Forming giant vesicles with controlled membrane composition, asymmetry, and contents
Forming giant vesicles with controlled membrane composition, asymmetry, and contents
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DOI：
10.1073/pnas.1016410108
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发表时间：
2011-06-07
影响因子：
11.1
通讯作者：
Fletcher, Daniel A.
中科院分区：
文献类型：
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作者：
Richmond, David L.;Schmid, Eva M.;Fletcher, Daniel A.
越来越多的知识，参与生物过程，如胞吞作用，胞吐作用，和运动的关键分子组成部分，使直接测试提出的机制模型重建。然而，目前用于从纯化的组分构建日益复杂的细胞结构和功能的技术在其创造模拟真实的细胞的物理和生物化学约束的条件的能力方面受到限制。在这里，我们提出了一个综合的方法，形成巨大的单层囊泡，同时控制（i）脂质组成和不对称性，（ii）定向膜蛋白掺入，和（iii）内部内容物。作为该方法的应用，我们构建了一个合成系统，其中膜蛋白被递送到巨囊泡的外部，模仿胞吐作用的方面。使用共聚焦荧光显微镜，我们可视化的小封装囊泡对接和混合膜组件与巨大的囊泡膜，导致先前封装的膜蛋白暴露于外部环境。这种用于产生巨大囊泡的方法可用于测试依赖于受限体积和复杂膜组成的生物过程模型，并且它可能有助于构建用于治疗和生物材料应用的功能系统。
Growing knowledge of the key molecular components involved in biological processes such as endocytosis, exocytosis, and motility has enabled direct testing of proposed mechanistic models by reconstitution. However, current techniques for building increasingly complex cellular structures and functions from purified components are limited in their ability to create conditions that emulate the physical and biochemical constraints of real cells. Here we present an integrated method for forming giant unilamellar vesicles with simultaneous control over (i) lipid composition and asymmetry, (ii) oriented membrane protein incorporation, and (iii) internal contents. As an application of this method, we constructed a synthetic system in which membrane proteins were delivered to the outside of giant vesicles, mimicking aspects of exocytosis. Using confocal fluorescence microscopy, we visualized small encapsulated vesicles docking and mixing membrane components with the giant vesicle membrane, resulting in exposure of previously encapsulated membrane proteins to the external environment. This method for creating giant vesicles can be used to test models of biological processes that depend on confined volume and complex membrane composition, and it may be useful in constructing functional systems for therapeutic and biomaterials applications.