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.

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.

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.

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.

The interplay between BAX and BAK tunes apoptotic pore growth to control mitochondrial-DNA-mediated inflammation.

BAX and BAK are key apoptosis regulators that mediate the decisive step of mitochondrial outer membrane permeabilization. However, the mechanism by which they assemble the apoptotic pore remains obscure. Here, we report that BAX and BAK present distinct oligomerization properties, with BAK organizing into smaller structures with faster kinetics than BAX. BAK recruits and accelerates BAX assembly into oligomers that continue to grow during apoptosis. As a result, BAX and BAK regulate each other as they co-assemble into the same apoptotic pores, which we visualize. The relative availability of BAX and BAK molecules thereby determines the growth rate of the apoptotic pore and the relative kinetics by which mitochondrial contents, most notably mtDNA, are released. This feature of BAX and BAK results in distinct activation kinetics of the cGAS/STING pathway with implications for mtDNA-mediated paracrine inflammatory signaling.

DRP1 interacts directly with BAX to induce its activation and apoptosis.

The apoptotic executioner protein BAX and the dynamin-like protein DRP1 co-localize at mitochondria during apoptosis to mediate mitochondrial permeabilization and fragmentation. However, the molecular basis and functional consequences of this interplay remain unknown. Here, we show that BAX and DRP1 physically interact, and that this interaction is enhanced during apoptosis. Complex formation between BAX and DRP1 occurs exclusively in the membrane environment and requires the BAX N-terminal region, but also involves several other BAX surfaces. Furthermore, the association between BAX and DRP1 enhances the membrane activity of both proteins. Forced dimerization of BAX and DRP1 triggers their activation and translocation to mitochondria, where they induce mitochondrial remodeling and permeabilization to cause apoptosis even in the absence of apoptotic triggers. Based on this, we propose that DRP1 can promote apoptosis by acting as noncanonical direct activator of BAX through physical contacts with its N-terminal region.

BCL-2-family protein tBID can act as a BAX-like effector of apoptosis.

During apoptosis, the BCL-2-family protein tBID promotes mitochondrial permeabilization by activating BAX and BAK and by blocking anti-apoptotic BCL-2 members. Here, we report that tBID can also mediate mitochondrial permeabilization by itself, resulting in release of cytochrome c and mitochondrial DNA, caspase activation and apoptosis even in absence of BAX and BAK. This previously unrecognized activity of tBID depends on helix 6, homologous to the pore-forming regions of BAX and BAK, and can be blocked by pro-survival BCL-2 proteins. Importantly, tBID-mediated mitochondrial permeabilization independent of BAX and BAK is physiologically relevant for SMAC release in the immune response against Shigella infection. Furthermore, it can be exploited to kill leukaemia cells with acquired venetoclax resistance due to lack of active BAX and BAK. Our findings define tBID as an effector of mitochondrial permeabilization in apoptosis and provide a new paradigm for BCL-2 proteins, with implications for anti-bacterial immunity and cancer therapy.

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.

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy.

Pore formation in regulated cell death.

The discovery of alternative signaling pathways that regulate cell death has revealed multiple strategies for promoting cell death with diverse consequences at the tissue and organism level. Despite the divergence in the molecular components involved, membrane permeabilization is a common theme in the execution of regulated cell death. In apoptosis, the permeabilization of the outer mitochondrial membrane by BAX and BAK releases apoptotic factors that initiate the caspase cascade and is considered the point of no return in cell death commitment. Pyroptosis and necroptosis also require the perforation of the plasma membrane at the execution step, which involves Gasdermins in pyroptosis, and MLKL in the case of necroptosis. Although BAX/BAK, Gasdermins and MLKL share certain molecular features like oligomerization, they form pores in different cellular membranes via distinct mechanisms. Here, we compare and contrast how BAX/BAK, Gasdermins, and MLKL alter membrane permeability from a structural and biophysical perspective and discuss the general principles of membrane permeabilization in the execution of regulated cell death.

MOMP in the absence of BH3-only proteins.

The minimum requirement for mitochondrial apoptosis has been controversial ever since the discovery of BCL-2 as a cell death regulator. In this issue ofGenes&Development, O'Neill and colleagues (pp. 973-988) end a long-standing debate by creating a cellular system free of BCL-2 family proteins, thereby identifying the outer mitochondrial membrane rather than BH3-only proteins as the only requirement for BAX/BAK activation and mitochondrial outer membrane permeabilization (MOMP).

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.

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.

Curcumin and NCLX inhibitors share anti-tumoral mechanisms in microsatellite-instability-driven colorectal cancer.

