Quantitative imaging and semiotic phenotyping of mitochondrial network morphology in live human cells.

The importance of mitochondria in tissue homeostasis, stress responses and human diseases, combined to their ability to transition between various structural and functional states, makes them excellent organelles for monitoring cell health. There is therefore a need for technologies to accurately analyze and quantify changes in mitochondrial organization in a variety of cells and cellular contexts. Here we present an innovative computerized method that enables accurate, multiscale, fast and cost-effective analysis of mitochondrial shape and network architecture from confocal fluorescence images by providing more than thirty features. In order to facilitate interpretation of the quantitative results, we introduced two innovations: the use of Kiviat-graphs (herein named MitoSpider plots) to present highly multidimensional data and visualization of the various mito-cellular configurations in the form of morphospace diagrams (called MitoSigils). We tested our fully automated image analysis tool on rich datasets gathered from live normal human skin cells cultured under basal conditions or exposed to specific stress including UVB irradiation and pesticide exposure. We demonstrated the ability of our proprietary software (named MitoTouch) to sensitively discriminate between control and stressed dermal fibroblasts, and between normal fibroblasts and other cell types (including cancer tissue-derived fibroblasts and primary keratinocytes), showing that our automated analysis captures subtle differences in morphology. Based on this novel algorithm, we report the identification of a protective natural ingredient that mitigates the deleterious impact of hydrogen peroxide (H2O2) on mitochondrial organization. Hence we conceived a novel wet-plus-dry pipeline combining cell cultures, quantitative imaging and semiotic analysis for exhaustive analysis of mitochondrial morphology in living adherent cells. Our tool has potential for broader applications in other research areas such as cell biology and medicine, high-throughput drug screening as well as predictive and environmental toxicology.

Quantitative assessment of mitochondrial morphology relevant for studies on cellular health and environmental toxicity.

Mitochondria are essential organelles that play crucial roles in cellular energy metabolism, calcium signaling and apoptosis. Their importance in tissue homeostasis and stress responses, combined to their ability to transition between various structural and functional states, make them excellent organelles for monitoring cellular health. Quantitative assessment of mitochondrial morphology can therefore provide valuable insights into environmentally-induced cell damage. High-content screening (HCS) provides a powerful tool for analyzing organelles and cellular substructures. We developed a fully automated and miniaturized HCS wet-plus-dry pipeline (MITOMATICS) exploiting mitochondrial morphology as a marker for monitoring cellular health or damage. MITOMATICS uses an in-house, proprietary software (MitoRadar) to enable fast, exhaustive and cost-effective analysis of mitochondrial morphology and its inherent diversity in live cells. We applied our pipeline and big data analytics software to assess the mitotoxicity of selected chemicals, using the mitochondrial uncoupler CCCP as an internal control. Six different pesticides (inhibiting complexes I, II and III of the mitochondrial respiratory chain) were tested as individual compounds and five other pesticides present locally in Occitanie (Southern France) were assessed in combination to determine acute mitotoxicity. Our results show that the assayed pesticides exhibit specific signatures when used as single compounds or chemical mixtures and that they function synergistically to impact mitochondrial architecture. Study of environment-induced mitochondrial damage has the potential to open new fields in mechanistic toxicology, currently underexplored by regulatory toxicology and exposome research. Such exploration could inform health policy guidelines and foster pharmacological intervention, water, air and soil pollution control and food safety.

Should we be afraid of dental robots? / Faut-il avoir peur des robots-dentistes ?

As it seems likely that France, at the forefront in the use of surgical robotic platforms, will authorize the deployment of dental robots in the short term, the purpose of this article is to question what is at stake in this technological revolution, for dental professionals, regulators but also (and above all) for patients: what awareness-raising will the intervention of machines bring? How does robotics reshuffle the cards of the care relationship? What are the ethical and public health issues? After having defined dental robots as non-humanoid, automated surgical devices with exclusive restorative vocation, we will see how the field of dental robotics currently oscillates between hopes and illusions. We will describe the new responsibilities and ethical requirements related to the introduction of these AI-powered technical objects, as well as the necessary safeguards that have to be implemented in order to avoid any moral buffer and to protect patients from any robotization (literally or metaphorically in the sense of 'zombification'). Finally, we will show that ultimately the question of robot-dentists is the natural evolution of unreasonable application of industrial processes to the rationalization of health and ectopic, neo-liberal practices shifting healthcare into a commercial commodity. Patients can participate in reversing this trend, by recalling that the centrality of the human person is the cornerstone of health professions.

