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      A genetically encoded probe for imaging nascent and mature HA-tagged proteins in vivo

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          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.

          Abstract

          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.

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          Most cited references 50

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          The green fluorescent protein.

          In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.
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            A general method for the covalent labeling of fusion proteins with small molecules in vivo.

            Characterizing the movement, interactions, and chemical microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic molecules capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small molecule to a protein, the formation of stable complexes between biarsenical compounds and peptides containing cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.
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              A general method to improve fluorophores for live-cell and single-molecule microscopy

              Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here, we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.
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                Author and article information

                Contributors
                Tim.Stasevich@colostate.edu
                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group UK (London )
                2041-1723
                3 July 2019
                3 July 2019
                2019
                : 10
                Affiliations
                [1 ]ISNI 0000 0004 1936 8083, GRID grid.47894.36, Department of Biochemistry and Molecular Biology, , Colorado State University, ; Fort Collins, CO 80523 USA
                [2 ]ISNI 0000 0001 2179 2105, GRID grid.32197.3e, Graduate School of Life Science and Technology, , Tokyo Institute of Technology, ; Yokohama, 226-8503 Japan
                [3 ]ISNI 0000 0001 2179 2105, GRID grid.32197.3e, Cell Biology Center, Institute of Innovative Research, , Tokyo Institute of Technology, ; Yokohama, 226-8503 Japan
                [4 ]ISNI 0000 0001 2179 2105, GRID grid.32197.3e, World Research Hub Initiative, Institute of Innovative Research, , Tokyo Institute of Technology, ; Yokohama, 226-8503 Japan
                Article
                10846
                10.1038/s41467-019-10846-1
                6610143
                31270320
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                Funding
                Funded by: KAKENHI JP18H05527
                Funded by: NIH R35GM119728 Colorado State University’s Office of the Vice President for Research Catalyst for Innovative Partnerships Program.
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                © The Author(s) 2019

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                cell biology, single-molecule biophysics

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