Buser Dominik P, Spiess Martin
Biozentrum, University of Basel;
Biozentrum, University of Basel.
J Vis Exp. 2019 Feb 21(144). doi: 10.3791/59111.
Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To study retrograde protein traffic, we have recently developed a versatile nanobody-based toolkit to analyze transport from the cell surface to the Golgi complex, either by fixed and live cell imaging, by electron microscopy, or biochemically. We engineered functionalized anti-green fluorescent protein (GFP) nanobodies - small, monomeric, high-affinity protein binders - that can be applied to cell lines expressing membrane proteins of interest with an extracellular GFP moiety. Derivatized nanobodies bound to the GFP reporters are specifically internalized and transported piggyback along the reporters' sorting routes. Nanobodies were functionalized with fluorophores to follow retrograde transport by fluorescence microscopy and live imaging, with ascorbate peroxidase 2 (APEX2) to investigate the ultrastructural localization of reporter-nanobody complexes by electron microscopy, and with tyrosine sulfation (TS) motifs to assess kinetics of trans-Golgi network (TGN) arrival. In this methodological article, we outline the general procedure to bacterially express and purify functionalized nanobodies. We illustrate the powerful use of our tool using the mCherry- and TS-modified nanobodies to analyze endocytic uptake and TGN arrival of cargo proteins.
蛋白质和膜从细胞表面运输到高尔基体及更远的地方对于体内平衡、细胞器特性和生理学至关重要。为了研究逆行蛋白运输，我们最近开发了一种基于通用纳米抗体的工具包，通过固定细胞成像、活细胞成像、电子显微镜或生物化学方法来分析从细胞表面到高尔基体复合体的运输。我们设计了功能化的抗绿色荧光蛋白（GFP）纳米抗体——小型、单体、高亲和力的蛋白结合物——可应用于表达带有细胞外GFP部分的感兴趣膜蛋白的细胞系。与GFP报告基因结合的衍生纳米抗体被特异性内化，并沿着报告基因的分选途径搭便车运输。纳米抗体用荧光团进行功能化，以便通过荧光显微镜和活细胞成像追踪逆行运输；用抗坏血酸过氧化物酶2（APEX2）通过电子显微镜研究报告基因-纳米抗体复合物的超微结构定位；用酪氨酸硫酸化（TS）基序评估反式高尔基体网络（TGN）到达的动力学。在这篇方法学文章中，我们概述了细菌表达和纯化功能化纳米抗体的一般程序。我们通过使用mCherry和TS修饰的纳米抗体来分析货物蛋白的内吞摄取和TGN到达，展示了我们工具的强大用途。
