Michèle Welby

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.

The spatial distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus varies during the cell cycle.

Recently, we have developed a photocrosslinking approach which uses anthracene as a photoactivatable group and which allows us to determine the lateral distribution of lipids in membranes quantitatively. In synchronous cultures of the gram‐positive bacterium Micrococcus luteus, this approach shows that the spatial distribution of phosphatidylglycerol and diamnnosyldiacylglycerol, the two major lipids in the bacterial membrane, varies greatly during the cell cycle. Minimum heterogeneity was observed during cell growth while maximum heterogeneity was detected during cell division.

Evidence for the incorporation of a fluorescent anthracene fatty acid into the membrane lipids of Micrococcus luteus

9-(2-Anthryl)-nonanoic acid, a newly synthesized photoactivable molecule, is shown to be incorporated into the membrane lipids of the bacterium Micrococcus luteus, through the regular metabolic pathway. This incorporation, which occurs at the sn-1 position exclusively and without any degradation or elongation of the anthracene fatty acid, is accompanied by an upward shift of the chain length of the other fatty acids.

Membrane partitioning of various δ-opioid receptor forms before and after agonist activations: The effect of cholesterol

Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment [Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168-9178]. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR*) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR* is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR* was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor-G-protein interaction.

Evidence for a homogeneous lateral distribution of lipids in a bacterial membrane: A photo cross-linking approach using anthracene as a photoactivable group

A new photo cross‐linking method has been developed for the study of the lateral distribution of lipids in natural membranes, which uses anthracene as a photoactivable group. This method, which rests on the potentiality of anthracene to form covalently bound dimers upon irradiation around 340–380 nm has been applied to the membrane lipids (dimannosyl diacylglycerol, phosphatidylglycerol, phosphatidylinositol) of the bacterium Micrococcus luteus. These glyco‐ and phospholipids were anthracene labelled by metabolically incorporating the synthetic 9‐(2‐anthryl)nonanoic acid. The following sequential procedure was used: (i) dimerization of the anthracene‐labelled lipids in the membrane by irradiation of the intact cells at 360 nm; (ii) extraction of the lipids and thin‐layer chromatography in the first dimension to separate the various lipid dimers from the monomers; (iii) partial dedimerization of the lipid dimers by illumination of the chromatogram at around 250–280 nm; (iv) chromatography in the second dimension to separate the native lipid monomers from the corresponding residual lipid dimers. On account of the occurrence of the 3 hetero dimers phosphatidylglycerol‐dimannosyl diacylglycerol, phosphatidylinositol‐dimannosyl diacylglycerol and phosphatidylglycerol‐phosphatidylinositol after irradiating the cells, it is concluded that in this bacterial membrane, dimannosyl diacylglycerol, phosphatidylglycerol and phosphatidylinositol are homogeneously distributed.

Probing the lateral organization of membranes: fluorescence repercussions of pyrene probe distribution.

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.

Occurrence of acylated proteins in the membrane of the bacterium Micrococcus luteus

The membrane of the Gram-positive bacterium Micrococcus luteus was shown to contain at least four proteins which can be acylated by palmitate. Experiments were carried out by incubating the cells in the presence of [14C]- or [3H]palmitic acid and by studying their distribution between membrane lipids and proteins. One of the acylated proteins of molecular weight around 38 kDa was clearly identified to be acylated by palmitate using a multi-step procedure. This included culture of the bacterium in the presence of [14C]palmitic acid, unlabeled acetate and unlabeled amino acids, followed by preparative polyacrylamide gel electrophoresis of membrane proteins after a careful delipidation step and, for given proteins, electroelution, acid hydrolysis and analysis of the fatty acids by gas chromatography. An overall analysis of the four acylated proteins extracted from bacteria cultured in the absence of exogeneous fatty acids reveals the presence, in situ, of palmitate as the main fatty acid, and of myristic, stearic and ararchidonic acids.

Use of New Fluorescent Lipids for the Study of the Organization and Dynamics of Lipids in Membranes

Consideration of pathophysiology of membrane disorder requires an understanding of the composition and structure of biomembranes. This is particularly true in case of lipids which are major components of biomembranes and whose chemical structure and organization can vary in response to changes in the cell diet or to lipid metabolism disorder.

Metabolic Incorporation of a New Fluorescent Anthracene Fatty Acid into the Membrane Lipids of Procaryotic and Eucaryotic Cells for Studying the Dynamic and Topology of Membranes

The fluid mosaic model for membranes supports the concept that both lipids and proteins are free to laterally diffuse in the bilayer, implying a random distribution of lipids and proteins within the layer. In fact, in the last decade, evidences have accumulated for the existence of heterogeneities in the distribution of the molecules as well as in their dynamical behaviour.