Lipid unsaturation promotes BAX and BAK pore activity during apoptosis.

BAX and BAK are proapoptotic members of the BCL2 family that directly mediate mitochondrial outer membrane permeabilition (MOMP), a central step in apoptosis execution. However, the molecular architecture of the mitochondrial apoptotic pore remains a key open question and especially little is known about the contribution of lipids to MOMP. By performing a comparative lipidomics analysis of the proximal membrane environment of BAK isolated in lipid nanodiscs, we find a significant enrichment of unsaturated species nearby BAK and BAX in apoptotic conditions. We then demonstrate that unsaturated lipids promote BAX pore activity in model membranes, isolated mitochondria and cellular systems, which is further supported by molecular dynamics simulations. Accordingly, the fatty acid desaturase FADS2 not only enhances apoptosis sensitivity, but also the activation of the cGAS/STING pathway downstream mtDNA release. The correlation of FADS2 levels with the sensitization to apoptosis of different lung and kidney cancer cell lines by co-treatment with unsaturated fatty acids supports the relevance of our findings. Altogether, our work provides an insight on how local lipid environment affects BAX and BAK function during apoptosis.

The regulation of the apoptotic pore-An immunological tightrope walk.

Apoptotic pore formation in mitochondria is the pivotal point for cell death during mitochondrial apoptosis. It is regulated by BCL-2 family proteins in response to various cellular stress triggers and mediates mitochondrial outer membrane permeabilization (MOMP). This allows the release of mitochondrial contents into the cytosol, which triggers rapid cell death and clearance through the activation of caspases. However, under conditions of low caspase activity, the mitochondrial contents released into the cytosol through apoptotic pores serve as inflammatory signals and activate various inflammatory responses. In this chapter, we discuss how the formation of the apoptotic pore is regulated by BCL-2 proteins as well as other cellular or mitochondrial proteins and membrane lipids. Moreover, we highlight the importance of sublethal MOMP in the regulation of mitochondrial-activated inflammation and discuss its physiological consequences in the context of pathogen infection and disease and how it can potentially be exploited therapeutically, for example to improve cancer treatment.

Ferroptosis in health and disease.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis.

Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1.

In cells, mitochondria undergo constant fusion and fission. An essential factor for fission is the mammalian dynamin-related protein 1 (Drp1). Dysregulation of Drp1 is associated with neurodegenerative diseases including Parkinson's, cardiovascular diseases and cancer, making Drp1 a pivotal biomarker for monitoring mitochondrial status and potential pathophysiological conditions. Here, we developed nanobodies (Nbs) as versatile binding molecules for proteomics, advanced microscopy and live cell imaging of Drp1. To specifically enrich endogenous Drp1 with interacting proteins for proteomics, we functionalized high-affinity Nbs into advanced capture matrices. Furthermore, we detected Drp1 by bivalent Nbs combined with site-directed fluorophore labelling in super-resolution STORM microscopy. For real-time imaging of Drp1, we intracellularly expressed fluorescently labelled Nbs, so-called chromobodies (Cbs). To improve the signal-to-noise ratio, we further converted Cbs into a "turnover-accelerated" format. With these imaging probes, we visualized the dynamics of endogenous Drp1 upon compound-induced mitochondrial fission in living cells. Considering the wide range of research applications, the presented Nb toolset will open up new possibilities for advanced functional studies of Drp1 in disease-relevant models.

Many paths lead to immunology.

While some people pore over the textbook and train through the classics of the field, many scientists come to immunology when they discover it intersecting with their "first love" interests. Five of these "accidental immunologists" tell us how they found their way to a fascination with the immune system.

Novel meriolin derivatives activate the mitochondrial apoptosis pathway in the presence of antiapoptotic Bcl-2.

Meriolin derivatives represent a new class of kinase inhibitors with a pronounced cytotoxic potential. Here, we investigated a newly synthesized meriolin derivative (termed meriolin 16) that displayed a strong apoptotic potential in Jurkat leukemia and Ramos lymphoma cells. Meriolin 16 induced apoptosis in rapid kinetics (within 2-3 h) and more potently (IC50: 50 nM) than the previously described derivatives meriolin 31 and 36 [1]. Exposure of Ramos cells to meriolin 16, 31, or 36 for 5 min was sufficient to trigger severe and irreversible cytotoxicity. Apoptosis induction by all three meriolin derivatives was independent of death receptor signaling but required caspase-9 and Apaf-1 as central mediators of the mitochondrial death pathway. Meriolin-induced mitochondrial toxicity was demonstrated by disruption of the mitochondrial membrane potential (ΔΨm), mitochondrial release of proapoptotic Smac, processing of the dynamin-like GTPase OPA1, and subsequent fragmentation of mitochondria. Remarkably, all meriolin derivatives were able to activate the mitochondrial death pathway in Jurkat cells, even in the presence of the antiapoptotic Bcl-2 protein. In addition, meriolins were capable of inducing cell death in imatinib-resistant K562 and KCL22 chronic myeloid leukemia cells as well as in cisplatin-resistant J82 urothelial carcinoma and 2102EP germ cell tumor cells. Given the frequent inactivation of the mitochondrial apoptosis pathway by tumor cells, such as through overexpression of antiapoptotic Bcl-2, meriolin derivatives emerge as promising therapeutic agents for overcoming treatment resistance.