BACKGROUND AND AIMS: Recent evidences highlight a role of the mitochondria calcium homeostasis in the development of colorectal cancer (CRC). To overcome treatment resistance, we aimed to evaluate the role of the mitochondrial sodium-calcium-lithium exchanger (NCLX) and its targeting in CRC. We also identified curcumin as a new inhibitor of NCLX. METHODS: We examined whether curcumin and pharmacological compounds induced the inhibition of NCLX-mediated mitochondrial calcium (mtCa2+) extrusion, the role of redox metabolism in this process. We evaluated their anti-tumorigenic activity in vitro and in a xenograft mouse model. We analyzed NCLX expression and associations with survival in The Cancer Genome Atlas (TCGA) dataset and in tissue microarrays from 381 patients with microsatellite instability (MSI)-driven CRC. RESULTS: In vitro, curcumin exerted strong anti-tumoral activity through its action on NCLX with mtCa2+ and reactive oxygen species overload associated with a mitochondrial membrane depolarization, leading to reduced ATP production and apoptosis. NCLX inhibition with pharmacological and molecular approaches reproduced the effects of curcumin. NCLX inhibitors decreased CRC tumor growth in vivo. Both transcriptomic analysis of TCGA dataset and immunohistochemical analysis of tissue microarrays demonstrated that higher NCLX expression was associated with MSI status, and for the first time, NCLX expression was significantly associated with recurrence-free survival. CONCLUSIONS: Our findings highlight a novel anti-tumoral mechanism of curcumin through its action on NCLX and mitochondria calcium overload that could benefit for therapeutic schedule of patients with MSI CRC.

TAT-RasGAP317-326 kills cells by targeting inner-leaflet-enriched phospholipids.

TAT-RasGAP317-326 is a cell-penetrating peptide-based construct with anticancer and antimicrobial activities. This peptide kills a subset of cancer cells in a manner that does not involve known programmed cell death pathways. Here we have elucidated the mode of action allowing TAT-RasGAP317-326 to kill cells. This peptide binds and disrupts artificial membranes containing lipids typically enriched in the inner leaflet of the plasma membrane, such as phosphatidylinositol-bisphosphate (PIP2) and phosphatidylserine (PS). Decreasing the amounts of PIP2 in cells renders them more resistant to TAT-RasGAP317-326, while reducing the ability of cells to repair their plasma membrane makes them more sensitive to the peptide. The W317A TAT-RasGAP317-326 point mutant, known to have impaired killing activities, has reduced abilities to bind and permeabilize PIP2- and PS-containing membranes and to translocate through biomembranes, presumably because of a higher propensity to adopt an α-helical state. This work shows that TAT-RasGAP317-326 kills cells via a form of necrosis that relies on the physical disruption of the plasma membrane once the peptide targets specific phospholipids found on the cytosolic side of the plasma membrane.

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.

Quantitative analysis of super-resolved structures using ASAP.

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.

DeepSinse: deep learning-based detection of single molecules.

MOTIVATION: Imaging single molecules has emerged as a powerful characterization tool in the biological sciences. The detection of these under various noise conditions requires the use of algorithms that are dependent on the end-user inputting several parameters, the choice of which can be challenging and subjective. RESULTS: In this work, we propose DeepSinse, an easily trainable and useable deep neural network that can detect single molecules with little human input and across a wide range of signal-to-noise ratios. We validate the neural network on the detection of single bursts in simulated and experimental data and compare its performance with the best-in-class, domain-specific algorithms. AVAILABILITYAND IMPLEMENTATION: Ground truth ROI simulating code, neural network training, validation code, classification code, ROI picker, GUI for simulating, training and validating DeepSinse as well as pre-trained networks are all released under the MIT License on www.github.com/jdanial/DeepSinse. The dSTORM dataset processing code is released under the MIT License on www.github.com/jdanial/StormProcessor. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Structural model of active Bax at the membrane.

Bax plays a central role in the mitochondrial pathway of apoptosis. Upon activation, cytosolic Bax monomers oligomerize on the surface of mitochondria and change conformation concertedly to punch holes into the outer membrane. The subsequent release of cytochrome c initiates cell death. However, the structure of membrane-inserted Bax and its mechanism of action remain largely unknown. Here, we propose a 3D model of active Bax at the membrane based on double electron-electron resonance (DEER) spectroscopy in liposomes and isolated mitochondria. We show that active Bax is organized at the membrane as assemblies of dimers. In addition to a stable dimerization domain, each monomer contains a more flexible piercing domain involved in interdimer interactions and pore formation. The most important structural change during Bax activation is the opening of the hairpin formed by helices 5 and 6, which adopts a clamp-like conformation central to the mechanism of mitochondrial permeabilization.