Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation
Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation
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DOI：
10.1021/acs.langmuir.7b02945
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发表时间：
2018-01-23
期刊：
影响因子：
3.9
通讯作者：
Daniel, Susan
中科院分区：
文献类型：
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作者：
Liu, Han-Yuan;Chen, Wei-Liang;Daniel, Susan
重构的支持脂质双层 (SLB) 被广泛用作体外细胞表面模型，因为它们与各种基于表面的分析技术兼容。然而，使用 SLB 作为细胞表面模型的挑战之一是膜组成的复杂性有限，包括跨膜蛋白的掺入和可能影响这些蛋白质活性的脂质多样性。此外，在 SLB 中保持跨膜蛋白的天然方向、功能和迁移率具有挑战性。在这里，我们利用细胞质膜囊泡和聚电解质刷之间的相互作用，从细胞表面萌芽的细胞质膜囊泡创建平面双层。这种方法促进膜蛋白和其他物质直接掺入平面双层，无需使用去污剂或重构，并保留膜成分。此外，聚电解质刷的结构充当平面双层和刚性支撑表面之间的缓冲垫，限制膜蛋白的胞质结构域与该表面的相互作用。单粒子追踪用于分析 GPI 连接的黄色荧光蛋白 (GPI-YFP) 和霓虹绿融合跨膜 P2X2 受体 (P2X2-neon) 的运动，并表明该平台在其天然膜环境中保留了超过 75% 的多次跨膜蛋白的迁移率。酶可及性测定证实蛋白质方向得以保留，并导致胞外结构域面向本体相，胞质侧面向支持物。由于这里介绍的平台保留了细胞质膜的复杂性并保留了蛋白质的方向和流动性，因此它是天然细胞表面的更好的代表性模拟，可能会在旨在了解细胞膜现象的生物测定中找到许多应用。
Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.