Lateral Phase Separation In Multicomponent Lipid Bilayers

In recent decades, researchers have proved that the lipid bilayer is heterogeneous. Lateral phase separation exists on the surface of bio-membrane, and specific lipids (GSLs, SM and Chol) form small lipid domains with biological function-"Rafts". Rafts participate into many vital biological activities, such as protein sorting, signal transduction and material transporting. To understand the mechanisms of these activities, model membranes are employed to simplify and mimic real membranes. The supported lipid bilayers (SLBs) are planar model membranes, which are quite suitable for AFM detection. We designed and modified the preparation method of SLBs and studied the morphological change with compositions, as well as the evolution of the morphologies. For more details:O The second chapter is about the efficient method of SLBs preparation. We first compare the ordinary methods including spin-coating, LB deposition and vesicle fusion, and found that result of vesicle fusion was in high quality but time-wasting. Thus, we transferred the preparation process into the small AFM fluid cell and kept the fusion process at high temperature, usually higher than Tm of all lipids. By these modification, we could accomplished SLBs fast without any ion additions. Besides, we also found out best practices in making vesicles. This method is highly efficient and reliable, and has high potential in research and application.O In Chapter3, the effect of monosialognglioside GM1concentration on the lateral phase separation in the SM/DOPC/Chol bilayers was studied by using atomic force microscopy. The results show that, with the increase of GM1mole fraction (x), the dominant composition of liquid-ordered (Lo) domains changes from SM to SM/GM1and finally to GM1. Meanwhile, the decrease of domain area (A) of the Lo phase with the increase of x follows a scaling law of A～x-3/2, for x>0.005, indicating that the domain growth is pinned with high GM1concentration. Results of in situ experiments of GM1insertion into SM/DOPC/cholesterol bilayers further supported our observations. In addition, we found that deceasing the content of cholesterol led to a higher critical concentration of the pinning effect of GM1on the phase separation. Here, we provided a general rule to predict the effect of GSLs on morphology by looking them as impurities rather than considering various interactions between lipids. Our results also solved the conflicts in literature.O In Chapter4, we studied the domain growth in multicomponent lipid bilayers. By heating and cooling circles, we gained the temperature of phase separation in SLBs. The melting lipid bilayers were under cooled to induce phase separation. We found that with little GM1in SM/DOPC/Chol bilayers, the domain growth after cooling is fast. Closer investigation on details shown three types of growth: isolated growth, merged growth and domain dissolving. The domain area, the shape factor, the distance between neighboring domains play critical roles in defining what types one domain adopted in bilayers. During these processes, the details of liquid-ordered domain growth demonstrated the Ld phase is a phase state between solid and liquid. We further prepared the effect of different GM1concentration on domain growth. Via the KJMA theory, we estimated the averaged diffusion rate in bilayers containing low and high GM1concentration. Results shown that the higher GM1concentration did disturb the domain growth. We studied the early stage of the evolution of domains, which is just a new field in international researches.O In the last chapter, we discussed the implement of the coexistence of two phase separation mechanisms of comb-coil PS-PI copolymers in PI selective solvents. The comb-coil A-B copolymer was proved to phase separate in two length scales in its bulk, that is between A/B segments and between comb part and coil chain. By strengthening the miscibility between PS and PI segments by selective solvents, we successfully achieved the coexistence of these two phase separation mechanisms by observing sphere micelles with two sizes in a single solution. Compared the results of DLS and AFM, we estimated the scales of the two sizes of the micelles. We also compared the effects of selectivity of the solvent, the branch length and the branch numbers on the binary size distribution, and found out that increasing these factors made the separation between micelles of different sizes.