Fluorescent probe partitioning in giant unilamellar vesicles of 'lipid raft' mixtures
Fluorescent probe partitioning in giant unilamellar vesicles of 'lipid raft' mixtures
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DOI：
10.1042/bj20100516
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发表时间：
2010-09-15
影响因子：
4.1
通讯作者：
Sharom, Frances J.
中科院分区：
文献类型：
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作者：
Juhasz, Janos;Davis, James H.;Sharom, Frances J.
筏状l（o）（液体有序）域在模型系统和细胞中使用显微镜技术的直接可视化，需要荧光探针与已知的分区偏好的一个阶段存在。然而，荧光探针可以显示不同的分配偏好在不同的脂质系统，也可以影响宿主脂质双层的相行为。因此，荧光探针的行为在定义的脂质双层系统与已知的相行为的详细了解是必不可少的，然后才可以用于识别域相态。使用巨大的单层囊泡组成的三元脂质混合物DOPC（1，2-dioleoyl-sn-glycero-3-phosphocholine）/DPPC（1，2-dipalmitoyl-sn-glycero-3-phosphocholine）/胆固醇，其中的相行为是已知的，我们研究了9种常用的荧光探针，使用共聚焦荧光显微镜。每个探针的分配偏好被分配的基础上的域面积分数的定量或通过使用一个良好的特征化的I（d）（液体无序）相标记。荧光探针进行了检查，单独和使用双重或三重标记的方法。大多数探针单独分配到I（d）相，而只有NAP（萘并[2，3-a]芘）和NBD-DPPE [1，2-二棕榈酰基-sn-甘油基-3-磷酸乙醇胺-N-（7-硝基-2-1，3-苯并恶二唑-4-基]优选I（o）相。我们发现Rh-DPPE（Lissamine（TM）罗丹明B-1，2-二棕榈酰-sn-甘油-3-磷酸乙醇胺）增加了混溶性转变温度T（混合）。有趣的是，DiIC（18）（1，1 ′-双十八烷基-3，3，3 ′，3 ′-四甲基吲哚羰花青高氯酸盐）的分配受Bodipy（R）-PC [2-（4，4-二氟-5，7-二甲基-4-硼杂-3a，4a-二氮杂-s-引达省-3-戊酰基）-1-六-癸酰基-sn-甘油基-3-磷酸胆碱]的影响。每种荧光探针的具体用途由其光稳定性、分配偏好、检测脂质相分离的能力和诱导的T（混合物）变化决定。我们证明了在给定的模型膜系统中测试特定荧光探针的重要性，而不是假设它标记特定的脂质相。
Direct visualization of raft-like l(o) (liquid-ordered) domains in model systems and cells using microscopic techniques requires fluorescence probes with known partitioning preference for one of the phases present. However, fluorescent probes may display dissimilar partitioning preferences in different lipid systems and can also affect the phase behaviour of the host lipid bilayer. Therefore a detailed understanding of the behaviour of fluorescent probes in defined lipid bilayer systems with known phase behaviour is essential before they can be used for identifying domain phase states. Using giant unilamellar vesicles composed of the ternary lipid mixture DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine)/cholesterol, for which the phase behaviour is known, we examined nine commonly used fluorescent probes using confocal fluorescence microscopy. The partitioning preference of each probe was assigned either on the basis of quantification of the domain area fractions or by using a well-characterized l(d) (liquid-disordered)-phase marker. Fluorescent probes were examined both individually and using dual or triple labelling approaches. Most of the probes partitioned individually into the l(d) phase, whereas only NAP (naphtho[2,3-a]pyrene) and NBD-DPPE [1,2-dipalmitoyl -sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1, 3-benzoxadiazol-4-yl] preferred the l(o) phase. We found that Rh-DPPE (Lissamine (TM) rhodamine B-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) increased the miscibility transition temperature, T(mix). Interestingly, the partitioning of DiIC(18) (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) was influenced by Bodipy (R)-PC [2-(4,4-difluoro-5, 7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexa-decanoyl-sn-glycero-3-phosphocholine]. The specific use of each of the fluorescent probes is determined by its photostability, partitioning preference, ability to detect lipid phase separations and induced change in T(mix). We demonstrate the importance of testing a specific fluorescent probe in a given model membrane system, rather than assuming that it labels a particular lipid phase.