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Mosaic composition of RIP1–RIP3 signalling hub and its role in regulating cell death

Abstract

RIP1 and RIP3, cell death mediators, form fibrous amyloids. How RIP1/RIP3 amyloidal oligomers assemble functional necrosomes and control cell death is largely unknown. Here we use super-resolution microscopy to directly visualize cellular necrosomes as mosaics of RIP1 and RIP3 oligomers. The small (initial) mosaic complexes are round, and the large mosaics are in a rod shape. RIP3 oligomers with sizes of tetramer or above are the domains in mosaics that allow MLKL, recruited by phosphorylated RIP3, to oligomerize for necroptosis. Unexpectedly, RIP1 autophosphorylation not only controls the ordered oligomerization of RIP1 but also is required for RIP1-initiated RIP3 homo-oligomerization in correct organization, which is indispensable for the formation of functional rod-shaped mosaics. Similarly, apoptosis initiated by enzymatically defective RIP3 requires the formation of rod-shaped mosaics of RIP3 and RIP1 oligomers. The revealing of nanoscale architecture of necrosomes here innovates our understanding of the structural and organizational basis of this signalling hub in cell death.

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Fig. 1: Nanoscale imaging analysis of necrosomes in necroptotic cells.
Fig. 2: RIP1–RIP3 mosaics are functional necrosomes.
Fig. 3: RHIM-dependent RIP3 oligomerization is required for MLKL oligomerization but dispensable for phosphorylation of RIP3 and MLKL.
Fig. 4: RIP3 oligomers with sizes of tetramer or above enable phosphorylated MLKL to form oligomers and subsequently trigger cell necroptosis.
Fig. 5: RIP1 kinase activity is required for RIP1 and RIP3 oligomerization in proper configuration that leads to the formation of functional rod-shaped complexes.
Fig. 6: RIP3 autophosphorylation is dispensable for the formation of rod-shaped complex.
Fig. 7: Apoptosis triggered by RIP3 kinase inhibition relies on the mosaic structure composed of RIP1 oligomer.

Data availability

The published structures of RIP1–RIP3 hetero-amyloid core (PDB 5V7Z), RIP1 kinase domain (PDB 4NEU), RIP1 death domain (PDB 6AC5) and RIP3 kinase domain (PDB 4M66) are accessible in the PDB (https://www.rcsb.org/) with respective IDs. Numerical source data giving rise to graphical representations and unprocessed images of blots are shown in source data figures. Detailed experimental procedures and additional data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

All Python scripts and Fiji macros for STORM image analysis are available in the online repository GitHub (https://github.com/xchenxmu/STORMing-the-necrosome).

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (81788101, 31420103910 and 81630042 to J.H.; 31871386, 31501115 and 32070736 to X.C.; 12090052 and 11874310 to J.S.), National Key R&D Program of China (2020YFA0803500 to J.H.), the 111 Project (B12001 to J.H.), the CAMS Innovation Fund for Medical Science (2019-I2M-5-062 to J.H.) and the China Postdoctoral Science Foundation (2016T90598 to X.C.). We thank L. Yao and C. Wu for help with electron microscopy; Q. Liu and Y. Fan (Bruker) for their assistance with confocal microscopy and AFM, respectively. We also acknowledge C. Zhou and L. Wang from Nikon for support and helpful discussions on super-resolution imaging.

Author information

Authors and Affiliations

Authors

Contributions

X.C. carried out the majority of experimental work with the help of W.W., Y.C. and H.C. R.Z. performed molecular modelling. J.Z., X.L., Y.Y. and J.S. carried out the analysis of super-resolution images. X.C. and J.H. designed experiments, interpreted data and wrote the manuscript. J.H. conceived and supervised the study.

Corresponding authors

Correspondence to Xin Chen or Jiahuai Han.

