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The proteins of the BCL-2 family are key regulators of mitochondrial apoptosis, acting as either promoters or inhibitors of cell death. The functional interplay and balance between the opposing BCL-2 family members control permeabilization of the outer mitochondrial membrane, leading to the release of activators of the caspase cascade into the cytosol and ultimately resulting in cell death. Despite considerable research, our knowledge about the mechanisms of the BCL-2 family of proteins remains insufficient, which complicates cell fate predictions and does not allow us to fully exploit these proteins as targets for drug discovery. Detailed understanding of the formation and molecular architecture of the apoptotic pore in the outer mitochondrial membrane remains a holy grail in the field, but new studies allow us to begin constructing a structural model of its arrangement. Recent literature has also revealed unexpected activities for several BCL-2 family members that challenge established concepts of how they regulate mitochondrial permeabilization. In this Review, we revisit the most important advances in the field and integrate them into a new structure–function-based classification of the BCL-2 family members that intends to provide a comprehensive model for BCL-2 action in apoptosis. We close this Review by discussing the potential of drugging the BCL-2 family in diseases characterized by aberrant apoptosis.BCL-2 proteins fulfil important functions in cell death as initiators, guardians and executioners of mitochondrial outer membrane permeabilization. Recent findings demonstrating complex interactions among BCL-2 proteins set forth a comprehensive model of BCL-2 action.

Equinatoxin II is a pore-forming protein of the actinoporin family. After membrane binding, it inserts its N-terminal α-helix and forms a protein/lipid pore. Equinatoxin II activity depends on the presence of sphingomyelin in the target membrane; however, the role of this specificity is unknown. On the other hand, sphingomyelin is considered an essential ingredient of lipid rafts and promotes liquid-ordered/liquid-disordered phase separation in model membranes that mimic raft composition. Here, we used giant unilamellar vesicles to simultaneously investigate the effect of sphingomyelin and phase separation on the membrane binding and permeabilizing activity of Equinatoxin II. Our results show that Equinatoxin II binds preferentially to the liquid-ordered phase over the liquid-disordered one and that it tends to concentrate at domain interfaces. In addition, sphingomyelin strongly enhances membrane binding of the toxin but is not sufficient for membrane permeabilization. Under the same experimental conditions, Equinatoxin II formed pores in giant unilamellar vesicles containing sphingomyelin only when liquid-ordered and -disordered phases coexisted. Our observations demonstrate the importance of phase boundaries for Equinatoxin II activity and suggest a double role of sphingomyelin as a specific receptor for the toxin and as a promoter of the membrane organization necessary for Equinatoxin II action.

Ferroptosis is a lytic, iron-dependent form of regulated cell death characterized by excessive lipid peroxidation and associated with necrosis spread in diseased tissues through unknown mechanisms. Using a novel optogenetic system for light-driven ferroptosis induction via degradation of the anti-ferroptotic protein GPX4, we show that lipid peroxidation and ferroptotic death can spread to neighboring cells through their closely adjacent plasma membranes. Ferroptosis propagation is dependent on cell distance and completely abolished by disruption of α-catenin-dependent intercellular contacts or by chelation of extracellular iron. Remarkably, bridging cells with a lipid bilayer or increasing contacts between neighboring cells enhances ferroptosis spread. Reconstitution of iron-dependent spread of lipid peroxidation between pure lipid, contacting liposomes provides evidence for the physicochemical mechanism involved. Our findings support a model in which iron-dependent lipid peroxidation propagates across proximal plasma membranes of neighboring cells, thereby promoting the transmission of ferroptotic cell death with consequences for pathological tissue necrosis spread. Roeck et al. show that ferroptosis spreads between neighboring cells via lipid peroxidation across adjacent membranes. Cell contact and extracellular iron are key factors, highlighting a potential mechanism for tissue necrosis propagation.

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.

The Bcl-2 proteins form a complex interaction network that controls mitochondrial permeabilization and apoptosis. The relative importance of different Bcl-2 complexes and their spatio-temporal regulation is debated. Using fluorescence cross-correlation spectroscopy to quantify the interactions within a minimal Bcl-2 network, comprised by cBid, Bax, and Bcl-xL, we show that membrane insertion drastically alters the pattern of Bcl-2 complexes, and that the C-terminal helix of Bcl-xL determines its binding preferences. At physiological temperature, Bax can spontaneously activate in a self-amplifying process. Strikingly, Bax also recruits Bcl-xL to membranes, which is sufficient to retrotranslocate Bax back into solution to secure membrane integrity. Our study disentangles the hierarchy of Bcl-2 complex formation in relation to their environment: Bcl-xL association with cBid occurs in solution and in membranes, where the complex is stabilized, whereas Bcl-xL binding to Bax occurs only in membranes and with lower affinity than to cBid, leading instead to Bax retrotranslocation. The permeabilization of the mitochondrial outer membrane to induce apoptosis is regulated by complex interactions between Bcl-2 family members. Here the authors develop a quantitative interactome of a membrane Bcl-2 network and identify a hierarchy of protein complexes in apoptosis induction.

