Lipid bilayers are fundamental structures of cell membranes, at which the transportation of materials and signals in and out of cell membranes take place. These reactions proceed through the actions of membrane proteins, and their proper structures and functions are retained in lipid bilayer membranes. It is necessary to reconstruct membrane proteins into artificial lipid membranes to investigate membrane proteins in vitro systems. In this study we investigate the fusion processes of proteoliposomes into solid-supported lipid bilayers (SLBs) by using an epi-fluorescence microscope (epi-FM) and an atomic force microscope (AFM).<br/>We prepared the SLB containing phosphatidylcholine (from chicken egg) (PC), phosphatidylethanolamine (from chicken egg) (PE), and cholesterol (Chol) at the ratio of 7:1:2 (w/w) (additionally 0.5 of dye-labeled lipid (BODIPY-H-PC) for fluorescence microscope observation) on a mica substrate by the vesicle fusion method. The lipid component is identical to that for previous ion channel studies using a black membrane [1]. The PC+PE+Chol-SLB had uniform fluorescence intensity in epi-FM images (Fig. 1a), and was continuous and fluid in the measurement of fluorescence recovery after photobleaching (FRAP). On the other hand, AFM observation showed that depressed domains with submicrometer lateral size existed in the SLB. We added the suspension of proteoliposomes, which was extracted from Chinese hamster ovary (CHO) cells, to the SLB. After incubating them at 37 °C for 30 min, the proteoliposomes fused to the SLB and appeared as dark domains (Fig. 1b). The majority the proteoliposome components were isolated from the artificial PC+PE+Chol-SLB. In situ epi-FM observation indicated that the fusion proceeded at specific sites in SLB, and the domains existed stably. AFM observation showed that the depressed domains worked as a specific site for the fusion of the proteoliposome.

Fusion processes of proteoliposomes into supported planar lipid bilayers

253rd National Meeting (2017)

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