André Lopez

Confined Diffusion Without Fences of a G-Protein-Coupled Receptor as Revealed by Single Particle Tracking

Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the micro -opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients.

Drastic changes in the fluorescence properties of NBD probes with the polarity of the medium: involvement of a TICT state?

Abstract The spectroscopic and photophysical properties of two 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) derivatives with different substituents on the nitrogen group are reported in 18 solvents. The solvatochromic shifts were analysed by correlating with polarity scales. The results, together with the help of modified neglect of diatomic overlap (MNDO) calculations, enable the polarity of the ground and first singlet excited states to be determined. Experiments based on variations in temperature and viscosity establish that the two probes undergo different de-excitation pathways. The possibilities of internal rotation leading to a twisted intramolecular charge transfer (TICT) state in the case of diethylamino-NBD are discussed. A study in binary solvent mixtures outlines specific solvent—solute interactions. Appropriate restrictions are emphasized on the utilization of NBD probes in biological fields.

Phthiocerol Dimycocerosates of M. tuberculosis Participate in Macrophage Invasion by Inducing Changes in the Organization of Plasma Membrane Lipids

Phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), in particular during the early step of infection when bacilli encounter their host macrophages. However, their cellular and molecular mechanisms of action remain unknown. Using Mtb mutants deleted for genes involved in DIM biosynthesis, we demonstrated that DIM participate both in the receptor-dependent phagocytosis of Mtb and the prevention of phagosomal acidification. The effects of DIM required a state of the membrane fluidity as demonstrated by experiments conducted with cholesterol-depleting drugs that abolished the differences in phagocytosis efficiency and phagosome acidification observed between wild-type and mutant strains. The insertion of a new cholesterol-pyrene probe in living cells demonstrated that the polarity of the membrane hydrophobic core changed upon contact with Mtb whereas the lateral diffusion of cholesterol was unaffected. This effect was dependent on DIM and was consistent with the effect observed following DIM insertion in model membrane. Therefore, we propose that DIM control the invasion of macrophages by Mtb by targeting lipid organisation in the host membrane, thereby modifying its biophysical properties. The DIM-induced changes in lipid ordering favour the efficiency of receptor-mediated phagocytosis of Mtb and contribute to the control of phagosomal pH driving bacilli in a protective niche.

Lipid domains and lipid/protein interactions in biological membranes

In the fluid mosaic model of membranes, lipids are organized in the form of a bilayer supporting peripheral and integral proteins. This model considers the lipid bilayer as a two-dimensional fluid in which lipids and proteins are free to diffuse. As a direct consequence, both types of molecules would be expected to be randomly distributed within the membrane. In fact, evidences are accumulating to indicate the occurrence of both a transverse and lateral regionalization of membranes which can be described in terms of micro- and macrodomains, including the two leaflets of the lipid bilayer. The nature of the interactions responsible for the formation of domains, the way they develop and the time- and space-scale over which they exist represent today as many challenging problems in membranology. In this report, we will first consider some of the basic observations which point to the role of proteins in the transverse and lateral regionalization of membranes. Then, we will discuss some of the possible mechanisms which, in particular in terms of lipid/protein interactions, can explain lateral heterogenities in membranes and which have the merit of providing a thermodynamic support to the existence of lipid domains in membranes.

Lipid lateral diffusion and membrane organization

Phospholipid; Photobleaching; Lateral diffusion; Membrane organization; Macrodomain; Microdomain

Role of Sterols in Modulating the Human μ-Opioid Receptor Function in Saccharomyces cerevisiae

This study provides evidence that the differences in membrane composition found from one cell type to another can represent a limiting factor to recovering the functionality of transmembrane proteins when expressed in heterologous systems. Restoring the properties of the human μ-opioid receptor in yeast (Saccharomyces cerevisiae), similar to those observed in native cells, was achieved by replacing ergosterol from yeast by cholesterol, which is normally found in mammalian plasma membranes. The results suggest that these two sterols have opposite effects with respect to the ligand binding function of the receptor. Ergosterol was found to constrain the μ-opioid receptor in an inactive state in yeast plasma membranes and cannot replace cholesterol in activating it. These data differ from previous works dealing with the function of related G-protein-coupled receptors (GPCR) in ergosterol-enriched membranes. This suggests that structural requirements of GPCR with respect to their modulation by lipid components differ from one protein to another. As a consequence, we assume that the presence of appropriate lipids around transmembrane proteins determines their function. This highlights the functional significance of lateral heterogeneities of membrane components within biological membranes.

