1. A genetically encoded probe for imaging nascent and mature HA-tagged proteins in vivo
- Author
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Zhao, N., Kamijo, K., Fox, P. D., Oda, H., Morisaki, T., Sato, Y., Kimura, Hiroshi, and Stasevich, T. J.
- Subjects
0301 basic medicine ,Recombinant Fusion Proteins/isolation & purification/*metabolism ,Embryo, Nonmammalian ,Neurons/metabolism ,General Physics and Astronomy ,02 engineering and technology ,Epitope ,Epitopes ,Protein biosynthesis ,lcsh:Science ,Zebrafish ,Neurons ,Multidisciplinary ,Chemistry ,Translation (biology) ,021001 nanoscience & nanotechnology ,Single Molecule Imaging ,3. Good health ,Cell biology ,0210 nano-technology ,Molecular Probes/*metabolism ,Cell type ,RNA, Messenger/genetics/metabolism ,Science ,Recombinant Fusion Proteins ,Green Fluorescent Proteins ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Single-molecule biophysics ,In vivo ,Cell Line, Tumor ,Animals ,RNA, Messenger ,Green Fluorescent Proteins/metabolism ,Staining and Labeling ,General Chemistry ,030104 developmental biology ,Cell culture ,Cytoplasm ,Molecular Probes ,Protein Biosynthesis ,Epitopes/*metabolism ,Single-Chain Antibodies/metabolism ,lcsh:Q ,Zebrafish/embryology ,Single-Chain Antibodies ,Embryo, Nonmammalian/metabolism - Abstract
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., Expression of genetically encoded antibodies for cell labelling is often limited by folding issues. Here, the authors engineer an anti-HA scFv antibody that works in the cellular environment and use it to track mRNA translation dynamics in living cells and to label proteins in live zebrafish embryos.
- Published
- 2019