Comme il est vraisemblable que la France, à la pointe dans l'utilisation des plateformes robotiques chirugicales, autorise à court terme le déploiement de robots-dentistes sur son territoire, l'objectif de cet article est d'interroger ce qui se joue dans cette révolution technologique, pour les professionnels du dentaire, les régulateurs, mais aussi (et surtout) pour les patients : quelle(s) prise(s) de conscience l'intervention de la machine et les mutations qui lui sont associées peuvent-elles amener ? Comment la robotique rebat-elle les cartes de la relation de soin ? Pour quels enjeux éthiques et de santé publique ? Après avoir défini les robots-dentistes comme des dispositifs chirurgicaux automatisés à vocation réparatrice, pour l'instant non anthropomorphes, nous verrons comment le domaine de la robotique dentaire oscille entre espoirs et illusions. Nous décrirons les nouvelles responsabilités et exigences éthiques liées à l'introduction de ces objets techniques équipés d'intelligence artificielle, ainsi que les nécessaires garde-fous à implémenter pour éviter toute distanciation morale et pour protéger la patientèle de toute robotisation (au sens propre ou au sens métaphorique de « zombification ¼). Nous montrerons enfin que la question du robot-dentiste n'est finalement que l'évolution naturelle de l'application déraisonnée de procédés industriels à la rationalisation de la santé et de pratiques néolibérales ectopiques conduisant à sa marchandisation. Les patients peuvent participer à l'infléchissement de cette tendance, en rappelant que la centralité de la personne humaine constitue la pierre angulaire des métiers de la santé.

Evidence for existence of an apoptosis-inducing BH3-only protein, sayonara, in Drosophila.

Cells need to sense stresses to initiate the execution of the dormant cell death program. Since the discovery of the first BH3-only protein Bad, BH3-only proteins have been recognized as indispensable stress sensors that induce apoptosis. BH3-only proteins have so far not been identified in Drosophila despite their importance in other organisms. Here, we identify the first Drosophila BH3-only protein and name it sayonara. Sayonara induces apoptosis in a BH3 motif-dependent manner and interacts genetically and biochemically with the BCL-2 homologous proteins, Buffy and Debcl. There is a positive feedback loop between Sayonara-mediated caspase activation and autophagy. The BH3 motif of sayonara phylogenetically appeared at the time of the ancestral gene duplication that led to the formation of Buffy and Debcl in the dipteran lineage. To our knowledge, this is the first identification of a bona fide BH3-only protein in Drosophila, thus providing a unique example of how cell death mechanisms can evolve both through time and across taxa.

Mitochondrial morphology is associated with respiratory chain uncoupling in autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is associated with unique changes in mitochondrial metabolism, including elevated respiration rates and morphological alterations. We examined electron transport chain (ETC) complex activity in fibroblasts derived from 18 children with ASD as well as mitochondrial morphology measurements in fibroblasts derived from the ASD participants and four typically developing controls. In ASD participants, symptoms severity was measured by the Social Responsiveness Scale and Aberrant Behavior Checklist. Mixed-model regression demonstrated that alterations in mitochondrial morphology were associated with both ETC Complex I+III and IV activity as well as the difference between ETC Complex I+III and IV activity. The subgroup of ASD participants with relative elevation in Complex IV activity demonstrated more typical mitochondrial morphology and milder ASD related symptoms. This study is limited by sample size given the invasive nature of obtaining fibroblasts from children. Furthermore, since mitochondrial function is heterogenous across tissues, the result may be specific to fibroblast respiration. Previous studies have separately described elevated ETC Complex IV activity and changes in mitochondrial morphology in cells derived from children with ASD but this is the first study to link these two findings in mitochondrial metabolism. The association between a difference in ETC complex I+III and IV activity and normal morphology suggests that mitochondrial in individuals with ASD may require ETC uncoupling to function optimally. Further studies should assess the molecular mechanisms behind these unique metabolic changes.Trial registration: Protocols used in this study were registered in clinicaltrials.gov as NCT02000284 and NCT02003170.

