Abstract
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.
Highlights
Fluorescent proteins (FPs) are workhorses in live-cell fluorescence microscopy due to the facile and specific labeling of the target proteins (Chudakov et al, 2010; Rodriguez et al, 2017)
UnaG protein has been used for imaging applications of mammalian cells such as local bilirubin quantification (Park et al, 2016), biomolecular interactions (To et al, 2016), and super-resolution visualization of subcellular structures (Kwon et al, 2020), there is no report that uses enhanced UnaG (eUnaG) in mammalian cells to the best of our knowledge
To demonstrate its potential to visualize the subcellular structures in mammalian cells, we fused eUnaG with various proteins such as Sec61β, Vim, clathrin light chain (CLC), Pex16, and cytosolic mCherry; transiently expressed the fusion proteins in Cos-7 cells; and observed the expression patterns (Figure 1A and Supplementary Figure 1)
Summary
Fluorescent proteins (FPs) are workhorses in live-cell fluorescence microscopy due to the facile and specific labeling of the target proteins (Chudakov et al, 2010; Rodriguez et al, 2017). The quality of the resultant super-resolution image is determined by two photophysical characteristics eUnaG for Single-Molecule Localization Microscopy of the fluorophores. The photon number emitted from the fluorescent state determines the localization precision of determining the centroid position of a single fluorophore (Thompson et al, 2002). The number of switching cycles and the fraction of time spent in the fluorescent state, termed as the on– off duty cycle, are related to the labeling density and the Nyquist resolution (Shroff et al, 2008). Most of the FPs offer lower photon numbers than the organic fluorophores, resulting in lower spatial resolutions (Patterson and Lippincott-Schwartz, 2002; Subach et al, 2009; Brakemann et al, 2011). FPs often show irreversible fluorescence transition that restricts the spot density or transition between two different emission states that complicates multicolor applications (McKinney et al, 2009)
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