Study On The Isolation And Reconstruction Of Lipid Rafts With Atomic Force Microscopy

The molecular interactions were studied in the prepared bilayers composed of phosphatidylcholine, phosphatidylethanolamine, cholesterol, sphingomyelin and ceramide. We analyzed the reason of the formation of different structures and function of the groups of each molecule in ordered to reconstruct the lipid rafts structure. We do the research about lipid rafts using preparing the lipid rafts from the cells and simulating in vitro, including the structure, compositions, and the physical and chemical property. Further study revealed the structure and function of lipid rafts.Methods: 1. we could obtain the mean molecular area and surface pressure isotherms of monolayer by LB technique and prepare the bilayers on the refresh mica keeping the certain surface pressure using Y type method. 2. Observation the structures of bilayers composed of different lipids by atomic force microscopy. 3. Detergent-resistant membranes fraction of human erythrocyte was isolated by Triton X-100 using sucrose density gradient centrifugation method.Results: 1. the single system, the monolayer of phosphatidylcholine showed two clear phase point, but for cholesterol and sphingomyelin we could not observe the phase point. The AFM experiments showed that the PC molecules could tend to form the complex structure, meanwhile, the SM appeared fiat, uniformly in shape. The cholesterol formed the steady sheet layer. According to the experiment, master the method of preparing the bilayers to establish foundation to further study the complex systems.2. For the binary system, the equimolar SM/PC, SM/PE, SM/Chol showed different character of structure. SM/DOPC could form bilayers with corrugated structures at the low surface pressure. When increased the surface pressure, the bilayers took on round microdomain structure, floating on the liquid PC. They could form the sheet layer as the surface pressure reached to the maximum. For the SM/PE system, at the low surface pressure, they could form microdomain structure with clear boundary, in the middle of it, there were many complexes. At the high surface pressure, they showed big flat sheet and condensed structures. For SM/Chol, there appeared uniform structure with covered mica about 60%. They could form the condensed structure with many small holes in it.3. For the ternary System, we chose the equimolar SM/Chol/PC, SM/Chol/PE and SM/Chol/ceramide mixtures as materials to prepare the bilayers. Meanwhile, we studied the effect of different cholesterol content in the equimolar SM/Chol binary bilayers. The results of experiments showed that large differences in the size and shape of domains in the different mixtures. For SM/Chol systems, the morphological analysis revealed that there was regular, fiat microdomains. Adding DOPC, there appeared regular, round microdomains structure, but for DOPE, it was difficult to form the regular microdomains. They could form lipid vesicle structure. When adding the ceramide, the images showed there were many granules on the mica. The structure was round with hollow on the center; it was similar with proposed lipid rafts. The results demonstrated that cholesterol could interact with SM of saturated lipid chain to form the microdomains structure. The microdomains structure became gradually larger and larger with the increasing cholesterol. In the end, they formed the stable sheet structures. Based on above research, for the more ingredients system, Atomic force microscopy had been used to study the effect of ceramide in the equimolar SM/DOPC/Chol ternary bilayers. The structure formed the complex firstly, and then evolved the particular net-shaped with the increasing of ceramide.4. The isolation and measurement of lipid rafts. Detergent-resistant membranes fraction of human erythrocyte was isolated by Triton X-100 using sucrose density gradient centrifugation method. The AFM images show that the single lipid raft existed in the form of particle complex with the diameter of 210 nm. The structure was round with hollow on the centre. The LB bilayers of isolated "ghost" showed that the stable slice formed with many complexes, which had certain structure property. The data of experiments demonstrated that the simulation of lipid rafts was similar with isolated lipid rafts in the aspect of morphology.Conclusions: The preparing of LB bilayers was relation with many ingredients, such as temperature, pH value, intensity and so on. The study showed the formations of certain structures were mainly due to the interaction of different functional groups of each molecule. Different functional groups made the molecules possess different hydrogen bond capacity to form the microdomain structure. In our systems, the sequence of cholesterol interaction with other molecules was ceramide>SM>DOPC. These microdomains that formed the lipid rafts complexes structure may have implications for cellular signaling.