Absence of fluid-ordered/fluid-disordered phase coexistence in ceramide/POPC mixtures containing cholesterol
Absence of fluid-ordered/fluid-disordered phase coexistence in ceramide/POPC mixtures containing cholesterol
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DOI：
10.1529/biophysj.105.077107
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发表时间：
2006-06-01
影响因子：
3.4
通讯作者：
Bagatolli, L. A.
中科院分区：
文献类型：
--
作者：
Fidorra, M.;Duelund, L.;Bagatolli, L. A.
用差示扫描量热法、傅里叶变换红外光谱、原子力显微镜和共聚焦/双光子激发荧光显微镜(包括Laurdan广义偏振函数像)研究了温度对猪脑神经酰胺和1-棕榈酰基-2-油酰基磷脂酰胆碱(POPC)在添加和不添加胆固醇前后的脂双层侧向结构的影响。对于不同的POPC/神经酰胺混合物(最高可达25mol%的神经酰胺)，观察到了一个宽的凝胶/流体相共存的温度区域，其特征是存在微米级的条状和花状的凝胶相区。这种观察到的相共存情况与之前报道的这种混合物的情况相反，在这种情况下，使用整体Laurdan广义极化(GP)测量声称没有凝胶/流体相共存。我们证明，当考虑Laurdan GP函数的可加性来检验用整体荧光测量获得的数据时，这种明显的差异(基于在显微镜和荧光计中获得的Laurdan GP数据之间的直接比较)消失了。在POPC/神经酰胺混合物中加入胆固醇显示出从凝胶/流体到凝胶/液体有序相共存的逐渐转变，实验中使用的不同实验技术表明了这一点。这最后的结果表明，在所研究的三元混合物中不存在流体有序/流体无序相共存，而在类似摩尔浓度下观察到的其他含神经酰胺基脂混合物(如用作典型RAFT模型膜的POPC/鞘磷脂/胆固醇)中的流体有序/流体无序相共存。此外，我们在三元混合物中观察到一个关键的胆固醇浓度，通过观察膜上的三个不同区域，该浓度产生了特殊的横向模式。
The effect of temperature on the lateral structure of lipid bilayers composed of porcine brain ceramide and 1-palmitoyl 2-oleoyl-phosphatidylcholine (POPC), with and without addition of cholesterol, were studied using differential scanning calorimetry, Fourier transformed infrared spectroscopy, atomic force microscopy, and confocal/two-photon excitation fluorescence microscopy (which included LAURDAN generalized polarization function images). A broad gel/fluid phase coexistence temperature regime, characterized by the presence of micrometer-sized gel-phase domains with stripe and flowerlike shapes, was observed for different POPC/ceramide mixtures (up to similar to 25 mol% ceramide). This observed phase coexistence scenario is in contrast to that reported previously for this mixture, where absence of gel/fluid phase coexistence was claimed using bulk LAURDAN generalized polarization (GP) measurements. We demonstrate that this apparent discrepancy (based on the direct comparison between the LAURDAN GP data obtained in the microscope and the fluorometer) disappears when the additive property of the LAURDAN GP function is taken into account to examine the data obtained using bulk fluorescence measurements. Addition of cholesterol to the POPC/ceramide mixtures shows a gradual transition from a gel/fluid to gel/liquid-ordered phase coexistence scenario as indicated by the different experimental techniques used in our experiments. This last result suggests the absence of fluid-ordered/fluid-disordered phase coexistence in the ternary mixtures studied in contrast to that observed at similar molar concentrations with other ceramide-base-containing lipid mixtures (such as POPC/sphingomyelin/cholesterol, which is used as a canonical raft model membrane). Additionally, we observe a critical cholesterol concentration in the ternary mixtures that generates a peculiar lateral pattern characterized by the observation of three distinct regions in the membrane.