Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215.
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215. Electronic address: mailto:
J Lipid Res. 2020 May;61(5):758-766. doi: 10.1194/jlr.RA119000565. Epub 2020 Jan 21.
The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.
细胞质膜中液体有序（Lo）脂质域（筏）的形成和性质仍知之甚少。这限制了我们操纵依赖有序脂质域的生物功能的能力。巨大的质膜囊泡（GPMVs）经历大规模的相分离，形成共存的 Lo 和液体无序脂质域。然而，用荧光显微镜检测到的 GPMVs 中的大规模相分离仅在低温下观察到。通过比较Förster 共振能量转移检测与荧光显微镜检测的域形成，我们发现纳米域，即纳米尺寸的域，在比大尺度相更高的温度下（高达 20°C）持续存在，直至生理温度。纳米域在较高温度下的持续存在与先前报道的理论计算一致。为了研究纳米域对脂质组成的敏感性，用胆固醇、磷脂或鞘磷脂组成在外质层中已被环糊精催化的脂质交换改变的哺乳动物细胞制备 GPMVs。在人工脂质囊泡中稳定或破坏有序域形成的脂质取代物对 GPMVs 中纳米域和大尺度相分离的热稳定性具有相似的影响，对于广泛的脂质组成，纳米域在比大尺度相更高的温度下持续存在。这表明，在生理条件下，质膜纳米域可能比大尺度相更容易形成。我们还得出结论，在完整细胞中进行的膜脂质取代能够调节质膜形成有序域的倾向。这意味着可以使用脂质取代来改变依赖有序域的生物学过程。
