A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo

Abstract

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

Figure 1

Development of CyOFP1, a cyan-excitable red fluorescent protein. (a) Normalized excitation and emission spectra of CyOFP1. (b) Sequence alignment of CyOFP1 and mNeptune1. Amino acids forming the chromophore are underlined. Interior mutations are in red and outer barrel mutations are colored pink. Mutations in the upper loop (when the barrel is oriented with termini pointing upwards) are colored gray. Amino acid numbering begins at −4 so that homologous sequences are numbered as in PDB file 3IP2 for the structure of Neptune. (c) Fluorescence images of HeLa CCL2 cells expressing CyOFP1 fused to various domains. For each fusion, the linker amino acid (aa) length is indicated in between the two domains, and the origin of the fusion partner and its normal subcellular location are indicated in parentheses. i, CyOFP1-18aa-actin (β-actin, actin cytoskeleton); ii, Cx43-7aa-CyOFP1 (rat α-1 connexin 43, gap junctions); iii, CytERM-17aa-CyOFP1 (rabbit cytochrome p450 aa1-29, endoplasmic reticulum); iv, CyOFP1-14aa-RhoB (human RhoB, endosomes); v, CyOFP1-5aa-CAAX (human c-Ha-Ras 20- aa farnesylation signal, plasma membrane); vi, Tractin-11aa-CyOFP1 (rat F-Tractin; actin cytoskeleton); vii, SiT-7aa-CyOFP1, human sialyltransferase aa1-45, Golgi apparatus); viii, COX8A-7aa-CyOFP1 (human cytochrome C oxidase subunit VIIIA; mitochondria); ix, CyOFP1-18aa-tubulin (human α-tubulin, microtubules). (d) Fluorescence images of CyOFP1-10aa-H2B (human histone 2B) in HeLa S3 cells; i, interphase; ii, prophase; iii, metaphase; iv, anaphase. Scale bars, 10 Mm.

Figure 2

Structural characterization of CyOFP0.5. (a) Cutaway view of CyOFP0.5 (left) and LSSmKate (right) with main chain in cartoon format and chromophores and their hydrogen-bonding partners in stick format. Chromophore and side-chain atoms are colored as carbon orange (CyOFP0.5) or pink (LSSmKate1), nitrogen blue, and oxygen red. In both proteins, the side chain of aa160 serves as a hydrogen bond donor and ESPT acceptor. (b) Proposed ESPT in CyOFP0.5. The proton from the phenolic hydroxyl group of the chromophore is transferred to the Lys160 after the chromophore excited by cyan light. Hydrogen bonds are represented as dashed black lines. (c) The CyOFP0.5 chromophore (top) is more coplanar than the LSSmKate1 chromophore (middle), and is engaged in two hydrogen bonds compared to one in LSSmKate1. The hydrogen bond partners of the CyOFP0.5 chromophore are similar to those of the DsRed chromphore (bottom).

Figure 3

Simultaneous dual-emission two-photon imaging of CyOFP1 with GFP-based reporters. (a) Two- photon excitation spectra of CyOFP1, GCaMP6s, EGFP, and fluorescein (as reference standard). Intensity is presented as thousands of counts per s per μM protein at 1 mW excitation. Error bars are standard deviation. n = 3 for CyOFP1, 4 for GCaMP6s, 2 for EGFP, and 4 for fluorescein. (b) Single optical section (upper row) and surface rendering (lower row) of tractin-CyOFP1 and cytosolic EGFP in MV3 melanoma cells acquired by two-photon Bessel-beam light-sheet microscopy. CyOFP1 is localized to the cortex of the cell and small membrane protrusions. (c) Fluorescence images of CyOFP1 and GCaMP6s in layer-2/3 pyramidal neurons in mouse brain V1 cortex in a single optical section acquired by two-photon excitation at 940 nm. (d) GCaMP6s responses of three mouse neurons co- expressing CyOFP1 in response to drifting gratings. Single sweeps (grey) and averages of 5 sweeps (red) are overlaid. Directions of grating motion (8 directions) are shown above traces (arrows).

Figure 4

Development of a BRET system with NanoLuc and CyOFP1. (a) NanoLuc and CyOFP1 are well matched for BRET, as the emission spectrum of NanoLuc overlaps the absorbance spectrum of CyOFP1. (b) Protein engineering flow chart leading to Antares. Numbers in parentheses refer to the first and last amino acid of the domain fragment contained in the fusion protein, with numbering as in Figure 1b. Linker sequences are indicated in single-letter amino acid code. Deletions of the C-terminus of NanoLuc yielded inactive enzyme and were not pursued further. (c) Emission spectra of the constructs depicted in b. Spectra were normalized to the NanoLuc emission peak at 460 nm. Higher 584-nm emission peaks indicate higher BRET efficiencies. (d) Comparison of emission spectra of purified Antares, NanoLuc, Orange Nano-lantern (ONL), and FLuc. Emissions from 100 μL of 2 nM protein immediately after addition of 20 μM substrate were measured, then normalized to peak emission of Antares.

Figure 5

Antares is superior to other reporters for BLI in mice. (a) Cells expressing Antares produce larger detectable bioluminescence signal in mouse phantoms than cells expressing other reporters. Cells expressing the indicated reporters with the indicated substrates were injected into a mouse phantom at 0.7-cm depth, and images were acquired in an IVIS Spectrum for 1 s in bioluminescence mode. Although peak pixel intensity of Antares was 46325 counts, the displayed intensity range was set to 0–5000 to allow visual confirmation of signal from all reporter proteins. (b) Quantitation of cellular bioluminescence in phantom mice. Total counts were normalized to co-expressed CFP intensity and then normalized to mean counts from Antares with FRZ. The second-brightest reporter-substrate combination, BRET6 with sCTZ, produced 21% of the detectable emission of Antares with FRZ. Error bars represent standard error of the mean (SEM) from 3 replicate measurements. (c) In mice, Antares produces more detectable signal upon IV administration of 0.33 μmol FRZ than FLuc upon IV or IP administration of 9.4 μmol luciferin. (d) Quantification of bioluminescence in living mice. Total counts were normalized to mean counts from Antares with FRZ. Error bars represent SEM of measurements from multiple mice (n = 5 for Antares, n = 6 for FLuc with IV luciferin, n = 16 for FLuc with IP luciferin).