A guideline on the molecular ecosystem regulating ferroptosis.

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.

7-Dehydrocholesterol is an endogenous suppressor of ferroptosis.

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.

Expanding roles of BCL-2 proteins in apoptosis execution and beyond.

The proteins of the BCL-2 family are known as key regulators of apoptosis, with interactions between family members determining permeabilisation of the mitochondrial outer membrane (MOM) and subsequent cell death. However, the exact mechanism through which they form the apoptotic pore responsible for MOM permeabilisation (MOMP), the structure and specific components of this pore, and what roles BCL-2 proteins play outside of directly regulating MOMP are incompletely understood. Owing to the link between apoptosis dysregulation and disease, the BCL-2 proteins are important targets for drug development. With the development and clinical use of drugs targeting BCL-2 proteins showing success in multiple haematological malignancies, enhancing the efficacy of these drugs, or indeed developing novel drugs targeting BCL-2 proteins is of great interest to treat cancer patients who have developed resistance or who suffer other disease types. Here, we review our current understanding of the molecular mechanism of MOMP, with a particular focus on recently discovered roles of BCL-2 proteins in apoptosis and beyond, and discuss what implications these functions might have in both healthy tissues and disease.

Exploring the Binding Affinity of the ARR2 GARP DNA Binding Domain via Comparative Methods.

Plants have evolved signaling mechanisms such as the multi-step phosphorelay (MSP) to respond to different internal and external stimuli. MSP responses often result in gene transcription regulation that is modulated through transcription factors such as B-type Arabidopsis response regulator (ARR) proteins. Among these proteins, ARR2 is a key component that is expressed ubiquitously and is involved in many aspects of plant development. Although it has been noted that B-type ARRs bind to their cognate genes through a DNA-binding domain termed the GARP domain, little is known about the structure and function of this type of DNA-binding domain; thus, how ARRs bind to DNA at a structural level is still poorly understood. In order to understand how the MSP functions in planta, it is crucial to unravel both the kinetics as well as the structural identity of the components involved in such interactions. For this reason, this work focusses on resolving how the GARP domain of ARR2 (GARP2) binds to the promoter region of ARR5, one of its native target genes in cytokinin signaling. We have established that GARP2 specifically binds to the ARR5 promoter with three different bi-molecular interaction systems-qDPI-ELISA, FCS, and MST-and we also determined the KD of this interaction. In addition, structural modeling of the GARP2 domain confirms that GARP2 entails a HTH motif, and that protein-DNA interaction most likely occurs via the α3-helix and the N-terminal arm of this domain since mutations in this region hinder ARR2's ability to activate transcription.

Pyroptosis Induction and Visualization at the Single-Cell Level Using Optogenetics.

Pyroptosis has been identified as a pro-inflammatory form of programmed cell death. It can be triggered by different stimuli including pathogen invasion or cell stress/danger signals releasing hundreds of proteins upon lysis that cause complex responses in neighboring cells. Pyroptosis is executed by the gasdermin (GSDM) family of proteins which, upon cleavage by caspases, form transmembrane pores that release cytokines to induce inflammation. However, despite the importance of gasdermins in the development of inflammatory diseases and cancer, a lot is still to be understood in the downstream consequences of this cell death pathway. Currently, conventional methods, such as drug treatments or chemically forced oligomerization, are limited in the spatiotemporal analysis of pyroptosis signaling in the cellular population, since all cells are primed for undergoing pyroptosis. Here, we provide a protocol for the application of a novel optogenetics tool called NLS_PhoCl_N-GSDMD_mCherry that enables precise temporal and spatial pyroptosis induction in a confocal microscopy setup, followed by imaging of the cell death process and subsequent quantitative analysis of the experiment. This tool opens new opportunities for the study of pyroptosis activation and of its effects on the bystander cell responses.

Mechanisms of BCL-2 family proteins in mitochondrial apoptosis.

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.

Calcium as a master regulator of ferroptosis and other types of regulated necrosis.

Regulation of proliferation and cell death is fundamental for organismal development and for restoring tissue homeostasis after biological stress. During the last years, several forms of regulated cell death have been discovered that share the loss of plasma membrane integrity as a common hallmark and that are collectively known as regulated necrosis (RN) pathways. During RN, plasma membrane damage is sensed by the cell by increases in the levels of intracellular calcium. Interestingly, cytosolic calcium influx can either lead to cell death or survival, given the versatile role of this ion in regulating multiple signaling processes. Among them, membrane repair enables the cells to tolerate the injury and, even in some conditions, survive. Here, we review calcium signaling in the context of RN pathways, with a focus on ferroptosis, a type of RN in which plasma membrane damage is elicited by the accumulation of oxidized lipids. In contrast, other forms of RN such as necroptosis and pyroptosis require dedicated pore-forming proteins for plasma membrane damage and cell death. We first focus on the current knowledge regarding the contribution of calcium to ferroptosis, and then illustrate the similarities and differences in calcium signaling with necroptosis and pyroptosis. Calcium signaling emerges as a key event in the cellular responses to membrane damage and in the regulation of cell death.

