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, 11 (1), 273

Bright Ligand-Activatable Fluorescent Protein for High-Quality Multicolor Live-Cell Super-Resolution Microscopy

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Bright Ligand-Activatable Fluorescent Protein for High-Quality Multicolor Live-Cell Super-Resolution Microscopy

Jiwoong Kwon et al. Nat Commun.

Abstract

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Repetitive on- and off-switching of holoUnaG by external bilirubin (BR) and light exposure.
a Scheme of BR-inducible fluorescence of UnaG. Crystal structures of apoUnaG, BR and holoUnaG are obtained from Protein Data Bank (PDB, ID: 4I3B). b Absorption (dashed lines) and fluorescence emission (solid lines) spectra of holoUnaG before bleaching (gray), after bleaching (red) and after recovery (blue). Abnormally large absorbance at < 460 nm in the blue dashed line may come from the additional BR at 1 µM for the recovery. c Repetitive photobleaching and fluorescence recovery of holoUnaG. Each picture was captured with 200 µW of 488-nm excitation light after indicated treatments for 60 min each. dg Widefield image and series of localization dataset of UnaG-Sec61β transiently expressed in a fixed Cos7 cell in different imaging buffer compositions. Raw data for single-molecule localization were recorded with 10 ms camera exposure time for 30,000 frames. The same field of view was observed sequentially from the left (d) to the right (g) under identical optical conditions for the localization datasets. h Normalized photon counts distribution and i the average number of localized molecules from the localization datasets in dg. The photon counts distributions were normalized by their own maximum counts. Scale bar: 2 µm. Error bars: standard deviations (n = 80). BR bilirubin, OS oxygen scavenger.
Fig. 2
Fig. 2. Kinetic study of the photo-switching process.
a Time-dependent fluorescence decay of holoUnaG under different irradiation intensity of the 488-nm laser source. The fluorescence intensities were recorded with 5-ms time resolution. Dotted lines are the averaged low decay curves, and solid lines indicate bi-exponential fits to the decay curves. b Off-switching rates (koff1 and koff2) of holoUnaG under various laser intensities obtained from a. Linear fits (solid lines) gave first-order kinetic rate constants for the bleaching reactions. c Off-switching rates at 300 W cm−2 of intensity in different imaging buffer supplements such as β-mercaptoethylamine (MEA), potassium iodide (KI) and ascorbic acid (Ascb). Only the oxygen scavenging systems based on glucose oxidase and catalase (GLOX and CAT) significantly slowed down the fluorescence decay, indicating that the major photo-degradation pathway of holoUnaG is the photo-oxidation. d Off-switching rates at 300 W cm−2 of intensity in different pH conditions. Little notable differences were observed. e Time-dependent fluorescence recovery of bleached holoUnaG in various concentration of external BR from 0.0 to 1.0 μM (dotted lines) and in photo-damaged BR (dmBR, purple dashed line). Solid lines indicate the fitting results using Supplementary Equation 14. The fluorescence intensities were recorded with 1-s time resolution. f On-switching rates (kon) of holoUnaG in different BR concentrations obtained from c, displayed with a linear fit. g Proposed two-state fluorescence switching model of holoUnaG. Light-induced oxidation of BR inside of holoUnaG by dissolved oxygen turns off the fluorescence, whereas the reverse reaction is purely affected by freely diffusing BR in solution. Thus, the switching rates in both directions can be controlled individually by either the light intensity or the BR concentration. Intensities in a and e were normalized by the initial value. Error bars: standard deviations (n = 5, from independent bulk measurements of pulled-down samples).
Fig. 3
Fig. 3. Separation and mass spectrometry analysis of the major photo-oxidation products.
a UV/vis chromatograms, at 405 nm, of photo-oxidation products of BR (OxBR) extracted from photobleached holoUnaG. For guidance, each chromatogram was offset by 0, 5 and 10 for irradiation times of 0 (gray), 10 (blue) and 20 min (red), respectively. Vertical black dashed line marks the retention time for BV obtained from a control experiment (Supplementary Fig. 4b, c). b An averaged mass spectrum for the retention time (RT) 9–11 min region of the LC–HRMS analysis. The most abundant ion species at m/z 315.1336 could correspond to the protonated ion ([M + H]+) of the possible oxidation product (M) inserted as an inset. P propionic acid (-CH2CH2COOH), V vinyl (-CH=CH2).
Fig. 4
Fig. 4. Applications to super-resolution microscopy of UnaG.
a Averaged photon numbers per frame from single holoUnaG as increasing the camera exposure time. b Distribution of photon counts per single switching event at 180 ms exposure time. c Comparison of the photon numbers per switching cycle for conventional super-resolution probes measured in an identical experimental setup, with 200 ms of camera exposure time. High illumination powers at 561 nm (mMaple3) or 488 nm (UnaG and Atto 488) were used so that most of the single fluorophores switched off in a single camera frame. As a result, UnaG gave more than 1200 photons, which stands between the mMaple3 and Atto 488. d Repetitive localization positions measured from surface-immobilized single UnaG molecules, projected in x axis and y axis in the lateral directions. Multiple localization distributions were aligned by their centroid positions obtained from Gaussian fits. The combined datasets were again fitted with Gaussian that yielded ~12 nm of localization precision and ~28 nm of full-width at half-maximum (FWHM) in lateral planes. The localization distributions were normalized by their own maximum counts. e Demonstration of SML imaging utilizing the photo-switchable nature of holoUnaG in various subcellular structures (left: ER, middle: vimentin filaments, right: clathrin-coated pits) in fixed Cos7 cells. f Close-up widefield and SML images from the yellow-boxed regions in e. Scale bars: 2 µm for e; 200 nm for f. Error bars: standard deviations (n = 5, each measurement contained more than 500 single-molecule information). CLC clathrin light chain.
Fig. 5
Fig. 5. Live-cell super-resolution microscopy.
a, b One-second-long SML images of the ER in a live Cos7 cell transfected with UnaG-Sec61β. a A far view with conventional widefield and SML images. The widefield image was taken immediately before super-resolution imaging. b A time series of closed-up SML images from the yellow-boxed region in a. Scale bars: 2 µm for a; 500 nm for b.
Fig. 6
Fig. 6. Two-color live-cell super-resolution microscopy.
a ER (green) and mitochondria (red) of a live Cos7 cell in two-color widefield (left) and SML (right) images. The cell was transfected with UnaG-Sec61β and subsequently stained with MitoTracker Red. The widefield image was taken immediately before super-resolution imaging. b Crosstalk analysis using singly labeled control samples imaged in the two emission channels of green and red split by a dichroic mirror and filtered by a single bandpass filter for each channel. The localizations numbers were normalized by the average count from the proper color channel. cf Mitochondrial matrix (green) and inner membrane (red) in two-color SML images of a live Cos7 cell transfected with Mito-UnaG and stained by MitoTracker Red. c A far view comparison between widefield (left) and SML (right) images. d Close-up comparison between widefield (left) and SML (right) images of yellow-boxed region in c. e, f Zoom-ins in the cyan- and white-boxed regions in c at 0 min (left) and after 5 min (right) with fusion (white arrows) and fission (yellow arrows) events. Scale bars: 2 µm for a, c; 200 nm for d, f. Error bars: standard deviations (n = 5, from independent SML images).

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