Necroptosis is a form of programmed cell death characterized by lack of caspase activity and a loss of plasma membrane integrity. Morphologically similar to necrosis, in the act of necroptosis, the plasma membrane is disrupted, causing release of cellular components to the extracellular fluid and an ensuing inflammatory response. Necroptosis proceeds via a regulated kinase cascade involving Receptor Interacting Protein Kinases RIPK1 and RIPK3. Mixed Lineage Kinase domain-Like protein (MLKL), a pseudokinase, is the final known obligate effector essential for the execution of necroptosis. Whilst the MLKL pseudokinase domain is incapable of catalysing phosphotransfer reactions, it is the site of RIPK3 phosphorylation. This phosphorylation event is thought to be integral to flipping a molecular switch regulated by the pseudokinase domain, resulting in activation of MLKL. Upon activation, MLKL oligomerises and translocates to the plasma membrane, and is there thought to play a destabilising role. Details of MLKL’s molecular mechanism of action, such as the stoichiometry of oligomerisation and how it interacts with the plasma membrane, remain unknown.
To understand better how MLKL might disrupt the plasma membrane as the cell undergoes necroptosis, we have performed a study on MLKL’s activity on Giant Unilamellar Vesicles (GUVs). GUVs approximate the size of a mammalian cell, and using confocal microscopy and fluorescently labelled MLKL protein we were able to monitor MLKL’s impact on the membrane in real time. We have shown that MLKL can bind to and permeabilise vesicles with low membrane curvature, and observed MLKL assembling into higher order structures on the membrane, not seen before using other model membrane systems.