Liquid domains in vesicles investigated by NMR and fluorescence microscopy
Liquid domains in vesicles investigated by NMR and fluorescence microscopy
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DOI：
10.1016/s0006-3495(04)74342-8
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发表时间：
2004-05-01
影响因子：
3.4
通讯作者：
Keller, SL
中科院分区：
文献类型：
--
作者：
Veatch, SL;Polozov, IV;Keller, SL
我们使用H-2-NMR， H-1-MAS NMR和荧光显微镜检测三种特定磷脂比例与30%胆固醇混合的不混溶性：2:1 DOPC/DPPC， 1:1 DOPC/DPPC和1:2 DOPC/DPPC。通过核磁共振和荧光显微镜观察到大规模（远大于160 nm）相分离为液体有序相（L-o）和液晶相（l - α）。通过拟合叠加的H-2-NMR谱，我们定量地确定L-o相强烈富集DPPC，适度富集胆固醇。在不同温度和膜成分下估计的领带线是基于H-2-NMR观察和先前发表的三元相图。H-2-和H-1-MAS核磁共振技术探测的长度尺度明显小于显微镜实验（亚微米与微米头皮），并且在混相转变附近观察到复杂的行为。巨型单层囊泡的荧光显微镜显示在混相转变下的微米级结构域。相比之下，多层囊泡的核磁共振显示，在混相转变下方存在更小（类似于80 nm）的畴，而大规模的脱混发生在较低的温度下，即T-low。在巨大单层囊泡中有序相的表面积分数的荧光显微镜测量中，T-low的转变也很明显。我们的结果再次强调了含胆固醇膜的复杂相行为，并为在类似膜中解释H-2-NMR实验提供了一个框架。
We use H-2-NMR, H-1-MAS NMR, and fluorescence microscopy to detect immiscibility in three particular phospholipid ratios mixed with 30% cholesterol: 2:1 DOPC/DPPC, 1:1 DOPC/DPPC, and 1:2 DOPC/DPPC. Large-scale (much greater than160 nm) phase separation into liquid-ordered (L-o) and liquid-crystalline (L-alpha) phases is observed by both NMR and fluorescence microscopy. By fitting superimposed H-2-NMR spectra, we quantitatively determine that the L-o phase is strongly enriched in DPPC and moderately enriched in cholesterol. Tie-lines estimated at different temperatures and membrane compositions are based on both H-2-NMR observations and a previously published ternary phase diagram. H-2- and H-1-MAS NMR techniques probe significantly smaller length scales than microscopy experiments (submicron versus micron-scalp), and complex behavior is observed near the miscibility transition. Fluorescence microscopy of giant unilamellar vesicles shows micrometer- scale domains below the miscibility transition. In contrast, NMR of multilamellar vesicles gives evidence for smaller (similar to80 nm) domains just below the miscibility transition, whereas large-scale demixing occurs at a lower temperature, T-low. A transition at T-low is also evident in fluorescence microscopy measurements of the surface area fraction of ordered phase in giant unilamellar vesicles. Our results reemphasize the complex phase behavior of cholesterol-containing membranes and provide a framework for interpreting H-2-NMR experiments in similar membranes.