Physiological Epidermal Growth Factor Concentrations Activate High Affinity Receptors to Elicit Calcium Oscillations

International audienceSignaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca 2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca 2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca 2+ signal quantitatively similar to the Ca 2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca 2+ channels that are not activated by internal Ca 2+ store depletion, while the nanomolar EGF response involved internal Ca 2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K + channels, the nanomolar response was insensitive to the blockade of these ion channels

TCR and CD28 Concomitant Stimulation Elicits a Distinctive Calcium Response in Naive T Cells

T cell activation is initiated upon ligand engagement of the T cell receptor (TCR) and costimulatory receptors. The CD28 molecule acts as a major costimulatory receptor in promoting full activation of naive T cells. However, despite extensive studies, why naive T cell activation requires concurrent stimulation of both the TCR and costimulatory receptors remains poorly understood. Here, we explore this issue by analyzing calcium response as a key early signaling event to elicit T cell activation. Experiments using mouse naive CD4+ T cells showed that engagement of the TCR or CD28 with the respective cognate ligand was able to trigger a rise in fluctuating calcium mobilization levels, as shown by the frequency and average response magnitude of the reacting cells compared with basal levels occurred in unstimulated cells. The engagement of both TCR and CD28 enabled a further increase of these two metrics. However, such increases did not sufficiently explain the importance of the CD28 pathways to the functionally relevant calcium responses in T cell activation. Through the autocorrelation analysis of calcium time series data, we found that combined but not separate TCR and CD28 stimulation significantly prolonged the average decay time (τ) of the calcium signal amplitudes determined with the autocorrelation function, compared with its value in unstimulated cells. This increasement of decay time (τ) uniquely characterizes the fluctuating calcium response triggered by concurrent stimulation of TCR and CD28, as it could not be achieved with either stronger TCR stimuli or by co-engaging both TCR and LFA-1, and likely represents an important feature of competent early signaling to provoke efficient T cell activation. Our work has thus provided new insights into the interplay between the TCR and CD28 early signaling pathways critical to trigger naive T cell activation

Mise en place d'un microscope de champ proche optique dédié à la Biologie

Les techniques de microscopie optiques dites classiques sont limitées en résolution latérale par le critère de Rayleigh. La microscopie en champ proche optique à balayage (Scanning Near-field Optical Microscopy: SNOM) repose sur l'emploi d'une sonde de taille nanométrique (sublongueur d'onde) comme nano-détecteur (SNOM en mode collection) ou comme nano-source (SNOM en mode transmission). Cette sonde est basée sur des leviers de microscopie à force atomique (Atomic Force microscopy: AFM) classiques en Nitrure de Silicium métallisés et comportant une ouverture de 80nm. Cette thèse est destinée à la mise en place d'un AFM couplé à un SNOM en vue de l'obtention d'images simultanées de la topographie et du signal optique issu d'échantillons fluorescents. Après avoir présenté à la fois les différentes techniques classiques dédiées à la détection de fluorescence ainsi que les techniques de champ proche, les trois bancs de mesures utilisés (fonctionnant en mode illumination ou en mode collection en transmission) sont détaillés. L'imagerie AFM nécessitant une fixation de l'échantillon sur son support, différents protocoles de fixation sont présentés et leurs efficacités discutées selon le type d'échantillon et au regard de résultats AFM. Des simulations électromagnétiques sur la transmission optique de la pointe selon qu'elle fonctionne comme source ou détecteur sont réalisées, permettant de valider le mode SNOM en illumination en transmission. Enfin, l'étude d'un SNOM du commerce et du SNOM mis en place est basée sur l'imagerie AFM et Fluorescence-SNOM sur trois échantillons types: des bactéries (Escherichia coli) exprimant une protéine fluorescente (EGFP: Enhanced Green Fluoresceent Protein), des billes fluorescentes de diamètre 100nm et enfin des brins d'ADN peignés sur lame de verreMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

International audienceOur goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)(1). Single-molecule videomicroscopy, offering millisecond and nanometric resolution(1-11), allows a detailed representation of membrane organization(12-14) by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis(15,16). Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.The nanometric accuracy(17) obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods(17) such as STORM18, PALM(19,20), RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales

Identification of a lysine-rich region of Fas as a raft nanodomain targeting signal necessary for Fas-mediated cell death.

