Spatiotemporal control of necroptotic cell death and plasma membrane recruitment using engineered MLKL domains

Abstract

Necroptosis is a form of programmed necrotic cell death in which a signaling cascade induces oligomerization of mixed lineage kinase domain-like (MLKL) protein, leading to plasma membrane rupture. Necroptotic cell death is recognized as important for protection against viral infection and has roles in a variety of diseases, including cancer and diabetes. Despite its relevance to health and disease states, many questions remain about the precise mechanism of necroptotic cell death, cellular factors that can protect cells from necroptosis, and the role of necroptosis in disease models. In this study, we engineered a light-activated version of MLKL that rapidly oligomerizes and is recruited to the plasma membrane in cells exposed to light, inducing rapid cell death. We demonstrate this tool can be controlled spatially and temporally, used in a chemical genetic screen to identify chemicals and pathways that protect cells from MLKL-induced cell death, and used to study signaling responses of non-dying bystander cells. In additional studies, we re-engineered MLKL to block its cell-killing capacity but retain light-mediated membrane recruitment, developing a new single-component optogenetic tool that allows modulation of protein function at the plasma membrane.