Using unnatural amino acids to selectively label proteins for cellular imaging: a cell biologist viewpoint

Abstract

Twenty-five years ago, GFP revolutionized the field of cell biology by enabling scientists to visualize, for the first time, proteins in living cells. However, when it comes to current, state-of-the-art imaging technologies, fluorescent proteins (such as GFP) have several limitations that result from their size and photophysics. Over the past decade, an elegant, alternative approach, which is based on the direct labeling of proteins with fluorescent dyes and is compatible with live-cell and super-resolution imaging applications, has been introduced. In this approach, an unnatural amino acid that can covalently bind a fluorescent dye is incorporated into the coding sequence of a protein. The protein of interest is thereby site-specifically fluorescently labeled inside the cell, eliminating the need for protein- or peptide-labeling tags. Whether this labeling approach will change cell biology research is currently unclear, but it clearly has the potential to do so. In this short review, a general overview of this approach is provided, focusing on the imaging of site-specifically labeled proteins in mammalian tissue culture cells, and highlighting its advantages and limitations for cellular imaging.

Keywords: bioorthogonal reactions; click chemistry; fluorescent dyes; genetic code expansion; light microscopy; noncanonical amino acids; protein labeling.

Fig. 1

Labeling cellular proteins through genetic code expansion (GCE) and click chemistry. (A) Incorporating an unAA during ribosomal translation. A UAG codon is inserted in‐frame into the amino acid sequence of a POI. A unique tRNA, which carries the complementary codon to the UAG and was charged with the unAA by a unique tRNA synthetase, recognizes the UAG codon and incorporates the unAA into the newly formed polypeptide. Translation then continues until the full‐length polypeptide is released from the ribosome, giving rise to the synthesis of a full‐length protein that carries the unAA at a specific site. (B) Bioorthogonal labeling via a click reaction. The unAA carries a chemical modification; in this case, a BCN moiety (red) that specifically and rapidly reacts with the tetrazine moiety (black) that is attached to a fluorescent dye (green). Consequently, the protein is directly labeled with a fluorescent dye at a specific site.

Fig. 2

Live‐cell imaging of proteins labeled via GCE and click chemistry. (A) Photobleaching experiments employing cellular proteins labeled with unAAs. Top panel: HEK293 cells expressing the HIV protein Env, which carries a BCN‐lysine at position 407. The cells were labeled with the cell‐impermeable dye Tet‐Cy5 and imaged using a spinning disk confocal microscope. Reproduced with permission from Ref. [30]. Bottom panel: COS7 cells expressing the endoplasmic reticulum marker ER^cb5TM, which was conjugated to a 14‐AA tag that carries BCN‐lysine. The cells were labeled with the cell‐permeable dye Tet‐TAMRA and imaged using a spinning disk confocal microscope. Reproduced from Ref. [36]. Scale bars, 10 μm. (B) SPT of EGFR in COS7 cells, imaged in TIRF mode over time. Top panel: The cells express EGFR, which carries BCN‐lysine at position 128, and are labeled with the cell‐impermeable dye Tet‐Cy3. Bottom panel: The cells express an EGFR‐GFP. In each case, individual particles obtained from the videos were segmented and tracked through time (right panels). Note that particles obtained for EGFR‐Cy3 were brighter than those obtained for EGFR‐GFP and that significantly more tracks were generated using this labeling approach. Reproduced from Ref. [34]. Scale bars, 10 μm.

Fig. 3

Super‐resolution microscopy imaging of proteins labeled via GCE and click chemistry. (A, B) SMLM performed on cellular proteins labeled with GCE and click chemistry. (A) Top panel: dSTORM imaging of NMDA receptors carrying a TCO‐lysine in position 392 and click‐labeled with Tet‐Cy5. Bottom panel: dSTORM imaging of NMDA receptors labeled with specific primary antibodies and Alexa 647 secondary antibodies. The right panels are zoomed‐in images of the areas marked with squares on the left panels. A wide‐field image is shown, for comparison, on the bottom right corner of the top left panel. Note that considerably more localizations were obtained using the click‐labeled NMDA receptor. Reproduced with permission from Ref. [33]. Scale bar, 2.5 μm. (B) dSTORM imaging of microtubules labeled with the microtubule‐binding protein EMTB carrying a TCO‐lysine at position 87 and click‐labeled with the cell‐permeable dye Tet‐HM‐SiR in COS7 cells. A wide‐field image is shown on the top left corner, for comparison. Reproduced from Ref. [24]. Scale bar, 1 μm. (C) Confocal (left) and STED (middle) images of the HIV protein Env, which carries a BCN‐lysine at position 407 and was labeled with Tet‐KK114 in HEK293 cells. Right panel: line intensity profiles obtained for an individual Env cluster (arrowheads in the left and middle panels), imaged via confocal microscopy or STED. The higher resolution obtained with STED can be clearly seen. Reproduced with permission from Ref. [30]. Scale bar, 1 μm. (D) SIM imaging of GCE‐labeled microtubules in COS7 cells. Microtubules, labeled in COS7 cells with tubulin that carry a BCN‐lysine at position 45 and Tet‐SiR, were imaged using wide‐field microscopy or SIM (left and middle panels, respectively). A zoomed‐in image of the red rectangle in the middle panel is shown in the top right panel. An intensity line profile across the region depicted by arrows in the top right panel is shown in the bottom right panel, demonstrating the ability to resolve fibers that are only 80 nm apart. Reproduced from Ref. [35]. Scale bars, 10 μm, zoomed‐in, 1 μm.