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Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Cell Biology thanks Sang-Hee Shim and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Necrosome is visualized as diffraction-limited puncta growing in the process of cell necroptosis.

a, Confocal images of HA-RIP3 and MLKL-Flag-reconstituted MLKL-KO HeLa cells treated with DMSO or TSZ (T: TNF, 10 ng/ml; S: Smac mimetic (SM-164), 100 nM; Z: zVAD-fmk, 20 μM), fixed and labeled for HA-RIP3 (green), RIP1 (purple) and MLKL-Flag (light blue). Red arrows in magnified images outlined by the white boxes emphasize the growing necrosome puncta. b-d, Intensity distribution of RIP1 (b, n = 39, 232, 453 for TSZ 0, 2, 4 h, respectively), RIP3 (c, n = 59, 940, 1772 for TSZ 0, 2, 4 h, respectively) or MLKL (d, n = 11, 278, 735 for TSZ 0, 2, 4 h, respectively) clusters detected in cells treated as in a. ‘n’ refers to the number of structures analyzed across two independent experiments. Box plots (b-d) show median and 25th to 75th percentiles, and whiskers indicate the minimum and maximum values. P values are determined by Kruskal-Wallis test with Dunn’s multiple comparisons test. Scale bars, 10 μm (original images) and 2 μm (magnified images). Statistical source data are provided in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 Analysis of single-color STORM images.

a, Representative histogram of localization precision of CF 647 calculated by LAMA algorithm31. b-e, Segmentation analysis of single-color STORM images (b). An example of RIP1 puncta (yellow) identified by machine learning-based algorithm (ilastik) in untreated HeLa cells expressing RIP3 is shown (c). The details of segmentation analysis are described in the Methods section. Area distribution (d) and ellipticity distribution (e) of RIP1 clusters were automatically quantified. Here, r value represents the effective diameter of the square with an equivalent area to the detected signals. The clusters with r value of more than 80 nm are TSZ-induced clusters and among them, those with an ellipticity of more than 2.00 are further regarded as rod-shaped structures. Scale bar, 100 nm. Statistical source data are provided in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 Rod-shaped structure is a general feature of cellular necrosome.

a, Single-color STORM images of endogenous RIP3 complexes revealed by using an anti-RIP3 antibody. Magnified images outlined by white boxes emphasize the rod-shaped RIP3 complexes only present in WT but not RIP3-deficient HT-29 cells treated with TSZ for 8 h. b, STORM images of RIP1 or RIP3 in HT-29 cells treated with TSZ (T: TNF, 10 ng/ml; S: Smac mimetic (SM-164), 100 nM; Z: zVAD-fmk, 20 μM) for indicated time. Magnified images are shown as inset figures, highlighting the RIP1 or RIP3 clusters during cell necroptosis. c, Percentage of rod-shaped structures detected by RIP1 or RIP3 staining in total enlarged clusters (RIP1, n = 1, 277, 334 for TSZ 0, 4, 8 h, respectively; RIP3, n = 8, 468, 409 for TSZ 0, 4, 8 h, respectively) by TSZ stimulation. The definitions of round- and rod-shaped structures are described in Extended Data Fig. 2d,e. d-g, Width (d) and length (f) distributions of RIP1 stained rods (n = 43, 61 for TSZ 4, 8 h, respectively) or width (e) and length (g) distributions of RIP3 stained rods (n = 76, 105 for TSZ 4, 8 h, respectively) in necroptotic HT-29 cells treated as in b. In c-g, ‘n’ refers to the number of structures analyzed across three independent experiments. Scale bars, 5 μm (original images in a and b) and 100 nm (magnified images in a and b). Statistical source data are provided in Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 Analysis of two-color STORM images.

a, Representative histogram of localization precision of CF 568 calculated by LAMA algorithm31. b, Two-color STORM images of the RIP1-RIP3 necrosomes in HA-RIP3 expressing HeLa cells treated with TSZ, and labeled for RIP1 and RIP3 (top, “both”), only for RIP3 (middle, “anti-HA only”) or only for RIP1 (bottom, “anti-RIP1 only”). Green indicates mouse secondary antibody conjugated CF 568, and purple indicates rabbit secondary antibody conjugated CF 647. Data are representative of four independent experiments. c, Cross-talk percentages between the two STORM color-channels determined from single-color-labeled necrosome shown in b. Data are mean ± SD of biological triplicates. d,e, Analysis workflow of two-color STORM images in this study (d). Clusters identified by ilastik in each channel were further assessed the possibility of co-clustering in a single channel or overlap between two channels. An example of cluster analysis of two-color STORM imaging is shown in e. The details of segmentation/overlap analysis of clusters are described in the Methods section. Scale bars, 200 nm (e) and 100 nm (b). Statistical source data are provided in Source Data Extended Data Fig. 4.

