Marie Erard

• PhD
• Professor (Full) at University of Paris-Sud

In 2023, the ImaBio consortium (imabio-cnrs.fr), an interdisciplinary life microscopy research group at the Centre National de la Recherche Scientifique, celebrated its 20th anniversary. ImaBio contributes to the biological imaging community through organization of MiFoBio conferences, which are interdisciplinary conferences featuring lectures and...

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...

AlphaFold2 (AF2) and RoseTTaFold (RF) have revolutionized structural biology, serving as highly reliable and effective methods for predicting protein structures. This article explores their impact and limitations, focusing on their integration into experimental pipelines and their application in diverse protein classes, including membrane proteins,...

Significance: Forces inside cells play a fundamental role in tissue growth, affecting important processes such as cancer cell migration or tissue repair after injury. Förster resonance energy transfer (FRET)-based tension sensors are a remarkable tool for studying these forces and should be made easier to use. Aim: We prove that absolute FRET ef...

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...

Significance Hydrogen peroxide plays an important role in the fine balance between physiological and pathological processes. To detect this diffusible small molecule, we expanded the scope of organic triggers in developing borinic acids as an alternative and more sensitive trigger than the most conventional boronate-based sensors. We discovered tha...

Yellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow...

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O 2 •− ), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91 phox and p22 phox ) forming the catalytic core, three cytosolic proteins (p67 phox , p47 phox and p40 phox ) a...

The key purpose of phagocytosis is the destruction of pathogenic microorganisms. The phagocytes exert a wide array of killing mechanisms that allow mastering the vast majority of pathogens. One of these mechanisms consists in the production of reactive oxygen species inside the phagosome by a specific enzyme, the phagocyte NADPH oxidase. This enzym...

Genetically-encoded Förster’s Resonance Energy Transfer (FRET) biosensors are indispensable tools to sense the spatiotemporal dynamics of signal transduction pathways. Investigating the crosstalk between different signalling pathways is becoming increasingly important to follow cell development and fate programs. To this end, FRET biosensors must b...

Phagosomal ROS generation is critical for our immune defense against microbial infections. Quantitative assessment of phagosomal ROS production is required to understand the complex relationship between the phagocyte and the microbe, in particular for pathogens that resist phagosomal destruction. ROS detection is difficult due to the transient natu...

A bimodal probe for protein detection that combines hyperpolarized ¹²⁹Xe NMR and fluorescence spectroscopy has been optimized. Fluorescence detection is performed by a ligand, CrAsH, that recognizes a target peptide fused to the specific protein. Detection through ¹²⁹Xe NMR spectroscopy is based on a cryptophane cage hosting xenon. More information...

A full understanding of biological phenomena involves sensitive and noninvasive detection. Herein, we report the optimization of a probe for intracellular proteins that combines the advantages of fluorescence and hyperpolarized ¹²⁹Xe NMR spectroscopy detection. The fluorescence detection part is composed of six residues containing a tetracysteine t...

Genetically-encoded Förster’s Resonance Energy Transfer (FRET) biosensors are indispensable tools to sense the spatiotemporal dynamics of signal transduction pathways. Investigating the crosstalk between different signalling pathways is becoming increasingly important to follow cell development and fate programs. To this end, FRET biosensors must b...

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, p47p...

Redox biology has become a major issue in numerous areas of physiology. Reactive oxygen species (ROS) have a broad range of roles from signal transduction to growth control and cell death. To understand the nature of these roles, accurate measurement of the reactive compounds is required. An increasing number of tools for ROS detection is available...

During the phagocytosis of pathogens by phagocyte cells, the NADPH oxidase complex is activated to produce superoxide anion, a precursor of microbial oxidants. The activated NADPH oxidase complex from phagocytes consists in two transmembrane proteins (Nox2 and p22(phox)) and four cytosolic proteins (p40(phox), p47(phox), p67(phox) and Rac1-2). In t...

