Finding the partner: FRET and beyond.

Given the importance of aberrant protein-protein interactions (PPIs) in disease, the recent drug discovery focuses on targeting the altered PPIs to treat the disease. In this context, identifying the atypical PPIs underlying the disease is critical for the development of diagnostics and therapeutics. Various biochemical, biophysical, and genetic methods have been reported to study PPIs. Here, we are giving a short account of those techniques with more emphasis on Förster resonance energy transfer (FRET), which can be used to monitor macromolecular interactions in live cells. Besides the basics of FRET, we explain the modifications of its application, like Single molecule FRET (smFRET), Fluorescence Lifetime Imaging Microscopy-FRET (FLIM-FRET), and photoswitching FRET. While smFRET is extensively used for evaluating the biology of nucleic acids and also to develop diagnostics, FLIM-FRET is widely exploited to study the PPIs underlying neurological disorders and cancer. Photoswitching FRET is a relatively newer technique and it has tremendous potential to unravel the significance of different PPIs. Besides these modifications, there are several advancements in the field by introducing new fluorophores. Identification of lanthanide chelates, quantum dots, and other nanoparticle fluorophores has revolutionized the applications of FRET in diagnostics and basic biology. Yet, these methods can be employed to study the interactions of only two molecules. Since the majority of the PPIs are multimeric complexes, we still need to improve our technologies to study these interactions in live cells in real-time.

pH sensitivity of YFPs is reduced upon AlphaRep binding: proof of concept in vitro and in living cells.

Yellow fluorescent proteins (YFPs) are commonly used in biology to track cellular processes, particularly as acceptors in experiments using the Förster Resonant Energy Transfer (FRET) phenomenon. However, their fluorescence intensity is strongly pH-dependent, limiting their utility in acidic environments. Here, we explore the pH sensitivity of YFPs upon binding with an artificial repeat protein (αRep) both in vitro and in living cells. We show that αRep binds to Citrine, with high affinity in the nanomolar range at physiological and acidic pHs, leading to increased thermal stability of the complex. Moreover, αRep binding reduces Citrine's pKa by 0.75 pH units, leading to a decreased sensitivity to pH fluctuations. This effect can be generalized to other YFPs as Venus and EYFP in vitro. An efficient binding of αRep to Citrine has also been observed in living cells both at pH 7.4 and pH 6. This interaction leads to reduced variations of Citrine fluorescence intensity in response to pH variations in cells. Overall, the study highlights the potential of αReps as a tool to modulate the pH sensitivity of YFPs, paving the way for future exploration of biological events in acidic environments by FRET in combination with a pH-insensitive cyan donor.

A novel insight into the molecular mechanism of human soluble guanylyl cyclase focused on catalytic domain in living cells.

Human soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. In this work, fluorescent proteins were employed to study the NO-induced sGC molecular mechanism via mutagenesis at the catalytic domain. The conformational change of sGC by mutant α1C595 was investigated in living cells through fluorescence lifetime imaging microscopy (FLIM). The results indicated that the NO-induced conformational change of the catalytic domain of sGC from "open to "closed" upon GTP-binding was regulated by the hydrogen (H)-bonding network of the catalytic domain. The mutation of C595 caused a big conformational change of catalytic domain with H-bond variation, which not only demonstrates the key role of the C595 site in the process of conformational change of the catalytic domain, but also reveals the regulatory mechanism of sGC at the catalytic domain. This finding would guide the design of small-molecule drugs targeting the catalytic domain to modulate sGC activity.

Visualization of in vivo protein-protein interactions in plants.

Molecular processes depend on the concerted and dynamic interactions of proteins, either by one-on-one interactions of the same or different proteins or by the assembly of larger protein complexes consisting of many different proteins. Here, not only the protein-protein interaction (PPI) itself, but also the localization and activity of the protein of interest (POI) within the cell is essential. Therefore, in all cell biological experiments, preserving the spatio-temporal state of one POI relative to another is key to understanding the underlying complex and dynamic regulatory mechanisms in vivo. In this review, we examine some of the applicable techniques to measure PPIs in planta as well as recent combinatorial advances of PPI methods to measure the formation of higher order complexes with an emphasis on in vivo imaging techniques. We compare the different methods and discuss their benefits and potential pitfalls to facilitate the selection of appropriate techniques by providing a comprehensive overview of how to measure in vivo PPIs in plants.

