Fig. 4

The biogenesis and release mechanisms of exosomes during necroptosis. Classical: The ESCRT-0 complex initiates intracellular sorting and recruits ESCRT-I. Collaboratively, ESCRT-I and ESCRT-II orchestrate membrane invagination, giving rise to early endosomes (ESEs). These ESEs undergo mutual fusion, maturing into late endosomes (LSEs). Necroptosis: In the initial stages of necroptosis, membrane receptors, such as tumor necrosis factor receptor 1 (TNFR1), death receptors, and Toll-like receptors (TLRs), are activated. This activation leads to the assembly of TNFR1-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), receptor-interacting serine/threonine-protein kinase 1 (RIPK1), and pro-caspase 8/10, culminating in the formation of the death-inducing signaling complex (DISC). Subsequently, procaspase 8/10 undergo cleavage to yield mature caspase 8/10, thereby activating caspase 3/7. Activated caspase 3/7, in turn, promotes LSE formation and cargo sorting and becomes encapsulated within LSEs. In the late stage of necroptosis, a complex is formed in which RIPK1/RIPK3 phosphorylates mixed lineage kinase domain-like protein (MLKL), and ZBP1 directly binds RIPK3 and MLKL through its RHIM domain. MLKL proceeds to generate pore complexes in the membrane, causing membrane disruption. Concurrently, MLKL aids in the formation and cargo sorting of LSEs, which encapsulate itself within the endosomal lumen. Eventually, LSEs undergo fusion, evolving into multivesicular bodies (MVBs). Through RIPK3/MLKL-mediated ESCRT-III phosphorylation and membrane invagination, MVBs give rise to exosomes. Alternatively, exosomes may be passively released due to rupture of the cell membrane or actively expelled through MLKL pores in the membrane. In addition to exosomes, apoptotic bodies are also released during necroptosis. (Black arrows: involved in necroptosis mechanisms; purple arrows: involved in exosome biogenesis; curved purple arrows: involved in the formation of exosome cargo)