Significance: Reactive oxygen species (ROS) fulfill numerous roles in biology ranging from signal transduction to the induction of cell death. To advance our understanding of these sometimes contradictory roles, quantitative, specific and sensitive ROS measurements are required. Recent advances: Several organic or genetically encoded probes were...

New fluorescent proteins (FPs) are constantly discovered from natural sources, and submitted to intensive engineering based on random mutagenesis and directed evolution. However, most of these newly developed FPs fail to achieve all the performances required for their bioimaging applications. The design of highly optimised FP-based reporters, simul...

10th European-Biophysical-Societies-Association (EBSA) European Biophysics Congress, Dresden, GERMANY, JUL 18-22, 2015

It is generally acknowledged that the popular cyan and yellow fluorescent proteins carried by genetically encoded reporters suffer from strong pH sensitivities close to the physiological pH range. We studied the consequences of these pH responses on the intracellular signals of model Förster resonant energy transfer (FRET) tandems and FRET-based re...

Cyan fluorescent proteins (CFPs) derived from Aequorea victoria green fluorescent protein are the most widely used Förster resonant energy transfer (FRET) donors in genetically encoded biosensors for live-cell imaging and bioassays. However, the weak and complex fluorescence emission of cyan variants, such as enhanced cyan fluorescent protein (ECFP...

Several studies have suggested that the V0 domain of the vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal...

Hepatocytes, which perform the main functions of the liver, are particularly vulnerable to toxic agents such as cadmium, an environmental pollutant. To identify the molecular targets for cadmium in hepatocytes, we have studied the effects of CdCl2 on the hybrid cell line WIF-B9 that exhibits stable structural and functional hepatocytic polarity. We...

Owing to their ability to be genetically expressed in live cells, fluorescent proteins have become indispensable markers in cellular and biochemical studies. These proteins can undergo a number of covalent chemical modifications that may affect their photophysical properties. Among other mechanisms, such covalent modifications may be induced by rea...

In the phagocytosis field, ROS production by the phagocyte NOX has been associated with pathogen killing for the last 50 years. Since the discovery of nonphagocyte NOX, numerous other roles for ROS production have been identified. Oxidative stress and ROS-mediated signaling have received much attention in recent years. Much lower concentrations of...

pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations ca...

Two covalently linked porphyrin-polyoxometalate hybrids have been prepared: an Anderson-type hexamolybdate [N(C(4)H(9))(4)](3)[MnMo(6)O(18){(OCH(2))(3)CNHCO(ZnTPP)}(2)] with two pendant zinc(ii)-tetraphenylporphyrins, and a Dawson-type vanadotungstate [N(C(4)H(9))(4)](5)H[P(2)V(3)W(15)O(59){(OCH(2))(3)CNHCO(ZnTPP)}] with one porphyrin. Electrochemi...

Cyan fluorescent proteins (CFPs) are widely used as FRET donors in genetically encoded biosensors for live cell imaging. Recently, cyan variants with greatly improved fluorescence quantum yields have been developed by large scale random mutagenesis. We show that the introduction of only two mutations, T65S and H148G, is able to confer equivalent pe...

Cyan fluorescent proteins (CFP) derived from Aequorea victoria GFP, carrying a tryptophan-based chromophore, are widely used as FRET donors in live cell fluorescence imaging experiments. Recently, several CFP variants with near-ultimate photophysical performances were obtained through a mix of site-directed and large scale random mutagenesis. To un...

Absorption spectra of CFP variants at basic, neutral and acid pHs. (A–E) Absorption spectra of the different CFP variants. (F) Model spectra obtained by linear combinations of the native and denatured spectra, showing the range of possible spectral shapes in the hypothesis of a two-state transition (see Text S1). The percentages indicate the relati...

Secondary structure content of CFP variants at neutral and acid pHs. Acid pHs mainly result in a loss of 10% of β-sheet, the main component of the native protein structure at pH 7.4. (TIF)

Dependence of the reversible bleaching rate constants of ECFP on the irradiance. (TIF)

Photoactivated return of ECFP fluorescence after transient photobleaching. The ECFP fluorescence was first bleached by maximum lamp power for less than 1 min. The return of fluorescence after switching off the illumination, was then monitored under different illumination regimes, and the different transient responses were normalized between minimum...

