Spontaneous and Reversible Switch from Amphiphilic to Oil-Like Structures

International audienceCurrent theories assume that the amphiphilicity of biological membranes is always preserved. We observed that two hydrogen-bonding lipid layers in contact can spontaneously and reversibly lose their amphiphilic structure and turn into an assembly of oily complexes. This result opens a new angle for understanding the reorganization of lipids during membrane fusion, since similar complexes could fill the troubling hydrophobic voids displayed in the current models. The unique tribological properties described here may also find application in the development of novel nanolubricants. Biological membranes are highly stable bilayer structures , whose integrity is provided by the amphiphilic character of their constituting molecules. The way by which the interaction between the hydrophobic parts combined with the water solubility of the hydrophilic groups produces these structures is now well understood [1]. Such membranes have a good mechanical cohesion that enables them to resist various stresses, and which is physiologically extremely important since membranes are used to wall off cells and to separate them into several intracellular compartments. The disruption of a bilayer structure and its transition to a nonbilayer structure does not spontaneously occur, although it is known to be an intermediate in the mechanism of membrane fusion, which takes place during many cellular processes: membrane trafficking, neurotransmitter release, infection by enveloped viruses [2,3]. To initiate membrane fusion, an energetic barrier has to be overcome in order to remove water molecules from the space between the two membranes, bend the membranes, and lead to some intermediate lipid structures. Within the cells, this energy is provided by a set of special proteins called fusion proteins: viral proteins [4] and SNARE proteins [5]. Bilayer cohesion can be lost, and membrane fusion can also occur in reconstituted systems by the addition of perturbing agents such as polymers [6] or calcium ions [7], which induce phase separation within the bilayers and locally enhance hydrophobic interactions, or by using lipids that attract each other strongly through, for example, electrostatic forces [8,9] or hydrogen bonds [10]. The structures adopted by the lipid assembly during bilayer disruption and fusion have not been fully characterized yet, although an intermediate called the fusion stalk is now considered as the most likely intermediate of membrane fusion [11-14]. This model assumes that the amphiphilic character of the lipid layers is conserved during the whole process even though the strong curvatures and voids that it implies are not always very realistic. We propose an alternate explanation: the existence of a transition state in which the lipid assembly could switch from its intrinsic amphiphilic character to a purely hydrophobic behavior. We show that such a transition can occur between several types of hydrogen-bonding lipid layers (nucleoside and nitrilotriacetate lipid layers). We analyze the phenomenon with the surface force apparatus (SFA) by studying the interaction between these lipid layers in contact, and observe that the pairing of hydrogen-bonding lipids that reside in opposite bilayers leads to the disruption of the classical monolayer structures and to the formation of an oil-like structure between the two surfaces. Interestingly, this phenomenon is reversible and the integrity of the monolayers can be recovered upon separation of the two surfaces. The oily structure of the matter between the two surfaces in contact is confirmed by applying Darcy's law, which relates a liquid viscosity to the velocity and the pressure. From force versus distance curves, we deduce the viscosity of this fluid, and the result is in good agreement with expected viscosity values of similar oils. We have performed force measurements in pure water between hydrogen-bonding lipid bilayers prepared by the Langmuir-Blodgett deposition technique on mica surfaces [15]. The depositions were done at a surface area slightly larger than the one at collapse to ensure maximum density of the layers. The first layer, made of dimyristoyl-phos-phatidyl-ethanolamine lipids (DMPE, from Avanti Polar Lipids), was used to render the mica hydrophobic. The second layer, made of lipids containing hydrogen-bonding groups in their polar head (synthesized as described elsewhere [16,17]) was deposited on the DMPE hydrophobic chains so that its headgroups were facing the aqueous solution. Three types of hydrogen-bonding lipids were used whose functional group was either thymidine (T), adenosine (A), or nitrilotriacetate (NTA). Nucleosides (A and T) are able to form two hydrogen bonds, whereas NTA groups can form up to six hydrogen bonds. In order to probe the effective contribution of hydrogen-bonding on the force curves, two sets of control lipids were also synthesized. In the first control lipid, the polar head resembles the T group except for a key H atom which is replaced by a methyl group, thus suppressing its hydrogen bond donor ability (MeT lipid). In the second control lipid, PRL 95, 218101 (2005) P H Y S I C A