International audienceFas interaction at the plasma membrane with its lipid and protein environment plays a crucial role in the early steps of Fas signalling induced by Fas ligand binding. Particularly, Fas localisation in the raft nanodomains, ezrin-mediated interaction with the actin cytoskeleton and subsequent internalization are critical steps in Fas-mediated cell death. We identified a lysine-rich region (LRR) in the cytoplasmic, membrane-proximal region of Fas as a key determinant modulating these initial events. Through a genetic approach, we demonstrate that Fas LRR represents another signal additional to palmitoylation targeting Fas to the raft nanodomains, and modulates Fas interaction with the cytoskeleton

Detection of long-range electrostatic interactions between charged molecules by means of fluorescence correlation spectroscopy

International audienceThe present paper deals with an experimental feasibility study concerning the detection of long- range intermolecular interactions through molecular diffusion behavior in solution. This follows previous analyses, theoretical and numerical, where it was found that inter-biomolecular long-range force fields of electrodynamic origin could be detected through deviations from Brownian diffusion. The suggested experimental technique was Fluorescence Correlation Spectroscopy (FCS). By con- sidering two oppositely charged molecular species in watery solution, that is, Lysozyme protein and a fluorescent dye molecule (Alexa488), the diffusion coefficient of the dye has been measured by means of the FCS technique at different values of the concentration of Lysozyme molecules, that is, at different average distances between the oppositely charged molecules. For the model consid- ered long-range interactions are built-in as electrostatic forces, the action radius of which can be varied by changing the ionic strength of the solution. The experimental outcomes clearly prove the detectability of long-range intermolecular interactions by means of the FCS technique. Molecular Dynamics simulations provide a clear and unambiguous interpretation of the experimental results

Ca²⁺ single-cell microscopy measurements induced by 20 pM EGF in COS-7 cells.

<p>A/Raster plot of normalized fluorescence intensity against time, grayscale coded according to fluorescence intensity. 20 pM EGF was applied 25 s after the start of the video time-lapse (white bar). B/Representative traces of fluorescence variation over time for four individual cells corresponding to the three classes of responses observed following 20 pM EGF application: from top to bottom panels, unresponsive cell (0 peak); cells displaying transient or sustained single response (1 peak); cell displaying oscillatory signals (>2 peaks). For each cell, the response is represented both as a grayscale coded raster plot (top, same representation as in A) and as line plot (bottom). C/Proportion of unresponsive (0 peak), single-peak responsive (1 peak) and oscillatory responsive (>2 peaks) cells following the addition of 20 pM EGF. D/Comparison of the average fluorescence signals in response to the addition of EGF-free buffer (n = 8 responsive cells over 41 tested, red trace) or of 20 pM EGF (n = 137 over 281 tested, black trace). Fluorescence signals were synchronized at the time the first fluorescence slope (time = 0 s), found by estimating the first derivative of the signal, and averaged over 150 s. E/Ca²⁺ signals are specifically triggered by EGFR activation. Population traces averaged over cells to which irrelevant (n = 32, black line) or antagonistic anti-EGFR (n = 19 cells, red line) antibodies were added (black bar) 200 s after the start of real-time fluorescence imaging. Empty and filled circles represent the median intensity during 176 s before and after the addition of antibodies respectively. EGF was applied 25 s after the start of the video time-lapse (white bar).</p

External Ca²⁺ dependence and sensitivity to K⁺ channel blocker charybdotoxin of EGF Ca²⁺ transients.

