Coupling mechanisms beween membrane leaflets

Biological membranes contain ordered and disordered domains. Small cholesterol containing ordered domains are called rafts. Ordered domains from the two monolayers are always in register as has been shown using the somewhat larger-sized liquid-ordered domains in artificial lipid bilayers. Even though the driving force behind such alignment has thus far remained enigmatic, it is fundamental for the formation of signaling platforms. Among the manifold forces implicated are friction at the membrane midplane, the mutual attraction of stiffed regions in the opposing leaflets, and the minimization of line tension between the somehow thicker ordered domains and the thinner disordered domains. The goal of the present application is to dissect the predictions of these hypotheses and thus to elucidate the molecular mechanism for lipid domain registration. We start by measuring the interaction energy between both leaflets. Therefore small ordered domains will be induced by polymer adsorption to one leaflet of free-standing planar bilayers and their slippage against the other leaflet will be measured by fluorescence correlation spectroscopy as a function of temperature. Both by fluorescence correlation spectroscopy and fluorescence imaging, the slippage of larger domains of raft forming lipids in one leaflet against non-raft lipids in both leaflets will be monitored and the dependency of the friction on different lipid species will be established. This enables us to separate whether, acting via overhang at the midplane, ordered domains in one leaflet may induce lipids in the other leaflet to adopt an ordered state or whether the macroscopic appearance of domains may be explained by line tension driven merger of invisibly small domains into larger ones. In addition, we will exploit both electrostriction and hydrostatic pressure to test the theoretical dependence of domain size on surface tension. To clarify the role of line tension we will use polymers to induce domains of different sizes in the two leaflets and we will measure how their interaction energy depends on the mismatch in their sizes. Last but not least we will test the calculated energies for the midplane interaction of (i) ordered with disordered and (i) ordered with ordered domains by carrying out similar experiments with membranes from bipolar lipids that do not exhibit midplane interactions. Throughout the whole project experimental and theoretical work will go hand in hand to ensure a proper mathematical description of membrane mechanics. We expect a molecular picture to emerge in which well characterized driving forces explain how lipid domain registration may occur in biological membranes.

Biological membranes harbor cholesterol containing areas that are often called rafts. They serve to conduct signals from outside the cell into the cytoplasm. The name originates from the observation that these somewhat thicker areas (domains) float within the membrane like rafts within a river. Despite their mobility the domains from both lipid monolayers must stay in contact. What is more, they must form a structural unit in order to fulfill their function. It was thus far unclear what glues these less than 200 nm wide domains from the two monolayers together, since a bilayer spanning element is not required. In the frame of the project we first showed that the coupling of domains from both membrane leaflets represents the energetically most favored configuration, because it minimizes the deformation of lipids on the domain boarder. In addition, ordered domains possess a higher rigidity than the rest of the membrane. Consequently they offer a higher resistance to thermal movements (undulations) of the lipid bilayer. As a result, the weekly bent areas from both monolayers overlap. To experimentally validate our theoretical considerations, we used lipids that are able to change their structure upon exposure to light with a specific wavelength. In one of the resulting structures the lipids are rigid and thicken the bilayer thereby forming domains. After having been photo-switched into the alternative structure, the photo-lipids dissolve the domains because they are now much softer and do no longer stick out of the lipid bilayer. We visualized the domains in the two monolayers by adding differently labeled lipids to each monolayer and monitored their fluorescence. In agreement with our theory, the domains from the two leafs were always in register even if their diameter did not exceed 40 nm. Since domains that small cannot be resolved by light microscopy, we used the size dependence of domain mobility to determine their diameter. The result suggests that domains do not evolve independently in the two monolayers, but evolve as one entity that right from the start spans both monolayers. We confirmed the hypothesis both by molecular dynamics simulation and in experiments with membranes that were formed from monolayers of different compositions. Thus, simple model membranes allowed to reveal the physical principles that serve to create the structural platforms for signal transduction across the cell membrane.