MLKL trafficking and accumulation at the plasma membrane control the kinetics and threshold for necroptosis

Abstract

Mixed lineage kinase domain-like (MLKL) is the terminal protein in the pro-inflammatory necroptotic cell death program. RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption, although the precise choreography of events is incompletely understood. Here, we use single-cell imaging approaches to map the chronology of endogenous human MLKL activation during necroptosis. During the effector phase of necroptosis, we observe that phosphorylated MLKL assembles into higher order species on presumed cytoplasmic necrosomes. Subsequently, MLKL co-traffics with tight junction proteins to the cell periphery via Golgi-microtubule-actindependent mechanisms. MLKL and tight junction proteins then steadily co-accumulate at the plasma membrane as heterogeneous micron-sized hotspots. Our studies identify MLKL trafficking and plasma membrane accumulation as crucial necroptosis checkpoints. Furthermore, the accumulation of phosphorylated MLKL at intercellular junctions accelerates necroptosis between neighbouring cells, which may be relevant to inflammatory bowel disease and other necroptosis-mediated enteropathies.

Stoking the Fire: How Dying Cells Propagate Inflammatory Signalling through Extracellular Vesicle Trafficking

“…An in-vitro kinetics analysis of EVs released by THP-1 human monocytes exposed to 56 °C heat stress demonstrated a time-dependent increase in EV release over a 45-min treatment period coinciding with an increase in LDH release, suggesting that EV generation occurred simultaneously to membrane lysis [ 30 ]. In this study, the heat-treated cells generated both large and medium EVs (isolated at 2000× g and 16,000× g centrifugation, respectively) and markedly fewer were isolated at 100,000× g , suggesting that the EVs were of nonexosomal origin [ 87 ]. In an in-vivo study on the role of EVs following major burns injury, analysis of blood samples of patients following thermal injury demonstrated elevated levels of circulating MVs that were predictive of mortality through their contribution to systemic inflammatory response syndrome (SIRS) [ 46 ], although the direct cause of this was not determined.…”

“…Necroptosis can be activated by several extracellular ligands including TNFα, IFNγ and LPS, which, when caspase 8 is downregulated or rendered inactive either pharmacologically or through viral inhibition, can initiate the formation of a RIPK1–RIPK3 signalling complex. This complex then facilitates phosphorylation of MLKL, enabling its active translocation to the plasma membrane and the formation of membrane pores that can release DAMPs into the extracellular space [ 87 , 97 ]. There are relatively few examples of EVs released by necroptotic cells, although those that have been reported have provided intriguing insights into their unique properties [ 31 , 36 ].…”

A toolbox for imaging RIPK1, RIPK3 and MLKL in mouse and human cells

“…As several other necroptotic proteins are lysine-rich, we considered whether the choice of fixative was a critical variable for robust immunodetection of MLKL, RIPK3 and RIPK1 in human and mouse cells. Accordingly, we compared the performance of 22 antibodies for immunofluorescent staining of human HT29 cells and mouse dermal fibroblasts (MDFs) -two cellular models that are well-characterized to undergo necroptosis when treated with TNF, Smacmimetic and IDN-6556 (herein referred to as TSI) 5,28,29,44,47 . To quantitatively gauge the performance of each antibody, their immunosignals were characterized in four ways: 1) A ratio of the immunofluorescent signals between a positive and negative control.…”

“…1e) and these specific signals represent the majority of all detectable signals ( Fig.1d). We recently developed another MLKL-specific antibody, clone 7G2 (source: in-house), that recognises an epitope centred on aF-aG loop the C-lobe of the human MLKL pseudokinase domain, which differs to the site recognised by the 10C2 clone 29 . Despite binding a distinct site in human MLKL to 10C2, the 7G2 clone shows similar specificity for human MLKL in immunoblot analyses and only yields specific immunofluorescent signals on methanol-fixed cells ( Fig.…”

“…In cellular contexts where the activities of the Inhibitors of Apoptosis proteins (IAPs) E3 Ubiquitin ligase family and the proteolytic apoptotic effector, Caspase-8, are depleted or compromised, necroptosis ensues. The precise choreography of necroptotic signaling is still emerging, although recent studies have defined key events and checkpoints in the pathway 26,27,28,29 . Following pathway induction, RIPK1 autophosphorylation prompts hetero-oligomerization with RIPK3 via an amyloid-forming motif in the region C-terminal to their kinase domains termed the RHIM (RIP Homotypic Interaction Motif) to form a cytoplasmic platform known as the necrosome 30,31,32 .…”