AUTHOR=Ko Sangyoon , Kwon Jiwoong , Shim Sang-Hee 

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

JOURNAL=Frontiers in Molecular Biosciences

VOLUME=Volume 8 - 2021

YEAR=2021

URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2021.647590

DOI=10.3389/fmolb.2021.647590

ISSN=2296-889X

ABSTRACT=We introduce 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 UnaG’s reversible fluorescence switching behavior and demonstrated the high photon outputs and high localization numbers in single-molecule localization microscopy (SMLM). In this work, we show 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 much better preserved vimentin filament structures than UnaG. eUnaG provides similarly high single-molecule photon count distribution to UnaG, thus also similarly high resolution in super-resolution images of various subcellular structures. The labeling 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.