Lipid Lateral Organization on Giant Unilamellar Vesicles Containing Lipopolysaccharides
Lipid Lateral Organization on Giant Unilamellar Vesicles Containing Lipopolysaccharides
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DOI：
10.1016/j.bpj.2011.01.012
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发表时间：
2011-02-16
影响因子：
3.4
通讯作者：
Bagatolli, Luis A.
中科院分区：
文献类型：
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作者：
Kubiak, Jakubs;Brewer, Jonathan;Bagatolli, Luis A.
我们开发了一种新的（据我们所知）协议来生成由单一脂多糖（LPS）物种和大肠杆菌极性脂质提取物的混合物组成的巨型单层囊泡（GUV）。选择极性头基大小不同的四种不同的 LPS（即 LPS smooth > LPS-Ra > LPS-Rc > LPS-Rd）来生成由不同 LPS/E 组成的 GUV。大肠杆菌极性脂质混合物。我们的程序包括两个主要步骤：1）、含有LPS的寡层脂质体的生成和纯化； 2)、在生理盐和pH条件下使用含有LPS的寡层囊泡电形成GUV。对 LPS 掺入膜模型（寡层囊泡和 GUV）的分析表明，LPS 的最终浓度低于初始大肠杆菌脂质/LPS 混合物的预期浓度。特别是，我们的协议允许 LPS-smooth 和 LPS-Ra 的掺入量不超过 15 mol%，LPS-Rc 和 LPS-Rd 的掺入量高达 25 mol%（相对于总脂质）。我们使用 GUV 来评估不同 LPS 物种对宿主膜（即大肠杆菌极性脂质提取物）横向结构的影响。罗丹明-DPPE 标记的 GUV 显示，含有高于 10 mol% 的 LPS-Rc 或 LPS-Rd 的 GUV 存在细长的微米大小的脂质结构域。 Laurdan GP 图像证实了这一发现，并表明这种特殊的横向情况对应于流体无序和凝胶（富含 LPS 富集）微米级结构域的共存，与用脂质 A 替换 LPS 时观察到的情况类似。对于包含较大极性头基（即 LPS-smooth 和 LPS-Ra）的 LPS，在 GUV 中探索的所有 LPS 浓度中都观察到不存在微米级结构域（最多类似于15摩尔%）。然而，荧光相关光谱（使用荧光标记的 LPS）和 Laurdan GP 实验在这些微观均质膜中表明存在尺寸低于我们显微镜分辨率（类似于 380 nm 径向）的 LPS 簇。我们的结果表明，LPS 可以在这些细菌模型膜中聚集成凝胶状结构域，并且这些结构域的大小取决于 LPS 的化学结构和浓度。
We developed a new (to our knowledge) protocol to generate giant unilamellar vesicles (GUVs) composed of mixtures of single lipopolysaccharide (LPS) species and Escherichia coli polar lipid extracts. Four different LPSs that differed in the size of the polar headgroup (i.e., LPS smooth > LPS-Ra > LPS-Rc > LPS-Rd) were selected to generate GUVs composed of different LPS/E. coli polar lipid mixtures. Our procedure consists of two main steps: 1), generation and purification of oligolamellar liposomes containing LPSs; and 2), electroformation of GUVs using the LPS-containing oligolamellar vesicles at physiological salt and pH conditions. Analysis of LPS incorporation into the membrane models (both oligolamellar vesicles and GUVs) shows that the final concentration of LPS is lower than that expected from the initial E. coli lipids/LPS mixture. In particular, our protocol allows incorporation of no more than 15 mol % for LPS-smooth and LPS-Ra, and up to 25 mol % for LPS-Rc and LPS-Rd (with respect to total lipids). We used the GUVs to evaluate the impact of different LPS species on the lateral structure of the host membrane (i.e., E. coli polar lipid extract). Rhodamine-DPPE-labeled GUVs show the presence of elongated micrometer-sized lipid domains for GUVs containing either LPS-Rc or LPS-Rd above 10 mol %. Laurdan GP images confirm this finding and show that this particular lateral scenario corresponds to the coexistence of fluid disordered and gel (LPS-enriched)-like micron-sized domains, in similarity to what is observed when LPS is replaced with lipid A. For LPSs containing the more bulky polar headgroup (i.e., LPS-smooth and LPS-Ra), an absence of micrometer-sized domains is observed for all LPS concentrations explored in the GUVs (up to similar to 15 mol %). However, fluorescence correlation spectroscopy (using fluorescently labeled LPS) and Laurdan GP experiments in these microscopically homogeneous membranes suggests the presence of LPS clusters with dimensions below our microscope's resolution (similar to 380 nm radial). Our results indicate that LPSs can cluster into gel-like domains in these bacterial model membranes, and that the size of these domains depends on the chemical structure and concentration of the LPSs.