Systematic compositional analysis of exosomal extracellular vesicles produced by cells undergoing apoptosis, necroptosis and ferroptosis.

Formation of extracellular vesicles (EVs) has emerged as a novel paradigm in cell-to-cell communication in health and disease. EVs are notably produced during cell death but it had remained unclear whether different modalities of regulated cell death (RCD) influence the biogenesis and composition of EVs. To this end, we performed a comparative analysis of steady-state (ssEVs) and cell death-associated EVs (cdEVs) following TNF-induced necroptosis (necEVs), anti-Fas-induced apoptosis (apoEVs), and ML162-induced ferroptosis (ferEVs) using the same cell line. For each RCD condition, we determined the biophysical and biochemical characteristics of the cell death-associated EVs (cdEVs), the protein cargo, and the presence of methylated ribosomal RNA. We found that the global protein content of all cdEVs was increased compared to steady-state EVs. Qualitatively, the isolated exosomal ssEVs and cdEVs, contained a largely overlapping protein cargo including some quantitative differences in particular proteins. All cdEVs were enriched for proteins involved in RNA splicing and nuclear export, and showed distinctive rRNA methylation patterns compared to ssEVs. Interestingly, necEVs and apoEVs, but strikingly not ferEVs, showed enrichment of proteins involved in ribosome biogenesis. Altogether, our work documents quantitative and qualitative differences between ssEVs and cdEVs.

A guide to the expanding field of extracellular vesicles and their release in regulated cell death programs.

Homeostasis disruption is visible at the molecular and cellular levels and may often lead to cell death. This vital process allows us to maintain the more extensive system's integrity by keeping the different features (genetic, metabolic, physiologic, and individual) intact. Interestingly, while cells can die in different manners, dying cells still communicate with their environment. This communication was, for a long time, perceived as only driven by the release of soluble factors. However, it has now been reconsidered with the increasing interest in extracellular vesicles (EVs), which are discovered to be released during different regulated cell death programs, with the observation of specific effects. EVs are game changers in the paradigm of cell-cell communication with tremendous implications in fundamental research with regard to noncell autonomous functions, as well as in biomarkers research, all of which are geared toward diagnostic and therapeutic purposes. This review is composed of two main parts. The first is a comprehensive presentation of the state of the art of the EV field at large. In the second part, we focus on EVs discovered to be released during different regulated cell death programs, also known as cell death EVs (cdEVs), and EV-associated specific effects on recipient cells in the context of cell death and inflammation/inflammatory responses.

Phase separation-based visualization of protein-protein interactions and kinase activities in plants.

Protein activities depend heavily on protein complex formation and dynamic posttranslational modifications, such as phosphorylation. The dynamic nature of protein complex formation and posttranslational modifications is notoriously difficult to monitor in planta at cellular resolution, often requiring extensive optimization. Here, we generated and exploited the SYnthetic Multivalency in PLants (SYMPL)-vector set to assay protein-protein interactions (PPIs) (separation of phases-based protein interaction reporter) and kinase activities (separation of phases-based activity reporter of kinase) in planta, based on phase separation. This technology enabled easy detection of inducible, binary and ternary PPIs among cytoplasmic and nuclear proteins in plant cells via a robust image-based readout. Moreover, we applied the SYMPL toolbox to develop an in vivo reporter for SNF1-related kinase 1 activity, allowing us to visualize tissue-specific, dynamic SnRK1 activity in stable transgenic Arabidopsis (Arabidopsis thaliana) plants. The SYMPL cloning toolbox provides a means to explore PPIs, phosphorylation, and other posttranslational modifications with unprecedented ease and sensitivity.

Spectrally Tunable Förster Resonance Energy Transfer-Based Biosensors Using Organic Dye Grafting.

