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.

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 dimannosyldiacylglycerol, 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.

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.

Transverse and lateral distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus.

The photodimerization of anthracene was used to investigate the transverse and lateral distribution of lipids in the membrane of the Gram-positive bacterium Micrococcus luteus. 9-(2-Anthryl)nonanoic acid (9-AN) is incorporated at a high rate into various membrane lipids of M. luteus. On irradiation of intact bacteria at 360 nm, anthracene-labeled lipids form stable photodimers which can be extracted and separated by thin-layer chromatography. We present here the results of a study on the distribution of two major lipids, phosphatidylglycerol (PG) and dimannosyldiacylglycerol (DMDG), within each leaflet of the membrane lipid bilayer. After metabolic incorporation of a tritiated derivative of 9-AN in M. luteus, the radioactivity associated with the photodimers issued from PG and DMDG was counted. In the bacterial membrane, the ratio of PG-DMDG heterodimer with respect to PG-PG and DMDG-DMDG homodimers is around half of what should be obtained for a homogeneous mixture of the two lipids. In order to find out whether this was due to an asymmetric distribution of the two lipids between the two membrane leaflets or a heterogeneous distribution of the two lipids within the same membrane leaflet, the transverse distribution of PG and DMDG was also investigated. This was carried out by following the kinetics of oxidation of the two lipids by periodic acid in the membrane of M. luteus protoplasts. PG predominated slightly in the outer layer (60%), while DMDG was found to be symmetrically distributed between the two leaflets. By itself, this lipid asymmetry cannot account for the lipid distribution determined from the photodimerization experiments. This indicates that PG and DMDG are not homogeneously distributed in the plane of the bacterial membrane.

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: dimerization of the anthracene-labelled lipids in the membrane by irradiation of the intact cells at 360 nm; extraction of the lipids and thin-layer chromatography in the first dimension to separate the various lipid dimers from the monomers; partial dedimerization of the lipid dimers by illumination of the chromatogram at around 250-280 nm; 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.

Glutamate excretion triggering mechanism: a reinvestigation of the surfactant-induced modification of cell lipids.

Lipid alterations induced by surfactants to trigger glutamate excretion were investigated with an industrial strain of Corynebacterium glutamicum. The lipid composition of this strain was determined for cultures in a synthetic medium and in a complex medium. A distribution of complex lipids between the cell wall and the cell membrane is proposed. Depending on growth conditions, 70-85% of the cell fatty acids had saturated chains in cells grown with surfactants. In the synthetic medium up to 80% of the straight-chain fatty acids may have come from the acylated surfactant added to the induce excretion. In industrial fermentation, the maximal excretion rate corresponded to the highest saturated fatty acid content of cells. It was shown by radioactive labelling in the synthetic medium that the addition of the acylated surfactant induced the degradation of more than 50% of the phospholipids. Some phospholipid synthesis occurred at that time using the surfactant (saturated) fatty acids, but the membrane did nor recover its previous phospholipid content. A model is proposed to explain the mechanism of glutamate excretion triggering by surfactants.

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.

Phase behaviour of cord factor and related bacterial glycolipid toxins. A monolayer study.

C-6 esters of methyl alpha-D-glucoside and C-6, C-6' 'diesters of alpha, alpha'-D-trehalose with C18 and C32 threo and erythro mycolic acids (from chemical source) and of C80-erythro-mycolic acid (from natural source) have been synthesized. Esters of a C32 deoxy analogue were prepared as well. Throughout a monolayer study at the air-water interface, these glycolipids are shown to form well organized phases in which the two hydrocarbon chains of mycoloyl residues must be in interaction. Compression isotherms of C32 esters suggested a transition between liquid-expanded and liquid-condensed states. Latent heats Qc and entropy changes delta S associated with these phase transitions as well as the critical temperature at which they occur have been measured. Within the monolayer, the molecular packing of these glycolipids depends on the presence of the hydroxyl group of mycoloyl residues and on its stereochemistry. In particular intermolecular hydrogen bonds between these groups are postulated in the case of the bis(C32-erythro-mycoloyl)-trehalose. On the other hand, short chain C18 esters form fluid phases (t greater than 10 degrees C) whereas very long chain C80 mycoloyl esters of trehalose exist in a condensed state (t = 20 degrees C). These glycolipids were found to interact strongly with dipalmitoylphosphatidylcholine and egg yolk lecithins (3-sn-phosphatidylcholine). Their phase behaviours are discussed in connection with hypotheses concerning the way they can interact with mitochondrial membranes.