Papers by andre cauchos lopez
Comparative study of the lateral motion of extrinsic probes and anthracene-labelled constitutive phospholipids in the plasma membrane of Chinese hamster ovary cells
European Journal of Biochemistry, 1988
9-(2-Anthryl)-nonanoic acid is a new photoactivatable fluorescent probe which has been designed f... more 9-(2-Anthryl)-nonanoic acid is a new photoactivatable fluorescent probe which has been designed for the study of the lateral diffusion and distribution of lipids in biological membranes by means of the anthracene photodimerization reaction. This anthracene fatty acid can be incorporated metabolically into the glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol) of Chinese hamster ovary (CHO) cells in culture. The diffusion coefficient of intrinsic lipids in the plasma membrane of these eukaryotic cells can thus be measured using the fluorescence recovery after a photobleaching technique, since illumination of the fluorescent anthracene groups yields non-fluorescent photodimers. For the sake of comparison, the extrinsic lipophilic probes 5-(N-hexadecanoyl)-aminofluorescein, 12-(9-anthroyloxy)-stearic acid, 9-(2-anthryl)-nonanoic acid and a synthetic anthracene-phosphatidylcholine were also used to label the plasma membrane of CHO cells. The diffusion coefficients for the extrinsic and intrinsic probes ranged over 1 - 2 x 10(-9) cm2/s. Small but significant differences were observed between the various probes reflecting differences they exhibit in size and polarity. All the extrinsic probes were free to diffuse, with a mobile fraction close to 100%. In contrast, a fractional recovery of only 75% was observed for the intrinsic anthracene-labelled phospholipids, suggesting that the anthracene fatty acid was metabolically incorporated into membrane lipid regions which were inaccessible to the extrinsic probes.
Probing the lateral organization of membranes: fluorescence repercussions of pyrene probe distribution
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2001
Phospholipids pyrene labeled are widely used to investigate dynamics and organizations of membran... more Phospholipids pyrene labeled are widely used to investigate dynamics and organizations of membranes. We studied pyrene probe lateral distribution by analyzing the variations of the molar absorption coefficient (epsilon) versus probe concentrations, in small unilamellar vesicles (SUV) made of phospholipids and/or glycolipids, with pyrene labeled phosphatidylcholine (PyPC) or phosphatidylglycerol (PyPG). The results were interpreted according to an infinite associative model. They indicated that an effective self-association process corresponding to K ranging from 30 to 100 M(-1) occurred with those probes incorporated in dimannosyl diacylglycerol (DMDG). In contrast, after SUV labeling of egg yolk phosphatidylcholine (EggPC) or phosphatidylglycerol (EggPG), K values < 1 M(-1) were determined. The corresponding percentages of various stacked forms of pyrene probes were calculated. They indicated that, for a 3% PyPG labeling, the monomer represented 21% of n-mers in DMDG and 94% in EggPC. The analysis of fluorescence experiments carried out on the same samples indicated that: (i) the fluorescence process of pyrene probes was generated by the monomers: and (ii) the excimer forming resulted from a diffusional encounter between one excited and one non-excited monomer. A correction of fluorescence data allowing a more correct interpretation of fluorescence measurements was proposed.
