Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.
J Am Chem Soc. 2013 Apr 17;135(15):5620-30. doi: 10.1021/ja310831m. Epub 2013 Apr 3.
The characterization of the lateral organization of components in biological membranes and the evolution of this arrangement in response to external triggers remain a major challenge. The concept of lipid rafts is widely invoked; however, direct evidence of the existence of these ephemeral entities remains elusive. We report here the use of secondary ion mass spectrometry (SIMS) to image the cholesterol-dependent cohesive phase separation of the ganglioside GM1 into nano- and microscale assemblies in a canonical lipid raft composition of lipids. This assembly of domains was interrogated in a model membrane system composed of palmitoyl sphingomyelin (PSM), cholesterol, and an unsaturated lipid (dioleoylphosphatidylcholine, DOPC). Orthogonal isotopic labeling of every lipid bilayer component and monofluorination of GM1 allowed generation of molecule specific images using a NanoSIMS. Simultaneous detection of six different ion species in SIMS, including secondary electrons, was used to generate ion ratio images whose signal intensity values could be correlated to composition through the use of calibration curves from standard samples. Images of this system provide the first direct, molecule specific, visual evidence for the colocalization of cholesterol and GM1 in supported lipid bilayers and further indicate the presence of three compositionally distinct phases: (1) the interdomain region; (2) micrometer-scale domains (d > 3 μm); (3) nanometer-scale domains (d = 100 nm to 1 μm) localized within the micrometer-scale domains and the interdomain region. PSM-rich, nanometer-scale domains prefer to partition within the more ordered, cholesterol-rich/DOPC-poor/GM1-rich micrometer-scale phase, while GM1-rich, nanometer-scale domains prefer to partition within the surrounding, disordered, cholesterol-poor/PSM-rich/DOPC-rich interdomain phase.
生物膜中成分的侧向组织特征以及这种排列结构对外界刺激的响应演变仍然是一个主要挑战。脂质筏的概念被广泛引用；然而，这些短暂实体存在的直接证据仍然难以捉摸。我们在这里报告使用二次离子质谱（SIMS）来成像神经节苷脂 GM1 中的胆固醇依赖性凝聚相分离，以形成经典脂质筏组成的纳米和微尺度组装体。在由棕榈酰鞘氨醇（PSM）、胆固醇和不饱和脂质（二油酰基磷脂酰胆碱，DOPC）组成的模型膜系统中，研究了这种结构域的组装。每个脂质双层成分的正交同位素标记和 GM1 的单氟化允许使用 NanoSIMS 生成分子特异性图像。SIMS 中同时检测六种不同的离子种类，包括二次电子，使用离子比图像生成，其信号强度值可以通过使用标准样品的校准曲线与组成相关联。该系统的图像提供了第一个直接的、分子特异性的、可视化证据，证明胆固醇和 GM1 在支撑脂质双层中的共定位，并且进一步表明存在三种组成上不同的相：（1）域间区域；（2）微米尺度的域（d > 3 μm）；（3）纳米尺度的域（d = 100nm 至 1μm）定位于微米尺度的域和域间区域内。富含 PSM 的纳米尺度域倾向于在更有序的、富含胆固醇/贫 DOPC/富含 GM1 的微米尺度相中分配，而富含 GM1 的纳米尺度域倾向于在周围无序的、贫胆固醇/富含 PSM/富含 DOPC 的域间相中分配。
