建構可控制膜蛋白面向之支撐式細胞膜

Abstract

膜蛋白在維持生物細胞機能中扮演著極為重要的角色，然而，膜蛋白脆弱的雙親性結構卻使得傳統上研究膜蛋白變得困難。即使將分離純化後的膜蛋白插入人工脂質體中作為仿生膜來研究膜蛋白，依然存在著難以準確控制膜蛋白在脂質體中的方向性及許多其他可能造成膜蛋白失活的缺點。因此，近來科學家們致力於發展既能保有膜蛋白活性結構及脂雙層膜的構型並能同時對其膜上蛋白進行研究的方法。在本研究中，我們將直接從海拉細胞（Hela cell）膜上發出的巨大細胞膜囊泡（GPMV）鋪成支撐式細胞膜平台，並展示這樣的膜平台能維持細胞膜及膜蛋白原有的方向性。同時，為了對膜平台上細胞膜兩側的膜蛋白進行雙向研究，我們發展出一套膜翻印技術來控制此平台上細胞膜的方向性：先將巨大細胞囊泡破在雲母片上形成支撐式細胞膜，再將此支撐式細胞膜翻印至玻璃基材上以翻轉膜的方向。我們利用海拉細胞中富含的一種穿膜蛋白──水通道蛋白3（Aquaporin 3），作為免疫染色的抗體標靶來檢測直接形成的支撐式細胞膜與翻印後的支撐式細胞膜中膜蛋白的面向。藉由分別會和針對水通道蛋白3在細胞膜內側之結構（即細胞質一側）或在細胞膜外側之結構之抗體的結合結果，可知當巨大細胞膜囊泡直接破裂在基材上時，水通道蛋白3中原本在細胞膜內側的結構將會朝上面向外界水溶液環境；而將膜翻印後，膜蛋白的方向性也會跟著翻轉，即變成原本在細胞膜外側的水通道蛋白結構朝上。有了這套翻膜技術，我們即可控制支撐式細胞膜的方向，並運用此特點來對細胞膜上內側與外側的蛋白質結構與性能來進行研究。

Membrane proteins play an important role in various cellular processes. However, it is still challenging to study them because of their amphiphilic properties. Even if using protein-reconstituted artificial liposomes as biomimetic membrane model, the difficulty of controlling protein-inserting orientation and undesired drawbacks which may make proteins denature still remain. Methods that can retain their structure and membrane topology information during their characterization are desirable for understanding their structure-function behavior. Here, we show the membrane orientation can be retained when we use giant plasma membrane vesicles (GPMVs) derived from Hela cells to form supported cell membrane platforms and also develop a blotting method to control the orientation of the deposited cell membrane in order to study membrane proteins from either the extracellular side or the cytoplasmic side. We used GPMVs to form the supported cell membrane platforms and blotted the deposited membrane on a mica surface onto a glass support to reverse the membrane orientation. We used Aquaporin 3 (AQP3), a native transmembrane protein from Hela cells, as a target to examine the topology of AQP3 in the directly-deposited and reversed membrane platforms. The immunostaining of antibodies targeting either the cyto-domain or ecto-domain of AQP3 showed that the intracellular side of the protein faced the bulk aqueous environment when the GPMVs spontaneously broke on the support, and that the protein orientation was reversed after blotting. With this blotting method, we can thus control the orientation of the supported cell membrane to study membrane protein functions and structures from either side of the cell plasma membrane.