Progress in Lipid Research, 1994
Rho-PE 3-Doxyl derivative of cholestane-3-one 2-(14-Carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxa... more Rho-PE 3-Doxyl derivative of cholestane-3-one 2-(14-Carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidineoxyl Dihydrosterculoylphosphatidylcholine Dilauroyl, dimyristoyl, dioleoyl, dipalmitoyl, distearoylphosphatidylcholine Egg yolk phosphatidylcholine Phosphatidylserine N-fluorescein-N '-phosphatidylethanolamine N-4-nitrobenzo-2-oxa-1,3-diazole laurate N-4-nitrobenzo-2-oxa-1,3-diazol-4-yl-phosphatidylethanolamine-Acyl-2-(N-4-nit robenzo-2-oxa-1,3-diazol)-aminohexanoyl (or aminocaproyl) and aminododecanoyl-phosphatidylcholine I-Acyl-2-[ 12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)]-aminododecanoyl-phosphatidylcholine,-phosphatidylethanolamine,-phosphatidylserine N I-cholesterylcarbamoyl-N s-(7-nitro-2,1,3-benzoxadiazol-4-yl)-3,6-dioxaoctane-1,8-diamine octadecyldimethyaniline N-doxylphosphatidylcholine 1-Palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine N-(1-pyrenyl-sulfonyl)dipalrnitoyl-L-alpha-phosphatidylethanolamine N-(1-pyrenyl-sulfonyl)dipalmitoyl-L-alpha-phosphatidylcholine Rhodamine-phosphatidylethanolamine
Phthiocerol Dimycocerosates of M. tuberculosis Participate in Macrophage Invasion by Inducing Changes in the Organization of Plasma Membrane Lipids
PLoS Pathogens, 2009
Microbiology, 1989
Addition of competence factor extracts to trigger competence in a culture of Streptococcus pneumo... more Addition of competence factor extracts to trigger competence in a culture of Streptococcus pneumoniae induced an increase in the intracellular pH and the Na+ content of the bacteria without any change in the K+ pool or in the membrane potential. These ionic shifts were concomitant with a stimulation of glycolysis that resulted in an enhanced ATP pool. Thus, in transforming conditions, at extracellular pH 7.8, competent bacteria presented a particularly high energetic state resulting from an increase in ApH and in the ATP pool, associated with an enhanced Na+ content. These features are discussed in the context of homeostasis regulation in response to an environmental stimulus.
Dynamic Confinement of NK2 Receptors in the Plasma Membrane
Journal of Biological Chemistry, 2004
Proteolipidic Composition of Exosomes Changes during Reticulocyte Maturation
Journal of Biological Chemistry, 2011
Journal of Biological Chemistry, 1996
Preferential binding to single-or double-stranded nucleic acids is important for the activity of ... more Preferential binding to single-or double-stranded nucleic acids is important for the activity of many proteins that process RNA and DNA. We have investigated the mechanism of strandedness discrimination with peptides derived from the putative DNA-binding domain of the RecA protein, a bacterial recombinase that modulates its affinity for single-stranded DNA by means of ATP binding and hydrolysis. Contributions of electrostatic and non-electrostatic interactions to binding of these peptides with polynucleotides were evaluated by fluorescence spectroscopy as a function of salt concentration and peptide charge. Binding of these peptides to single-and double-stranded nucleic acids was dominated by non-electrostatic interactions. Small electrostatic contributions selectively enhanced peptide complexation with single-stranded nucleic acids. Similar results were observed in control experiments carried out with tripeptides containing charged and aromatic amino acid residues. It was possible to modify the strandedness preference of peptide-polynucleotide complexes by changing electrostatic contributions to the binding free energy. These observations suggest a mechanism whereby some proteins that interact with DNA or RNA might determine and regulate their relative affinity for single-and double-stranded nucleic acids.
Journal of Biological Chemistry, 2000
This study provides evidence that the differences in membrane composition found from one cell typ... more 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-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-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.
CD4 Interacts Constitutively with Multiple CCR5 at the Plasma Membrane of Living Cells
Journal of Biological Chemistry, 2007
CD4 and CCR5 Constitutively Interact at the Plasma Membrane of Living Cells
Journal of Biological Chemistry, 2006
FEBS Letters, 2006
Previous single-molecule studies have shown a longterm diffusion superimposed to a short-term con... more Previous single-molecule studies have shown a longterm diffusion superimposed to a short-term confinement of the human mu opioid (hMOP) receptors at the surface of heterologous cells. However, additional ensemble average measurements are required to reach a complete understanding of the undergoing process. Here, we analyse, by fluorescence recovery after photobleaching measurements, the lateral diffusion of fully functional T7-EGFP-hMOP receptors in neuroblastoma SH-SY5Y cells naturally expressing a low level of the wild-type receptor. Experiments carried out at variable observation radii demonstrate the restriction of the receptors diffusion to sub-micrometer sized domains. Furthermore, consistently with the long-term singlemolecule data, the domains are found permeable.
FEBS Letters, 1989
It is shown that investigating the lateral motion of lipids in biological membranes can provide u... more It is shown that investigating the lateral motion of lipids in biological membranes can provide useful information on membrane lateral organization. After labeling membranes with extrinsic or intriinsic lipophihc fluorescent probes, fluorescence recovery aRer photobleaching experiments strongly suggests that specialized cells like spermatozoa, eggs and epithelia exhibit surface membrane regionalization or macrocompartmentation and that lateral microheterogeneities or lipid microdomains exist in the plasma membrane of many cellular systems.