Source data

Extended Data Fig. 5 Two-color STORM imaging of rod-shaped necrosomes.

a, Two-color STORM of necrosomes showing the spatial localization of RIP1-RIP3 in HA-RIP3 and MLKL-Flag-expressing MLKL-KO HeLa cells treated with TSZ. b-d, Viability of HT-29 (b), LS 174 T (c) or THP-1 (d) cells treated as indicated. THP-1 cells were primed by 100 nM PMA. Data are represented as mean ± SD of biological triplicates. e, Two-color STORM images of RIP1 and RIP3 clusters in cells from (b-d). f, Two-color STORM of necrosomes showing the spatial localization of RIP3-MLKL in HA-RIP3 and MLKL-Flag-expressing MLKL-KO HeLa cells treated with TSZ. g-j, Two-color STORM images showing the localizations of pRIP3 (g) or pMLKL (i) and RIP3 in WT and RIP3-deficient (g) or MLKL-deficient (i) HT-29 cells in the presence of TSZ. Area of pRIP3 (n = 163, 90 structures analyzed across three independent experiments for Round or Rod, respectively, h) or pMLKL (n = 237, 237 structures analyzed across three independent experiments for Round or Rod, respectively, j) puncta in round-/rod-shaped necrosome were calculated. STORM images are representative of three independent experiments. Data in h and j are mean ± SEM. All P values are determined by two-tailed, unpaired t-test. Scale bars, 5 μm (original images in g and i) and 100 nm (a, e, f and magnified images in g and i). Statistical source data are provided in Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 RIP1 is dispensable for RIP3 rods formation by overexpressed RIP3 and RIP3 overexpression-induced necroptosis.

a, Single-color STORM image of RIP3 complex by Dox-induced RIP3 in RIP1-deficient HeLa cells (left) and two-color STORM image of TSZ-induced RIP1-RIP3 rod in HA-RIP3 expressing HeLa cells (right). b-f, Flag-RIP3-WT or Flag-RIP3-RHIMMut expression was induced in RIP1-deficient HeLa cells by addition of Dox. The RIP3-containing complex was immunoprecipitated with an anti-Flag antibody and visualized by using negative stain EM (b). Cell viability was measured based on quantitation of the ATP present (c). Data are represented as mean ± SD of biological triplicates. Confocal images of RIP3 are shown (d) and the fraction of cells with RIP3 puncta were calculated (e). n = 75–324 cells pooled by two independent experiments. Cell lysates were collected and analyzed by immunoblotting for the indicated proteins under reducing or non-reducing conditions (f). Scale bars, 10 μm (d) and 100 nm (a and b). Unprocessed blots and statistical source data are provided in Source Data Extended Data Fig. 6.

Source data

Extended Data Fig. 7 Disordered RIP1 complexes by non-phosphorylated RIP1.

a, HA-RIP3-expressing HeLa cells were treated with TSZ or TSZ plus Nec-1. Cell lysates were collected and subjected to western blot analysis for the indicated proteins. b-e, Viability of HA-RIP3 expressing HeLa (b), HT-29 (c), LS 174 T (d) and THP-1 (e) cells treated as indicated. THP-1 cells were primed by 100 nM PMA. Data are represented as mean ± SD of biological triplicates. f,g, Confocal images of RIP1 and RIP3 (f) and area distribution of RIP1 clusters (g, n = 2015 for TSZ and n = 1131 for TSZ + Nec-1) in cells treated as in b. ‘n’ refers to the number of structures analyzed across two independent experiments. h, Flag-RIP1 WT or mutants were overexpressed in RIP1-KO HeLa cells with or without Nec-1 for 24 h. Cell lysates were collected and subjected to western blot analysis for the indicated proteins. i, Confocal images of Flag-RIP1 WT or mutants in cells treated as in h. The experiment was performed twice with similar results. Scale bars, 10 μm. Unprocessed blots and statistical source data are provided in Source Data Extended Data Fig. 7.

