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Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy

We introduced enhanced UnaG (eUnaG), a ligand-activatable fluorescent protein, for conventional and super-resolution imaging of subcellular structures in the mammalian cells. eUnaG is a V2L mutant of UnaG with twice brighter bulk fluorescence. We previously discovered the reversible fluorescence swi...

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Autores principales: Ko, Sangyoon, Kwon, Jiwoong, Shim, Sang-Hee
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8093618/
https://www.ncbi.nlm.nih.gov/pubmed/33959634
http://dx.doi.org/10.3389/fmolb.2021.647590
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author Ko, Sangyoon
Kwon, Jiwoong
Shim, Sang-Hee
author_facet Ko, Sangyoon
Kwon, Jiwoong
Shim, Sang-Hee
author_sort Ko, Sangyoon
collection PubMed
description We introduced enhanced UnaG (eUnaG), a ligand-activatable fluorescent protein, for conventional and super-resolution imaging of subcellular structures in the mammalian cells. eUnaG is a V2L mutant of UnaG with twice brighter bulk fluorescence. We previously discovered the reversible fluorescence switching behavior of UnaG and demonstrated the high photon outputs and high localization numbers in single-molecule localization microscopy (SMLM). In this study, we showed that the fluorescence of eUnaG can be switched off under blue-light illumination, while a high concentration of fluorogenic ligands in the buffer can efficiently restore the fluorescence, as in UnaG. We demonstrated the capacity of eUnaG as an efficient protein label in mammalian cells, as well as for SMLM by utilizing its photoswitchable nature. While cytosolic UnaG proteins showed aggregated patches and fluorescence reduction at high expression levels, eUnaG-labeled protein targets successfully formed their proper structures in mammalian cells without notable distortion from the endogenous structure in the majority of transiently expressing cells. In particular, eUnaG preserved the vimentin filament structures much better than the UnaG. eUnaG provided similarly high single-molecule photon count distribution to UnaG, thus also similarly high resolution in the super-resolution images of various subcellular structures. The sampling coverage analysis of vimentin filaments in SMLM images showed the improvement of labeling efficiency of eUnaG. eUnaG is a high-performance fluorescent protein for fluorescence and single-molecule localization imaging in green emission with minimal labeling artifact.
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spelling pubmed-80936182021-05-05 Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy Ko, Sangyoon Kwon, Jiwoong Shim, Sang-Hee Front Mol Biosci Molecular Biosciences We introduced enhanced UnaG (eUnaG), a ligand-activatable fluorescent protein, for conventional and super-resolution imaging of subcellular structures in the mammalian cells. eUnaG is a V2L mutant of UnaG with twice brighter bulk fluorescence. We previously discovered the reversible fluorescence switching behavior of UnaG and demonstrated the high photon outputs and high localization numbers in single-molecule localization microscopy (SMLM). In this study, we showed that the fluorescence of eUnaG can be switched off under blue-light illumination, while a high concentration of fluorogenic ligands in the buffer can efficiently restore the fluorescence, as in UnaG. We demonstrated the capacity of eUnaG as an efficient protein label in mammalian cells, as well as for SMLM by utilizing its photoswitchable nature. While cytosolic UnaG proteins showed aggregated patches and fluorescence reduction at high expression levels, eUnaG-labeled protein targets successfully formed their proper structures in mammalian cells without notable distortion from the endogenous structure in the majority of transiently expressing cells. In particular, eUnaG preserved the vimentin filament structures much better than the UnaG. eUnaG provided similarly high single-molecule photon count distribution to UnaG, thus also similarly high resolution in the super-resolution images of various subcellular structures. The sampling coverage analysis of vimentin filaments in SMLM images showed the improvement of labeling efficiency of eUnaG. eUnaG is a high-performance fluorescent protein for fluorescence and single-molecule localization imaging in green emission with minimal labeling artifact. Frontiers Media S.A. 2021-04-20 /pmc/articles/PMC8093618/ /pubmed/33959634 http://dx.doi.org/10.3389/fmolb.2021.647590 Text en Copyright © 2021 Ko, Kwon and Shim. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Ko, Sangyoon
Kwon, Jiwoong
Shim, Sang-Hee
Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy
title Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy
title_full Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy
title_fullStr Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy
title_full_unstemmed Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy
title_short Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy
title_sort enhanced unag with minimal labeling artifact for single-molecule localization microscopy
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8093618/
https://www.ncbi.nlm.nih.gov/pubmed/33959634
http://dx.doi.org/10.3389/fmolb.2021.647590
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