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, 137 (14), 4602-5

Genetic Code Expansion Enables Live-Cell and Super-Resolution Imaging of Site-Specifically Labeled Cellular Proteins

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Genetic Code Expansion Enables Live-Cell and Super-Resolution Imaging of Site-Specifically Labeled Cellular Proteins

Chayasith Uttamapinant et al. J Am Chem Soc.

Abstract

Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

Figures

Scheme 1
Scheme 1. Fluorophore Labeling of Cellular Proteins via Genetic Encoding of Unnatural Amino Acids and Diels–Alder Cycloaddition
Figure 1
Figure 1
Efficient BCNK-dependent expression of cytoskeletal proteins and specific labeling with a tetrazine-fluorophore. (A) HEK293T cells were transfected with plasmids encoding (Pyl tRNACUA)4/BCNKRS and (Pyl tRNACUA)4/POI (POI = protein of interest = actinD4TAG or K118TAG, vimentinN116TAG or E187TAG) and grown in the presence or absence of 1 mM BCNK for 48 h. Actin was HA-tagged, while vimentin was c-myc-tagged. (B) Analyses of labeling specificity. HEK cells were transfected and grown with/without BCNK as in (A). After removal of excess BCNK, cells were treated with 400 nM tet1-CFDA for 20 min, then washed for 2 h before lysis. Lysates were resolved and their fluorescence visualized. Cells were transfected with plasmids encoding (Pyl tRNACUA)4/BCNKRS and (Pyl tRNACUA)4 in the omit reporter controls. Loading controls for both (A) and (B) are shown in Figure S5.
Figure 2
Figure 2
STED, SIM, and STORM imaging of EGFR in HEK293T cells. (A) Confocal image of EGFR labeled with BCNK and tet2-ATTO488 and corresponding STED image of the same cell. Line-profile analysis of the indicated membrane segment shows resolution improvement of STED over confocal microscopy. (B) Deconvolved widefield fluorescence image of EGFR labeled as in (A), corresponding SIM image of the same cell and line-profile analysis of the indicated membrane segment. (C) Widefield fluorescence image of EGFR-GFP labeled with TCOK and tet2-Alexa Fluor 647, corresponding STORM image of the same cell and cross-sectional analysis of the indicated membrane segment. ROI = region of interest.
Figure 3
Figure 3
STORM imaging of vimentin (N116TAG) and actin (K118TAG) in COS-7 cells. (A) Widefield fluorescence images of vimentin labeled with BCNK and tet1-SiR and corresponding STORM images. Images in the second row are zoom-ins of the boxed regions in the first row. (B) Cross-sectional distribution of localizations along a vimentin filament segment from a STORM image (Figure S9) and its Gaussian fit, which gives FWHM of 70 nm. (C) Widefield images of actin labeled with BCNK and tet1-SiR and corresponding STORM images. Third and fourth columns are zoom-in images of the boxed regions in the first and second columns, respectively. (D) Cross-sectional distribution of localizations along two nearby actin filaments in box D. The two filaments are separated by ∼125 nm. (E) Cross-sectional distribution of localizations along a nuclear actin filament segment from box E and its corresponding Gaussian fit, which gives FWHM of 92 nm. Larger versions of all images are shown in Figure S10.

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References

    1. Bates M.; et al. Science 2007, 317, 1749. - PMC - PubMed
    1. Betzig E.; et al. Science 2006, 313, 1642. - PubMed
    1. Willig K. I.; et al. Nature 2006, 440, 935. - PubMed
    1. Hofmann M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17565. - PMC - PubMed
    1. Gustafsson M. G. J. Microsc. 2000, 198, 82. - PubMed

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