Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen-activating tags based on the fluorescence-activating and absorption shifting tag (FAST) that are compatible with two-color, live-cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development and the development of split complementation systems capable of detecting multiple protein–protein interactions by live-cell fluorescence microscopy.
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All data generated during this study are included in this published article (and its Supplementary Information files) or are available from the corresponding author on reasonable request.
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We thank K.D. Wittrup, for providing us with the pCTCON2 vector and the EBY100 yeast strain for the yeast display selection. We also thank the flow cytometry facility Cytométrie Imagerie Saint-Antoine of UMS LUMIC at the Faculty of Medicine of Sorbonne University and, particularly, A. Munier for her assistance. This work has been supported by the European Research Council (ERC-2016-CoG-724705 FLUOSWITCH to A.G., ERC StG 714688 NanoCellActivity to P.D. and ERC-2019-CoG-863869 FUSION to S.P.-P.), the Wellcome Trust Core Award (203141 to S.P.-P.), and the Research Foundation-Flanders (G0B8817N to P.D. and 1514319N to B.M.).
The authors declare the following competing financial interest: A.G. is cofounder and hold equity in Twinkle Bioscience/The Twinkle Factory, a company commercializing the FAST technology.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
A yeast displayed library of FAST variants was screened in presence of both HMBR and HBR-3,5DOM in order to identify redFAST and greenFAST.
Comparison of the KDs (in μM) for HMBR and HBR-3,5DOM of FAST (blue star) and clones selected from FACS (a) and variants constructed through rational design (b, filled markers). Green dots: greenFAST selection, magenta dots: redFAST selection.
a, b, Affinities of greenFAST and redFAST for a) HMBR and b) HBR-3,5DOM. Mean of n = 3, represented as mean ± sem, protein concentration 100 nM. (c, d) Determination of molar absorptivity for c) greenFAST and d) redFAST with their cognate fluorogen by forward titration and standardization with FAST (protein concentration, 40 µM). e, f, Determination of quantum yield for e) greenFAST and d) redFAST with their cognate fluorogen by reciprocal dilution using FAST:fluorogen as a standard (protein concentration, 40 µM).
Yeast cells expressing FAST (a), greenFAST (b) and greenFAST (c) were analyzed by flow cytometry in the presence of either only HMBR (5 μM) or HBR-3,5DOM (10 μM) or in the presence of both (5 μM HMBR and 10 μM HBR-3,5DOM). Efficient induction of protein expression was verified through independent labeling with an Alexa488-conjugated antibody. The analysis was done typically on about 110,000 cells. Supplementary Figure 1 exemplifies the gating strategy used. d, Representative confocal micrograph (n = 5 from 1 experiment) of a mixture of yeast cells expressing greenFAST (green) and redFAST (magenta) in the presence of 5 μM HMBR and 10 μM HBR-3,5DOM. Scale bar 10 μm.
a, b, comparison of greenFAST and FAST photostability in different illumination conditions. Both were expressed as H2B fusions in HEK293T and imaged with 10 μM HMBR. Images taken every a) 1 s and b) 10 s with 1.27 μs pixel dwell, excitation with 488 nm laser. c, d, Comparison of c) FAST and d) greenFAST photostability as a function of fluorogen concentration at 13 kW/cm2 for 488 nm laser, 1.27 μs pixel dwell, images taken every 1 s. 100 images were acquired followed by 60 s in the dark before acquisition was restarted. e, redFAST and FAST expressed in the cytosol in HEK293T cells labeled with 10 μM HBR-3,5DOM were illuminated with 4.9 kW/cm2 for 541 nm laser, 1.27 μs pixel dwell, images taken every 1 s.
a–d, Representative micrograph (n = 8 from 3 experiments) of U2OS cells stably expressing a FUCCI cell cycle sensor. a, Design of a mammalian cell cycle sensor. Tracking of individual cell cycles is possible through stable expression of redFAST-hCdt(30–120) and greenFAST-hGem(1–120). b–d, Cell A can be tracked S/G2 (b, 40 mins) through division (c, 6 hrs 10 mins) while Cell B (c, top arrow) can be tracked through the G1-S transition (d, 19 hrs 50 mins). Images were taken every 5 mins. 5 μM HMBR and 10 μM HBR-3,5DOM. Scale bars 10 μm.
Zebrafish embryos were incubated with 5 μM fluorogen during 1 h at 50% epiboly or overnight (ON) from 50% epiboly to 24 hpf. The graph shows the percentage of embryos with no defect (white), axis defects (grey), or dead (black) at 48 hpf. Controls untreated and treated with DMSO only were performed. The number (n) of embryos used for each condition is indicated.
a, b, Representative micrographs (n = 3 from 3 experiments) of HEK293T cells co-expressing FK506-binding protein (FKBP) fused to CFAST11 and FKBP-rapamycin-binding domain of mammalian target of rapamycin (FRB) fused to either greenNFAST (a) or redNFAST (b) were labeled with both 5 μM HMBR and 10 μM HBR-3,5DOM, and imaged before and after addition of 500 nM rapamycin. The green channel shows HMBR fluorescence, while the magenta channel shows HBR-3,5DOM fluorescence. Graphs show the temporal evolution of the fluorescence intensity of individual cells (n = 7 and 8) after rapamycin addition. c, d, Representative micrographs (n = 3 from 3 experiments) of HEK293T cells co-expressing FKBP fused to CFAST11 and FBBP fused to either greenNFAST (c) or redNFAST (d) treated with 100 nM AP1510 and labeled with both 5 μM HMBR and 10 μM HBR-3,5DOM. Cells were then imaged before and after the addition of 1.1 μM rapamycin. The green channel shows HMBR fluorescence, while the magenta channel shows HBR-3,5DOM fluorescence. Graphs show the temporal evolution of the fluorescence intensity of individual cells (n = 8 and 11) after rapamycin addition.
DNA and protein sequences of greenFAST, redFAST; Supplementary Fig. 1 and Tables 1–12.
Zebrafish embryos were injected with redFAST-zGem(1–100)-P2A-greenFAST-zCdt1(1–190) mRNA at the one-cell stage, and timelapse imaging was performed starting from the 256-cell stage on embryos incubated with 5 μM of HMBR and 5 μM of HBR-3,5DOM. Scale bar is 20 μm. Images every 5 min.
Whole-zebrafish embryo imaging. Zebrafish embryos were injected with redFAST-zGem(1–100)-P2A-greenFAST-zCdt1(1–190) mRNA at the one-cell stage, and timelapse imaging was performed starting from the 256-cell stage on embryos incubated with 5 μM of HMBR and 5 μM of HBR-3,5DOM. Scale bar is 100 μm.
Detection of FKBP–FKBP homodimer encoded by split-redFAST followed by detection of FRB–FKBP detected by split-greenFAST using timelapse imaging in HEK293T cells labeled with 5 μM of HMBR and 10 μM of HBR-3,5DOM. First, the cells were treated with 100 nM of AP1510 (imaged every 5 min), and then at t = 140 min AP1510 was removed and 1 µM of rapamycin was added (imaged every 30 s). Scale bar, 20 μm.
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Tebo, A.G., Moeyaert, B., Thauvin, M. et al. Orthogonal fluorescent chemogenetic reporters for multicolor imaging. Nat Chem Biol 17, 30–38 (2021). https://doi.org/10.1038/s41589-020-0611-0