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. 2023 Jun;618(7967):1065-1071.
doi: 10.1038/s41586-023-05991-z. Epub 2023 May 17.

Structural basis of NINJ1-mediated plasma membrane rupture in cell death

Morris Degen #  1 , José Carlos Santos #  2 , Kristyna Pluhackova #  3 , Gonzalo Cebrero  1 , Saray Ramos  2 , Gytis Jankevicius  1 , Ella Hartenian  2 , Undina Guillerm  4 , Stefania A Mari  5 , Bastian Kohl  1 , Daniel J Müller  5 , Paul Schanda  4 , Timm Maier  1 , Camilo Perez  1 , Christian Sieben  6   7 , Petr Broz  8 , Sebastian Hiller  9
Affiliations

Structural basis of NINJ1-mediated plasma membrane rupture in cell death

Morris Degen et al. Nature. 2023 Jun.

Abstract

Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1-7. Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-18 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Polymerization kinetics of plasma membrane NINJ1.
a,b, Western blot analysis of endogenous GSDMD and NINJ1 in primed BMDMs after nigericin stimulation (Nig) for 1.5 h (a) or for 2, 5, 15, 25, 55 or 85 min (b) followed by treatment with the membrane-impermeable BS3 crosslinker for 5 min. FL, full length. Of note, tubulin, used here as a loading control, is crosslinked owing to BS3 entry through GSDMD pores under nigericin-treated conditions. Time in b is the total incubation time for nigericin and BS3 treatment. Short exp., short exposure. For gel source data, see Supplementary Fig. 1. c, LDH release from primed BMDMs after nigericin stimulation. d, Time-lapse fluorescence confocal microscopy of HeLa cells co-expressing hNINJ1–GFP and opto-casp1 following photo-activation. Images show the NINJ1–GFP fluorescence at the basal plane of the cell and the influx of DRAQ7 (maximum (max.) projection from a z-stack) to track plasma membrane permeabilization. Time was normalized to the onset of increase in DRAQ7 nuclear fluorescence. White arrows indicate regions that are enlarged in the insets. Scale bar, 10 µm. e–g, Normalized quantification of the distribution inhomogeneity of NINJ1–GFP (e), HATMD–GFP (f) or E-cadherin–GFP (g) at the basal plane of cells, and DRAQ7 nuclear fluorescence intensity after photoactivation of opto-casp1 (F). The distribution inhomogeneity at each time point (Dt) was normalized to the distribution inhomogeneity at the initial time point of the experiment (Di). Data are mean ± s.d. Data are representative of 2 (b), 3 (a) or 14 (d) independent experiments, or pooled from 2 independent experiments performed in triplicate (c) or at least 10 (e–g) independent experiments. Source data
Fig. 2
Fig. 2. Super-resolution imaging of NINJ1 assemblies.
a, Wide-field imaging of DmrB–Casp4tg HeLa cells expressing hNINJ1–GFP and GSDMD used for STORM microscopy in b–e. Cells were left untreated or stimulated with B/B homodimerizer 3 h before fixation and labelling with Alexa Fluor 647-conjugated anti-GFP nanobodies. Scale bar, 50 µm. b–e, STORM super-resolution imaging of hNINJ1–GFP in cells from a using TIRF illumination of the basal plane. b, Left, untreated or B/B-stimulated cells expressing hNINJ1–GFP. Scale bar, 10 µm. b, Right, STORM super-resolution reconstruction of hNINJ1–GFP labelled with Alexa Fluor 647-conjugated anti-GFP nanobodies. The indicated outlined regions are magnified on the right. Scale bar, 500 nm. c, Gallery of hNINJ1–GFP clusters found in pyroptotic cells. The small clusters are also observed in non-activated cells. Scale bars, 500 nm. d, Radius of gyration (Rg) and eccentricity (Ecc) for each identified hNINJ1 cluster. Plots show the distribution of all identified clusters from three independent experiments. The lines indicate median values. Statistical analysis based on the median Rg and Ecc of each experiment using Student’s unpaired two-sided t-test. **P < 0.01, ***P < 0.001. e, Overview STORM reconstruction of assemblies in B/B-stimulated cells expressing hNINJ1–GFP including filamentous structures. Two filaments are highlighted with magenta arrowheads. The indicated regions are magnified on the right. Scale bars, 1 µm. Data in a–c,e are representative of at least three independent experiments. Source data
Fig. 3
Fig. 3. Cryo-EM structure of NINJ1 filament.
