Department of Pathology and Molecular Medicine, Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Rm A315 Botterell Hall, Kingston, ON, K7L 3N6, Canada.
J Fluoresc. 2010 Jan;20(1):401-5. doi: 10.1007/s10895-009-0548-x. Epub 2009 Oct 13.
Internalization and intracellular trafficking of membrane proteins are now recognized as essential mechanisms that contribute to a number of cellular processes. Current methods lack the ability to specifically label the plasma membrane of a live cell, follow internalization of labeled membrane molecules, and conclusively differentiate newly formed membrane-derived vesicles from pre-existing endocytic or secretory structures in the cytoplasm. Here, we detail a visualization method for surface biotinylation of plasma membrane-derived vesicles that allows us to follow their progress from membrane to cytosol at specific time points. Using the transmembrane receptor RET as a model, we demonstrate how this method can be applied to identify plasma membrane-derived vesicle maturation, determine RET's presence within these structures, and monitor RET's recycling to the cell surface. This method improves on static and less discriminatory methods, providing a tool for analysis of real-time vesicle trafficking that is applicable to many systems.
现在人们已经认识到，膜蛋白的内化和细胞内转运是许多细胞过程的重要机制。目前的方法缺乏特异性标记活细胞质膜的能力，无法跟踪标记的膜分子的内化，也无法明确区分新形成的膜衍生小泡与细胞质中预先存在的内吞或分泌结构。在这里，我们详细介绍了一种用于质膜衍生小泡的表面生物素化的可视化方法，该方法可使我们在特定时间点从质膜追踪到胞质中的小泡。我们使用跨膜受体 RET 作为模型，展示了如何应用这种方法来识别质膜衍生小泡的成熟过程，确定 RET 在这些结构中的存在，并监测 RET 向细胞表面的再循环。该方法改进了静态和非鉴别性方法，为分析实时囊泡运输提供了一种工具，适用于许多系统。
