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

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

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

Enhancement of Satellite Free-Space Laser Communication Systems Using Machine Learning Methods

Laser communication systems have many advantages over conventional radio frequency (RF) systems. Using light instead of RF supports significantly higher communication data rates. However, such optical payloads are currently mainly used in non-optimised SatCom systems, and thus their full capabilities remain unexploited. In response to this Craft Prospect with CGI UK is focused on upgrading optical systems by using machine learning (ML) methods to improve link acquisition of laser communication terminals (LCTs) and to manage networked laser SatCom system outages

Accuracy-enhanced solar resource maps of South Africa

Paper presented to the 3rd Southern African Solar Energy Conference, South Africa, 11-13 May, 2015.SolarGIS is a global database of solar resource and meteorological parameters, developed and operated by GeoModel Solar. This database is updated daily by real-time satellite, atmospheric and meteorological data inputs. The aim of presented work was accuracy enhancement of solar resource data for South Africa, Lesotho and Swaziland. This was achieved by regional adaptation of SolarGIS solar model with data measured at fourteen high-standard solar measuring stations sourced by Eskom, GeoSUN Africa, SAURAN, STERG and Ripasso Energy. The accuracy-enhancement procedure is based on correlation of the ground measurements with the satellite-based SolarGIS model and determination of correction coefficients for model inputs. Use of these coefficients reduced systematic deviation of the input aerosol data, which is key factor determining the model accuracy in Southern Africa. The user uncertainty of the longterm estimate based on adapted data is in the range of ±5% to ±7.5% for DNI, and ±3% to ±4% for GHI. The model now delivers more accurate high-resolution solar resource time series, which helps reducing financial risk and improving engineering quality of the solar power plants. The presented maps show longterm yearly averages of Direct Normal Irradiation (DNI) and Global Horizontal Irradiation (GHI) with 1-km spatial resolution. They are calculated by aggregation of sub-hourly modeled time series, representing a period 1994 to 2013. The maps are accessible from http://www.sauran.net/. High resolution data can be accessed from http://solargis.info.cf201

Payload Testing of a Weak Coherent Pulse Quantum Key Distribution Module for the Responsive Operations on Key Services (ROKS) Mission

Quantum Key Distribution (QKD) missions currently in development for space are expanding in number due to the increasing need for more secure means of encryption combined with the range limitations of terrestrial QKD. Most of these new missions are using smaller satellites to test their payloads. The ROKS (Responsive Operations for Key Services) mission is one such mission. It will utilize a 6U CubeSat bus and is set to launch in Q4 2022. A breadboard model of a 785nm weak coherent pulse quantum source module designed for ROKS, named JADE, was tested within a lab testbench environment with the mission’s systems represented by breadboard models with equivalent components. JADE’s optical module was miniaturized to be compatible with the limited payload volumes of these small classes of satellites. Lab-based testbench characterization of JADE’s ability to emit quantum pulses with four polarization states that propagate through the beam steering system for analysis by a receiver box was demonstrated. Future work will focus on further shrinking the JADE module down to less than 1/3U size, increasing the interoperability of the module with standard interfaces for both CubeSats and SmallSats, and adding further capabilities and full environmental testing qualification to JADE

There is no simple model of the plasma membrane organizationyy

Ever since technologies enabled the characterization of eukaryotic plasma membranes, heterogeneities in the distributions of its constituents were observed. Over the years this led to the proposal of various models describing the plasma membrane organization such as lipid shells, picket-and-fences, lipid rafts, or protein islands, as addressed in numerous publications and reviews. Instead of emphasizing on one model we in this review give a brief overview over current models and highlight how current experimental work in one or the other way do not support the existence of a single overarching model. Instead, we highlight the vast variety of membrane properties and components, their influences and impacts. We believe that highlighting such controversial discoveries will stimulate unbiased research on plasma membrane organization and functionality, leading to a better understanding of this essential cellular structure