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

Dendritic cell aggresome-like induced structures are dedicated areas for ubiquitination and storage of newly synthesized defective proteins

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control antigen processing and presentation. One of these mechanisms is the sorting of polyubiquitinated proteins in large cytosolic aggregates called dendritic cell aggresome-like induced structures (DALIS). DALIS formation and maintenance are tightly linked to protein synthesis. Here, we took advantage of an antibody recognizing the antibiotic puromycin to follow the fate of improperly translated proteins, also called defective ribosomal products (DRiPs). We demonstrate that DRiPs are rapidly stored and protected from degradation in DALIS. In addition, we show that DALIS contain the ubiquitin-activating enzyme E1, the ubiquitin-conjugating enzyme E225K, and the COOH terminus of Hsp70-interacting protein ubiquitin ligase. The accumulation of these enzymes in the central area of DALIS defines specific functional sites where initial DRiP incorporation and ubiquitination occur. Therefore, DCs are able to regulate DRiP degradation in response to pathogen-associated motifs, a capacity likely to be important for their immune functions

A Novel ZAP-70 Dependent FRET Based Biosensor Reveals Kinase Activity at both the Immunological Synapse and the Antisynapse

Many hypotheses attempting to explain the speed and sensitivity with which a T-cell discriminates the antigens it encounters include a notion of relative spatial and temporal control of particular biochemical steps involved in the process. An essential step in T-cell receptor (TCR) mediated signalling is the activation of the protein tyrosine kinase ZAP-70. ZAP-70 is recruited to the TCR upon receptor engagement and, once activated, is responsible for the phosphorylation of the protein adaptor, Linker for Activation of T-cells, or LAT. LAT phosphorylation results in the recruitment of a signalosome including PLCγ1, Grb2/SOS, GADS and SLP-76. In order to examine the real time spatial and temporal evolution of ZAP-70 activity following TCR engagement in the immune synapse, we have developed ROZA, a novel FRET-based biosensor whose function is dependent upon ZAP-70 activity. This new probe not only provides a measurement of the kinetics of ZAP-70 activity, but also reveals the subcellular localization of the activity as well. Unexpectedly, ZAP-70 dependent FRET was observed not only at the T-cell -APC interface, but also at the opposite pole of the cell or “antisynapse”

Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS)

International audienceWe introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells

Recent Developments in Fluorescence Correlation Spectroscopy for Diffusion Measurements in Planar Lipid Membranes

Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support

Analyse structurale et fonctionnelle du gene SRP de Saccharomyces cerevisiae codant pour une proteine riche en residu serine : etude de sa regulation transcriptionnelle positive par le glucose

SIGLECNRS T 60227 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc