Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, 80523, USA.
Graduate School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, 226-8503, Japan.
Nat Commun. 2019 Jul 3;10(1):2947. doi: 10.1038/s41467-019-10846-1.
To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo.
为了拓展活细胞成像工具包，我们设计了一种单链可变片段，它在体内以高亲和力和特异性结合线性 HA 表位。这种被称为 HA frankenbody 的探针可以点亮多种颜色的 HA 标记的核、细胞质、膜和线粒体蛋白，适用于多种细胞类型。HA frankenbody 还可以在活细胞中进行最先进的单分子实验，我们通过在 U2OS 细胞中追踪单个 HA 标记的组蛋白和在 U2OS 细胞和神经元中追踪单个 mRNA 翻译动力学来证明这一点。结合 SunTag，我们还可以同时追踪两种 mRNA 种类，证明现在可以仅使用遗传编码工具进行比较的单分子翻译研究。最后，我们使用 HA frankenbody 精确地定量发育中的斑马鱼胚胎中 HA 标记蛋白的表达。HA frankenbody 的多功能性使其成为体内蛋白质动态成像的强大工具。