<p>A/Proportion of cells responding (grey bar) or not responding (white bar) to 2 nM EGF in 3 mM extracellular Ca²⁺ (n = 24) or in 0 mM Ca^2+/1 mM EGTA (n = 28) in the extracellular medium. B/Fluorescence intensity signaling of individual cells (each represented by a different color) during the application of 2 nM EGF (white bar) when 3 mM Ca²⁺ was present (n = 24). The averaged population signal is shown as a thick black trace. C/Fluorescence intensity of individual cells (each represented by a different color) during the application of 2 nM EGF (white bar) when Ca²⁺ was removed and 1 mM EGTA was added to the extracellular medium (n = 28). The averaged population signal is shown as a thick black trace. D/Average of all cell signals during 2 nM EGF application, synchronized at the time of the first fluorescence peak and averaged for 150 sec, when 3 mM Ca²⁺ was present (black line, n = 24) or when Ca²⁺ was removed from and 1 mM EGTA was added to the extracellular medium (red line, n = 28). E/Proportion of cells responding (grey bar) or not responding (white bar) to 20 pM EGF in 3 mM extracellular Ca²⁺ (n = 13) or in 0 mM Ca^2+/1 mM EGTA (n = 11) in the extracellular medium. F/Fluorescence intensity of individual cells (each represented by a different color) during the application of 20 pM EGF (white bar) when 3 mM Ca²⁺ was present (n = 13). The averaged population signal is shown as a thick black trace. G/Fluorescence intensity of individual cells (each represented by a different color) during the application of 20 pM EGF (white bar) when Ca²⁺ was removed from and 1 mM EGTA was added to the extracellular medium (n = 11). The averaged population signal is shown as a thick black trace. H/Average of all cell signals during 20 pM EGF application, synchronized at the time the first fluorescence peak and for 150 sec, when 3 mM Ca²⁺ was present (black line, n = 13) or when Ca²⁺ was removed from and 1 mM EGTA was added to the extracellular medium (red line, n = 11). I/Proportion of cells responding (grey bar) or not responding (white bar) to 2 nM EGF in the absence (0, n = 24/27) or in the presence (100, n = 16/19) of 100 nM charybdotoxin (chx) in the extracellular medium. J/Proportion of cells responding (grey bar) or not responding (white bar) to 20 pM EGF in the absence (0, n = 16/22) or in the presence (100, n = 6/22) of 100 nM charybdotoxin (chx) in the extracellular medium.</p

Image analysis protocol to study Ca²⁺ signaling.

<p>A/Image of fluo5F calcium-dependent fluorescence after addition of 2 nM EGF. ROIs were drawn over two COS-7 cells (red and blue circles) and 3 areas outside cells (grey circles) in the same visual field. Scale bar: 100 µm. B/Raw fluorescence intensity (<i>F</i>) as a function of time for the two cell ROIs (red and blue lines), and the three background ROIs (grey lines) shown in A. 2 nM EGF application (represented by a white bar) was performed 25 s after the start of the video time-lapse. C/Grayscale-coded raster plot of fluorescence intensity over time of 41 control cells in response to buffer application (<i>i.e.</i> in the absence of EGF). Buffer was added 25 s after the start of the video time-lapse (as indicated by a white bar). D/Data (upper graph) obtained in cells subjected to control application of buffer in the absence of EGF (same data as in C). Background fluorescence (<i>F_bkg</i>), evaluated by averaging the fluorescence of three areas outside cells, was subtracted from the signal (<i>F_cell</i>), measured in 32 cells showing no fluorescence peak throughout the entire video time-lapse. The average fluorescence did not exhibit a flat baseline due to photobleaching. An <i>F_bleach</i> term was determined from a single exponential fit (lower graph) to the average of 32 traces calculated from <i>F_cell</i> -<i>F_bkg</i>/<i>F</i>(0) where <i>F</i>(0) is the average of the 25 images preceding buffer application (white bar, 25 s after the start of the video time-lapse). E/Normalized fluorescence intensity (Δ<i>F</i>/<i>F</i>) as a function of time for the two cell ROIs (red and blue lines) shown in A. EGF (white bar) was added to cells 25 s after the start of the video time-lapse. F/Histogram of fluorescence intensity values from the 32 control cells where no peak was detected when buffer was added. A centered value (<i>t</i>0) and a standard deviation (<i>SD</i>) were extracted from the Gaussian fit (red line) of the distribution and a threshold value (<i>th</i>) was set as <i>t</i>0+3 <i>SD</i> = 0.23, and was used for the detection of significant responses in further experiments.</p