Source data

Extended Data Fig. 8 Effect of RIP3 kinase inhibitors on the necrosome formation.

a, Confocal images of the RIP1-RIP3 necrosomes in RIP3-expressing HeLa cells treated with TSZ or TSZ plus RIP3 kinase inhibitors (GSK’840 or Dabra). Purple indicates RIP1 and green is RIP3. b, Area histogram of RIP3 puncta in cells from a. n = 973–3524 clusters from 10 fields. c-f, Flag-RIP3 was co-overexpressed with PPM1B WT or its inactive form (R179G) in HEK293T cells. Cell lysates were collected and subjected to western blot analysis for the indicated proteins. Data are representative of three independent experiments (c). Representative confocal images of RIP3 and PPM1B-WT or PPM1B-R179G from two independent experiments (d). STORM images (e) and length distribution (f) of RIP3 rods in the presence of PPM1B WT or R179G mutant (n = 699 for WT and n = 589 for R179G). ‘n’ refers to the number of structures analyzed across two independent experiments. Box plots show median and 25th to 75th percentiles, and whiskers indicate the minimum and maximum values. P values are determined by two-tailed, unpaired t-test. Scale bars, 10 μm (a and d) and 100 nm (e). Unprocessed blots and statistical source data are provided in Source Data Extended Data Fig. 8.

Source data

Extended Data Fig. 9 The effects of expression of mRIP3 WT or mutants in HEK293T cells.

a-c, Flag-mRIP3 WT or mutants (K51A, D161N, or RHIMMut) were expressed in HEK293T cells with or without 5 μM GSK’872. Confocal (a) and STORM (c) images of RIP1-RIP3 complexes are shown. The fraction of cells with RIP3 puncta in a were calculated (b). n = 97–156 cells pooled by two independent experiments. Data in c are representative of three independent experiments. Scale bars, 10 μm (a) and 100 nm (c). Statistical source data are provided in Source Data Extended Data Fig. 9.

Source data

Extended Data Fig. 10 Model of the assembly and function of mosaic RIP1-RIP3 rods.

a, A hypothetic model of RIP1-RIP3 mosaics: (I) Bidirectional β sheets resulted from RHIM-dependent RIP1/RIP3 homo- or hetero-interactions; (II) The β sheets further form helical necrosome rods with about 47 nm in width and 33 nm in pitch length; (III) RIP1/RIP3 homo-oligomers distribute heterogeneously in the rod-shaped necrosome, exhibiting a mosaic pattern. b, Upon necroptosis stimuli, RIP1 is autophosphorylated and this modification retains RIP1 assembly in a proper manner. Ordered RIP1 interaction initiates ordered recruitment of downstream RIP3, leading to the formation of helical rod-shaped necrosomes. Necrosomal RIP1- and RIP3-homo-oligomers distribute as a mosaic pattern, and RIP3 oligomers with sizes equal to or more than tetramer license phosphorylated MLKL to oligomerize for necroptosis. RHIM domain-mediated interaction rather than RIP3 autophosphorylation/MLKL recruitment is indispensable for the formation of rod-shaped necrosomes. In addition, the mosaic RIP1-RIP3 rods are essential for apoptosis by recruiting FADD/Caspase-8 when RIP3 kinase activity was eliminated by GSK’872 or by kinase-dead mutations. Disrupting the ordered RIP1-RIP3 rod-shaped structures could impair the recruitment/activation of downstream molecules. Thus, the ordered RIP1-RIP3 mosaic rod functions as a bidirectional module for the signaling of necroptosis and apoptosis under certain conditions.

Supplementary information

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Unprocessed western blots.

Source Data Extended Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 8

Unprocessed western blots.

Source Data Extended Data Fig. 9

Statistical source data.

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Chen, X., Zhu, R., Zhong, J. et al. Mosaic composition of RIP1–RIP3 signalling hub and its role in regulating cell death. Nat Cell Biol 24, 471–482 (2022). https://doi.org/10.1038/s41556-022-00854-7

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