Low-order MLKL oligomers induce irregular membrane defects in necroptosis

Abstract

MLKL is a key executioner of necroptosis, a lytic form of regulated cell death associated with inflammatory pathologies. Upon activation by RIPK3-mediated phosphorylation, MLKL oligomerizes and translocates to the plasma membrane to induce permeabilization through yet unknown mechanisms. By imaging single particles of MLKL at the plasma membrane of necroptotic cells, here we show that, upon activation, MLKL transitions from a diffuse cytosolic distribution to discrete, low-mobility assemblies before cell bursting. This is accompanied by oligomerization into a mixture of species ranging from monomers up to hexamers. We directly visualize MLKL-induced membrane damage using atomic force microscopy imaging in supported bilayers, which reveals the formation of irregular membrane openings whose edges are only partially decorated with protein. Our findings support a mechanism in which low-order self-assemblies of MLKL disrupt plasma membrane integrity and stabilize membrane defect edges without organizing into defined pore structures, in contrast to membrane permeabilization in other forms of cell death. This reframes necroptosis execution and dissects membrane insertion of MLKL oligomers for potential targeting.