Apoptosis and necrosis are two fundamentally distinct forms of cell death. While apoptosis is characterized by caspase-mediated proteolytic cleavage that drives its execution and provides robust biochemical markers, the terminal events of necrotic cell death, including necroptosis, pyroptosis and ferroptosis, remain poorly understood. In particular, it is unclear what might be the execution-phase biochemical processes in necrosis. The lack of specific markers for terminal necrosis makes it difficult to define when and where irreversible cell death occurs in physiological and pathological contexts.
In this study, the authors combined super-resolution optical diffraction tomography (ODT) with biochemical and proteomic approaches to systematically analyze necrotic cell death. At the single-cell level, they observed that even under the same pyroptosis-inducing conditions, individual cells can diverge into distinct fates, undergoing either apoptosis or necrosis (Fig. 1). This finding highlights previously unappreciated heterogeneity in cell death decisions and elevates the understanding of cell death regulation to single-cell resolution.
Focusing on necrosis, the study revealed that canonical death pathways are sufficient to initiate cell death, but the terminal stage involves downstream events occurring after plasma membrane rupture. Through N-terminomic analysis, the authors identified a large number of proteolytic cleavage events that occur specifically in ruptured necrotic cells. These cleavages are predominantly located at the C-termini of arginine (Arg) and lysine (Lys) residues, indicating the involvement of extracellular trypsin-like proteases.
Importantly, these proteolytic events do not contribute to the initiation of cell death but instead take place after membrane rupture, when extracellular proteases gain access to intracellular substrates. The resulting large-scale and highly specific protein cleavage drives nuclear envelope disruption, genomic DNA degradation (Fig. 2). Functionally, this process promotes efficient phagocytic clearance of necrotic cell remnants and helps limit autoimmune responses. Together, these findings demonstrate that plasma membrane rupture does not mark the completion of necrotic cell death; rather, terminal execution requires extracellular protease–mediated, site-specific proteolytic evens with important biological consequences.
Finally, leveraging the specificity of these cleavage events, the authors developed monoclonal antibodies that recognize neo-protein fragments generated by necrotic cleavage. These antibodies serve as novel tools for detecting terminal necrotic events both in vitro and in vivo, enabling precise investigation of necrosis in disease contexts.
This study provides two key conceptual advances in our understanding of cell death. First, it reveals that even under identical death-inducing conditions, individual cells can adopt distinct fates, thereby highlighting an unexpected level of heterogeneity and establishing cell death regulation at single-cell resolution. Second, it demonstrates that plasma membrane rupture alone might not represent the terminal endpoint of necrosis; instead, necrosis includes a downstream execution phase characterized by extracellular protease-mediated proteolysis.
DOI: 10.15302/vita.2026.04.0023