Experimental Cell Research, 1987
The mobility characteristics of lipids were studied in the plasmalemma of dissociated presumptive... more The mobility characteristics of lipids were studied in the plasmalemma of dissociated presumptive ectodermal cells from embryos of Pleurodeles Walt1 at different stages of development, from early blastula to early neurula, using a Fluorescence Recovery After Photobleaching technique (FRAP), after incorporation of the lipophilic fluorescent probe SN-(hexadecanoyl)-aminofluoresceine (HEDAF) into the cell plasma membrane. At all stages of development, fluorescence recovery was found to extrapolate to lOO%, which suggested that the lipid phase in these plasma membranes can be regarded as dynamically homogeneous (no immobilized fraction). It appears as a continuum over a wide cell surface area, in which lipids are free to move laterally. The lateral diffusion coefficient of the probe, obtained from statistical analysis of the fluorescence recovery data, was found to decrease significantly from blastula to gastrula, slightly increasing at the neurula stage. These changes in the dynamic properties of the lipid probe HEDAF during gastrulation suggest that the lipid phase of the plasma membrane of these ectodermal cells undergo structural changes. The results lend support to the idea that the plasma membrane of these cells is actively involved in the morphogenetic movements which characterize the development of the embryo. @ 1987 Academic PESS, hc.
Functional membrane diffusion of G-protein coupled receptors
European Biophysics Journal, 2007
European Biophysics Journal, 1998
In this paper we show that FRAP experiments at variable beam radii provide an experimental approa... more 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 D app and the real diffusion coefficient D 0 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.
Influence of obstacles on lipid lateral diffusion: computer simulation of FRAP experiments and application to proteoliposomes and biomembranes
European Biophysics Journal, 1994
Fluorescence Recovery After Photobleaching experiments were simulated using a computer approach i... more Fluorescence Recovery After Photobleaching experiments were simulated using a computer approach in which a membrane lipid leaflet was mimicked using a triangular lattice obstructed with randomly distributed immobile and non-overlapping circular obstacles. Influence of the radius r and area fraction c of these obstacles and of the radius R of the observation area on the relative diffusion coefficient D* (Eq. (1)) and mobile fraction M was analyzed. A phenomenological equation relating D* to r and c was established. Fitting this equation to the FRAP data we obtained with the probe NBD-PC embedded in bacteriorhodopsin/egg-PC multilayers suggests that this transmembrane protein rigidifies the surrounding lipid phase over a distance of about 18 A (approximately equal to two lipid layers) from the protein surface. In contrast, analysis of published diffusion constants obtained for lipids in the presence of gramicidin suggests that in terms of lateral diffusion, this relatively small polypeptide does not significantly affect the surrounding lipid phase. With respect to the mobile fraction M, and for point obstacles above the percolation threshold, an increase in R led to a decrease in M which can be associated with the existence of closed domains whose average size and diffusion properties can be determined. Adaptation of this model to the re-interpretation of the FRAP data obtained by Yechiel and Edidin (J Cell Biol (1987) 115:755-760) for the plasma membrane of human fibroblasts consistently leads to the suggestion that the lateral organization of this membrane would be of the confined type, with closed lipid domains of approximately equal to 0.5 microns 2 in area.
Chemistry and Physics of Lipids, 1994
In the fluid mosaic model of membranes, lipids are organized in the form of a bilayer supporting ... more 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 tobe 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.
Membrane functional organisation and dynamic of μ-opioid receptors
Cellular and Molecular Life Sciences, 2009
The activation and signalling activity of the membrane mu-opioid receptor (MOP-R) involve interac... more The activation and signalling activity of the membrane mu-opioid receptor (MOP-R) involve interactions among the receptor, G-proteins, effectors and many other membrane or cytosolic proteins. Decades of investigation have led to identification of the main biochemical processes, but the mechanisms governing the successive protein-protein interactions have yet to be established. We will need to unravel the dynamic membrane organisation of this complex and multifaceted molecular machinery if we are to understand these mechanisms. Here, we review and discuss advances in our understanding of the signalling mechanism of MOP-R resulting from biochemical or biophysical studies of the organisation of this receptor in the plasma membrane.
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Papers by andre cauchos lopez