Department of Medical Physics, Juravinski Cancer Centre at Hamilton Health Sciences, Ontario, Canada.
Biochem J. 2010 Sep 15;430(3):415-23. doi: 10.1042/BJ20100516.
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 rhodamine B-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) increased the miscibility transition temperature, T(mix). Interestingly, the partitioning of DiIC18 (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) was influenced by Bodipy-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.
使用显微镜技术直接观察模型系统和细胞中的筏状 l(o)（有序液体）域，需要具有已知对存在相之一的分配偏好的荧光探针。然而，荧光探针在不同的脂质系统中可能表现出不同的分配偏好，并且还可能影响宿主脂质双层的相行为。因此，在将荧光探针用于识别域相状态之前，必须详细了解它们在具有已知相行为的特定脂质双层系统中的行为。使用由三元脂质混合物 DOPC（1,2-二油酰基-sn-甘油-3-磷酸胆碱）/ DPPC（1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱）/胆固醇组成的巨大的单层囊泡，对于其相行为是已知的，我们使用共焦荧光显微镜检查了九种常用的荧光探针。每个探针的分配偏好是基于域面积分数的定量确定的，或者是使用经过良好表征的 l(d)（无序液体）相标记物确定的。荧光探针分别进行了检查，也使用了双标记或三标记方法。大多数探针单独分配到 l(d)相，而只有 NAP（萘[2,3-a]芘）和 NBD-DPPE [1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-（7-硝基-2-1,3-苯并恶二唑-4-基]优先选择 l(o)相。我们发现 Rh-DPPE（Lissamine rhodamine B-1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺）增加了混合转变温度 T(mix)。有趣的是，DiIC18（1,1'-十八烷基-3,3,3',3'-四甲基吲哚碳菁高氯酸盐）的分配受 Bodipy-PC [2-（4,4-二氟-5,7-二甲基-4-硼-3a,4a-二氮杂-s-茚并-3-戊酰基）-1-十六酰基-sn-甘油-3-磷酸胆碱]的影响。每种荧光探针的特定用途取决于其光稳定性、分配偏好、检测脂质相分离和诱导 T(mix)变化的能力。我们证明了在特定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。