Super-resolution microscopy allows imaging of cellular structures with high throughput and detail. However, the efficient and quantitative analysis of images generated is challenging with existing tools. Here, we develop ASAP (automated structures analysis program) to enable rapid and automated detection, classification and quantification of super-resolved structures. We validate ASAP on ground truth data and demonstrate its broad applicability by analyzing images of nucleoporins, TORC1 complexes, endocytic vesicles and Bax pores.

The BH3-only class of Bcl-2 family proteins triggers mitochondrial apoptosis. Several mechanisms are used to restrain the pro-apoptotic activity of these proteins. Dynein light chain (DYNLL) 1 and 2 has been proposed to negatively regulate the activity of Bim and Bmf, respectively, and the Bim–DYNLL1 interaction leads to the formation of large protein complexes on mitochondria. Here we found that Bim and Bmf interact with both isoforms of DYNLL (DYNLL1 and DYNLL2). DYNLL1/2 not only induced homo-dimerization of Bim and Bmf but also led to the formation of ternary complexes (Bim–DYNLL–Bmf), both in cell-free and in cellular systems. DYNLL-induced oligomerization stabilized Bmf in cultured cells and inhibited its degradation by the ubiquitin-independent 20S proteasome in a cell-free system. Surprisingly, overexpression of wild-type Bmf but not of a DYNLL-binding-deficient mutant induced degradation of endogenous Bim in different cell lines, but both variants sensitized to apoptosis. Mutant Bmf incapable of interacting with anti-apoptotic Bcl-2 proteins and of inducing apoptosis still caused Bim degradation. These results suggest that Bmf overexpression-induced Bim degradation is not due to the displacement of Bim from anti-apoptotic Bcl-2 proteins but a direct consequence of the modulation of Bim–DYNLL association. A peptide derived from the DYNLL-binding domain of Bim also led to the degradation of Bim as well as of its preferred binding partner Mcl-1. Thus DYNLL regulates the mitochondrial pathway of apoptosis by determining the stability of Bmf, Bim, and Mcl-1 proteins.

Bax is a Bcl-2 protein crucial for apoptosis initiation and execution, whose active conformation is only partially understood. Dipolar EPR spectroscopy has proven to be a valuable tool to determine coarse-grained models of membrane-embedded Bcl-2 proteins. Here we show how the combination of spectroscopically distinguishable nitroxide and gadolinium spin labels and Double Electron-Electron Resonance can help to gain new insights into the quaternary structure of active, membrane-embedded Bax oligomers. We show that attaching labels bulkier than the conventional MTSL may affect Bax fold and activity, depending on the protein/label combination. However, we identified a suitable pair of spectroscopically distinguishable labels, which allows to study complex distance networks in the oligomers that could not be disentangled before. Additionally, we compared the stability of the different spin-labeled protein variants in E . coli and HeLa cell extracts. We found that the gem -diethyl nitroxide-labeled Bax variants were reasonably stable in HeLa cell extracts. However, when transferred into human cells, Bax was found to be mislocalized, thus preventing its characterization in a physiological environment. The successful use of spectroscopically distinguishable labels on membrane-embedded Bax-oligomers opens an exciting new path towards structure determination of membrane-embedded homo- or hetero-oligomeric Bcl-2 proteins via EPR.

Ferroptosis, an intricately regulated form of cell death characterized by uncontrolled lipid peroxidation, has garnered substantial interest since this term was first coined in 2012. Recent years have witnessed remarkable progress in elucidating the detailed molecular mechanisms that govern ferroptosis induction and defence, with particular emphasis on the roles of heterogeneity and plasticity. In this Review, we discuss the molecular ecosystem of ferroptosis, with implications that may inform and enable safe and effective therapeutic strategies across a broad spectrum of diseases. In this Review, Dai, Stockwell, Kroemer, Tang and colleagues offer a comprehensive discussion of the molecular regulation of ferroptosis and highlight how this may be potentially leveraged for therapeutic benefit for disease treatment.