H-bonding networks in the chromophore cavity of ECFP, Cerulean and GFP. (A) ECFP structure of Lelimousin et al (2WSN, solid, grey) [40] and Bae et al (1OXD, transparent, red) [39], reproduced from the Main Section, (B) Cerulean structure of Lelimousin et al (2WSO, solid, grey) and Malo et al (2Q57, transparent, red) [29], the latter displaying an a...

I. Spectral analyses of CFP variants at different pHs. II. Modeling and analysis of photobleaching experiments. III. Structural analyses of the chromophore environment. (DOC)

Apparent photobleaching and recovery of ECFP fluorescence under variable illumination conditions. Experiments designed to reproduce the results of [14]. See Text S1. (TIF)

Fluorescence emission spectra of CFP variants at basic, neutral and acid pHs. (A–E) Fluorescence emission spectra of the different CFP variants. (F) Model spectra obtained by linear combinations of the native and denatured spectra, showing the range of possible spectral shapes in the hypothesis of a two-state transition. The percentages indicate th...

Dependence of the amplitude of reversible bleaching of ECFP on the irradiance. (TIF)

Reversible bleaching parameters of purified and cytosolic CFP variants. The elementary rate constants of reversible photobleaching koff and photoactivated return kon were determined from experimental data on agarose beads as described in Text S1. (DOC)

Reversible photobleaching of cytosolic CFPs expressed in living MDCK cells. Experimental conditions were identical to those used for purified proteins. Each curve is an average of 4 to 6 decays collected from different cell individuals. Continuous lines are best fits to the model Fnorm = y0+y1t+y2 exp(−t/τRev). (TIF)

Irreversible bleaching of cytosolic CFPs expressed in living MDCK cells. (A) Constant irradiation at 0.2 W/cm2 was applied while camera images were taken every 20 s. Each curve is the average of 4 to 6 decays collected from different cell individuals. Continuous lines are best fits of the decays to a simple exponential model with time constant τIrr...

Stereogram of the overlapped structures of mTurquoise and SCFP3A in the CFP chromophore region. The structures of mTurquoise (2YE0, solid), and SCFP3A (2YDZ, transparent) [15] were aligned along the protein backbones. All heavy atoms and water molecules located within 9 Å of the CG atom of the chromophore are displayed. RMSD calculations were perfo...

Complementary time-resolved fluorescence parameters of CFP variants. Correlation of the CFP fluorescence quantum yields with the integrated pre-exponential amplitude (aL) and position (τL) of the longest lifetime peak in fluorescence lifetime distributions. (DOC)

Production of ROS by the leukocyte NADPH oxidase is essential for the destruction of pathogenic bacteria inside phagosomes. The enzyme is a complex of cytosolic and membranous subunits that need to assemble upon activation. Biochemical data suggest that the complex is renewed continuously during activity. Furthermore, it is generally assumed that c...

Phagocytes produce large quantities of reactive oxygen species for pathogen killing; however, the kinetics and amplitude of ROS production on the level of individual phagosomes are poorly understood. This is mainly due to the lack of appropriate methods for quantitative ROS detection with microscopic resolution. We covalently attached the ROS-sensi...

The tendency of GFP-like fluorescent proteins to dimerize in vitro is a permanent concern as it may lead to artifacts in FRET imaging applications. However, we have found recently that CFP and YFP (the couple of GFP variants mostly used in FRET studies) show no trace of association in the cytosol of living cells up to millimolar concentrations. In...

New complexes based on a coordination interaction between a pyridyl-porphyrin (namely 5,10,15-tritolyl-20-(4-pyridyl)porphyrin, 5,10,15-tritolyl-20-(3-pyridyl)porphyrin or 5,10,15-triphenyl-20-(4-pyridyl)porphyrin) and a Keggin-type polyoxometalate (α-[MSiW11O39]6−, M=Co2+ or Ni2+) are formed in solution. The formation of these complexes is clearly...

