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.

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.

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.

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.

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.

Necroptosis: (Last) Message in a Bubble.

RIPK3 kinase-mediated phosphorylation of MLKL pseudokinase is the execution event of necroptosis. Two independent reports-in Immunity (Yoon et al., 2017) and Cell (Gong et al., 2017)-reveal that MLKL affects homeostatic membrane trafficking and necroptosis-enhanced bubble formation involving interaction with the ESCRT machinery.

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.