a, Cryo-EM micrograph showing filamentous hNINJ1 (white arrows) along representative 2D classes. Scale bar, 25 nm. Micrograph representative of 13,124 micrographs from one dataset. b, Organization of hNINJ1 filaments with helices represented as tubes and each subunit shown in a colour gradient (yellow–green–purple). The main interaction interfaces I, II and III are shown below. c, A single hNINJ1 filament subunit, comprising helices α1–α4, with surface representation outlined in light grey. d, Lipophilicity and charge distribution of the hNINJ1 filament. e, Permeability of hNINJ1 proteoliposomes at different protein:lipid molar ratios. Data are mean + s.d. (n = 3 independent experiments). Statistical analysis by one-way ANOVA. ****P < 0.0001. Source data
Fig. 4
Fig. 4. The mechanism of NINJ1-mediated PMR.
a, Three subunits of filamentous hNINJ1 with overview of residues selected for mutagenesis study (intermolecular interactions, magenta; intramolecular interaction, purple; membrane interactions, green). b, Schematic representation of the residues selected for mutagenesis. c, Cytotoxicity upon overexpression of wild-type (WT) or mutant mNINJ1 in HEK 293T cells. d, Permeability of proteoliposomes containing wild-type and mutant mNINJ1 compared with protein-free liposomes (empty). Data are mean + s.d. (n = 3). e, Release of LDH in primary Ninj1–/– BMDMs reconstituted with wild-type mNINJ1 or different mNINJ1 mutants upon nigericin treatment (1.5 h). Reconstitution with the empty vector and non-transduced Ninj1–/– BMDMs (–) were used as controls. f, Cytotoxicity upon B/B treatment in HeLa cells co-expressing DmrB–caspase-4 and wild-type or mutant mNINJ1. Killing score corresponds to the cytotoxicity, measured by LDH release, normalized against wild-type mNINJ1 control (c) or mock-treated controls (f). Statistical analysis in c–f by individual comparison to the control condition highlighted in bold. Data are mean + s.d. and data are pooled from two independent experiments performed in triplicate (c; for K45Q, K45Q, A47L, D53A, V82W, L121W, T123L and A138L mutants), three independent experiments performed in triplicate (c; for mock, WT and V82F, I84F, Q91A, G95L and A138L mutants), and representative of two independent experiments performed in triplicate (c; for L121F mutant), pooled from two independent experiments performed in triplicate (f) or representative of two independent experiments performed in triplicate (e). In c,e,f, multiple plates were used to test all mutants, thus control conditions were included in each of the plates. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001; NS, not significant. P values by one-way ANOVA with Dunnet’s multiple comparisons tests. g, Structural model of NINJ1-mediated membrane rupture. Non-activated NINJ1 is randomly distributed in the plasma membrane (PM). Upon activation, NINJ1 polymers lyse the membrane, resulting in the release of cytosolic content (red). Source data
Extended Data Fig. 1
Extended Data Fig. 1. Inflammasome activation induces NINJ1-dependent PMR downstream of GSDMD.
a, Topology model of non-activated NINJ1 in the plasma membrane (PM). b, Immunofluorescence microscopy of NINJ1 in BMDMs. Scale bars, 15 µm. c, Western blot analysis of NINJ1 expression in wild-type (WT), Gsdmd–/– and Ninj1–/– BMDMs. d, Release of LDH or IL-1β in primed WT, Gsdmd–/– and Ninj1–/– BMDMs stimulated with Nigericin (1 h), transfected with poly(dA:dT) (1 h) or LPS (5 h), or infected with S. Typhimurium (1.5 h). Uptake of propidium iodide (PI) was measured every 10 min after the different stimulations. e, Western blot analysis of endogenous GSDMD in primed BMDMs after Nigericin stimulation for 1 h followed by treatment with BS3 crosslinker for 30 min. f, Time-lapse fluorescence microscopy of primed WT and Ninj1–/– BMDMs upon transfection with LPS. Pyroptotic cells lost membrane integrity (acquisition of PI, a membrane-impermeable DNA dye) and were labeled by annexin-V (labels phosphatidylserine exposure to the outer leaflet of the plasma membrane). Plasma membrane (PM) blebs observed during pyroptosis (white arrows) collapsed in a NINJ1-dependent manner. DIC, differential interference contrast. Scale bars, 20 µm. Graphs show the mean ± SD and data are representative from 2 (b,c,e,f) or pooled from 2 independent experiments performed in triplicate (d). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. P-values were calculated using one-way ANOVA with multiple comparisons Dunnett tests. For gel source data, see Supplementary Fig. 2. Source data
Extended Data Fig. 2
Extended Data Fig. 2. NINJ1 quickly polymerizes at the plasma membrane upon pyroptosis activation.
