Graduate Group in Biophysics, University of California, Berkeley, CA 94720, USA.
Proc Natl Acad Sci U S A. 2011 Jun 7;108(23):9431-6. doi: 10.1073/pnas.1016410108. Epub 2011 May 18.
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.
对参与内吞作用、外排作用和运动等生物过程的关键分子成分的认识不断增加，使得通过重构来直接测试提出的机制模型成为可能。然而，目前从纯化的成分构建越来越复杂的细胞结构和功能的技术在创造能够模拟真实细胞物理和生化限制的条件方面能力有限。在这里，我们提出了一种形成具有同时控制（i）脂质组成和不对称性，（ii）定向膜蛋白掺入和（iii）内部内容的巨大单室囊泡的综合方法。作为该方法的应用，我们构建了一个模拟外排作用某些方面的合成系统，其中膜蛋白被递送到巨大囊泡的外部。使用共聚焦荧光显微镜，我们观察到小的封装囊泡与巨囊泡膜对接并混合膜成分，导致先前封装的膜蛋白暴露于外部环境中。这种制备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的模型，并且对于构建用于治疗和生物材料应用的功能性系统可能有用。
