Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes
Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes
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DOI：
10.1016/j.bbamem.2012.03.007
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发表时间：
2012-07-01
影响因子：
3.4
通讯作者：
Schwille, Petra
中科院分区：
文献类型：
--
作者：
Sezgin, Erdinc;Levental, Ilya;Schwille, Petra
已经开发了几种以共存液体无序（Ld）和有序（Lo）脂质相为特征的简化膜模型来模拟细胞膜的异质组织，从而帮助我们理解有序脂质蛋白纳米结构域（称为“筏”）的性质和功能作用。尽管其复杂性大大降低，但使用模型膜对局部脂质环境进行定量表征并非易事，并且与细胞膜的相似之处并不总是显而易见的。同样，各种荧光标记的脂质类似物已被用于研究体外膜组织和功能，尽管这些探针相对于其天然对应物的生物活性通常仍未表征。对于筏偏好的脂质（“筏脂质”，例如鞘脂和甾醇）来说尤其如此，其结构域偏好是其分子结构的严格功能，因此容易受到荧光标记的破坏。在这里，我们分析了合成巨单层囊泡（GUV）和细胞源巨质膜囊泡（GPMV）中多种荧光筏脂质类似物的相分配。我们观察到复杂的分配行为取决于标签大小、极性、电荷和位置、脂质头基和膜组成。 GPMV 中的几种筏脂质类似物分配到有序相，而完全合成的 GUV 则相反，在完全合成的 GUV 中，大多数筏脂质类似物错误分配到无序相。这种行为与合成系统中共存相之间的有序差异大大增强有关。此外，标记后，脂质的分配和配体结合都会受到干扰：虽然霍乱毒素 B 在 Lo 相中结合未标记的 GM1，但它仅在 Ld 相中结合荧光标记的 GM1。通过受激发射损耗 (STED) 纳米显微镜对完整细胞质膜进行的荧光相关光谱 (FCS) 一致地揭示了筏脂质类似物的恒定水平的有限扩散，其分配行为差异很大，表明这些现象具有不同的物理化学基础。 (C) 2012 Elsevier B.V. 保留所有权利。
Several simplified membrane models featuring coexisting liquid disordered (Ld) and ordered (Lo) lipid phases have been developed to mimic the heterogeneous organization of cellular membranes, and thus, aid our understanding of the nature and functional role of ordered lipid-protein nanodomains, termed "rafts". In spite of their greatly reduced complexity, quantitative characterization of local lipid environments using model membranes is not trivial, and the parallels that can be drawn to cellular membranes are not always evident. Similarly, various fluorescently labeled lipid analogs have been used to study membrane organization and function in vitro, although the biological activity of these probes in relation to their native counterparts often remains uncharacterized. This is particularly true for raft-preferring lipids ("raft lipids", e.g. sphingolipids and sterols), whose domain preference is a strict function of their molecular architecture, and is thus susceptible to disruption by fluorescence labeling. Here, we analyze the phase partitioning of a multitude of fluorescent raft lipid analogs in synthetic Giant Unilamellar Vesicles (GUVs) and cell-derived Giant Plasma Membrane Vesicles (GPMVs). We observe complex partitioning behavior dependent on label size, polarity, charge and position, lipid headgroup, and membrane composition. Several of the raft lipid analogs partitioned into the ordered phase in GPMVs, in contrast to fully synthetic GUVs, in which most raft lipid analogs mis-partitioned to the disordered phase. This behavior correlates with the greatly enhanced order difference between coexisting phases in the synthetic system. In addition, not only partitioning, but also ligand binding of the lipids is perturbed upon labeling: while cholera toxin B binds unlabeled GM1 in the Lo phase, it binds fluorescently labeled GM1 exclusively in the Ld phase. Fluorescence correlation spectroscopy (FCS) by stimulated emission depletion (STED) nanoscopy on intact cellular plasma membranes consistently reveals a constant level of confined diffusion for raft lipid analogs that vary greatly in their partitioning behavior, suggesting different physicochemical bases for these phenomena. (C) 2012 Elsevier B.V. All rights reserved.