Proteolipidic composition of exosomes changes during reticulocyte maturation.

During the orchestrated process leading to mature erythrocytes, reticulocytes must synthesize large amounts of hemoglobin, while eliminating numerous cellular components. Exosomes are small secreted vesicles that play an important role in this process of specific elimination. To understand the mechanisms of proteolipidic sorting leading to their biogenesis, we have explored changes in the composition of exosomes released by reticulocytes during their differentiation, in parallel to their physical properties. By combining proteomic and lipidomic approaches, we found dramatic alterations in the composition of the exosomes retrieved over the course of a 7-day in vitro differentiation protocol. Our data support a previously proposed model, whereby in reticulocytes the biogenesis of exosomes involves several distinct mechanisms for the preferential recruitment of particular proteins and lipids and suggest that the respective prominence of those pathways changes over the course of the differentiation process.

Characterization of membrane domains by FRAP experiments at variable observation areas.

Abstract In this paper we show that FRAP experiments at variable beam radii provide an experimental approach for investigating membrane organization and dynamics, with great potential for identifying micrometer-sized domains and determining their size and the diffusion coefficient of the lipid and protein molecules they contain. Monte Carlo simulations of FRAP experiments at variable beam radii R on models of compartmentalized membranes have allowed us to establish the relationships (i) between the mobile fraction M of a diffusing particle and the size r of the domains, and (ii) between the apparent diffusion coefficient Dapp and the real diffusion coefficient D0 of this particle inside the domains. Furthermore, in its present stage of development, this approach allows us to specify whether these domains are strictly closed or not. This approach was first validated on an experimental model of a strictly compartmentalized membrane consisting of a monolayer of apposed spherical phospholipid bilayers supported by silica beads of known radius (0.83 μm). To prevent fusion between the spherical bilayers 5 mol% of a polymer-grafted phospholipid was added to the lipids. Analysis of the M versus R data yielded a radius r of 0.92±0.09 μm for the spherical bilayers, close to that of the supporting silica beads. When applied to the experimental data available for lipids and proteins in the plasma membrane of living cells, this approach suggests the existence of domains within these membranes with a radius of about 0.4 – 0.7 μm for the lipids and 0.25 μm for the proteins. These domains are not strictly closed and they are believed to be delineated by fluctuating barriers which are more or less permeable to lipid and protein molecules.

New approach to metabolism of 5'-deoxy-5-fluorouridine in humans with fluorine-19 NMR

SummaryThe metabolism of 5′-deoxy-5-fluorouridine (5′dFUrd), an antitumor fluoropyrimidine, has been investigated in human biofluids (blood, plasma, urine) using a new method: fluorine-19 NMR spectrometry. This method allows direct study of the biological sample and simultaneous identification of all the fluorinated metabolites. In the blood of a patient treated with 5′dFUrd during a 6-h continuous perfusion, we observed unmetabolized 5′dFUrd, 5-fluorouracil, 5,6-dihydrofluorouracil, and another metabolite which has not previously been reported α-fluoro-β-alanine. The two major metabolites in urine are unmetabolized 5′dFUrd and α-fluoroβ-alanine.

CD4 and CCR5 Constitutively Interact at the Plasma Membrane of Living Cells A CONFOCAL FLUORESCENCE RESONANCE ENERGY TRANSFER-BASED APPROACH

Human immunodeficiency virus entry into target cells requires sequential interactions of the viral glycoprotein envelope gp120 with CD4 and chemokine receptors CCR5 or CXCR4. CD4 interaction with the chemokine receptor is suggested to play a critical role in this process but to what extent such a mechanism takes place at the surface of target cells remains elusive. To address this issue, we used a confocal microspectrofluorimetric approach to monitor fluorescence resonance energy transfer at the cell plasma membrane between enhanced blue and green fluorescent proteins fused to CD4 and CCR5 receptors. We developed an efficient fluorescence resonance energy transfer analysis from experiments carried out on individual cells, revealing that receptors constitutively interact at the plasma membrane. Binding of R5-tropic HIV gp120 stabilizes these associations thus highlighting that ternary complexes between CD4, gp120, and CCR5 occur before the fusion process starts. Furthermore, the ability of CD4 truncated mutants and CCR5 ligands to prevent association of CD4 with CCR5 reveals that this interaction notably engages extracellular parts of receptors. Finally, we provide evidence that this interaction takes place outside raft domains of the plasma membrane.