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动态光诱导模型膜上的蛋白质图案。

Dynamic Light-Induced Protein Patterns at Model Membranes.

机构信息

Institute of Physiological Chemistry and Pathobiochemistry, University of Münster.

Institute of Physiological Chemistry and Pathobiochemistry, University of Münster;

出版信息

J Vis Exp. 2024 Feb 23(204). doi: 10.3791/66531.

Abstract

The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell polarization, migration, and division. Thus, methods to recruit proteins to model membranes with subcellular resolution and high temporal control are essential when reproducing and controlling such processes in synthetic cells. Here, a method is described for fabricating light-regulated reversible protein patterns at lipid membranes with high spatiotemporal precision. For this purpose, we immobilize the photoswitchable protein iLID (improved light-inducible dimer) on supported lipid bilayers (SLBs) and on the outer membrane of giant unilamellar vesicles (GUVs). Upon local blue light illumination, iLID binds to its partner Nano (wild-type SspB) and allows the recruitment of any protein of interest (POI) fused to Nano from the solution to the illuminated area on the membrane. This binding is reversible in the dark, which provides dynamic binding and release of the POI. Overall, this is a flexible and versatile method for regulating the localization of proteins with high precision in space and time using blue light.

摘要

蛋白质在细胞膜上的精确定位和激活在特定时间会引发许多细胞过程,包括细胞极化、迁移和分裂。因此,当在合成细胞中复制和控制这些过程时,用亚细胞分辨率和高时间控制来募集蛋白质到模型膜的方法是必不可少的。在这里,描述了一种用于在脂质膜上以高时空精度制造光调控的可逆蛋白质图案的方法。为此,我们将光可切换蛋白 iLID(改良的光诱导二聚体)固定在支撑脂质双层(SLB)和巨大的单室脂质体(GUV)的外膜上。局部蓝光照射后,iLID 与它的伴侣 Nano(野生型 SspB)结合,并允许从溶液中招募到膜上被照亮区域的任何与 Nano 融合的感兴趣的蛋白质(POI)。在黑暗中,这种结合是可逆的,这提供了 POI 的动态结合和释放。总的来说,这是一种使用蓝光以高精度在空间和时间上调节蛋白质定位的灵活和通用的方法。

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