Arabidopsis PII Proteins Form Characteristic Foci in Chloroplasts Indicating Novel Properties in Protein Interaction and Degradation.

The PII protein is an evolutionary, highly conserved regulatory protein found in both bacteria and higher plants. In bacteria, it modulates the activity of several enzymes, transporters, and regulatory factors by interacting with them and thereby regulating important metabolic hubs, such as carbon/nitrogen homeostasis. More than two decades ago, the PII protein was characterized for the first time in plants, but its physiological role is still not sufficiently resolved. To gain more insights into the function of this protein, we investigated the interaction behavior of AtPII with candidate proteins by BiFC and FRET/FLIM in planta and with GFP/RFP traps in vitro. In the course of these studies, we found that AtPII interacts in chloroplasts with itself as well as with known interactors such as N-acetyl-L-glutamate kinase (NAGK) in dot-like aggregates, which we named PII foci. In these novel protein aggregates, AtPII also interacts with yet unknown partners, which are known to be involved in plastidic protein degradation. Further studies revealed that the C-terminal component of AtPII is crucial for the formation of PII foci. Altogether, the discovery and description of PII foci indicate a novel mode of interaction between PII proteins and other proteins in plants. These findings may represent a new starting point for the elucidation of physiological functions of PII proteins in plants.

Quantitative live-cell imaging and 3D modeling reveal critical functional features in the cytosolic complex of phagocyte NADPH oxidase.

Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. However, uncontrolled ROS production contributes to inflammation, making NADPH oxidase a major drug target. It consists of two membranous (Nox2 and p22phox) and three cytosolic subunits (p40phox, p47phox, and p67phox) that undergo structural changes during enzyme activation. Unraveling the interactions between these subunits and the resulting conformation of the complex could shed light on NADPH oxidase regulation and help identify inhibition sites. However, the structures and the interactions of flexible proteins comprising several well-structured domains connected by intrinsically disordered protein segments are difficult to investigate by conventional techniques such as X-ray crystallography, NMR, or cryo-EM. Here, we developed an analytical strategy based on FRET-fluorescence lifetime imaging (FLIM) and fluorescence cross-correlation spectroscopy (FCCS) to structurally and quantitatively characterize NADPH oxidase in live cells. We characterized the inter- and intramolecular interactions of its cytosolic subunits by elucidating their conformation, stoichiometry, interacting fraction, and affinities in live cells. Our results revealed that the three subunits have a 1:1:1 stoichiometry and that nearly 100% of them are present in complexes in living cells. Furthermore, combining FRET data with small-angle X-ray scattering (SAXS) models and published crystal structures of isolated domains and subunits, we built a 3D model of the entire cytosolic complex. The model disclosed an elongated complex containing a flexible hinge separating two domains ideally positioned at one end of the complex and critical for oxidase activation and interactions with membrane components.

Vesicular PtdIns(3,4,5)P3 and Rab7 are key effectors of sea urchin zygote nuclear membrane fusion.

Regulation of nuclear envelope dynamics is an important example of the universal phenomena of membrane fusion. The signalling molecules involved in nuclear membrane fusion might also be conserved during the formation of both pronuclear and zygote nuclear envelopes in the fertilised egg. Here, we determine that class-I phosphoinositide 3-kinases (PI3Ks) are needed for in vitro nuclear envelope formation. We show that, in vivo, PtdIns(3,4,5)P3 is transiently located in vesicles around the male pronucleus at the time of nuclear envelope formation, and around male and female pronuclei before membrane fusion. We illustrate that class-I PI3K activity is also necessary for fusion of the female and male pronuclear membranes. We demonstrate, using coincidence amplified Förster resonance energy transfer (FRET) monitored using fluorescence lifetime imaging microscopy (FLIM), a protein-lipid interaction of Rab7 GTPase and PtdIns(3,4,5)P3 that occurs during pronuclear membrane fusion to create the zygote nuclear envelope. We present a working model, which includes several molecular steps in the pathways controlling fusion of nuclear envelope membranes.

