Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 138 (43), 14423-14433

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells

Affiliations

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells

Tao Peng et al. J Am Chem Soc.

Abstract

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

Figures

Figure 1
Figure 1
Scheme for site-specific fluorescence labeling and imaging of intracellular proteins of interest (POI) in live cells using unnatural amino acid incorporation via genetic code expansion and bioorthogonal tetrazine ligation reaction.
Figure 2
Figure 2
Structures of (A) unnatural amino acids and (B) tetrazine-fluorophores 1-11 used in this study.
Figure 3
Figure 3
Comparative evaluation of UAAs and tetrazine-fluorophores for fluorescence labeling of IFITM3 in live cells using ingel fluorescence analysis. Hela cells expressing HA-IFITM3-F8UAA were labeled with (A, B) green tetrazine-fluorophores 1-5 (λex = 488 nm, λem = 510 nm), (C, D) orange tetrazine-fluorophores 6-7 (λex = 557 nm, λem = 576 nm), or (E, F) red tetrazine-fluorophores 8-11 (λex = 645 nm, λem = 661 nm) and lysed for in-gel fluorescence analysis (top panel) and Commassie Blue staining (CB, bottom pannel) after brief wash. 50 μM concentrations of CpK, exo-BCNK, and 2’-aTCOK were used, while 4’-eTCOK was supplemented into the media at 1 mM concentration. (B, D, and F) Bar graphs showing the fluorescence labeling efficiency of HA-IFITM3-F8UAA with tetrazine-fluorophores. Fluorescence intensity of every IFITM3 band was quantified and normalized to the most intense band of the corresponding gel, the intensity of which is set to 1. Data from three independent replicates were quantified and averaged for plotting the graphs. Data are mean ± S.E.M., n = 3. Representative gels are shown in (A), (C), and (E).
Figure 4
Figure 4
Bioorthogonal fluorescence imaging of HA-IFITM3-F8UAA with tetrazine-fluorophores versus immunofluorescence staining. Hela cells expressing HA-IFITM3-F8UAA in the presence of (A) BocK (50 μM) or (B) 2’-aTCOK (50 μM) were labeled with H-Tz-BODIPY-FL, H-Tz-rhodamine, or H-Tz-SiR (500 nM, 0.5 h) under physiological conditions, briefly washed, and subjected to anti-HA immunofluorescence staining. DAPI (blue) was used to stain nuclei. Images were acquired with confocal microscopy. Scale bars = 10 μm.
Figure 5
Figure 5
Live cell bioorthogonal fluorescence imaging of HA-IFITM3-F8UAA with tetrazine-fluorophores. Hela cells expressing HA-IFITM3-F8UAA and GFP- or mCherry-LAMP1 were labeled with H-Tz-BODIPY-FL, H-Tz-rhodamine, or H-Tz-SiR (500 nM, 0.5 h) under physiological conditions, briefly washed, and directly analyzed with confocal microscopy. (A) HA-IFITM3-F8UAA was expressed in the presence of BocK (50 μM). (B) HA-IFITM3-F8UAA was expressed in the presence of 2’-aTCOK (50 μM). Hoechst (blue) was used to stain nuclei. Scale bars = 10 μm.
Figure 6
Figure 6
Live cell bioorthogonal fluorescence imaging of HA-IFITM3-F8UAA localization with H-Tz-BODIPY-FL. Hela cells were transfected with (A) HA-IFITM3-F8TAG or (B) HA-IFITM3-F8TAG-Y20F and mCherry-tagged endocytic markers in the presence of 2’-aTCOK (50 μM), labeled with H-Tz-BODIPY-FL (250 nM, 0.5 h) under physiological conditions, briefly washed, and directly analyzed with confocal microscopy. Rab5, Rab7, LAMP1, and CellMask were used as early endosome, later endo-some, lysosome, and plasma membrane markers, respectively. For plasma membrane staining, cells were transfected only with IFITM3 plasmids, labeled, and stained with CellMask before imaging. Hoechst (blue) was used to stain nuclei. Pearson's correlation coefficients were shown in the merged images to evaluate the co-localization of IFITM3 (green) with markers (red). 10-20 cells were used to calculate Pearson's correlation coefficients for each sample. Scale bars = 10 μm.
Figure 7
Figure 7
Time-lapse imaging of the fusion process of IFITM-containing vesicle with dextran particles. IFITM3-F8aTCOK was labeled with H-Tz-BODIPY-FL and dextran particles are labeled with pHrodo Red. Images were acquired every 30 s. The time points shown on the figure are relative to the first image in the series. Scale bars = 1 μm.
Figure 8
Figure 8
Live cell bioorthogonal fluorescence imaging of benchmark intracellular proteins. HEK293T cells were transfected with mCherry-tagged plasmids as indicated in the presence of 2’-aTCOK (50 μM), labeled with H-Tz-SiR (500 nM, 0.5 h) under physiological conditions, briefly washed, and directly analyzed with confocal microscopy. Hoechst (blue) was used to stain nuclei. Scale bars = 10 μm.

Similar articles

See all similar articles

Cited by 19 PubMed Central articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback