Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells*
Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells*
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DOI：
10.1074/mcp.m114.044016
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发表时间：
2015-01
影响因子：
7
通讯作者：
B. Traenkle;F. Emele;R. Anton;O. Poetz;R. S. Häussler;J. Maier;P. Kaiser;Armin M Scholz;S. Nueske;Andrea Buchfellner;Tina Romer;U. Rothbauer
中科院分区：
文献类型：
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作者：
B. Traenkle;F. Emele;R. Anton;O. Poetz;R. S. Häussler;J. Maier;P. Kaiser;Armin M Scholz;S. Nueske;Andrea Buchfellner;Tina Romer;U. Rothbauer
β-catenin是典型Wnt通路的关键组分，在许多发育和体内平衡过程中起着至关重要的作用。β-连环蛋白的不同任务是通过其亚细胞定位和参与多蛋白复合物来协调的。为了更好地了解β-连环蛋白在活细胞中的作用，我们从骆驼类的重链抗体中提取了一组新的单域抗体，称为纳米抗体。我们选择了识别β-catenin的n端、核心或c端结构域的纳米体，并将这些新的高亲和力结合物作为捕获分子应用于内源性β-catenin复合物的夹心免疫测定和共免疫沉淀。此外，我们通过将纳米体的结合部分与荧光蛋白结合，设计了细胞内功能的抗β-连环蛋白染色体体。利用这些染色体体，我们首次能够可视化内源性β-连环蛋白在活细胞中的亚细胞定位和核易位。此外，染色体信号使我们能够利用高含量成像实时追踪可扩散的、低磷酸化的β-catenin在复合处理下的积累。本研究中表征的抗β-连环蛋白纳米体和色体是一种多功能工具，可以在生化和细胞生物学检测中使用一种新颖独特的方法来监测亚细胞β-连环蛋白的动态。
β-catenin is the key component of the canonical Wnt pathway and plays a crucial role in a multitude of developmental and homeostatic processes. The different tasks of β-catenin are orchestrated by its subcellular localization and participation in multiprotein complexes. To gain a better understanding of β-catenin's role in living cells we have generated a new set of single domain antibodies, referred to as nanobodies, derived from heavy chain antibodies of camelids. We selected nanobodies recognizing the N-terminal, core or C-terminal domain of β-catenin and applied these new high-affinity binders as capture molecules in sandwich immunoassays and co-immunoprecipitations of endogenous β-catenin complexes. In addition, we engineered intracellularly functional anti-β-catenin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. For the first time, we were able to visualize the subcellular localization and nuclear translocation of endogenous β-catenin in living cells using these chromobodies. Moreover, the chromobody signal allowed us to trace the accumulation of diffusible, hypo-phosphorylated β-catenin in response to compound treatment in real time using High Content Imaging. The anti-β-catenin nanobodies and chromobodies characterized in this study are versatile tools that enable a novel and unique approach to monitor the dynamics of subcellular β-catenin in biochemical and cell biological assays.