledge the Nikon Imaging Centre at KCL and the NIHR Comprehensive Biomedical Research Centre at Guy's and St Thomas’ NHS Foundation Trust for access to core equipment. We thank the staff of the Wolfson Bioimaging Facility for their support. We thank J. Martin-Serrano for gifts of plasmids and cells.
The authors declare no competing financial interests.
Extended data figures and tables
a, b, Immunofluorescence analysis of HeLa cells stained with anti-tubulin, anti-CHMP2A or anti-CHMP2B and DAPI (a). Images in a are representative of two acquired images per field of view. Cells in b were treated with control or CHMP2A-targeting siRNA; images representative of four (control) or two (CHMP2A siRNA) acquired images. c, Deconvolved projections of HeLa cells stained with anti-CHMP2A and DAPI, corresponding to stills from Supplementary Video 1. Images representative of two deconvolved image series. d, HeLa cells stably expressing GFP–CHMP4B were imaged live during the anaphase to telophase transition. Telophase frames at 30-s intervals are presented, corresponding to stills from Supplementary Video 2. Images representative of four acquisitions. e, Immunofluorescence analysis of human diploid fibroblasts stained with anti-CHMP2A, anti-tubulin and DAPI, images representative of three acquired cells per cell cycle phase. f, g, Immunofluorescence analysis of HeLa cells stained with anti-CHMP2A, DAPI and either anti-mAb414 (f) or anti-LaminA/C (g), images representative of five acquired cells. Arrowheads indicate regions of formed nuclear pores or lamina as indicated. h, Quantification of abnormal nuclei (the presence of multiple lobes, micronuclei, lamina ingression or invagination) in HeLa cells transfected with the indicated siRNA and stained with anti-LaminA/C (1,300 cells over 5 experiments quantified per treatment; data are mean ± s.d.). Images representative of three (control, CHMP2A siRNA) or two (LEM4 siRNA) acquired fields of view and resolved cell lysates were examined by western blotting with anti-CHMP2A, anti-CHMP2B or anti-GAPDH antisera as indicated. Scale bars, 10 µm.
Extended Data Figure 2 Correlative light and electron microscopy (CLEM) of endogenous CHMP2A localization in telophase NE.
a–c, Phase-contrast (a), correlative immunofluorescence (b) and transmission electron microscopy of HeLa cells stained with anti-CHMP2A, detected by Alexa-594-fluoronanogold and DAPI. Boxed region in a is shown in b; boxed region in b is shown in c. In all cases, images representative of three cells prepared for CLEM. d, 3D rendering of tomographic reconstruction of forming NE from boxed region in c and Fig. 1d; a single example of a nucleo-cytoplasmic channel was selected for 3D rendering. e–g, Z-slices extracted from tomographic reconstructions of forming NE depicting CHMP2A localization to isolated vesicles (e, i) and nucleo-cytoplasmic channels (arrows in e, ii, f, g) at the indicated Z-heights Localization of CHMP2A to nucleo-cytoplasmic channels was observed in three independent cells; data from a second cell are presented in Extended Data Fig. 3. Note CHMP2A localization to nucleo-cytoplasmic channels is distinct from nuclear pores (asterisk in f). h, Quantification of CHMP2A labelling from two independently prepared cells. Channels were defined as discontinuities up to 80 nm, and gaps were defined as discontinuities over 80 nm. Distances of the gold-particles from channels or gaps were measured on the tomograms in three-dimensions and plotted as a histogram. Most (74.4%) of the gold label was found within 150 nm of nucleo-cytoplasmic channels, and most (70.6%) of the gold label was found more than 150 nm from the larger gaps in the NE. Scale bars, 10 µm (b) and 200 nm (f, g).
a–c, Phase-contrast (a), correlative immunofluorescence (b) and transmission electron microscopy (c) of a second HeLa cell stained with anti-CHMP2A, detected by Alexa-594-fluoronanogold and DAPI. Boxed region in a is shown in b; boxed region in b is shown in c. d, Z-slices extracted from tomographic reconstruction of forming NE from boxed region in c depicting CHMP2A-localization to nucleo-cytoplasmic channels at the indicated Z-heights. Arrow indicates nucleo-cytoplasmic channel. Images in all cases representative of 3 cells processed for CLEM, quantification of gold localization given in Extended Data Fig. 2H. Scale bars, 24 µm (b), 1 µm (c) and 200 nm (d).