Agonist binding to the NMDA receptor drives movement of its cytoplasmic domain without ion flow.

The NMDA receptor (R) plays important roles in brain physiology and pathology as an ion channel. Here we examine the ion flow-independent coupling of agonist to the NMDAR cytoplasmic domain (cd). We measure FRET between fluorescently tagged cytoplasmic domains of GluN1 subunits of NMDARs expressed in neurons. Different neuronal compartments display varying levels of FRET, consistent with different NMDARcd conformations. Agonist binding drives a rapid and transient ion flow-independent reduction in FRET between GluN1 subunits within individual NMDARs. Intracellular infusion of an antibody targeting the GluN1 cytoplasmic domain blocks agonist-driven FRET changes in the absence of ion flow, supporting agonist-driven movement of the NMDARcd. These studies indicate that extracellular ligand binding to the NMDAR can transmit conformational information into the cell in the absence of ion flow.

Lipid-protein interactions: Lessons learned from stress.

Biological membranes are essential for normal function and regulation of cells, forming a physical barrier between extracellular and intracellular space and cellular compartments. These physical barriers are subject to mechanical stresses. As a consequence, nature has developed proteins that are able to transpose mechanical stimuli into meaningful intracellular signals. These proteins, termed Mechanosensitive (MS) proteins provide a variety of roles in response to these stimuli. In prokaryotes these proteins form transmembrane spanning channels that function as osmotically activated nanovalves to prevent cell lysis by hypoosmotic shock. In eukaryotes, the function of MS proteins is more diverse and includes physiological processes such as touch, pain and hearing. The transmembrane portion of these channels is influenced by the physical properties such as charge, shape, thickness and stiffness of the lipid bilayer surrounding it, as well as the bilayer pressure profile. In this review we provide an overview of the progress to date on advances in our understanding of the intimate biophysical and chemical interactions between the lipid bilayer and mechanosensitive membrane channels, focusing on current progress in both eukaryotic and prokaryotic systems. These advances are of importance due to the increasing evidence of the role the MS channels play in disease, such as xerocytosis, muscular dystrophy and cardiac hypertrophy. Moreover, insights gained from lipid-protein interactions of MS channels are likely relevant not only to this class of membrane proteins, but other bilayer embedded proteins as well. This article is part of a Special Issue entitled: Lipid-protein interactions.

Syntenin-1 and ezrin proteins link activated leukocyte cell adhesion molecule to the actin cytoskeleton.

Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both homotypic interactions with other ALCAM molecules and heterotypic interactions with the surface receptor CD6 expressed at the T cell surface. Despite biochemical and biophysical evidence of a dynamic association between ALCAM and the actin cytoskeleton, no detailed information is available about how this association occurs at the molecular level. Here, we exploit a combination of complementary microscopy techniques, including FRET detected by fluorescence lifetime imaging microscopy and single-cell force spectroscopy, and we demonstrate the existence of a preformed ligand-independent supramolecular complex where ALCAM stably interacts with actin by binding to syntenin-1 and ezrin. Interaction with the ligand CD6 further enhances these multiple interactions. Altogether, our results propose a novel biophysical framework to understand the stabilizing role of the ALCAM supramolecular complex engaged to CD6 during dendritic cell-T cell interactions and provide novel information on the molecular players involved in the formation and signaling of the immunological synapse at the dendritic cell side.

The spatiotemporal regulation of the Keap1-Nrf2 pathway and its importance in cellular bioenergetics.

