Kanwa Nishu, Reverte-López María, Schwille Petra
Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry.
Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry;
J Vis Exp. 2025 Jun 20(220). doi: 10.3791/68530.
Biomimetic lipid membranes in the form of giant unilamellar vesicles (GUVs) are commonly used to mimic cellular membrane behavior because of the ease of protein reconstitution inside GUVs, visualization, as well as understanding cellular membrane-protein dynamics. However, cell membranes comprise lipid rafts (or domains) arising from the presence of different lipids in the cellular membrane. Such increased complexity in model systems can be incorporated to result into phase separated GUVs, where lipid composition can be finely tuned. While encapsulation methods for the generation of homogeneous GUVs are widely known, methods to encapsulate proteins within phase separated GUVs are limited. Here, this protocol presents a simplified one-pot production of phase separated GUVs, comprised of liquid-disordered (Ld) and liquid ordered (Lo) domains, efficiently encapsulating different cytoskeletal proteins, i.e., FtsZ and actin, making the method a versatile tool for minimal cell production. Specifically, this approach uses an emulsion transfer protocol to produce GUVs with a high encapsulation efficiency. In this method, a lipid-monolayer is first generated by emulsifying a protein solution in a lipid/oil mixture, where lipids of varying phase transition temperatures are chosen to yield phase separation in the resultant GUVs. This emulsion is transferred gently on top of a lipid-in-oil solution in another tube, resulting in the formation of a water-oil interface. The solution is then centrifuged at elevated temperatures (ideally at 37 °C to retain protein activity), after which GUVs are collected for imaging. This method simplifies the in vitro reconstitution of cytoskeletal proteins within phase separated GUVs without using a cumbersome laboratory setup, and thus serves as a convenient method for studying the mechanics of cytoskeletal-membrane interactions in confinement.
巨型单层囊泡（GUVs）形式的仿生脂质膜由于易于在GUVs内部进行蛋白质重构、可视化以及理解细胞膜 - 蛋白质动力学，因而常用于模拟细胞膜行为。然而，细胞膜包含由细胞膜中不同脂质的存在而产生的脂筏（或结构域）。模型系统中这种增加的复杂性可以被纳入以产生相分离的GUVs，其中脂质组成可以被精细调节。虽然用于生成均匀GUVs的封装方法广为人知，但将蛋白质封装在相分离的GUVs内的方法却很有限。在此，本方案展示了一种简化的一锅法生产相分离的GUVs，其由液相无序（Ld）和液相有序（Lo）结构域组成，能够有效封装不同的细胞骨架蛋白，即FtsZ和肌动蛋白，使该方法成为用于最小细胞生产的通用工具。具体而言，这种方法使用乳液转移方案来生产具有高封装效率的GUVs。在该方法中，首先通过在脂质/油混合物中乳化蛋白质溶液来生成脂质单层，其中选择具有不同相变温度的脂质以在所得的GUVs中产生相分离。将该乳液轻轻转移到另一管油包脂质溶液的顶部，导致形成水 - 油界面。然后在升高的温度（理想情况下为37°C以保持蛋白质活性）下离心该溶液，之后收集GUVs用于成像。该方法简化了相分离的GUVs内细胞骨架蛋白的体外重构，而无需使用繁琐的实验室设置，因此是研究受限条件下细胞骨架 - 膜相互作用机制的便捷方法。