a, Quantification of CHMP2A recruitment to the telophase NE or the midbody from Fig. 2c (n = 3, 10 cells (midbody or telophase) scored per experiment). b, Quantification of cytokinetic failure from cells treated with the indicated siRNA (300 cells were quantified per experiment, from three independent experiments). Data are mean ± s.d.
a–d, β-galactosidase activity of yeast co-transformed with the indicated Gal4 (ESCRT)- and VP16-fused proteins (n = 2). e, Resolved cell lysates and glutathione-bound fractions from 293T cells transfected with the indicated fusion proteins were examined by western blotting with anti-GFP (n = 3). f, β-galactosidase activity of yeast co-transformed with the indicated Gal4- and VP16-fused proteins (n = 3). g, Microscale thermophoresis experiments detailing binding of CHMP2A to GST (n = 4), His–UFD1 (n = 5) or His–UFD1(1–257) (n = 4). As no reduction in thermophoresis signal was observed for GST or His–UFD1(1–257) across the concentration range, we present here the average thermophoresis signal change at equivalent protein concentrations for these three proteins, normalized to zero at the concentration in capillary 1. h, Alexa-647-labelled CHMP2A, His–UFD1 and His–UFD1(1–257) were examined by infrared imaging or Coomassie staining. Data are mean ± s.d.
Extended Data Figure 6 UFD1 depletion does not affect ESCRT-dependent receptor degradation, lentivirus release or cytokinetic abscission.
a, Resolved cell lysates of HeLa cells transfected with the indicated siRNA were examined by western blotting with anti-UFD1 or anti-HSP90 antisera. b, Resolved lysates of human diploid fibroblasts transfected with the indicted siRNA and treated for the indicated times with epidermal growth factor (20 ng ml−1) were examined by western blotting with anti-EGFR, anti-UFD1 and anti-GAPDH antisera. EGFR degradation was quantified by densitometry (n = 3). c, Resolved cell lysates from 293T cells transfected with the indicated HIV-1 based lentiviral plasmids, a virally packaged GFP-plasmid, and the indicated siRNA were examined by western blotting with anti-p24 capsid, -HSP90, -TSG101, -CHMP2A, -CHMP2B and -UFD1 antibodies. Viral supernatants were collected and used to infect target HeLa cells. Resolved virions present in the 293T supernatant were examined by western blotting with anti-p24 capsid. Resolved lysates of infected HeLa cells were examined by western blotting with anti-GFP. Virion release was the ratio of released to cellular p24 capsid, as quantified by densitometry (n = 2); infectivity was quantified as GFP signal in target cells, as quantified by densitometry (n = 2). d, siRNA-transfected HeLa cells were fixed and stained with anti-tubulin. Multinucleate cells (n = 5) or cells connected by midbodies (n = 5) were scored visually, 300 cells scored per experiment. Data are mean ± s.d.
a, b, Timelapse microscopy analysis and quantification of NE-rim formation in HeLa cells stably expressing YFP–LAP2β and mCh–H2B and treated with the indicated siRNA. Scale bars, 10 µm. Time for rim formation post anaphase onset given (mins) (control, 8.53 ± 0.09, 226 cells analysed over 8 independent experiments; CHMP2A-1, 7.60 ± 0.09, 205 cells analysed over 7 independent experiments; CHMP2A-2, 6.86 ± 0.12, 37 cells analysed over 2 independent experiments; CHMP2B, 6.92 ± 0.09, 79 cells analysed over 4 independent experiments; CHMP2A and CHMP2B, 6.84 ± 0.13, 50 cells analysed over 2 independent experiments; CHMP4B, 7.07 ± 0.14, 44 cells analysed over 2 independent experiments; UFD1, 9.2 ± 0.18, 39 cells analysed over 3 independent experiments). Data are mean ± s.e.m. (in minutes). Images representative of the indicated number of cell analysed. c, Resolved cell lysates from a were analysed by western blotting with the indicated antisera.