The Kelch-like ECH associated protein 1 (Keap1)-NF-E2 p45-related factor 2 (Nrf2) pathway regulates networks of proteins that protect against the cumulative damage of oxidants, electrophiles and misfolded proteins. The interaction between transcription factor Nrf2 and its main negative cytoplasmic regulator Keap1 follows a cycle whereby the protein complex sequentially adopts two conformations: 'open', in which Nrf2 binds to one monomer of Keap1, followed by 'closed', in which Nrf2 interacts with both members of the Keap1 dimer. Electrophiles and oxidants (inducers) are recognized by cysteine sensors within Keap1, disrupting its ability to target Nrf2 for ubiquitination and degradation. Consequently, the protein complex accumulates in the 'closed' conformation, free Keap1 is not regenerated and newly synthesized Nrf2 is stabilized to activate target-gene transcription. The prevailing view of the Keap1-Nrf2 pathway, for which there exists a wealth of experimental evidence, is that it lies at the heart of cellular defence, playing crucial roles in adaptation and survival under conditions of stress. More recently, the significance of Nrf2 in intermediary metabolism and mitochondrial physiology has also been recognized, adding another layer of cytoprotection to the repertoire of functions of Nrf2. One way by which Nrf2 influences mitochondrial activity is through increasing the availability of substrates (NADH and FADH2) for respiration. Another way is through accelerating fatty acid oxidation (FAO). These findings reinforce the reciprocal relationship between oxidative phosphorylation and the cellular redox state, and highlight the key role of Nrf2 in regulating this balance.

Immunoprecipitation and FRET-FLIM to Determine Metabolons on the Plant ER.

Metabolons are protein complexes that contain all the enzymes necessary for a metabolic pathway but also scaffolding proteins. Such a structure allows efficient channeling of intermediate metabolites form one active site to the next and is highly advantageous for labile or toxic intermediates. Here we describe two methods currently used to identify metabolons via protein-protein interaction methodology: immunoprecipitations using GFP-Trap®_A beads to find novel interaction partners and potential metabolon components and FRET-FLIM to test for and quantify protein-protein interactions in planta.

Cytoplasmic localization of Mdm2 in cells expressing mutated NPM is mediated by p53.

Specific C-terminal nucleophosmin (NPM) mutations are related to the acute myeloid leukaemia and cause mistargeting of mutated NPM (NPMmut) to the cytoplasm. Consequently, multiple NPM-interacting partners, e.g., the tumour suppressor p53, become also mislocalized. We found that ubiquitin ligase Mdm2 mislocalizes to the cytoplasm in the presence of NPMmut as well. Since p53 interacts with Mdm2, we searched for the NPMmut-p53-Mdm2 complex and interactions of its constituents in live cells and cell lysates using fluorescently tagged proteins, fluorescence lifetime imaging and immunoprecipitation. We proved existence of the ternary complex, which likely adopts a chain-like configuration. Interaction between Mdm2 and NPMmut was not detected, even under conditions of upregulated Mdm2 and p53 induced by Actinomycin D. We assume that p53 serves in the complex as a bridging link between Mdm2 and NPMmut. This conclusion was supported by disruption of the Mdm2-p53 interaction by Nutlin-3A, which resulted in relocalization of Mdm2 to the nucleus, while both NPMmut and p53 remained in the cytoplasm. Importantly, silencing of p53 also prevented mislocalization of Mdm2 in the presence of NPMmut.

The LC3B FRET biosensor monitors the modes of action of ATG4B during autophagy in living cells.

