Rapid analysis of Förster resonance energy transfer by two-color global fluorescence correlation spectroscopy: Trypsin proteinase reaction

AbstractIn this study we introduce the combination of two-color global fluorescence correlation spectroscopy (2CG-FCS) and Förster resonance energy transfer (FRET) as a very powerful combination for monitoring biochemical reactions on the basis of single molecule events. 2CG-FCS, which is a new variation emerging from the family of fluorescence correlation spectroscopy, globally analyzes the simultaneously recorded auto- and cross-correlation data from two photon detectors monitoring the fluorescence emission of different colors. Overcoming the limitations inherent in mere auto- and cross-correlation analysis, 2CG-FCS is sensitive in resolving and quantifying fluorescent species that differ in their diffusion characteristics and/or their molecular brightness either in one or both detection channels. It is able to account for effects that have often been considered as sources of severe artifacts in two-color and FRET measurements, the most prominent artifacts comprising photobleaching, cross talk, or concentration variations in sample preparation. Because of its very high statistical accuracy, the combination of FRET and 2CG-FCS is suited for high-throughput applications such as drug screening. Employing beam scanning during data acquisition even further enhances this capability and allows measurement times of <2s. The improved performance in monitoring a FRET sample was verified by following the protease cleavage reaction of a FRET-active peptide. The FRET-inactive subpopulation of uncleaved substrate could be correctly assigned, revealing a substantial portion of inactive or missing acceptor label. The results were compared to those obtained by two-dimensional fluorescence intensity distribution analysis

Specific lipid recruitment by the retroviral gag protein upon HIV-1 assembly : from model membranes to infected cells

The retroviral Gag protein targets the plasma membrane of infected cells for viral particle formation and release. The matrix domain (MA) of Gag is myristoylated for membrane anchoring but also contains a highly basic region that recognizes acidic phospholipids. Gag targets lipid molecules at the inner leaflet of the plasma membrane including phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) and cholesterol. Here, we addressed the question whether HIV-1 Gag was able to trap PI(4,5)P2 and/or other lipids during HIV-1 assembly in silico, in vitro on reconstituted membranes and in cellulo at the plasma membrane of the host CD4+ T cells. In silico, we could observe the first PI(4,5)P2 preferential recruitment by HIV-1 MA or Gag while protein docked on artificial membranes. In vitro, using biophysical techniques, we observed the specific trapping of PI(4,5)P2, and, to a lesser extent, cholesterol and the exclusion of sphingomyelin, during HIV-1 myr(-)Gag self-assembly on LUVs and SLBs. Finally, in infected living CD4+ T cells, we measured lipid dynamics within and away from HIV-1 assembly sites using super-resolution stimulated emission depletion (STED) microscopy coupled with scanning Fluorescence Correlation Spectroscopy (sSTED-FCS). The analysis of HIV-1 infected CD4+ T lymphocytes revealed that, upon virus assembly, HIV-1 is able to specifically trap PI(4,5)P2, and cholesterol but not phosphatidylethanolamine (PE) or sphingomyelin (SM) at the cellular membrane. Furthermore, analyzing CD4+ T cells expressing only HIV-1 Gag protein showed that Gag is the main driving force restricting the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. Our data provide the first direct evidence showing that HIV-1 Gag creates its own specific lipid environment for virus assembly by selectively recruiting lipids to generate PI(4,5)P2/cholesterol-enriched nanodomains favoring virus assembly, and that HIV-1 does not assemble on pre-existing lipid domains

Estrategias de marketing cinematográfico aplicadas por Tondero Producciones. Estudio de las películas Guerrero y Solos

Analiza los diferentes momentos y componentes del marketing mundial aplicado en dos películas nacionales producidas por la compañía realizadora Tondero y cómo dicha comunicación varia dependiendo del público y tipo de explotación. Para el análisis se eligió las películas Solos y Guerrero, al ser películas con cortes, públicos y modelos de distribución distintos y al estar actualmente en su proceso publicitario

Управління трудовим потенціалом при створенні інноваційної продукції

Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries

Symmetric coupling of four spin-1/2 systems

We address the non-binary coupling of identical angular momenta based upon the representation theory for the symmetric group. A correspondence is pointed out between the complete set of commuting operators and the reference-frame-free subsystems. We provide a detailed analysis of the coupling of three and four spin-1/2 systems and discuss a symmetric coupling of four spin-1/2 systems.Comment: 20 pages, no figure

HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

HIV-1 Gag protein assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly PI(4,5)P2, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites were determined using super-resolution microscopy coupled with scanning fluorescence correlation spectroscopy in living cells. Analysis of HIV-1–infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2 and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data showed that Gag is the main driving force to restrict the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. This is the first direct evidence highlighting that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol as a membrane nanoplatform for virus assembly