Domain Registration in Raft-Mimicking Lipid Mixtures Studied Using Polymer-Tethered Lipid Bilayers

Abstract

The degree of domain registration in a liquid-ordered/liquid-disordered phase-separating lipid mixture consisting of 1-stearoyl-2-oleoyl-sn-3-phosphocholine, egg sphingomyelin, and cholesterol (molar mixing ratio of 1:1:1) was studied using three different planar lipid bilayer architectures distinguished by their bilayer-substrate distance d using epifluorescence microscopy. The bilayer systems, which were built layer by layer using Langmuir-Blodgett/Schaefer film depositions, included a solid-supported bilayer (d approximately 15 A) and two polymer-supported bilayers with d approximately 30 A and d approximately 58 A, respectively. Complete domain registration between Langmuir-Blodgett and Schaefer monolayer domains was observed for d approximately 58 A but not in the cases when d approximately 15 A and d approximately 30 A. Building the bilayer layer by layer guaranteed that any preexisting domains were not in registration initially; our data show that the domain registration observed was not caused by lipid flip-flop or by lateral rearrangement of preexisting large-scale domains. Instead, additional studies on bilayer systems with asymmetric lipid composition indicate that preexisting domains in the Langmuir-Blodgett monolayer induce the formation of completely registered domains in the opposite Schaefer monolayer. This study provides insight into possible biophysical mechanisms of transbilayer domain coupling. Our findings support the concept that the formation of transbilayer signaling platforms based on registered raft domains may occur without the active involvement of membrane-spanning proteins.

Undulations Drive Domain Registration from the Two Membrane Leaflets

“…4). Substrate proximity hampers the collective motion of lipids (30,31) while reducing the mobility of individual lipids by only two-to fivefold (32,33). Thus, domains in the substrate-adhering leaflet are completely immobile laterally.…”

Tuning lipid mixtures to induce or suppress domain formation across leaflets of unsupported asymmetric bilayers

“…Inserting a Ն58-Å polymer cushion between a supported bilayer and its substrate brings domains into registration but still produces results distinct from our unsupported bilayers. For example, domains in supported bilayers are induced from the leaflet near the substrate to the far leaflet but never in reverse, and a uniform leaflet does not suppress domains in a phase-separated leaflet (12). We surmount experimental challenges of supported bilayers by producing asymmetric, unsupported membranes.…”

“…Some researchers argue that only proteins should be able to induce domains across leaflets (9). Other groups, including our own, have observed what seems to be domain induction across leaflets of protein-free membranes deposited on solid surfaces, but generally these efforts have been frustrated by experimental difficulties (7,(10)(11)(12).…”

Lipid Domain Co-localization Induced by Membrane Undulations

“…Interestingly, when both leaflets of the bilayer membrane contain lipid domains, they are often found co-localized across the leaflets (7)(8)(9)(10), implying the presence of a thermodynamic interleaflet coupling, L > 0, defined via DF ¼ ÀLDA o , where DF and DA o denote the changes in the system free energy and domain overlap, respectively. To explain this observed registration between domains, several physical mechanisms have been proposed, such as cholesterol flip-flop and dynamic chain interdigitation (11) and complex interplay between chain entropic and energetic effects (12).…”