a,b, Schematic representation of the experimental setup used to analyze NINJ1 polymerization at the plasma membrane upon optogenetic induction of GSDMD-driven pyroptosis in single cells: HeLa cells expressing Opto-Casp1 (red dots) are photo-activated with 488 nm light to induce rapid Opto-Casp1 clustering and caspase activation, triggering GSDMD pore formation and influx of DRAQ7 (a membrane-impermeable DNA dye) (a). Time-lapse fluorescence confocal microscopy allows to follow NINJ1-GFP localization and clustering in different Z-planes of the cell (b). c—e, Time-lapse fluorescence confocal microscopy images (basal or central plane of cells) of HeLa cells co-expressing Opto-Casp1 and hNINJ1-GFP (c), HATMD-GFP (d) or E-cadherin-GFP (e), after photo-activation. DRAQ7 influx (maximum projection from a Z-stack) shows plasma membrane permeabilization during cell death. Time was normalized to the onset of increase in DRAQ7 nuclear fluorescence. Scale bars correspond to 10 µm and white arrows point to inset images. Data are representative from fourteen (c), eleven (d) or ten (e) independent experiments.
Extended Data Fig. 3
Extended Data Fig. 3. Cell death triggers NINJ1 polymerization at the plasma membrane and induces PMR in a cell-intrinsic manner.
a, Immunofluorescence microscopy of endogenous NINJ1 in primed BMDMs upon stimulation with Nigericin (1 h), or poly(dA:dT) transfection (1 h) (to induce pyroptosis), or in naïve cells stimulated with TNF + SM (SMAC-mimetic AZD 5582) for 16 h (to induce apoptosis). Scale bars, 10 µm. b, LDH release in WT, Gsdmd–/– or Ninj1–/– BMDMs stimulated with TNF + SM (16 h). c, Schematic representation of the experimental setup to assess if NINJ1-driven PMR acts in a cell-intrinsic (in Cis) or -extrinsic (in Trans) manner: primed WT BMDMs were co-cultured with Casp11–/– (C11–/–) BMDMs previously incubated with CFSE. Co-cultured cells were then transfected with purified LPS and the incorporation of propidium iodide (PI) in dying cells (dashed contour) was quantified in both cell types by fluorescence microscopy. Different co-cultures were prepared (WT + WTCFSE-pos; C11–/– + C11–/–CFSE-pos; WT + C11–/–CFSE-pos; C11–/– + WTCFSE-pos), and the percentage of cells with PI positive nuclei was quantified in the CFSE-negative and -positive cells. Graphs show the mean ± SD and data are representative from two independent experiments (a), or pooled from two independent experiments performed in triplicate (b,c). *** P < 0.001; ns, not significant. P-values were calculated using one-way ANOVA with multiple comparisons Dunnett tests. Source data
Extended Data Fig. 4
Extended Data Fig. 4. NINJ1-GFP cluster size and resolution after density-based clustering.
a, Release of LDH in HeLa cells expressing DmrB-Casp4 upon activation with different doses of B/B homodimerizer for 3 h. Graph shows the mean ± SD and data are representative from two independent experiments performed in triplicate. b, Absence of reorganization of the control protein HATMD-GFP upon cell death. HeLa cells co-expressing DmrB-Casp4 and HATMD-GFP were untreated (UT) or stimulated with B/B homodimerizer and imaged using TIRF microscopy to visualize the membrane organization of HATMD-GFP. c, Density-based clustering of NINJ1 clusters, correlating their FRC resolution and size (Rg). Clustering was performed in multiple regions of interest (ROIs) across multiple cells, with equal total area for each condition. Rg and FRC are plotted against the number of localizations per cluster for each identified cluster. For plotting and further analysis in panels d–f, only clusters with more than 100 localizations were used. d, Histogram of cluster sizes (Rg). e, Distribution of cluster per area cell surface. Analysed were 23/72 cells from 2/4 independent experiments (UT/+BB). Lines indicate mean values. f, Extended gallery of large NINJ1-GFP structures found in pyroptotic cells. Gallery of clusters identified after stimulation (as in Fig. 2c) and selected by the cyan quadrant (Rg > 200 nm, localizations > 500) in c. Scale bar, 2 µm. Source data
Extended Data Fig. 5
Extended Data Fig. 5. Structure determination of hNINJ1 filaments.