a, Schematic of nuclear envelope integrity assay. b, Control-siRNA-treated HeLa cells reporting nucleo-cytoplasmic partitioning using the GFP–NLS–βGal assay, average NE compartmentalization from 20 cells presented. Nucleo-cytoplasmic partitioning stabilizes at 85 min (indicated by arrow). c, Immunofluorescence analysis of HeLa cells stably expressing YFP–LAP2β, transfected with the indicated siRNA then stained with anti-mAb414 and DAPI (n = 3). Scale bars, 10 µm. d, Mask used to quantify nuclear pore formation by image-based flowcytometry (Imagestream). e, Imagestream analysis of HeLa cells transfected with the indicated siRNA, then stained with anti-mAb414 and DAPI. Nuclear pore intensity quantified by mask described in d. Representative images from two independent experiments, histogram and population averages displayed, graphical quantification of NPC intensity from the indicated number of gated cells (control, 3,045; CHMP2A-1, 1,256; CHMP2A-2, 2,152; CHMP2B, 5,237; UFD1-1, 4,146; UFD1-3, 4,325). Data are mean ± s.d.
a, Quantification of NE sealing from siRNA-treated cells as in Fig. 4b (control, 140 cells from 7 independent experiments; UFD1-1, 60 cells from 3 independent experiments, P = 0.044; UFD1-3, 60 cells from 3 independent experiments, P = 0.021; CHMP2B 40 cells from 2 independent experiments; two-tailed Student's t-test was used to assess significance at the 85-min time point). b, Resolved cell lysates from a were analysed by western blotting with the indicated antisera. c, NE integrity assay as performed with cells stably expressing mCh–H2B and GFP–NLS and transfected with the indicated siRNA. Differences in nucleo-cytoplasmic partitioning was assessed after plateau at the 65-min time point using a two-tailed Student's t-test (control, 79 cells from 4 independent experiments, CHMP2A-1, 60 cells from 3 independent experiments, P = 0.048; CHMP2A-2, 52 cells from 3 independent experiments, P = 0.011; CHMP3, 28 cells from 3 independent experiments, P = 0.028). d, e, HeLa cells stably expressing mCh–H2B and GFP–NLS were transfected with the indicated siRNA and imaged live. 60 min after anaphase onset, cytoplasmic signal was photo-ablated (T = 0) and recovery of cytoplasmic signal from the nuclear pool was calculated for the indicated conditions (cytoplasmic:nuclear ratio of GFP–NLS was normalized to T = 0; control, 21 cells from 4 independent experiments; CHMP2A-1, 24 cells from 4 independent experiments, P = 0.04; CHMP2A-2, 23 cells from 4 independent experiments, P = 0.05; CHMP3, 15 cells from 3 independent experiments, P = 0.004, two-tailed Student's t-test was used to assess significance after 10 min). Scale bars, 10 µm. f, Scoring of multinucleate and midbody-connected HeLa cells transfected with the indicated siRNA and stained with anti-tubulin and DAPI (300 cells analysed per condition, n = 4). Data are mean ± s.e.m. (a, c, d) and mean ± s.d. (f).
a, Presentation of reconstructed tomograms from Fig. 4d. b, CHMP2A-depleted cells exhibited more non-NPC discontinuities per unit area, while the number of NPC per unit area was constant. Tomograms as described in Fig. 4d, e were scored for discontinuities. The internal diameter of NPCs was slightly reduced in CHMP2A-depleted cells (control, 84 ± 7.6 nm, CHMP2A-1, 74 ± 8.8 nm; CHMP2A-2, 74 ± 5.7 nm). c, Schematic depicting topological equivalent of ESCRT-III-dependent membrane fusion events.
Deconvolved 3D reconstruction of HeLa cells stained with anti-CHMP2A and DAPI and analysed by widefield microscopy, from Extended Data Figure 1C. (AVI 2486 kb)
Movie of GFP-CHMP4B localisation during the anaphase to telophase transition, from Extended Data Figure 1D. (AVI 1460 kb)
3D reconstruction of HeLa cells stained with anti-CHMP2A and DAPI and analysed by correlative light and electron tomography, From Figure 1D. (MOV 16147 kb)
3D reconstruction of HeLa cells stained with anti-CHMP2A and DAPI and analysed by correlative light and electron tomography, as depicted in Figure 1D and Extended Data Figure 2. (MOV 15633 kb)
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Olmos, Y., Hodgson, L., Mantell, J. et al. ESCRT-III controls nuclear envelope reformation. Nature 522, 236–239 (2015). https://doi.org/10.1038/nature14503
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