Real-Time Growth Kinetics Analysis of Macromolecular Assemblies in Cells with Single Molecule Resolution.

Single molecule fluorescence microscopy has the unique advantage to provide real-time information on the spatiotemporal assembly of individual protein complexes in cellular membranes. This includes the assembly of proteins into oligomer species of numerous copy numbers. However, there is a need for improved tracing analysis of the real-time growth kinetics of these assemblies in cells with single molecule resolution. Here, we present an automated analysis software to accurately measure the real-time kinetics of assembly of individual high-order oligomer complexes. Our software comes with a simple Graphical User Interface (GUI), is available as a source code and an executable, and can analyze a full data set of several hundred to thousand molecules in less than 2 minutes. Importantly, this software is suitable for the analysis of intracellular protein oligomers, whose stoichiometry is usually more difficult to quantify due to variability in signal detection in the different areas of the cell. We validated our method with simulated ground-truth data and time-lapse images of diffraction-limited oligomeric assemblies of BAX and BAK proteins on mitochondria of cells undergoing apoptosis. Our approach provides the broad community of biologists with a fast, user-friendly tool to trace the compositional evolution of macromolecular assemblies, and potentially model their growth for a deeper understanding of the structural and biophysical mechanisms underlying their functions.

Mitochondrial pores at the crossroad between cell death and inflammatory signaling.

Mitochondria are cellular organelles with a major role in many cellular processes, including not only energy production, metabolism, and calcium homeostasis but also regulated cell death and innate immunity. Their proteobacterial origin makes them a rich source of potent immune agonists, normally hidden within the mitochondrial membrane barriers. Alteration of mitochondrial permeability through mitochondrial pores thus provides efficient mechanisms not only to communicate mitochondrial stress to the cell but also as a key event in the integration of cellular responses. In this regard, eukaryotic cells have developed diverse signaling networks that sense and respond to the release of mitochondrial components into the cytosol and play a key role in controlling cell death and inflammatory pathways. Modulating pore formation at mitochondria through direct or indirect mechanisms may thus open new opportunities for therapy. In this review, we discuss the current understanding of the structure and molecular mechanisms of mitochondrial pores and how they function at the interface between cell death and inflammatory signaling to regulate cellular outcomes.

Structural Insights into Polymer-Bounded Lipid Nanodiscs.

Membrane proteins are an essential part of signaling and transport processes and are targeted by multiple drugs. To isolate and investigate them in their native state, polymer-bounded nanodiscs have become valuable tools. In this study, we investigate the lipid model system dimyristoyl-phosphocholine (DMPC) with the nanodisc-forming copolymers styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA). Using small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS), we studied the influence of polymer concentration and temperature on the nanodisc structure. In Tris buffer, the size of nanodiscs formed with SMA is smaller compared to DIBMA at the same polymer ratio. In both cases, the size decreases monotonically with increasing polymer concentration, and this effect is more pronounced when using SMA. Measurements at temperatures (T) between 5 and 30 °C in phosphate buffer showed an incomplete solubilization at high T even at polymer/lipid ratios above that required for complete lipid solubilization. For DIBMA, the nanodiscs developed at lower temperatures are stable and the net repulsion increases, while for SMA, the individual nanodiscs possess smaller sizes and are less affected by T. However, using DLS, one can observe SMA agglomerates at low T. Interestingly, for both polymers, no drastic changes of the observable parameters (radius and bilayer thickness) are seen upon cooling, which would indicate a sharp (first-order) phase transition from liquid-crystalline to gel, but only gradual changes. Hence, we conclude that the transition from a gel toward a liquid-crystalline lipid phase proceeds over a broad T-range compared to a continuous lipid bilayer. These results can pave the way toward the development of better protocols for studying membrane proteins stabilized in this type of membrane mimics.

Reversible Live-Cell Labeling with Retro-engineered HaloTags Enables Long-Term High- and Super-Resolution Imaging.

Self-labeling enzymes (SLE) such as the HaloTag have emerged as powerful tools in high and super-resolution fluorescence microscopy. Newly developed fluorogenic SLE substrates enable imaging in the presence of excess dye. To exploit this feature for reversible labeling, we engineered two variants of HaloTag7 with restored dehalogenase activity. Kinetic studies in vitro showed different turnover kinetics for reHaloTagS (≈0.006 s-1 ) and reHaloTagF (≈0.055 s-1 ). Imaging by confocal and stimulated emission depletion microscopy yielded 3-5-time enhanced photostability of reHaloTag labeling. Prominently, single molecule imaging with reHaloTags enabled controlled and stable labeling density over extended time periods. By combination with structured illumination, simultaneous visualization of single molecule diffusion and organellar dynamics was achieved. These applications highlight the potential of reHaloTag labeling for pushing the limits of advanced fluorescence microscopy techniques.

The Important Role of Membrane Fluidity on the Lytic Mechanism of the α-Pore-Forming Toxin Sticholysin I.

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.