Although several mechanisms of macroautophagy/autophagy have been dissected in the last decade, following this pathway in real time remains challenging. Among the early events leading to its activation, the ATG4B protease primes the key autophagy player MAP1LC3B/LC3B. Given the lack of reporters to follow this event in living cells, we developed a Förster's resonance energy transfer (FRET) biosensor responding to the priming of LC3B by ATG4B. The biosensor was generated by flanking LC3B within a pH-resistant donor-acceptor FRET pair, Aquamarine-tdLanYFP. We here showed that the biosensor has a dual readout. First, FRET indicates the priming of LC3B by ATG4B and the resolution of the FRET image makes it possible to characterize the spatial heterogeneity of the priming activity. Second, quantifying the number of Aquamarine-LC3B puncta determines the degree of autophagy activation. We then showed that there are pools of unprimed LC3B upon ATG4B downregulation, and the priming of the biosensor is abolished in ATG4B knockout cells. The lack of priming can be rescued with the wild-type ATG4B or with the partially active W142A mutant, but not with the catalytically dead C74S mutant. Moreover, we screened for commercially-available ATG4B inhibitors, and illustrated their differential mode of action by implementing a spatially-resolved, broad-to-sensitive analysis pipeline combining FRET and the quantification of autophagic puncta. Finally, we uncovered the CDK1-dependent regulation of the ATG4B-LC3B axis at mitosis. Therefore, the LC3B FRET biosensor paves the way for a highly-quantitative monitoring of the ATG4B activity in living cells and in real time, with unprecedented spatiotemporal resolution.Abbreviations: Aqua: aquamarine; ATG: autophagy related; AURKA: aurora kinase A; BafA1: bafilomycin A1; CDK1: cyclin dependent kinase 1; DKO: double knockout; FLIM: fluorescence lifetime imaging microscopy; FP: fluorescence protein; FRET: Förster's resonance energy transfer; GABARAP: GABA type A receptor-associated protein; HBSS: Hanks' balanced salt solution; KO: knockout; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NSC: NSC 185058; PE: phosphatidylethanolamine; SKO: single knockout; TKO: triple knockout; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type; ZPCK: Z-L-phe chloromethyl ketone.

Quantitative Imaging of Genetically Encoded Fluorescence Lifetime Biosensors.

Genetically encoded fluorescence lifetime biosensors have emerged as powerful tools for quantitative imaging, enabling precise measurement of cellular metabolites, molecular interactions, and dynamic cellular processes. This review provides an overview of the principles, applications, and advancements in quantitative imaging with genetically encoded fluorescence lifetime biosensors using fluorescence lifetime imaging microscopy (go-FLIM). We highlighted the distinct advantages of fluorescence lifetime-based measurements, including independence from expression levels, excitation power, and focus drift, resulting in robust and reliable measurements compared to intensity-based approaches. Specifically, we focus on two types of go-FLIM, namely Förster resonance energy transfer (FRET)-FLIM and single-fluorescent protein (FP)-based FLIM biosensors, and discuss their unique characteristics and benefits. This review serves as a valuable resource for researchers interested in leveraging fluorescence lifetime imaging to study molecular interactions and cellular metabolism with high precision and accuracy.

Substrate microtopographies induce cellular alignment and affect nuclear force transduction.

Cellular physiology has been mainly studied by using two-dimensional cell culture substrates which lack in vivo-mimicking extracellular environment and interactions. Thus, there is a growing need for more complex model systems in life sciences. Micro-engineered scaffolds have been proven to be a promising tool in understanding the role of physical cues in the co-regulation of cellular functions. These tools allow, for example, probing cell morphology and migration in response to changes in chemo-physical properties of their microenvironment. In order to understand how microtopographical features, what cells encounter in vivo, affect cytoskeletal organization and nuclear mechanics, we used direct laser writing via two-photon polymerization (TPP) to fabricate substrates which contain different surface microtopographies. By combining with advanced high-resolution spectral imaging, we describe how the constructed grid and vertical line microtopographies influence cellular alignment, nuclear morphology and mechanics. Specifically, we found that growing cells on grids larger than 10 × 20 µm2 and on vertical lines increased 3D actin cytoskeleton orientation along the walls of microtopographies and abolished basal actin stress fibers. In concert, the nuclei of these cells were also more aligned, elongated, deformed and less flattened, indicating changes in nuclear force transduction. Importantly, by using fluorescence lifetime imaging microscopy for measuring Förster resonance energy transfer for a genetically encoded nesprin-2 molecular tension sensor, we show that growing cells on these microtopographic substrates induce lower mechanical tension at the nuclear envelope. To conclude, here used substrate microtopographies modulated the cellular mechanics, and affected actin organization and nuclear force transduction.

Genetically encoded dual fluorophore reporters for graded oxygen-sensing in light microscopy.

Hypoxia is an essential regulator of cell metabolism, affects cell migration and angiogenesis during development and contributes to a wide range of pathological conditions. Multiple techniques to assess hypoxia through oxygen-imaging have been developed. However, significant limitations include low spatiotemporal resolution, limited tissue penetration of exogenous probes and non-dynamic signals due to irreversible probe-chemistry. First genetically-encoded reporters only partly overcame these limitations as the green and red fluorescent proteins (GFP/RFP) families require molecular oxygen for fluorescence. For the herein presented ratiometric and FRET-FLIM reporters dUnORS and dUnOFLS, we exploited oxygen-dependent maturation in combination with the hypoxia-tolerant fluorescent-protein UnaG. For ratiometric measurements, UnaG was fused to the orange large Stokes Shift protein CyOFP1, allowing excitation with a single light-source, while fusion of UnaG with mOrange2 allowed FRET-FLIM analysis. Imaging live or fixed cultured cells for calibration, we applied both reporters in spheroid and tumor transplantation-models and obtained graded information on oxygen-availability at cellular resolution, establishing these sensors as promising tools for visualizing oxygen-gradients in-vivo.

Investigating Plant Protein-Protein Interactions Using FRET-FLIM with a Focus on the Actin Cytoskeleton.

The study of protein-protein interactions is fundamental to understanding how actin-dependent processes are controlled through the regulation of actin-binding proteins by their interactors. FRET-FLIM (Förster resonance energy transfer-fluorescence lifetime imaging microscopy) is a sensitive bioimaging method to detect protein-protein interactions in living cells through measurement of FRET, facilitated by the interactions of fluorophore-tagged fusion protein. As a sensitive and noninvasive method for the spatiotemporal visualization of dynamic protein-protein interactions, FRET-FLIM holds several advantages over other methods of protein interaction assays. FRET-FLIM has been widely employed to characterize many plant protein interactions, including interactions between actin-regulatory proteins and their binding partners. As we increasingly understand the plant actin cytoskeleton to coordinate a diverse number of complex functions, the study of actin-regulatory proteins and their interactors becomes increasingly technically challenging. Sophisticated and sensitive in vivo methods such as FRET-FLIM are likely to be crucial to the study of protein-protein interactions as more complex and challenging hypotheses are addressed.

Characterization of Organellar-Specific ABA Responses during Environmental Stresses in Tobacco Cells and Arabidopsis Plants.

Abscisic acid (ABA) is a critical phytohormone involved in multifaceted processes in plant metabolism and growth under both stressed and nonstressed conditions. Its accumulation in various tissues and cells has long been established as a biomarker for plant stress responses. To date, a comprehensive understanding of ABA distribution and dynamics at subcellular resolution in response to environmental cues is still lacking. Here, we modified the previously developed ABA sensor ABAleon2.1_Tao3 (Tao3) and targeted it to different organelles including the endoplasmic reticulum (ER), chloroplast/plastid, and nucleus through the addition of corresponding signal peptides. Together with the cytosolic Tao3, we show distinct ABA distribution patterns in different tobacco cells with the chloroplast showing a lower level of ABA in both cell types. In a tobacco mesophyll cell, organellar ABA displayed specific alterations depending on osmotic stimulus, with ABA levels being generally enhanced under a lower and higher concentration of salt and mannitol treatment, respectively. In Arabidopsis roots, cells from both the meristem and elongation zone accumulated ABA considerably in the cytoplasm upon mannitol treatment, while the plastid and nuclear ABA was generally reduced dependent upon specific cell types. In Arabidopsis leaf tissue, subcellular ABA seemed to be less responsive when stressed, with notable increases of ER ABA in epidermal cells and a reduction of nuclear ABA in guard cells. Together, our results present a detailed characterization of stimulus-dependent cell type-specific organellar ABA responses in tobacco and Arabidopsis plants, supporting a highly coordinated regulatory network for mediating subcellular ABA homeostasis during plant adaptation processes.

Consequences of the constitutive NOX2 activity in living cells: Cytosol acidification, apoptosis, and localized lipid peroxidation.

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O2•-), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91phox and p22phox) forming the catalytic core, three cytosolic proteins (p67phox, p47phox and p40phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components. A chimeric protein, called 'Trimera', composed of the essential domains of the cytosolic proteins p47phox (aa 1-286), p67phox (aa 1-212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy.