构建具有可控方向的支撑细胞膜。,Scientific Reports

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构建具有可控方向的支撑细胞膜。
Scientific Reports ( IF 3.8 ) Pub Date : 2019-02-26 , DOI: 10.1038/s41598-019-39075-8
Shao-Wei Lyu , Jou-Fang Wang , Ling Chao

膜蛋白在各种细胞过程中发挥着重要作用。在表征过程中能够保留其结构和膜拓扑信息的方法对于理解其结构功能行为是可取的。在这里，我们使用巨型质膜囊泡（GPMV）形成支撑的细胞膜，并开发一种印迹方法来控制沉积的细胞膜的方向，以便研究细胞外或细胞质侧的膜蛋白。我们表明，直接沉积的膜可以保留膜的方向，并且可以将云母上沉积的膜吸到玻璃上以反转膜的方向。我们使用水通道蛋白 3 (AQP3)（Hela 细胞中丰富的天然跨膜蛋白）作为靶标来检查直接沉积和反向膜平台中的细胞膜方向。针对 AQP3 细胞结构域或胞外结构域的抗体的免疫染色表明，当 GPMV 在支持物上自发破裂时，细胞膜的细胞内侧面向大量水环境，并且印迹后膜方向发生逆转。通过这种印迹方法，我们可以控制支持的细胞膜的方向，以研究细胞质膜两侧的膜蛋白功能和结构。

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Constructing Supported Cell Membranes with Controllable Orientation.

Membrane proteins play important roles in various cellular processes. Methods that can retain their structure and membrane topology information during their characterization are desirable for understanding their structure-function behavior. Here, we use giant plasma membrane vesicles (GPMVs) to form the supported cell membrane and develop a blotting method to control the orientation of the deposited cell membrane in order to study membrane proteins from either the extracellular or the cytoplasmic sides. We show that the membrane orientation can be retained in the directly-deposited membrane and the deposited membrane on mica can be blotted onto glass to reverse the membrane orientation. We used Aquaporin 3 (AQP3), an abundant native transmembrane protein in Hela cells, as a target to examine the cell membrane orientation in the directly-deposited and reversed membrane platforms. The immunostaining of antibodies targeting either the cyto-domain or ecto-domain of AQP3 shows that the intracellular side of the cell membrane faced the bulk aqueous environment when the GPMVs spontaneously ruptured on the support and that the membrane 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.

更新日期：2019-02-26

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