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1.  Physiological Epidermal Growth Factor Concentrations Activate High Affinity Receptors to Elicit Calcium Oscillations 
PLoS ONE  2014;9(9):e106803.
Signaling 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 Ca2+ 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 Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ 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 Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ 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.
PMCID: PMC4179260  PMID: 25265278
2.  Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS) 
PLoS Computational Biology  2013;9(9):e1003245.
We 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.
Author Summary
The adaptive immune response to pathogen invasion requires the stimulation of lymphocytes by antigen-presenting cells. We hypothesized that investigating the dynamics of the T lymphocyte activation by monitoring intracellular calcium fluctuations might help explain the high specificity and selectivity of this phenomenon. However, the quantitative and exhaustive analysis of calcium fluctuations by video microscopy in the context of cell-to-cell contact is a tough challenge. To tackle this, we developed a complete solution named MAAACS (Methods for Automated and Accurate Analysis of Cell Signals), in order to automate the detection, cell tracking, raw data ordering and analysis of calcium signals. Our algorithm revealed that, when in contact with antigen-presenting cells, T lymphocytes generate oscillating calcium signals and not a massive and sustained calcium response as was originally thought. We anticipate our approach providing many more new insights into the molecular mechanisms triggering adaptive immunity.
PMCID: PMC3784497  PMID: 24086124
3.  A Novel ZAP-70 Dependent FRET Based Biosensor Reveals Kinase Activity at both the Immunological Synapse and the Antisynapse 
PLoS ONE  2008;3(1):e1521.
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”.
PMCID: PMC2211399  PMID: 18231606
4.  Single-Fluorophore Diffusion in a Lipid Membrane over a Subwavelength Aperture 
Journal of Biological Physics  2006;32(1):SN1-SN4.
We use submicrometer apertures milled in an aluminium film to study the diffusion dynamics of β-Bodipy-FL-C5-HPC (Bodipy-PC) fluorophores in a lipid dioleoylphosphatidylcholine (DOPC) multilayer. The observation volume is limited by the aperture diameter, well below the optical wavelength. This spatial resolution improvement comes together with an enhancement of the detected fluorescence per molecule as compared to an open sample, with a significant increase up to 3.5 times.
PMCID: PMC3022499  PMID: 19669430
fluorescence correlation spectroscopy; confocal microscopy; nano-apertures; lipid bilayer
5.  Dendritic cell aggresome-like induced structures are dedicated areas for ubiquitination and storage of newly synthesized defective proteins 
The Journal of Cell Biology  2004;164(5):667-675.
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.
PMCID: PMC2172164  PMID: 14981091
DRiPs; DALIS; puromycin; dendritic cells; antigen processing

Results 1-5 (5)