Pannexin-1 Channels govern the Generation of Necroptotic small Extracellular Vesicles

Abstract

The activation of mixed lineage kinase-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3) controls the execution of necroptosis, a regulated form of necrosis that occurs in apoptosis-deficient conditions. Active oligomerized MLKL triggers the exposure of phosphatidylserine residues on the cell surface and disrupts the plasma membrane integrity by forming lytic pores. MLKL also governs the biogenesis and shedding of proinflammatory small extracellular vesicles (EVs) during the early steps of necroptosis, however the molecular basis is unknown. Here, we find that MLKL oligomers activate plasma membrane Pannexin-1 (PANX1) channels, concomitantly to the loss of phosphatidylserine asymmetry. This plasma membrane "leakiness" requires the Rab GTPase Rab27 isoforms, which usher the small EVs to their release. Conversely, PANX1 knockdown disorganized the small EVs machinery and precludes vesicles extrusion. These data identify a novel signaling nexus between MLKL, Rab27, and PANX1, and propose ways to interfere with small EV generation.allows the pseudokinase to insert into the PM and compromise cell-membrane integrity leading to the cell's demise [10][11][12][13][14].Shortly before PM collapses, MLKL oligomers trigger the exposure of phosphatidylserine (PS) residues at the cell surface, acting as "eat-me" signals for the surrounding immune cells [15,16]. However, cells with phosphorylated MLKL and flipped PS are not necessarily committed to death and can "resuscitate" provided the stimulus is removed in a timely manner [16,17]. MLKL also governs the dynamic biogenesis and release of small extracellular vesicles (EVs) during the early stages of necroptosis by cells which are otherwise viable [15,18]. These membrane vesicles containing cytosolic material emerge as potent vectors of intercellular communication [19]. How MLKL controls these many functions continues to be elucidated. Hints may come from the discovery that active MLKL targets intracellular organelles in addition to the PM [3], and was demonstrated to regulate constitutive endosomal trafficking [18]. Furthermore, MLKL silencing causes a drastic reduction in the number of nascent intracellular small EVs, also called intraluminal vesicles (ILVs), which are no longer expelled into the extracellular space [18].The mechanism by which MLKL licenses the biogenesis and the release of small EVs out of the cells remains poorly understood. Here, we find that MLKL oligomers also promote the activation of PANX1 hemichannels via the Rab27 small GTPase subfamily during the early stages of necroptosis. Although this Rab27-PANX1 axis appears dispensable for the proper execution of the necroptotic program, it tightly regulates the generation of small EVs. RESULTS MLKL promotes "Leakiness" of the Plasma Membrane during NecroptosisHT-29 colon cancer cells, which constitute a classical model for necroptosis [4,5] were treated with TNFa (T) in the presence of the pan-caspase inhibitor Q-VD-OPh (Q), together with the Smac mimetic Birinapant (S) [20]. Necroptosis was then v...