Fluorescent probe partitioning in GUVs of binary phospholipid mixtures: Implications for interpreting phase behavior
Fluorescent probe partitioning in GUVs of binary phospholipid mixtures: Implications for interpreting phase behavior
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DOI：
10.1016/j.bbamem.2011.09.006
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发表时间：
2012-01-01
影响因子：
3.4
通讯作者：
Sharom, Frances J.
中科院分区：
文献类型：
--
作者：
Juhasz, Janos;Davis, James H.;Sharom, Frances J.
已知膜脂的相行为会影响许多整合蛋白的组织和功能。巨型单层囊泡（GUV）提供了一个非常有用的模型系统，可以使用荧光成像检查脂质相分离的细节。二元和三元脂质混合物的 GUV 中域的可视化需要对存在的相之一具有分配偏好的荧光探针。为了避免在解释这些脂质混合物的相行为时可能出现的陷阱，需要对这些研究中使用的荧光探针进行足够彻底的表征。现在很明显，荧光探针在脂质相之间显示出不同的分配偏好，具体取决于特定的脂质宿主系统。在这里，我们展示了使用一组荧光探针和共焦荧光显微镜来检查 1,2-二油酰-sn-甘油-3-磷酸胆碱 (DOPC)/1,2-二棕榈酰-sn-甘油-3-磷酸胆碱 (DPPC) 二元混合物的 GUV 中的相分离的好处。研究发现，在很宽的温度范围（14-25℃）下，由 40:60 mol% DOPC/DPPC 组成的 GUV 表面上，斑块和原纤维凝胶相域与液体无序 (Id) 域共存。荧光脂质 12-二棕榈酰-sn-甘油-3-磷酸乙醇胺-N-(7-硝基-2-1,3-苯并恶二唑-4-基 (NBD-DPPE)，被证明是用于原纤维结构域可视化的最有效探针。在 Lissamine (TM) 罗丹明 B 1,2-二十六酰基-sn-甘油-3-磷酸乙醇胺存在下(Rh-DPPE)，我们无法检测原纤维结构域。总体而言，我们表明选择不同的荧光探针作为脂质相报告基因可能会导致对 DOPC/DPPC 混合物相行为的截然不同的解释。(C) 2011 Elsevier B.V. 保留所有权利。
The phase behavior of membrane lipids is known to influence the organization and function of many integral proteins. Giant unilamellar vesicles (GUVs) provide a very useful model system in which to examine the details of lipid phase separation using fluorescence imaging. The visualization of domains in GUVs of binary and ternary lipid mixtures requires fluorescent probes with partitioning preference for one of the phases present To avoid possible pitfalls when interpreting the phase behavior of these lipid mixtures, sufficiently thorough characterization of the fluorescent probes used in these studies is needed. It is now evident that fluorescent probes display different partitioning preferences between lipid phases, depending on the specific lipid host system. Here, we demonstrate the benefit of using a panel of fluorescent probes and confocal fluorescence microscopy to examine phase separation in GUVs of binary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Patch and fibril gel phase domains were found to co-exist with liquid disordered (Id) domains on the surface of GUVs composed of 40:60 mol% DOPC/DPPC, over a wide range of temperatures (14-25 degrees C). The fluorescent lipid, 12-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl (NBD-DPPE), proved to be the most effective probe for visualization of fibril domains. In the presence of Lissamine (TM) rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rh-DPPE) we were unable to detect fibril domains. This fluorophore also affected the partitioning behavior of other fluorescent probes. Overall, we show that the selection of different fluorescent probes as lipid phase reporters can result in very different interpretation of the phase behavior of DOPC/DPPC mixtures. (C) 2011 Elsevier B.V. All rights reserved.