Fig. 3: Rod-shaped RIP1-RIP3 architecture in necrosomes enables signal amplification and threshold responses.

a Diagram illustrating a basic model depicting higher-order complexes assembly by two components. b Effects of varying the number of B (n) or A (m) molecules on output, with one component fixed at 1 within the subunit. Green arrows indicate changes in output signal and regulatory behavior with increasing input intensity as n or m varies. c Impact of diverse assembly stoichiometries on output (upper panel) and regulatory behavior (lower panel). The sensitivity coefficient quantifies the threshold response by measuring the reduction in stimulus intensity required to decrease the output from its maximum (20 a.u.). d Schematic of necrosome assembly in MLKL-KO HeLa cells. Immunoblots are representative of three independent experiments. p-RIP3 was quantified by band intensity. The kinetic model (Supplementary Table 2) recapitulates experimental RIP3 phosphorylation dynamics. e Model predictions of how RIP3:RIP1 stoichiometries in necrosomes affect necroptotic signal output. f Predicted effect of reduced RIP1 expression on RIP3 phosphorylation (upper panel), alongside experimental validation using literature data⁹ (lower panel). p-RIP3 was quantified by band intensity and RIP1 levels were controlled by shRNA-mediated knockdown. g Schematic of MAP7-based calibration system created in BioRender (https://BioRender.com/zaak4bk).HeLa cells expressing Flag-MAP7-HA or Flag-MAP7-P2A-Flag-MAP7-HA (“2Flag-MAP7-HA”) are expected to display Flag:HA stoichiometries of 1:1 and 2:1, respectively. Dual-color confocal (h) and STORM (j) images of HeLa cells expressing Flag-MAP7-HA or 2Flag-MAP7-HA, immunolabeled with anti-Flag (purple) and anti-HA (green) antibodies. Quantification of fluorescence intensity (i; n = 40 fields per group) and localization counts (k; n = 45 structures per group) confirmed Flag:HA ratios matched expected values. Insets show dual-labeled tubulin with calculated Flag:HA ratios. l Three-color STORM of necrosomes in HA-RIP1/Flag-RIP3–expressing RIP1-MLKL-DKO HeLa cells treated with TSZ for 4 h, labeled for HA-RIP1 (green), Flag-RIP3 (purple), and p-RIP3 (yellow). Insets highlight necrosomes with calculated Flag:HA ratios and p-RIP3 localizations. m Quantification of RIP3:RIP1 stoichiometry and relative p-RIP3 levels (localizations) in individual necrosomes (n = 89). Images in (h, j, l) are representative of two independent experiments. Scale bars: 10 μm (h, overviews in j and l) and 300 nm (insets in j and l). Source data are provided as a Source Data file.