Computational strategies to combat COVID-19: useful tools to accelerate SARS-CoV-2 and coronavirus research.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel virus of the family Coronaviridae. The virus causes the infectious disease COVID-19. The biology of coronaviruses has been studied for many years. However, bioinformatics tools designed explicitly for SARS-CoV-2 have only recently been developed as a rapid reaction to the need for fast detection, understanding and treatment of COVID-19. To control the ongoing COVID-19 pandemic, it is of utmost importance to get insight into the evolution and pathogenesis of the virus. In this review, we cover bioinformatics workflows and tools for the routine detection of SARS-CoV-2 infection, the reliable analysis of sequencing data, the tracking of the COVID-19 pandemic and evaluation of containment measures, the study of coronavirus evolution, the discovery of potential drug targets and development of therapeutic strategies. For each tool, we briefly describe its use case and how it advances research specifically for SARS-CoV-2. All tools are free to use and available online, either through web applications or public code repositories. Contact:evbc@unj-jena.de.

KCTD: A new gene family involved in neurodevelopmental and neuropsychiatric disorders.

The underlying molecular basis for neurodevelopmental or neuropsychiatric disorders is not known. In contrast, mechanistic understanding of other brain disorders including neurodegeneration has advanced considerably. Yet, these do not approach the knowledge accrued for many cancers with precision therapeutics acting on well-characterized targets. Although the identification of genes responsible for neurodevelopmental and neuropsychiatric disorders remains a major obstacle, the few causally associated genes are ripe for discovery by focusing efforts to dissect their mechanisms. Here, we make a case for delving into mechanisms of the poorly characterized human KCTD gene family. Varying levels of evidence support their roles in neurocognitive disorders (KCTD3), neurodevelopmental disease (KCTD7), bipolar disorder (KCTD12), autism and schizophrenia (KCTD13), movement disorders (KCTD17), cancer (KCTD11), and obesity (KCTD15). Collective knowledge about these genes adds enhanced value, and critical insights into potential disease mechanisms have come from unexpected sources. Translation of basic research on the KCTD-related yeast protein Whi2 has revealed roles in nutrient signaling to mTORC1 (KCTD11) and an autophagy-lysosome pathway affecting mitochondria (KCTD7). Recent biochemical and structure-based studies (KCTD12, KCTD13, KCTD16) reveal mechanisms of regulating membrane channel activities through modulation of distinct GTPases. We explore how these seemingly varied functions may be disease related.

TRIM17 and TRIM28 antagonistically regulate the ubiquitination and anti-apoptotic activity of BCL2A1.

BCL2A1 is an anti-apoptotic member of the BCL-2 family that contributes to chemoresistance in a subset of tumors. BCL2A1 has a short half-life due to its constitutive processing by the ubiquitin-proteasome system. This constitutes a major tumor-suppressor mechanism regulating BCL2A1 function. However, the enzymes involved in the regulation of BCL2A1 protein stability are currently unknown. Here, we provide the first insight into the regulation of BCL2A1 ubiquitination. We present evidence that TRIM28 is an E3 ubiquitin-ligase for BCL2A1. Indeed, endogenous TRIM28 and BCL2A1 bind to each other at the mitochondria and TRIM28 knock-down decreases BCL2A1 ubiquitination. We also show that TRIM17 stabilizes BCL2A1 by blocking TRIM28 from binding and ubiquitinating BCL2A1, and that GSK3 is involved in the phosphorylation-mediated inhibition of BCL2A1 degradation. BCL2A1 and its close relative MCL1 are thus regulated by common factors but with opposite outcome. Finally, overexpression of TRIM28 or knock-out of TRIM17 reduced BCLA1 protein levels and restored sensitivity of melanoma cells to BRAF-targeted therapy. Therefore, our data describe a molecular rheostat in which two proteins of the TRIM family antagonistically regulate BCL2A1 stability and modulate cell death.

Database and Bioinformatic Analysis of BCL-2 Family Proteins and BH3-Only Proteins.

BCL-2 proteins correspond to a structurally, functionally, and phylogenetically heterogeneous group of regulators that play crucial roles in the life and death of animal cells. Some of these regulators also represent therapeutic targets in human diseases including cancer. In the omics era, there is great need for easy data retrieval and fast analysis of the molecular players involved in cell death. In this chapter, we present generic and specific computational resources (such as the reference database BCL2DB) as well as bioinformatics tools that can be used to investigate BCL-2 homologs and BH3-only proteins.

KCTD7 deficiency defines a distinct neurodegenerative disorder with a conserved autophagy-lysosome defect.

OBJECTIVE: Several small case series identified KCTD7 mutations in patients with a rare autosomal recessive disorder designated progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis (CLN14). Despite the name KCTD (potassium channel tetramerization domain), KCTD protein family members lack predicted channel domains. We sought to translate insight gained from yeast studies to uncover disease mechanisms associated with deficiencies in KCTD7 of unknown function. METHODS: Novel KCTD7 variants in new and published patients were assessed for disease causality using genetic analyses, cell-based functional assays of patient fibroblasts and knockout yeast, and electron microscopy of patient samples. RESULTS: Patients with KCTD7 mutations can exhibit movement disorders or developmental regression before seizure onset, and are distinguished from similar disorders by an earlier age of onset. Although most published KCTD7 patient variants were excluded from a genome sequence database of normal human variations, most newly identified patient variants are present in this database, potentially challenging disease causality. However, genetic analysis and impaired biochemical interactions with cullin 3 support a causal role for patient KCTD7 variants, suggesting deleterious alleles of KCTD7 and other rare disease variants may be underestimated. Both patient-derived fibroblasts and yeast lacking Whi2 with sequence similarity to KCTD7 have impaired autophagy consistent with brain pathology. INTERPRETATION: Biallelic KCTD7 mutations define a neurodegenerative disorder with lipofuscin and lipid droplet accumulation but without defining features of neuronal ceroid lipofuscinosis or lysosomal storage disorders. KCTD7 deficiency appears to cause an underlying autophagy-lysosome defect conserved in yeast, thereby assigning a biological role for KCTD7. Ann Neurol 2018;84:774-788.

Whi2 is a conserved negative regulator of TORC1 in response to low amino acids.

Yeast WHI2 was originally identified in a genetic screen for regulators of cell cycle arrest and later suggested to function in general stress responses. However, the function of Whi2 is unknown. Whi2 has predicted structure and sequence similarity to human KCTD family proteins, which have been implicated in several cancers and are causally associated with neurological disorders but are largely uncharacterized. The identification of conserved functions between these yeast and human proteins may provide insight into disease mechanisms. We report that yeast WHI2 is a new negative regulator of TORC1 required to suppress TORC1 activity and cell growth specifically in response to low amino acids. In contrast to current opinion, WHI2 is dispensable for TORC1 inhibition in low glucose. The only widely conserved mechanism that actively suppresses both yeast and mammalian TORC1 specifically in response to low amino acids is the conserved SEACIT/GATOR1 complex that inactivates the TORC1-activating RAG-like GTPases. Unexpectedly, Whi2 acts independently and simultaneously with these established GATOR1-like Npr2-Npr3-Iml1 and RAG-like Gtr1-Gtr2 complexes, and also acts independently of the PKA pathway. Instead, Whi2 inhibits TORC1 activity through its binding partners, protein phosphatases Psr1 and Psr2, which were previously thought to only regulate amino acid levels downstream of TORC1. Furthermore, the ability to suppress TORC1 is conserved in the SKP1/BTB/POZ domain-containing, Whi2-like human protein KCTD11 but not other KCTD family members tested.

Comment on "Sterilizing immunity in the lung relies on targeting fungal apoptosis-like programmed cell death".

Shlezinger et al (Reports, 8 September 2017, p. 1037) report that the common fungus Aspergillus fumigatus, a cause of aspergillosis, undergoes caspase-dependent apoptosis-like cell death triggered by lung neutrophils. However, the technologies they used do not provide reliable evidence that fungal cells die via a protease signaling cascade thwarted by a fungal caspase inhibitor homologous to human survivin.

Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins.

The morphology of a population of mitochondria is the result of several interacting dynamical phenomena, including fission, fusion, movement, elimination and biogenesis. Each of these phenomena is controlled by underlying molecular machinery, and when defective can cause disease. New understanding of the relationships between form and function of mitochondria in health and disease is beginning to be unraveled on several fronts. Studies in mammals and model organisms have revealed that mitochondrial morphology, dynamics and function appear to be subject to regulation by the same proteins that regulate apoptotic cell death. One protein family that influences mitochondrial dynamics in both healthy and dying cells is the Bcl-2 protein family. Connecting mitochondrial dynamics with life-death pathway forks may arise from the intersection of Bcl-2 family proteins with the proteins and lipids that determine mitochondrial shape and function. Bcl-2 family proteins also have multifaceted influences on cells and mitochondria, including calcium handling, autophagy and energetics, as well as the subcellular localization of mitochondrial organelles to neuronal synapses. The remarkable range of physical or functional interactions by Bcl-2 family proteins is challenging to assimilate into a cohesive understanding. Most of their effects may be distinct from their direct roles in apoptotic cell death and are particularly apparent in the nervous system. Dual roles in mitochondrial dynamics and cell death extend beyond BCL-2 family proteins. In this review, we discuss many processes that govern mitochondrial structure and function in health and disease, and how Bcl-2 family proteins integrate into some of these processes.

Systemic Delivery of Tumor-Targeted Bax-Derived Membrane-Active Peptides for the Treatment of Melanoma Tumors in a Humanized SCID Mouse Model.

Melanoma is a highly metastatic and deadly form of cancer. Invasive melanoma cells overexpress integrin αvß3, which is a well-known target for Arg-Gly-Asp-based (RGD) peptides. We developed a sophisticated method to synthetize milligram amounts of a targeted vector that allows the RGD-mediated targeting, internalization, and release of a mitochondria-disruptive peptide derived from the pro-apoptotic Bax protein. We found that 2.5 µM Bax[109-127] was sufficient to destabilize the mitochondria in ten different tumor cell lines, even in the presence of the anti-apoptotic Bcl2 protein, which is often involved in tumor resistance. This pore-forming peptide displayed antitumor activity when it was covalently linked by a disulfide bridge to the tetrameric RAFT-c[RGD]4-platform and after intravenous injection in a human melanoma tumor model established in humanized immuno-competent mice. In addition to its direct toxic effect, treatment with this combination induced the release of the immuno-stimulating factor monocyte chimoattractant protein 1 (MCP1) in the blood and a decrease in the level of the pro-angiogenic factor FGF2. Our novel multifunctional, apoptosis-inducing agent could be further customized and assayed for potential use in tumor-targeted therapy.

Unexpected cross-reactivity of anti-cathepsin B antibodies leads to uncertainties regarding the mechanism of action of anti-CD20 monoclonal antibody GA101.

GA101, also known as obinutuzumab or Gazyva (Gazyvaro), is a glycoengineered type II humanized antibody that targets the CD20 antigen expressed at the surface of B-cells. This novel anti-CD20 antibody is currently assessed in clinical trials with promising results as a single agent or as part of therapeutic combinations for the treatment of B-cell malignancies. Detailed understanding of the mechanisms of GA101-induced cell death is needed to get insight into possible resistance mechanisms occurring in patients. Although multiple in vitro and in vivo mechanisms have been suggested to describe the effects of GA101 on B-cells, currently available data are ambiguous. The aim of our study was to clarify the cellular mechanisms involved in GA101-induced cell death in vitro, and more particularly the respective roles played by lysosomal and mitochondrial membrane permeabilization. Our results confirm previous reports suggesting that GA101 triggers homotypic adhesion and caspase-independent cell death, two processes that are dependent on actin remodeling and involve the production of reactive oxygen species. With respect to lysosomal membrane permeabilization (LMP), our data suggest that lack of specificity of available antibodies directed against cathepsin B may have confounded previously published results, possibly challenging current LMP-driven model of GA101 action mode.

Quantification and Characterization of UVB-Induced Mitochondrial Fragmentation in Normal Primary Human Keratinocytes.

UV irradiation is a major environmental factor causing skin dryness, aging and cancer. UVB in particular triggers cumulative DNA damage, oxidative stress and mitochondrial dysfunction. The objective of our study was to provide both qualitative and quantitative analysis of how mitochondria respond to UVB irradiation in normal human epidermal keratinocytes (NHEK) of healthy donors, with the rationale that monitoring mitochondrial shape will give an indication of cell population fitness and enable the screening of bioactive agents with UVB-protective properties. Our results show that NHEK undergo dose-dependent mitochondrial fragmentation after exposure to UVB. In order to obtain a quantitative measure of this phenomenon, we implemented a novel tool for automated quantification of mitochondrial morphology in live cells based on confocal microscopy and computational calculations of mitochondrial shape descriptors. This method was used to substantiate the effects on mitochondrial morphology of UVB irradiation and of knocking-down the mitochondrial fission-mediating GTPase Dynamin-related protein 1 (DRP1). Our data further indicate that all the major mitochondrial dynamic proteins are expressed in NHEK but that their level changes were stronger after mitochondrial uncoupler treatment than following UVB irradiation or DRP1 knock-down. Our system and procedures might be of interest for the identification of cosmetic or dermatologic UVB-protective agents.

Redefining the BH3 Death Domain as a 'Short Linear Motif'.

B cell lymphoma-2 (BCL-2)-related proteins control programmed cell death through a complex network of protein-protein interactions mediated by BCL-2 homology 3 (BH3) domains. Given their roles as dynamic linchpins, the discovery of novel BH3-containing proteins has attracted considerable attention. However, without a clearly defined BH3 signature sequence the BCL-2 family has expanded to include a nebulous group of nonhomologous BH3-only proteins, now justified by an intriguing twist. We present evidence that BH3s from both ordered and disordered proteins represent a new class of short linear motifs (SLiMs) or molecular recognition features (MoRFs) and are diverse in their evolutionary histories. The implied corollaries are that BH3s have a broad phylogenetic distribution and could potentially bind to non-BCL-2-like structural domains with distinct functions.

Differential mechanisms of asparaginase resistance in B-type acute lymphoblastic leukemia and malignant natural killer cell lines.

Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an important drug for treating patients with acute lymphoblastic leukaemia (ALL) and natural killer (NK) cell lymphoma. Although different native or pegylated ASNase-based chemotherapy are efficient, disease relapse is frequently observed, especially in adult patients. The neo-synthesis of Asn by asparagine synthetase (AsnS) following ASNase treatment, which involves the amino acid response and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways, is believed to be the basis of ASNase-resistance mechanisms. However, AsnS expression has not emerged as an accurate predictive factor for ASNase susceptibility. The aim of this study was to identify possible ASNase sensitivity/resistance-related genes or pathways using a new asparaginase, namely a pegylated r-crisantaspase, with a focus on classic Asn-compensatory responses and cell death under conditions of Asn/L-glutamine limitation. We show that, for B-ALL cell lines, changes in the expression of apoptosis-regulatory genes (especially NFκB-related genes) are associated with ASNase susceptibility. The response of malignant NK cell lines to ASNase may depend on Asn-compensatory mechanisms and other cellular processes such as cleavage of BCL2A1, a prosurvival member of the Bcl-2 protein family. These results suggest that according to cellular context, factors other than AsnS can influence ASNase susceptibility.

[From dualism to multiplicity: seeing BCL-2 family proteins and cell death with new eyes]. / Du duel au pluriel: réflexions sur les protéines régulatrices d'apoptose de la famille BCL-2 et sur la mort cellulaire.

The concept of cell death has many links to the concept of death itself, defined as the opposite of life. Achievements obtained through research on apoptosis have apparently allowed us to transcend this Manichean view. Death is no longer outside, but rather inside living systems, as a constitutive force at work within the living matter. Whereas the death of cells can be positive and breed "creation" (e.g. during morphogenesis), its dysregulation can also cause or contribute to fatal diseases including cancer. It is tempting to apply this biological discourse to illuminate the relations between life and death, taken in general terms, but does this generalization actually hold? Is this discourse not essentially a metaphor? If cell death is considered as a vital aspect of various biological processes, then are we not faced with some vitalistic conception of death? Are there one or more meanings to the word "death"? Does the power to self-destruct act in opposition to other key features of living entities, or rather in juxtaposition to them? In this article, we first describe how the field of cell death has been developed on the basis of perceived and built dichotomies, mirroring the original opposition between life and death. We detail the limitations of the current paradigm of apoptosis regulation by BCL-2 family proteins, which nicely illustrate the problem of binary thinking in biology. Last, we try to show a way out of this dualistic matrix, by drawing on the notions of multiplicity, complexity, diversity, evolution and contingency.