a, Negative Stain TEM micrograph of purified hNINJ1 ring assemblies (white arrows). Scale bar, 50 nm. Micrograph representative of n = 14 independent experiments. b, Mass photometry measurements of purified hNINJ1 in presence of detergent. c, Western Blot of purified hNINJ1 sample used for cryo-EM structure determination. For gel source data, see Supplementary Fig. 3. d, Cryo-EM processing workflow. e, Distribution of orientations over azimuth and elevation angles for particles included in the calculation of the final map (n = 709,840 particles). f, Gold standard Fourier shell correlation (GSFSC) plot for final helical refinement of hNINJ1 dataset. g, Modeled helices α1−α4 shown individually within the experimentally determined cryo-EM density (grey mesh). h, Collection of views of the hNINJ1 double filament model.
Extended Data Fig. 6
Extended Data Fig. 6. Structural and functional analysis of NINJ1.
a, 15N R2 transverse relaxation rate measurements of GB1-hNINJ1–82 in the absence of any membrane-mimicking environment show that the N-terminal ~40 residues are highly flexible, while the remainder has limited flexibility. b, Negative stain micrograph of purified hNINJ1∆1–36 showing ring assemblies (white arrows), scale bar corresponds to 50 nm. Micrograph representative of n = 2 independent experiments. c, Overlay of 20 Alphafold predictions of hNINJ1 with the experimentally determined structure. d, Snapshots at 0 µs and 1 µs of an AA simulation of a hypothetical filament, where the experimental monomer was replaced by the Alphafold-predicted monomer (n = 1). The backbone of helices α3 and α4 was restrained to study the propagation of helices α1 and α2. e, Residue pairs with co-evolutionary coupling displayed on the filament structure of hNINJ1. Shown are all pairs among the 100 most significant couplings, that are closer in space in the filament than in the monomer. A list of these pairs is given in Supplementary Table 2. f, Hydrophobic interaction occurring between hNINJ1 filaments. g, Relative fluorescence traces of hNINJ1 proteoliposomes with different lipid to protein molar ratios, as indicated. Star indicates the addition of dithionite. Arrow indicates timepoint of Triton X-100 addition. Triplicates are shown. Source data
Extended Data Fig. 7
Extended Data Fig. 7. Stability of NINJ1 polymers probed by molecular dynamics simulation.
a, Snapshot after 1 µs of an all-atom simulation of linear NINJ1 oligomers on both edges of a membrane stripe (n = 2). b, Snapshot after 20 µs of a CG MD simulation of linear filaments capping the edges of a membrane stripe (n = 2). c, Root mean square deviations (RMSD) over simulation time of the backbone of linear pentamers attached to the membrane patches. Average RMSD and SEM over individual, unique pentamers and simulations (n = 4 for AA and n = 8 for CG) are given. d, Average structure and B-factors (in Å2) of the hNINJ139–152 backbone (n = 20) in simulations (a). Structures were aligned to helices α3 and α4 prior analysis. e, Representative snapshots of CG MD simulations of the hNINJ139–152 double filament after 50 µs in absence (left, n = 2) and presence (right, n = 2) of DDM. The black box highlights the simulation unit cell. Periodic filament copies were added on right and left. f, Representative snapshot after 40 µs of a CG simulation of a circular hNINJ139–152 45-mer in a lipid bilayer (n = 4). Insets on the right show the protein packing in a straight (bottom) and in a curved part of the polymer (top). g, Time evolution of the average inner area of rings made of 45 hNINJ139–152 (n = 4) or hNINJ176–152 (n = 3) relative to the area at t = 3 µs. The shaded areas show SEM over individual simulations. h, Snapshots at 150 µs of four independent CG MD simulations of 45-mer hNINJ139-152 (n = 4). i, Starting structure, collapse at 3 µs, as well as three snapshots at 150 µs of independent CG MD simulations of 45-mer hNINJ176–152 rings, i.e. missing helices α1 and α2 (n = 3). Lipids are shown as grey sticks, with phosphates highlighted as spheres. hNINJ1 protomers are randomly colored yellow, green or purple with the exception of c, where the purple-green-yellow coloring scales with the B-factor (purple low B-factor, yellow large B-factor). Source data
Extended Data Fig. 8
Extended Data Fig. 8. Analysis of NINJ1 single-point mutants.
