Necroptosis is a form of regulated necrosis mediated by the formation of the necrosome, composed of the RIPK1/RIPK3/MLKL complex. Here, we developed a proximity ligation assay (PLA) that allows in situ visualization of necrosomes in necroptotic cells and in vivo. Using PLA assay, we show that necrosomes can be found in close proximity to the endoplasmic reticulum (ER). Furthermore, we show that necroptosis activates ER stress sensors, PERK, IRE1α, and ATF6 in a RIPK1-RIPK3-MLKL axis–dependent manner. Activated MLKL can be translocated to the ER membrane to directly initiate the activation of ER stress signaling. The activation of IRE1α in necroptosis promotes the splicing of XBP1, and the subsequent incorporation of spliced XBP1 messenger RNA (mRNA) into extracellular vesicles (EVs). Finally, we show that unlike that of a conventional ER stress response, necroptosis promotes the activation of unfolded protein response (UPR) sensors without affecting their binding of GRP78. Our study reveals a signaling pathway that links MLKL activation in necroptosis to an unconventional ER stress response.

坏死性凋亡是由 RIPK1/RIPK3/MLKL 复合物组成的坏死体形成介导的一种调节性坏死。在这里，我们开发了一种邻近连接测定法 (PLA)，它允许在 necroptotic 细胞和体内对坏死体进行原位可视化。使用 PLA 测定，我们表明可以在内质网 (ER) 附近发现坏死体。此外，我们发现坏死性凋亡以 RIPK1-RIPK3-MLKL 轴依赖的方式激活 ER 应力传感器、PERK、IRE1α 和 ATF6。激活的 MLKL 可以转移到 ER 膜以直接启动 ER 应激信号的激活。坏死性凋亡中 IRE1α 的激活促进了 XBP1 的剪接，并随后将剪接的 XBP1 信使 RNA (mRNA) 掺入到细胞外囊泡 (EV) 中。最后，我们表明，与传统的 ER 应激反应不同，坏死性凋亡促进了未折叠蛋白反应 (UPR) 传感器的激活，而不影响它们与 GRP78 的结合。我们的研究揭示了一种将坏死性凋亡中的 MLKL 激活与非常规 ER 应激反应联系起来的信号通路。