Measuring lipid asymmetry in planar supported bilayers by fluorescence interference contrast microscopy
Measuring lipid asymmetry in planar supported bilayers by fluorescence interference contrast microscopy
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DOI：
10.1021/la047654w
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发表时间：
2005-02-15
期刊：
影响因子：
3.9
通讯作者：
Tamm, LK
中科院分区：
文献类型：
--
作者：
Crane, JM;Kiessling, V;Tamm, LK
作为生物细胞膜的模型，具有不对称分布的工程支持的脂类双层具有很大的科学和实用价值。原则上，可以通过Langmuir-Blodgett/Schafer(Lb/LS)或Langmuir-Blodgett/Vf(Lb/VF)技术(Kalb等人)来制备不对称支撑的脂质双层。毕奇姆。生物群落。学报1992,1103,307-316)。然而，在生物相关的脂质双层中的不对称性的保留从来没有在这些系统中被实验检验过。在目前的工作中，我们开发了一种基于荧光干涉对比显微镜(FLIC)的技术来测量支撑双层中的脂质不对称性。我们比较了含胆固醇和不含胆固醇的双层膜在液体有序相(L(O))和液体无序相(L(D))中的最终不对称程度。在所检测的五种不同的荧光脂质探针中，1，2-dipalmitoyl-phosphatidylethanolamine-N-[lissamine罗丹明是在荧光显微镜下研究复杂组成和相的负载型双层膜的最佳方法。由LB/LS方法制备的不对称标记双层膜一旦完成，最多只有70%-80%是不对称的。在LO或ID相的LB/LS双层中，胆固醇增加了相对单层之间的脂质混合程度。对于最初的单层，使用系链聚合物载体并没有改善所得到的双层中的脂类不对称性。然而，在使用或不使用系链聚合物载体的情况下，不对称的LB/VF双层保留了几乎100%的不对称标记。通过荧光显微镜观察发现，L_b/L_s双层膜中心的脂质混合对L(D)-L(O)相共存的出现有很大的影响。
There is substantial scientific and practical interest in engineering supported lipid bilayers with asymmetric lipid distributions as models for biological cell membranes. In principle, it should be possible to make asymmetric supported lipid bilayers by either the Langmuir-Blodgett/Schafer (LB/LS) or Langmuir-Blodgett/vesicle fusion (LB/VF) techniques (Kalb et al. Biochim. Biophys. Acta 1992, 1103, 307-316). However, the retention of asymmetry in biologically relevant lipid bilayers has never been experimentally examined in any of these systems. In the present work, we developed a technique that is based on fluorescence interference contrast (FLIC) microscopy to measure lipid asymmetry in supported bilayers. We compared the final degree of lipid asymmetry in LB/LS and LB/VF bilayers with and without cholesterol in liquid-ordered (l(o)) and liquid-disordered (l(d)) phases. Of five different fluorescent lipid probes that were examined, 1,2-dipalmitoyl-phosphatidylethanolamine-N-[lissamine rhodamine 13] was the best for studying supported bilayers of complex composition and phase by FLIC microscopy. An asymmetrically labeled bilayer made by the LB/LS method was found to be at best 70-80% asymmetric once completed. In LB/LS bilayers of either lo Or Id phase, cholesterol increased the degree of lipid mixing between the opposing monolayers. The use of a tethered polymer support for the initial monolayer did not improve lipid asymmetry in the resulting bilayer. However, asymmetric LB/VF bilayers retained nearly 100% asymmetric label, with or without the use of a tethered polymer support. Finally, lipid mixing across the center of LB/LS bilayers was found to have drastic effects on the appearance of l(d)-l(o) phase coexistence as shown by epifluorescence microscopy.