Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging

Surface molecules, distributed in diverse patterns and clusters on cell membranes, influence vital functions of living cells. It is therefore important to understand their molecular surface organisation under different physiological and pathological conditions. Here, we present a model-free, quantitative method to determine the distribution of cell surface molecules based on TIRF illumination and super-resolution optical fluctuation imaging (SOFI). This SOFI-based approach is robust towards single emitter multiple-blinking events, high labelling densities and high blinking rates. In SOFI, the molecular density is not based on counting events, but results as an intrinsic property due to the correlation of the intensity fluctuations. The effectiveness and robustness of the method was validated using simulated data, as well as experimental data investigating the impact of palmitoylation on CD4 protein nanoscale distribution in the plasma membrane of resting T cells.Comment: 9 pages, 3 figures plus Supplementary Informatio

First Steps in the Cross-Comparison of Solar Resource Spatial Products in Europe

International audienceYearly sum of global irradiation is compared from six spatial (map) databases: ESRA, PVGIS, Meteonorm, Satel-Light, HelioClim-2, and NASA SSE. This study does not identify the best database, but in a relative cross-comparison it points out to the areas of higher variability of outputs. Two maps are calculated to show an average of the yearly irradiation for horizontal surface together with the standard deviation that illustrates the combined effect of differences between the databases at the regional level. Differences at the local level are analysed on a set of 37 randomly selected points: global irradiation is calculated from subset of databases for southwards inclined (at 34°) and 2-axis tracking surfaces. Differences at the regional level indicate that within 90% of the study area the uncertainty of yearly global irradiation estimates (expressed by standard deviation) does not exceed 7% for horizontal surface, 8.3% for surface inclined at 34°, and 10% for 2-axis tracking surface. Higher differences in the outputs from the studied databases are found in complex climate conditions of mountains, along some coastal zones and in areas where solar radiation modelling cannot rely on sufficient density and quality of input dat

MESoR - Management and exploitation of solar resource knowledge

CD-ROMKnowledge of the solar resource is essential for the planning and operation of solar energy systems. A number of data bases giving information on solar resources have been developed over the past years. The result is a fragmentation of services each having each own mechanism of access and all are giving different results due to different methods, input data and base years. The project MESoR, co-funded by the European Commission, reduces the associated uncertainty by setting up standard benchmarking rules and measures for comparing the data bases, user guidance to the application of resource data and unifying access to various data bases

Solar Atlas for the Southern and Eastern Mediterranean

International audienceSouthern and eastern Mediterranean regions are prone to production of electricity by solar systems. The solar resource is the "fuel" of such systems and its availability is a key economic parameter in system design. Even though the southern and eastern Mediterranean region is served by several commercial data providers, in a public domain, so far only coarse resolution (100 km) data or data with limited temporal coverage is available. For more rapid development of policies and to attract the industrial interest in this region a more enhanced and easy to access free information is needed. The project will bring high resolution (1 km), long term coverage of at least 15 years data on the available solar resources for the region covering the countries Syria, Jordan, Israel, Lebanon, Egypt, Libya, Tunisia, Algeria, Morocco, Palestine National Authority, Mauretania and Turkey. The resource data will be derived from Earth Observation satellite data, based on published and transparent methodologies and the data will be validated with existing ground measurements in the region. The database will be provided by SOLEMI and Helioclim-3 (SoDa) sources - Global Horizontal Irradiation (GHI) and Direct Normal Irradiation (DNI). The data will be made available via a distributed information system which will ensure the ease of access to the data. The free access to the data will include historical, annual and monthly averages, and more detailed data products and services will remain the domain of commercial data providers. This paper will show the first prototype of the user interface for an easy web access to the solar radiation as well as ancillary geographical data. With the presentation of this paper we aim to encourage potential users to give us feedback on the further development

Functional single-cell analysis of T-cell activation by supported lipid bilayer-tethered ligands on arrays of nanowells

Supported lipid bilayers are an important biomolecular tool for characterizing immunological synapses. Immobilized bilayers presenting tethered ligands on planar substrates have yielded both spatio-temporal and structural insights into how T cell receptors (TCRs) reorganize during the initial formation of synapses upon recognition of peptide antigens bound to major histocompatibility complex (MHC) molecules. The prototypical configuration of these assays, however, limits the extent to which the kinetics and structure of the supramolecular activation clusters of the synapse (that occur in seconds or minutes) can be related to subsequent complex cellular responses, such as cytokine secretion and proliferation, occurring over hours to days. Here we describe a new method that allows correlative measures of both attributes with single-cell resolution by using immobilized lipid bilayers and tethered ligands on the surface of dense arrays of subnanoliter wells. This modification allows each nanowell to function as an artificial antigen-presenting cell (APC), and the synapses formed upon contact can be imaged by fluorescence microscopy. We show that the lipid bilayers remain stable and mobile on the surface of the PDMS, and that modifying the ligands tethered to the bilayer alters the structure of the resulting synapses in expected ways. Finally, we demonstrate that this approach allows the subsequent characterization of secreted cytokines from the activated human T cell clones by microengraving in both antigen- and pan-specific manners. This new technique should allow detailed investigations on how biophysical and structural aspects of the synapse influence the activation of individual T cells and their complex functional responses.National Institute of Allergy and Infectious Diseases (U.S.) (5P01AI045757)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051