The NADPH-oxidase of phagocytic cells is a multicomponent enzyme that generates superoxide. It comprises a membrane flavocytochrome b558 and four cytosolic proteins; p67phox, p47phox, p40phox and Rac. The NADPH-binding site of this complex was shown to be located on the flavocytochrome b558. However, a number of studies have suggested the presence...

The modifications induced by reactive oxygen species (ROS) on fluorescent proteins (FPs) may have important implications for live cell fluorescence imaging. Using quantitative gamma-radiolysis, we have studied the ROS-induced biochemical and photophysical perturbations on recombinant cyan fluorescent protein (CFP). After oxidation by the OH radical...

Fluorescent proteins (FPs) are essential for live cell studies using fluorescence microscopy. To date, the molecular basis for FPs' irreversible photobleaching and the nature of the associated photoproducts are a matter of debate. Mass spectrometry, which should be an ideal technique for the structural dissection of FPs, cannot be harnessed efficie...

Recent advances in microscopy techniques and the development of many different colour variants of the GFP family of proteins allow for a more direct analysis of protein function in live cells. The advantage of genetically coded fluorescent protein probes is often offset by their photophysical properties which usually make them very sensitive to cel...

The photophysical study of platinum carbonyl clusters shows that these clusters exhibit double emitting properties in the visible and near-infrared spectral region for single excitation wavelength. When deposited on glass, they self-assemble into long nanowires or spherical aggregates which still exhibit fluorescence properties analogous to those o...

We have studied the fluorescence decays of the purified enhanced cyan fluorescent protein (ECFP, with chromophore sequence Thr-Trp-Gly) and of its variant carrying the single H148D mutation characteristic of the brighter form Cerulean. Both proteins exhibit highly complex fluorescence decays showing strong temperature and pH dependences. At neutral...

It has been previously established that a lesion created by a microcapillary in the membrane of a single aerobic cell (from skin or immune origin) was sufficient to induce a local membrane depolarization and the ensuing release of oxidative bursts. Their kinetic and quantitative features reveal the activity of cell constitutive enzymes, namely, NAD...

Store-operated calcium entry (SOCE) is a key regulator in the activation of leukocytes. 3,5-Bistrifluoromethyl pyrazole (BTP) derivatives have been identified recently as inhibitors of T lymphocyte activation. The inhibitory effect of one of these compounds, N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxa...

A significant number of exocytosis events recorded with amperometry demonstrate a prespike feature termed a "foot" and this foot has been correlated with messengers released via a transitory fusion pore before full exocytosis. We have compared amperometric spikes with a foot with spikes without a foot at chromaffin cells and found that the probabil...

Vesicular exocytosis is important in the communication between cells in complex organisms. It controls the release of specific chemical or biochemical messengers stored in the emitting cell, which elicit a response upon detection by the target cells. Secretion of a messenger molecule (a neurotransmitter) was measured electrochemically, which allowe...

The electrochemical signature of peroxynitrite oxidation is reported for the first time, and its mechanism discussed in the light of data obtained by steady-state and transient voltammetry at microelectrodes. Peroxynitrite is an important biological species generated by aerobic cells presumably via the near diffusion-limited coupling of nitric oxid...

Positioning an ultramicroelectrode at micrometric distances from an isolated living cell ensures that any electroactive material released by the cell is collected and analyzed by the electrode surface. The film of extracellular fluid comprised between the cell and the electrode surfaces defines an artificial synaptic cleft of a few hundred femtolit...

Carbon fiber platinized ultramicroelectrodes placed within micrometres of a single living cell are used to monitor cellular events. This artificial synapse is used here to collect and examine the very nature of the massive oxidative bursts produced by human fibroblasts when their membrane is locally depolarized by a puncture made with a micrometre...

Fluorescent proteins of the green fluorescent protein (GFP) family have given birth to a vast array of targetable optical sensors that play a major role in the deciphering of live cell chemistry. These genetically encoded reporters benefit from unequaled specificity and integration into the cellular machinery, while they gather withing less than 10...