Biosensors based on Förster resonance energy transfer (FRET) have revolutionized cellular biology by allowing the direct measurement of biochemical processes in situ. Many genetically encoded sensors make use of fluorescent proteins that are limited in spectral versatility and that allow few ways to change the spectral properties once the construct has been created. In this work, we developed genetically encoded FRET biosensors based on the chemigenetic SNAP and HaloTag domains combined with matching organic fluorophores. We found that the resulting constructs can display comparable responses, kinetics, and reversibility compared to their fluorescent protein-based ancestors, but with the added advantage of spectral versatility, including the availability of red-shifted dye pairs. However, we also find that the introduction of these tags can alter the sensor readout, showing that careful validation is required before applying such constructs in practice. Overall, our approach delivers an innovative methodology that can readily expand the spectral variety and versatility of FRET-based biosensors.

Keeping Cell Death Alive: An Introduction into the French Cell Death Research Network.

Since the Nobel Prize award more than twenty years ago for discovering the core apoptotic pathway in C. elegans, apoptosis and various other forms of regulated cell death have been thoroughly characterized by researchers around the world. Although many aspects of regulated cell death still remain to be elucidated in specific cell subtypes and disease conditions, many predicted that research into cell death was inexorably reaching a plateau. However, this was not the case since the last decade saw a multitude of cell death modalities being described, while harnessing their therapeutic potential reached clinical use in certain cases. In line with keeping research into cell death alive, francophone researchers from several institutions in France and Belgium established the French Cell Death Research Network (FCDRN). The research conducted by FCDRN is at the leading edge of emerging topics such as non-apoptotic functions of apoptotic effectors, paracrine effects of cell death, novel canonical and non-canonical mechanisms to induce apoptosis in cell death-resistant cancer cells or regulated forms of necrosis and the associated immunogenic response. Collectively, these various lines of research all emerged from the study of apoptosis and in the next few years will increase the mechanistic knowledge into regulated cell death and how to harness it for therapy.

Plasma membrane perforation by GSDME during apoptosis-driven secondary necrosis.

Secondary necrosis has long been perceived as an uncontrolled process resulting in total lysis of the apoptotic cell. Recently, it was shown that progression of apoptosis to secondary necrosis is regulated by Gasdermin E (GSDME), which requires activation by caspase-3. Although the contribution of GSDME in this context has been attributed to its pore-forming capacity, little is known about the kinetics and size characteristics of this. Here we report on the membrane permeabilizing features of GSDME by monitoring the influx and efflux of dextrans of different sizes into/from anti-Fas-treated L929sAhFas cells undergoing apoptosis-driven secondary necrosis. We found that GSDME accelerates cell lysis measured by SYTOX Blue staining but does not affect the exposure of phosphatidylserine on the plasma membrane. Furthermore, loss of GSDME expression clearly hampered the influx of fluorescently labeled dextrans while the efflux happened independently of the presence or absence of GSDME expression. Importantly, both in- and efflux of dextrans were dependent on their molecular weight. Altogether, our results demonstrate that GSDME regulates the passage of compounds together with other plasma membrane destabilizing subroutines.

Characteristic ERK1/2 signaling dynamics distinguishes necroptosis from apoptosis.

ERK1/2 involvement in cell death remains unclear, although many studies have demonstrated the importance of ERK1/2 dynamics in determining cellular responses. To untangle how ERK1/2 contributes to two cell death programs, we investigated ERK1/2 signaling dynamics during hFasL-induced apoptosis and TNF-induced necroptosis in L929 cells. We observed that ERK1/2 inhibition sensitizes cells to apoptosis while delaying necroptosis. By monitoring ERK1/2 activity by live-cell imaging using an improved ERK1/2 biosensor (EKAR4.0), we reported differential ERK1/2 signaling dynamics between cell survival, apoptosis, and necroptosis. We also decrypted a temporally shifted amplitude- and frequency-modulated (AM/FM) ERK1/2 activity profile in necroptosis versus apoptosis. ERK1/2 inhibition, which disrupted ERK1/2 signaling dynamics, prevented TNF and IL-6 gene expression increase during TNF-induced necroptosis. Using an inducible cell line for activated MLKL, the final executioner of necroptosis, we showed ERK1/2 and its distinctive necroptotic ERK1/2 activity dynamics to be positioned downstream of MLKL.

Quantifying single-cell ERK dynamics in colorectal cancer organoids reveals EGFR as an amplifier of oncogenic MAPK pathway signalling.

Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC.

Punching Holes in Cellular Membranes: Biology and Evolution of Gasdermins.

The gasdermin (GSDM) family has evolved as six gene clusters (GSDMA-E and Pejvakin, PJVK), and GSDM proteins are characterized by a unique N-terminal domain (N-GSDM). With the exception of PJVK, the N-GSDM domain is capable of executing plasma membrane permeabilization. Depending on the cell death modality, several protease- and kinase-dependent mechanisms directly regulate the activity of GSDME and GSDMD, the two most widely expressed and best-studied GSDMs. We provide an overview of all GSDMs in terms of biological function, tissue expression, activation, regulation, and structure. In-depth phylogenetic analysis reveals that GSDM genes show many gene duplications and deletions, suggesting that strong evolutionary forces and a unique position of the PJVK gene are associated with the occurrence of complex inner-ear development in vertebrates.

Ionizing radiation results in a mixture of cellular outcomes including mitotic catastrophe, senescence, methuosis, and iron-dependent cell death.

Radiotherapy is commonly used as a cytotoxic treatment of a wide variety of tumors. Interestingly, few case reports underlined its potential to induce immune-mediated abscopal effects, resulting in regression of metastases, distant from the irradiated site. These observations are rare, and apparently depend on the dose used, suggesting that dose-related cellular responses may be involved in the distant immunogenic responses. Ionizing radiation (IR) has been reported to elicit immunogenic apoptosis, necroptosis, mitotic catastrophe, and senescence. In order to link a cellular outcome with a particular dose of irradiation, we performed a systematic study in a panel of cell lines on the cellular responses at different doses of X-rays. Remarkably, we observed that all cell lines tested responded in a similar fashion to IR with characteristics of mitotic catastrophe, senescence, lipid peroxidation, and caspase activity. Iron chelators (but not Ferrostatin-1 or vitamin E) could prevent the formation of lipid peroxides and cell death induced by IR, suggesting a crucial role of iron-dependent cell death during high-dose irradiation. We also show that in K-Ras-mutated cells, IR can induce morphological features reminiscent of methuosis, a cell death modality that has been recently described following H-Ras or K-Ras mutation overexpression.

Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM.

Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM.

Novel Reporter for Faithful Monitoring of ERK2 Dynamics in Living Cells and Model Organisms.

Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues.

From FRET imaging to practical methodology for kinase activity sensing in living cells.

Biological processes are intrinsically dynamic. Although traditional methods provide valuable insights for the understanding of many biological phenomena, the possibility of measuring, quantifying, and localizing proteins within a cell, a tissue, and even an embryo has revolutionized our train of thoughts and has encouraged scientists to develop molecular tools for the assessment of protein or protein complex dynamics within their physiological context. These ongoing efforts rest on the emergence of biophotonic techniques and the continuous improvement of fluorescent probes, allowing precise and reliable measurements of dynamic cellular functions. The march of the "in vivo biochemistry" has begun, already yielding breathtaking results.

Recombination between polioviruses and co-circulating Coxsackie A viruses: role in the emergence of pathogenic vaccine-derived polioviruses.

