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To better understand animal cell plasma membranes, we studied simplified models, namely four-component lipid bilayer mixtures. Here we describe the domain size transition in the region of coexisting liquid-disordered (Ld) + liquid-ordered (Lo) phases. This transition occurs abruptly in composition space with domains increasing in size by two orders of magnitude, from tens of nanometers to microns. We measured the line tension between coexisting Ld and Lo domains close to the domain size transition for a variety of lipid mixtures, finding that in every case the transition occurs at a line tension of ∼0.3 pN. A computational model incorporating line tension and dipole repulsion indicated that even small changes in line tension can result in domains growing in size by several orders of magnitude, consistent with experimental observations. We find that other properties of the coexisting Ld and Lo phases do not change significantly in the vicinity of the abrupt domain size transition.

On model lipid membranes exhibiting coexisting liquid-ordered + liquid-disordered domains, the domains can be either micron-size “macro-domains” or tens of nanometer-size “nano-domains”. In the four-component mixture DSPC/DOPC/POPC/Chol, the size of the domains can be controlled by varying the ratio of DOPC and POPC within the two-phase regime, which we have found increase the Ld/Lo line tension as the DOPC fraction increases. This study shows that the partitioning of a probe is affected far more by the changes in the Ld/Lo lipid composition as DOPC and POPC are interchanged than by the domain size. Thus, phase-separation at the nanoscale is fundamentally the same as phase-separation at the macroscale concerning partitioning behavior.

We report the phase diagram for the four-component DSPC/DOPC/POPC/Chol mixture and compare it to our recent findings for the DSPC/DOPC/SOPC/Chol mixture. Four-component mixtures allow exploring the transition from macroscopic-to-nanoscopic domains. We have found modulated phase morphology in a particular region of composition within the liquid-ordered (Lo) and liquid-disordered (Ld) coexistence region in DSPC/DOPC/POPC/Chol and DSPC/DOPC/SOPC/Chol mixtures. Overall, we conclude the following: (1) Phase diagrams are very similar with shifts observed in phase boundaries; (2) Comparing the phase behavior of the two mixtures, the striking difference is in the compositional location where modulated phases are seen: the SOPC-containing mixture requires much higher DOPC concentration to form modulated phases. This observation is consistent with lower line tension in the SOPC-containing mixtures as compared to the POPC-containing mixtures; (3) By controlling lipid composition, we observe distinct types of modulated liquid-liquid phase morphologies, including linear, irregular, and angular features in GUVs. These studies show that both the size and morphology of membrane rafts are controlled by the mixture composition and the type of low-melting lipid in mixtures with high-melting lipid and cholesterol.View Large Image | View Hi-Res Image | Download PowerPoint Slide

In multi-component lipid mixtures, macro-domains can be directly observed with light microscopy, but nano-domains cannot. Monte-Carlo simulations of liquid-disordered + liquid-ordered phase separation, using a competing-interactions model, can simulate various types of phase morphologies including nano-domains. These simulations capture a more realistic picture of nano-domains as irregular clusters rather than smooth, round disks. On a curved vesicle, when line tension and the competing interaction of bending energy are of similar magnitude, modulated phase patterns occur Current work focuses on studying the various morphologies when competing interactions of line tension, bending energy, and dipole-dipole repulsion form nano-domains.