Brown Angela C, Towles Kevin B, Wrenn Steven P
Department of Chemical and Biological Engineering, Drexel University, 3141 Chestnut Street, Philadelphia, Pennsylvania 19104.
Langmuir. 2007 Oct 23;23(22):11188-96. doi: 10.1021/la7006342. Epub 2007 Sep 22.
The heterogeneity of cell membranes, specifically the presence of lipid rafts, has been hypothesized to play a role in a large number of cellular processes. Although extensive work has been carried out to show the function of lipid rafts in these processes, the characterization of lipid rafts has proven to be extremely difficult. It is known that raft size is relevant to the function of cellular processes and that raft coalescence may be a driving factor for these processes; however, it remains unclear what factors influence raft size and coalescence in natural cell membranes. In this work, we study two ternary model phospholipid and cholesterol systems using two steady-state fluorescent techniques to detect and characterize membrane domains. Domain size is determined through the use of a model to relate experimental Förster resonance energy transfer (FRET) measurements to domain size. Domains in the range of 3-15 nm were detected in a dioleoylphosphatidylcholine-dipalmitoylphosphatidylcholine-cholesterol (DOPC-DPPC-Chol) system, while only a very small region containing domains was detected in a 1-palmitoyl-2-oleoyl-phosphatidylcholine-dipamitoylphosphatidylcholine-cholesterol (POPC-DPPC-Chol) system. In addition, the polarity-dependent emission maximum shift of the acceptor 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DAN-PC) was used to detect the type of liquid phase(s) present in the membrane. It was found that, even in the case in which no two-phase coexistence was observed (POPC-DPPC-Chol), two liquid phases are present, although not necessarily in coexistence. These steady-state fluorescent techniques provide a method for detecting the presence of very small domains in model membranes and provide previously inaccessible detail about the phase behavior of these two systems.
细胞膜的异质性，特别是脂筏的存在，被认为在大量细胞过程中发挥作用。尽管已经进行了大量工作来展示脂筏在这些过程中的功能，但事实证明，对脂筏进行表征极其困难。已知脂筏大小与细胞过程的功能相关，并且脂筏聚结可能是这些过程的驱动因素；然而，尚不清楚哪些因素会影响天然细胞膜中脂筏的大小和聚结。在这项工作中，我们使用两种稳态荧光技术研究了两个三元模型磷脂和胆固醇系统，以检测和表征膜结构域。通过使用一个模型将实验性的福斯特共振能量转移（FRET）测量结果与结构域大小相关联，从而确定结构域大小。在二油酰磷脂酰胆碱 - 二棕榈酰磷脂酰胆碱 - 胆固醇（DOPC - DPPC - Chol）系统中检测到了3 - 15纳米范围内的结构域，而在1 - 棕榈酰 - 2 - 油酰 - 磷脂酰胆碱 - 二棕榈酰磷脂酰胆碱 - 胆固醇（POPC - DPPC - Chol）系统中仅检测到了一个包含结构域的非常小的区域。此外，受体1 - 肉豆蔻酰 - 2 - [12 - [(5 - 二甲基氨基 - 1 - 萘磺酰基)氨基]十二烷酰基] - sn - 甘油 - 3 - 磷酸胆碱（DAN - PC）的极性依赖性发射最大值位移被用于检测膜中存在的液相类型。结果发现，即使在未观察到两相共存的情况下（POPC - DPPC - Chol），也存在两种液相，尽管不一定共存。这些稳态荧光技术提供了一种检测模型膜中非常小的结构域存在的方法，并提供了关于这两个系统相行为的以前无法获得的细节。
