Cell membranes contain various kinds of lipids and have lipid bilayer as the fundamental structure. The organization of lipids leads to domain formation, which is responsible for fundamental cellular functions. Ternary “HLC” lipid bilayer consisting of a high transition temperature (High-Tm) lipid, a Low-Tm lipid, and ~30 mol% cholesterol (Chol), is the representative model for lipid raft. Recently, we reported microdomain formation in “LLC” lipid bilayer, comprising Low-Tm phosphatidylethanolamine (PE), Low-Tm phosphatidylcholine (PC) and Chol, which is a unique phenomenon. The microdomains are enriched with polyunsaturated PE and function as fusion site of proteoliposomes, which is vital for the reconstitution of membrane proteins into artificial lipid bilayer. In this study, we aim to investigate the mechanism of the microdomain formation induced by polyunsaturated PE. 1,2-didocosahexaenoyl-PE (22:6-22:6PE), 1,2-dilinolenoyl-PE (18:3-18:3PE), 1-palmitoyl-2-docosahexaenoyl-PE (16:0-22:6PE), 1,2-dioleoyl-PE (18:1-18:1PE) and 1-palmitoyl-2-linoleoyl-PE (16:0-18:2PE) were used. We prepared supported lipid bilayer (SLB) comprising PE, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), and Chol at a molar ratio of CPE:67 − CPE:33 (CPE = 9–22 mol%), using the vesicle fusion method. Two fluorescence-tagged lipids, Rb-DPPE and Bis-bodipy-FL-PC were incorporated. The SLBs were observed with fluorescence microscopy and atomic force microscopy (AFM). Nanomechanical properties of SLB was evaluated by AFM force spectroscopy. PE+POPC+Chol-SLBs of all the PEs were formed at all CPE. In 18:3-18:3PE+POPC+Chol-SLB at = 22 mol%, dark and bright spots were observed in the Rb-DPPE and Bis-bodipy-FL-PC images, respectively, indicating microdomain formation. In the same SLB, the microdomains were observed as depression with depth of ~2 nm in the AFM topographies. We evaluated the area fraction of the microdomains (θ) and its dependence on CPE and unsaturation degree of PE. Microdomains were not observed at the lower unsaturation degree of PE. At higher unsaturation degree of PE, θ increased with CPE. Additionally, 18:3-18:3PE and 16:0-22:6PE have identical total unsaturation degree, but the latter showed distinctly lower θ than the former. The AFM force-distance curves showed the microdomain and the surrounding SLB were at Ld- and Lo-like states. Segregation of polyunsaturated PE drives the Ld-like microdomain formation in the Lo-like region enriched with PC and Chol.

Non-raft microdomain formation in cholesterol-containing lipid bilayers induced by polyunsaturated phosphatidylethanolamine

ACS Spring 2022

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