Ten outbreaks of poliomyelitis caused by pathogenic circulating vaccine-derived polioviruses (cVDPVs) have recently been reported in different regions of the world. Two of these outbreaks occurred in Madagascar. Most cVDPVs were recombinants of mutated poliovaccine strains and other unidentified enteroviruses of species C. We previously reported that a type 2 cVDPV isolated during an outbreak in Madagascar was co-circulating with coxsackieviruses A17 (CA17) and that sequences in the 3' half of the cVDPV and CA17 genomes were related. The goal of this study was to investigate whether these CA17 isolates can act as recombination partners of poliovirus and subsequently to evaluate the major effects of recombination events on the phenotype of the recombinants. We first cloned the infectious cDNA of a Madagascar CA17 isolate. We then generated recombinant constructs combining the genetic material of this CA17 isolate with that of the type 2 vaccine strain and that of the type 2 cVDPV. Our results showed that poliovirus/CA17 recombinants are viable. The recombinant in which the 3' half of the vaccine strain genome had been replaced by that of the CA17 genome yielded larger plaques and was less temperature sensitive than its parental strains. The virus in which the 3' portion of the cVDPV genome was replaced by the 3' half of the CA17 genome was almost as neurovirulent as the cVDPV in transgenic mice expressing the poliovirus cellular receptor gene. The co-circulation in children and genetic recombination of viruses, differing in their pathogenicity for humans and in certain other biological properties such as receptor usage, can lead to the generation of pathogenic recombinants, thus constituting an interesting model of viral evolution and emergence.

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements.

Frequency-domain fluorescence lifetime imaging microscopy (FLIM) has become a commonly used technique to measure lifetimes in biological systems. However, lifetime measurements are strongly dependent on numerous experimental parameters. Here, we describe a complete calibration and characterization of a FLIM system and suggest parameter optimization for minimizing measurement errors during acquisition. We used standard fluorescent molecules and reference biological samples, exhibiting both single and multiple lifetime components, to calibrate and evaluate our frequency domain FLIM system. We identify several sources of lifetime precision degradation that may occur in FLIM measurements. Following a rigorous calibration of the system and a careful optimization of the acquisition parameters, we demonstrate fluorescence lifetime measurements accuracy and reliability. In addition, we show its potential on living cells by visualizing FRET in CHO cells. The proposed calibration and optimization protocol is suitable for the measurement of multiple lifetime components sample and is applicable to any frequency domain FLIM system. Using this method on our FLIM microscope enabled us to obtain the best fluorescence lifetime precision accessible with such a system.

Impact of exogenous sequences on the characteristics of an epidemic type 2 recombinant vaccine-derived poliovirus.

Pathogenic circulating vaccine-derived polioviruses (cVDPVs) have become a major obstacle to the successful completion of the global polio eradication program. Most cVDPVs are recombinant between the oral poliovirus vaccine (OPV) and human enterovirus species C (HEV-C). To study the role of HEV-C sequences in the phenotype of cVDPVs, we generated a series of recombinants between a Madagascar cVDPV isolate and its parental OPV type 2 strain. Results indicated that the HEV-C sequences present in this cVDPV contribute to its characteristics, including pathogenicity, suggesting that interspecific recombination contributes to the phenotypic biodiversity of polioviruses and may favor the emergence of cVDPVs.

In vivo characterisation of the human UCP3 gene minimal promoter in mice tibialis anterior muscles.

Transcriptional mechanisms controlling human UCP3 gene expression in skeletal muscle remain poorly understood. Experiments based on plasmid electrotransfer into tibialis anterior muscle of C57/BL6 male mice were set up in order to functionally analyze the hUCP3 gene promoter. These transfection experiments showed that a 6300 bp region upstream of the transcription initiation site was sufficient to mediate maximal promoter activity. Further analyses with a series of 5(')-deleted constructs demonstrated that the hUCP3 gene minimal promoter was located between nucleotides -284 and -40. Furthermore, an essential region was identified between nucleotides -284 and -224. The analysis of this region revealed a putative response element for PPAR located between nucleotides -281 and -269. Finally, mutations of potential cis-acting elements within the hUCP3 minimal promoter showed the presence of two TATA boxes (-198/-194 and -45/-41) required for constitutive UCP3 gene expression. To our knowledge, this is the first time that molecular characterization of the UCP3 promoter has been achieved using an in vivo experimental model.