A genetically encoded fluorescent probe in mammalian cells

Abstract

Fluorescent reporters are useful in vitro and in vivo probes of protein structure, function, and localization. Here we report that the fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), can be site-specifically incorporated into proteins in mammalian cells in response to the TAG codon with high efficiency using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. We further demonstrate that Anap can be used to image the subcellular localization of proteins in live mammalian cells. The small size of Anap, its environment-sensitive fluorescence, and the ability to introduce Anap at specific sites in the proteome by simple mutagenesis make it a unique and valuable tool in eukaryotic cell biology.

Figure 1

Structure and normalized absorption (purple) and fluorescence (Deep blue, in EtOAc; light blue, in DMSO; Cyan, in water) spectra of Anap.

Figure 2

Evaluation of the activity of Anap specific tRNA_C-UA^EcLeu/AnapRS in mammalian cells. (A) The construction of pAnap and pEGFP plamids. Identities of different genetic elements used in these constructs are shown. (B) Expression of EGFP-40-Anap in HEK293 cells in the presence (left) and absence (right) of 0.5 mM Anap, achieved by co-transfection of pAnap and pEGFP, analyzed by fluorescence microscopy. (B) ESI-MS analysis of purified EGFP-40-Anap mutant. Expected molecular weight: 29773 Da; observed: 29775 Da.

Figure 3

Nuclear localization of Histone-H3 (Thr59-Anap)-mCherry (C-terminal) fusion protein in HEK293 (panel A), HeLa (panel B) and CHO (panel C) cells, visualized using Anap (first column, pseudo-colored in blue, superimposed with the bright field image) or mCherry (second column, pseudo-colored in red, superimposed with the bright field image) as fluorescent reporters by laser scanning confocal microscopy. A composite image of these is shown in the third column. Scale bars, 10 μm.

Figure 4

Subcellular localization of endoplasmic reticulum (ER) and golgi-resident proteins in human cell-lines visualized using Anap (first column, pseudo-colored in blue, superimposed with the bright field image) or mCherry (second column, pseudo-colored in red, superimposed with the bright field image) as fluorescent reporters by confocal microscopy: (A) ER-resident Grp94 (Ser127-Anap)-mCherry (C-terminal) in HeLa cells. (B) ER-resident Grp94 (Leu7-Anap)-mCherry (C-terminal) in HeLa cells. (C) Golgi resident GalT1 (Ser3-Anap)-mCherry (C-terminal) in HEK293 cells. Scale bars: 10 μm.