Galectin-3 Mediates Cross-Talk between K-Ras and Let-7c Tumor Suppressor microRNA

International audienceBACKGROUND: Galectin-3 (Gal-3) and active (GTP-bound) K-Ras contribute to the malignant phenotype of many human tumors by increasing the rate of cell proliferation, survival, and migration. These Gal-3-mediated effects result from a selective binding to K-Ras.GTP, causing increased nanoclustering in the cell membrane and leading to robust Ras signaling. Regulation of the interactions between Gal-3 and active K-Ras is not fully understood. METHODS AND FINDINGS: To gain a better understanding of what regulates the critical interactions between these two proteins, we examined the role of Gal-3 in the regulation of K-Ras by using Gal-3-knockout mouse embryonic-fibroblasts (Gal-3-/- MEFs) and/or Gal-3/Gal-1 double-knockout MEFs. We found that knockout of Gal-3 induced strong downregulation (∼60%) of K-Ras and K-Ras.GTP. The downregulation was somewhat more marked in the double-knockout MEFs, in which we also detected robust inhibition(∼50%) of ERK and Akt activation. These additional effects are probably attributable to inhibition of the weak interactions of K-Ras.GTP with Gal-1. Re-expression of Gal-3 reversed the phenotype of the Gal-3-/- MEFs and dramatically reduced the disappearance of K-Ras in the presence of cycloheximide to the levels seen in wild-type MEFs. Furthermore, phosphorylation of Gal-3 by casein kinase-1 (CK-1) induced translocation of Gal-3 from the nucleus to the cytoplasm and the plasma membrane, leading to K-Ras stabilization accompanied by downregulation of the tumor suppressor miRNA let-7c, known to negatively control K-Ras transcription. CONCLUSIONS: Our results suggest a novel cross-talk between Gal-3-mediated downregulation of let 7c microRNA (which in turn negatively regulates K-Ras transcription) and elucidates the association among Gal-3 let-7c and K-Ras transcription/translation, cellular compartmentalization and activity

Dual Role of CD4 in Peripheral T Lymphocytes

The interaction of T-cell receptors (TCRs) with self- and non-self-peptides in the major histocompatibility complex (MHC) stimulates crucial signaling events, which in turn can activate T lymphocytes. A variety of accessory molecules further modulate T-cell signaling. Of these, the CD4 and CD8 coreceptors make the most critical contributions to T cell sensitivity in vivo. Whereas, CD4 function in T cell development is well-characterized, its role in peripheral T cells remains incompletely understood. It was originally suggested that CD4 stabilizes weak interactions between TCRs and peptides in the MHC and delivers Lck kinases to that complex. The results of numerous experiments support the latter role, indicating that the CD4-Lck complex accelerates TCR-triggered signaling and controls the availability of the kinase for TCR in the absence of the ligand. On the other hand, extremely low affinity of CD4 for MHC rules out its ability to stabilize the receptor-ligand complex. In this review, we summarize the current knowledge on CD4 in T cells, with a special emphasis on the spatio-temporal organization of early signaling events and the relevance for CD4 function. We further highlight the capacity of CD4 to interact with the MHC in the absence of TCR. It drives the adhesion of T cells to the cells that express the MHC. This process is facilitated by the CD4 accumulation in the tips of microvilli on the surface of unstimulated T cells. Based on these observations, we suggest an alternative model of CD4 role in T-cell activation

Identifying Individual T Cell Receptors of Optimal Avidity for Tumor Antigens.

Cytotoxic T cells recognize, via their T cell receptors (TCRs), small antigenic peptides presented by the major histocompatibility complex (pMHC) on the surface of professional antigen-presenting cells and infected or malignant cells. The efficiency of T cell triggering critically depends on TCR binding to cognate pMHC, i.e., the TCR-pMHC structural avidity. The binding and kinetic attributes of this interaction are key parameters for protective T cell-mediated immunity, with stronger TCR-pMHC interactions conferring superior T cell activation and responsiveness than weaker ones. However, high-avidity TCRs are not always available, particularly among self/tumor antigen-specific T cells, most of which are eliminated by central and peripheral deletion mechanisms. Consequently, systematic assessment of T cell avidity can greatly help distinguishing protective from non-protective T cells. Here, we review novel strategies to assess TCR-pMHC interaction kinetics, enabling the identification of the functionally most-relevant T cells. We also discuss the significance of these technologies in determining which cells within a naturally occurring polyclonal tumor-specific T cell response would offer the best clinical benefit for use in adoptive therapies, with or without T cell engineering