Necroptosis is a caspase-independent form of programmed cell death. It is best known as a defensive measure against infectious agents that can inhibit apoptosis, however, necroptosis also participates in sterile pathologies such as ischaemia-reperfusion injury and autoinflammatory disease. Intracellular necroptotic signalling culminates in the activation of the protein, mixed lineage kinase domain-like (MLKL), which then translocates to the plasma membrane and causes cell death by compromising membrane integrity.
Much attention has been paid to the upstream events which govern MLKL activation and the terminal event whereby MLKL disrupts membrane integrity. In contrast, the mechanisms that traffic MLKL to the plasma membrane are entirely unknown. To address this gap in knowledge, we have devised immunofluorescence-based techniques to track and quantify the activation and trafficking of endogenous human MLKL during epithelial necroptosis at the single-cell level.
Via this approach, we have defined the rate-limiting mechanisms that control the trafficking of MLKL from the cytosol to the plasma membrane. Interestingly, MLKL shares the same trafficking pathway as the tight junction-associated protein, ZO-1 and co-accumulates at tight junctions in the plasma membrane during epithelial necroptosis. Furthermore, once at the plasma membrane, we find that intact tight junctions potently counteract the ability of MLKL to cause epithelial necroptosis, whereas tight junction disassembly sensitises epithelial cells to necroptosis. Hence, the trafficking and membranolytic activity of MLKL is mechanistically linked to tight junctions during epithelial necroptosis.
Collectively, our work uncovers two new checkpoints in necroptosis: one that regulates the kinetics of necroptosis, and one that controls the amount of MLKL needed to trigger necroptosis in epithelial cells. These checkpoints represent novel targets for anti-necroptotic therapy.