Pyroptosis, necroptosis, and ferroptosis are well-characterized forms of regulated necrosis that have been associated with human diseases. During regulated necrosis, plasma membrane damage facilitates the movement of ions and molecules across the bilayer, which finally leads to cell lysis and release of intracellular content. Therefore, these types of cell death have an inflammatory phenotype. Each type of regulated necrosis is mediated by a defined machinery comprising protein and lipid molecules. Here, we discuss how the interaction and reshaping of these cellular components are essential and distinctive processes during pyroptosis, necroptosis, and ferroptosis. We point out that although the plasma membrane is the common target in regulated necrosis, different mechanisms of permeabilization have emerged depending on the cell death form. Pore formation by gasdermins (GSDMs) is a hallmark of pyroptosis, while mixed lineage kinase domain-like (MLKL) protein facilitates membrane permeabilization in necroptosis, and phospholipid peroxidation leads to membrane damage in ferroptosis. This diverse repertoire of mechanisms leading to membrane permeabilization contributes to define the specific inflammatory and immunological outcome of each type of regulated necrosis. Current efforts are focused on new therapies that target critical protein and lipid molecules on these pathways to fight human pathologies associated with inflammation.

Actinoporins have emerged as archetypal α-pore-forming toxins (PFTs) that promote the formation of pores in membranes upon oligomerization and insertion of an α-helix pore-forming domain in the bilayer. These proteins have been used as active components of immunotoxins, therefore, understanding their lytic mechanism is crucial for developing this and other applications. However, the mechanism of how the biophysical properties of the membrane modulate the properties of pores generated by actinoporins remains unclear. Here we studied the effect of membrane fluidity on the permeabilizing activity of sticholysin I (St I), a toxin that belongs to the actinoporins family of α-PFTs. To modulate membrane fluidity we used vesicles made of an equimolar mixture of phosphatidylcholine (PC) and egg sphingomyelin (eggSM), in which PC contained fatty acids of different acyl chain lengths and degrees of unsaturation. Our detailed single-vesicle analysis revealed that when membrane fluidity is high, most of the vesicles are partially permeabilized in a graded manner. In contrast, more rigid membranes can be either completely permeabilized or not, indicating an all-or-none mechanism. Altogether, our results reveal that St I pores can be heterogeneous in size and stability, and that these properties depend on the fluid state of the lipid bilayer. We propose that membrane fluidity at different regions of cellular membranes is a key factor to modulate the activity of the actinoporins, which has implications for the design of different therapeutic strategies based on their lytic action.

Summary: Ferroptosis is an iron-dependent form of regulated necrosis associated with lipid peroxidation. Despite its key role in the inflammatory outcome of ferroptosis, little is known about the molecular events leading to the disruption of the plasma membrane during this type of cell death. Here we show that a sustained increase in cytosolic Ca ²⁺ is a hallmark of ferroptosis that precedes complete bursting of the cell. We report that plasma membrane damage leading to ferroptosis is associated with membrane nanopores of few nanometers in radius and that ferroptosis, but not lipid peroxidation, can be delayed by osmoprotectants. Importantly, Ca ²⁺ fluxes during ferroptosis correlate with the activation of ESCRT-III-mediated membrane repair, which counterbalances the kinetics of cell death and modulates the inflammatory signature of ferroptosis. Our findings with ferroptosis provide a unifying concept that sustained high levels of cytosolic Ca ²⁺ prior to plasma membrane disruption are a common feature of regulated necrosis and position ESCRT-III as a general protective mechanism in these inflammatory cell death pathways.