Fabrice Dumas

Is the protein/lipid hydrophobic matching principle relevant to membrane organization and functions?

Biological membranes are complex and well‐organized multimolecular assemblies composed of a wide variety of protein and lipid molecular species. If such a diversity in protein and lipid polar headgroup structures may easily be related to a large panel of functions, the wide dispersion in acyl chain length and structure which the lipids display is more difficult to understand. It is not required for maintaining bilayer assembly and fluidity. Direct information on the lateral distribution of these various molecular species, on their potential specificity for interaction between themselves and with proteins and on the functional implications of these interactions is also still lacking. Because hydrophobic interactions play a major role in stabilizing membrane structures, we suggest considering the problem from the point of view of the matching of the hydrophobic surface of proteins by the acyl chains of the lipids. After a brief introduction to the hydrophobic matching principle, we will present experimental results which demonstrate the predictive power of the current theories and then, we will introduce the new and important concept of protein/lipid sorting in membranes. Finally, we will show how the hydrophobic matching condition may play a key role in the membrane organization and function.

Molecular sorting of lipids by bacteriorhodopsin in dilauroylphosphatidylcholine/distearoylphosphatidylcholine lipid bilayers

A combined experimental and theoretical study is performed on binary dilauroylphosphatidylcholine/distearoylphosphatidylcholine (DLPC/DSPC) lipid bilayer membranes incorporating bacteriorhodopsin (BR). The system is designed to investigate the possibility that BR, via a hydrophobic matching principle related to the difference in lipid bilayer hydrophobic thickness and protein hydrophobic length, can perform molecular sorting of the lipids at the lipid-protein interface, leading to lipid specificity/selectivity that is controlled solely by physical factors. The study takes advantage of the strongly nonideal mixing behavior of the DLPC/DSPC mixture and the fact that the average lipid acyl-chain length is strongly dependent on temperature, particularly in the main phase transition region. The experiments are based on fluorescence energy transfer techniques using specifically designed lipid analogs that can probe the lipid-protein interface. The theoretical calculations exploit a microscopic molecular interaction model that embodies the hydrophobic matching as a key parameter. At low temperatures, in the gel-gel coexistence region, experimental and theoretical data consistently indicate that BR is associated with the short-chain lipid DLPC. At moderate temperatures, in the fluid-gel coexistence region, BR remains in the fluid phase, which is mainly composed of short-chain lipid DLPC, but is enriched at the interface between the fluid and gel domains. At high temperatures, in the fluid phase, BR stays in the mixed lipid phase, and the theoretical data suggest a preference of the protein for the long-chain DSPC molecules at the expense of the short-chain DLPC molecules. The combined results of the experiments and the calculations provide evidence that a molecular sorting principle is active because of hydrophobic matching and that BR exhibits physical lipid selectivity. The results are discussed in the general context of membrane organization and compartmentalization and in terms of nanometer-scale lipid-domain formation.

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.

CD4 Interacts Constitutively with Multiple CCR5 at the Plasma Membrane of Living Cells A FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING AT VARIABLE RADII APPROACH

The entry of human immunodeficiency virus into target cells requires successive interactions of the viral envelope glycoprotein gp120 with CD4 and the chemokine receptors CCR5 or CXCR4. We previously demonstrated, by Förster resonance energy transfer experiments, the constitutive association of CD4 and CCR5 at the surface of living cells. We therefore speculated that this interaction may correlate with compartmentalization of CD4 and CCR5 within the plasma membrane. Here, we characterize the lateral distribution, the dynamics, and the stoichiometry of these receptors in living cells stably expressing CD4 and/or CCR5 by means of fluorescence recovery after photobleaching at variable radii experiments. We found that (i) these receptors expressed alone are confined into 1-μm-sized domains, (ii) CD4-CCR5 associations occur outside and inside smaller domains, and (iii) these interactions involve multiple CCR5 molecules per CD4.

Out of America: tracing the genetic footprints of the global diffusion of maize

Maize was first domesticated in a restricted valley in south-central Mexico. It was diffused throughout the Americas over thousands of years, and following the discovery of the New World by Columbus, was introduced into Europe. Trade and colonization introduced it further into all parts of the world to which it could adapt. Repeated introductions, local selection and adaptation, a highly diverse gene pool and outcrossing nature, and global trade in maize led to difficulty understanding exactly where the diversity of many of the local maize landraces originated. This is particularly true in Africa and Asia, where historical accounts are scarce or contradictory. Knowledge of post-domestication movements of maize around the world would assist in germplasm conservation and plant breeding efforts. To this end, we used SSR markers to genotype multiple individuals from hundreds of representative landraces from around the world. Applying a multidisciplinary approach combining genetic, linguistic, and historical data, we reconstructed possible patterns of maize diffusion throughout the world from American “contribution” centers, which we propose reflect the origins of maize worldwide. These results shed new light on introductions of maize into Africa and Asia. By providing a first globally comprehensive genetic characterization of landraces using markers appropriate to this evolutionary time frame, we explore the post-domestication evolutionary history of maize and highlight original diversity sources that may be tapped for plant improvement in different regions of the world.

The transmembrane protein bacterioopsin affects the polarity of the hydrophobic core of the host lipid bilayer.

Influence of the transmembrane protein bacterioopsin (the retinal-free form of bacteriorhodopsin) on the polarity of egg-phosphatidylcholine bilayers was studied by means of a steady-state and time-resolved fluorescence approach exploiting the solvatochromic properties of the 2-anthroyl fluorophore. Introduced in phosphatidylcholine molecules in the form of 8-(2-anthroyl)octanoic acid, this fluorophore probed the hydrocarbon core of the lipid bilayer. As previously shown (E. Pérochon et al., Biochemistry 31 (1992) 7672-7682), water molecules were detected in this region of the terminal part of the lipid acyl chains. Their number was considerably reduced upon addition of bacterioopsin to the lipids. This was assessed by a blue shift in the fluorescence emission spectra of the probe and a marked decrease in the fractional population of fluorophores interacting with water, to the benefit of those experiencing a hydrophobic environment. In agreement with current theories, this decrease in the hydration of the bilayer may be linked to an increase in the acyl chain order and a decrease in the lateral diffusion coefficient of lipids near the protein. The data obtained at high protein concentration accounts for a protein/lipid interface which is much less hydrated than the hydrophobic core of a protein-free lipid bilayer.

Consequences of Hydrophobic Matching on the Lateral Distribution of Lipids Around Bacteriorhodopsin Reconstituted in DLPC/DSPC Mixtures

To test the hypothesis that differences in lipid bilayer and membrane protein hydrophobic thicknesses can brings about a mechanism of lipid molecular sorting by proteins, computer simulations and Fluorescence Resonance Energy Transfer experiments (between NBD-labeled lipids as donor and retinal as acceptor) were carried out on Bacteriorhodopsin reconstituted in DLPC/DSPC mixtures. This study took advantage of the non-ideal mixing behavior of DLPC and DSPC and the fact that the average lipid acyl-chain length depends on temperature. At low temperature, in the gel-gel coexistence region, BR is found associated with the short-chain lipid DLPC. At moderate temperature, in the fluid-gel coexistence region, BR still shows preference for DLPC but now stands at the fluid-gel boundary. At high temperature, in the fluid-fluid phase, the theoretical data shows preference of BR for the longchain DSPC at the expense of the short-chain DLPC molecules.