a, Comparison of hNINJ1 and mNINJ1 sequences. Orange background shows identical residues. Secondary structure is indicated on top. In the structured part (residues 44–138), 2 amino acids differ and 93 are identical. Arrows indicate selected single residues that were mutated in this work. b, c, Western blot analysis of mNINJ1 expression (b) and LDH release (c) in HEK 293T cells transiently transfected with WT or different mNINJ1 mutants. mNINJ1 expression was induced by adding doxycycline (Dox) for 48 h. d, Triplicates of fluorescence traces of WT-NINJ1, mutants, and empty liposomes. Star indicates the addition of dithionite. Arrow indicates timepoint of Triton X-100 addition. e, Western blot analysis of mNINJ1 expression in Ninj1–/– primary BMDMs transduced with a retroviral vector expressing WT or different mNINJ1 mutants. Transduction with the empty vector or non-transduced Ninj1–/– cells (–) were used as controls. f, LDH release and western blot analysis of mNINJ1 expression in HeLa stably expressing FLAG-GSDMD-V5 and Dox-inducible DmrB-Casp4, upon transient transfection with Dox-inducible mNINJ1 and induction of protein expression for the indicated time points. g, LDH release in HeLa cells co-expressing Dox-inducible DmrB-Casp4 and Dox-inducible WT or different mNINJ1 mutants. Cells were treated with Dox for 16 h, followed by treatment with B/B homodimerizer. B/B untreated cells were also used as control. h, Western blot analysis of mNINJ1 and GSDMD expression in HeLa cells stably co-expressing FLAG-GSDMD-V5 and Dox-inducible DmrB-Casp4, and transiently expressing WT or different mNINJ1 mutants. i, Western blot analysis of GSDMD processing in HeLa cells stably co-expressing FLAG-GSDMD-V5 and Dox-inducible DmrB-Casp4, and transiently expressing WT or different mNINJ1 mutants, upon caspase-4 activation with B/B homodimerizer. Cell lysates and supernatants were combined and analyzed. In (h,i) mNINJ1 expression was induced with Dox for 16 h. Graphs show the mean ± SD and data are representative from at least two independent experiments performed in triplicate. For gel source data, see Supplementary Fig. 4. Source data
Extended Data Fig. 9
Extended Data Fig. 9. Interactions of the NINJ1 N-terminal helices with membranes.
a, Quartz crystal microbalance with dissipation (QCM-D) experiments. Frequency change upon addition of GB1-hNINJ1(1–81) to supported lipid bilayer membranes containing only DOPC (black), 80% DOPC/20% DOPS (orange) or 60% DOPC/40% DOPS (red). Duplicate experiments with independently produced protein and lipid bilayers are denoted with squares and circles. The lines denote fits of a Hill equation. b, Liposome composition-dependent dye release by hNINJ140–60 peptide or its scrambled sequence version. Data are means of n = 4 replicates for 3.75 µM and n = 5 replicates for the other concentrations, with error bars denoting SD. Significance was calculated using Student’s unpaired two-sided t-test. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant. c, Residue-specific membrane interaction probability of hNINJ120–152 from CG MD simulations (n = 10). Membrane with PS only in the cytosolic leaflet, corresponding to a healthy cell, are shown in green, while membrane with PS in both leaflets, corresponding to activated cell death signal, is shown as grey bars. The N-terminal adhesion motif (NAM) is highlighted. The error bars denote SEM over the individual simulations (n = 10). Significance was calculated using Student’s paired two-sided t-test. * P < 0.05, ** P < 0.01; *** P < 0.001. d, Sequence alignment of hNINJ1 and hNINJ2. Conserved residues are highlighted in orange. The secondary structure of hNINJ1 is shown on top. Source data
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