Luminescent proteins for high-speed single-cell and whole-body imaging

The use of fluorescent proteins has revolutionized our understanding of biological processes. However, the requirement for external illumination precludes their universal application to the study of biological processes in all tissues. Although light can be created by chemiluminescence, light emission from existing chemiluminescent probes is too weak to use this imaging modality in situations when fluorescence cannot be used. Here we report the development of the brightest luminescent protein to date, Nano-lantern, which is a chimera of enhanced Renilla luciferase and Venus, a fluorescent protein with high bioluminescence resonance energy transfer efficiency. Nano-lantern allows real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence and sensitive tumour detection in freely moving unshaved mice. We also create functional indicators based on Nano-lantern that can image Ca2+, cyclic adenosine monophosphate and adenosine 5′-triphosphate dynamics in environments where the use of fluorescent indicators is not feasible. These luminescent proteins allow visualization of biological phenomena at previously unseen single-cell, organ and whole-body level in animals and plants.

Relative signal change [cAMP] (M) a, Domain structure of Nano-lantern (cAMP). Three affinity variants were developed using two kinds of cAMP-binding domains and different linker lengths. b, Normalized spectra of Nano-lantern (cAMP) variants with or without cAMP. Emission spectra measurement was performed at least in triplicate, and the averaged data are shown. c, Relative brightness of recombinant Nano-lantern and Nano-lantern (cAMP) variants, with or without cAMP. d, cAMP titration curves of the Nano-lantern (cAMP) variants. Measurements in c and d were performed at least in triplicate, and the averaged data and standard deviations are shown. e, Normalized spectra of FRET-based cAMP indicators using the same cAMP-binding domain of Nano-lantern (cAMP1.6), with or without cAMP. a, A fluorescence image of Dictyostelium discoideum cells expressing Nano-lantern (cAMP1.6). b, Luminescence images of Dictyostelium discoideum cells expressing Nano-lantern (cAMP1.6) before and after the addition of 100 μM cAMP. Scale bars, 0.2 mm. c, A typical pattern of time course of the intensity at ROIs indicated as colored boxes in (a). This is a representative data from three replicates. Typical patterns of luminescent signals change of wild-type (AX2) or adelylate cyclase A mutant (ACA) cells expressing Nano-lantern (cAMP1.6) recorded by spectrophotometer. Transient increase of luminescent signal upon cAMP stimulation (1 μM) was lost in ACA mutant. This is a representative data from three replicates.   a, Domain structure of Nano-lantern (ATP) variants. b, Normalized spectra of Nano-lantern (ATP) variants with or without ATP. Emission spectra measurements were performed at least in triplicate, and the averaged data are shown. c, ATP titration curves of Nano-lantern (ATP) variants. Measurements in c were performed at least in triplicate, and the averaged data and standard deviations are shown. To date several chemiluminescent proteins have been used for bioimaging, including, beetle luciferase 6 , Gaussia luciferase 32,33 , and Renilla luciferase 9, 34 . From these candidates, to make an artificial protein with the optimal BRET efficiency, we chose the improved RLuc, RLuc8 4 , as the BRET donor. RLuc8 is suitable for structural and functional imaging in living cells because it does not require co-factors such as ATP or Mg 2+ to catalyze its substrate, unlike beetle luciferase 35 .
Furthermore it is not secreted into the extracellular space, like Gaussia luciferase 33 , and it has a higher turnover rate than beetle luciferase. We first performed random mutagenesis on RLuc8 to screen for brighter variants. Among >6,000 candidates, we identified the S257G mutant whose recombinant protein displays 1.3 times more luminescence signal in the whole emission than Rluc8 ( Fig. 1b).

Selection of the appropriate BRET acceptor for RLuc8
We next searched for a suitable acceptor for RLuc8. In a previous report 9 , EYFP was used as the BRET acceptor for RLuc8 (eBAF-Y), showing a better BRET efficiency than EGFP. However, the improved EYFP variant, Venus, is frequently used as the acceptor in FRET-based indicators instead of EYFP 36 . We examined both EYFP and Venus as an RLuc8 acceptor 10 ; these FPs exhibit identical patterns of absorption and emission, but have different molar absorption coefficients. An essential factor for BRET efficiency is the rate constant of the energy transfer, k T (r), from donor to acceptor, which can be theoretically calculated as follows: where Q D is the luminescent quantum yield of the donor, n is the refractive index of the medium, N is Avogadro's number, r is the distance between donor and acceptor, τ D is the lifetime of the donor, J(λ) is the overlap integral, which expresses the degree of spectral overlap between the donor emission and acceptor absorption, and κ 2 is the orientation factor 37 . Among these parameters, the spectral property of the BRET acceptor only affects J(λ), which is expressed as follows: We further compared other properties that could affect the BRET efficiency. Venus is superior to EYFP in maturation speed, which increases the yield of the fusion protein. In addition, Venus is less sensitive to H + and Cl -, which increases the protein's stability in an acidic environment or one of high ionic strength.

Optimization of the relative orientation and distance between donor and acceptor
To greatly change the relative orientation and distance between the donor and acceptor chromophore, we tested a circularly permutated (cp) Venus series and C-terminus 10-amino acid truncated version of Venus (VenusC10) instead of the Venus moiety in VRL0.0 ( Supplementary Fig. S1). Among them, VenusC10-RLuc8 (VRL10.0) and cp229Venus-RLuc8 exhibited the largest and second largest 530/480 nm ratio (6.3 and 5.3, respectively). These data show in this case that relative distance was dominant over relative orientation. We further tested the flexible N-terminus 1-6 amino acid truncated versions of RLuc8, by replacing the RLuc8 in VRL10.0. The differences in their peak ratios were small, but VRL10.3 had the highest peak ratio (8.7, Supplementary Table S1) among them. To compare the brightness of RLuc8, eBAF-Y, and VRL10.3, we integrated each emission spectrum from 400-650 nm. VRL10.3 displayed an increase in integrated light intensity that was 4.1 and 2.2 times that of RLuc8 and eBAF-Y, respectively (Fig. 1b). Finally, we introduced the S257G mutation into the RLuc8 moiety of VRL10.3. The resulting protein, VRL10.3_S257G, was named Nano-lantern.

Supplementary Note 2 Consideration of the insertion site for Nano-lantern-based indicators
To develop functional indicators using Nano-lantern, we used a strategy based on the complementation of split luciferase (CSL) mechanism 13,38,39 . CSL-based indicators are composed of inter-or intra-molecularly split luciferase fused with "bio-active molecule recognition protein motif(s)," whose conformational change reconstitutes the catalytic activity of the split-Luc in a target-molecule-dependent fashion ( Supplementary Fig. S3a).  40 and a previous report 13 , we chose two insertion sites ( Supplementary Fig. S3b). One was the 91/92 position (Ser-Tyr) located at the loop between a -sheet (72-76th) and -helix (93-106th) in the hydrolase domain, which had already been examined 13 . The other was 228/229, located at the connecting loop between the hydrolase and cap domain, which was expected to be flexible due to its amino acid composition (Gly-Gly) 39 .

Design and properties of Nano-lantern (Ca 2+ )
To experimentally validate these insertion sites, we first constructed CaM-M13@91_RLuc8_S257G, in which the CaM-M13 motif of YC3.60 was inserted into the 91/92 position of RLuc8_S257G. The purified protein showed a change in luminescence intensity with a 110% dynamic range upon Ca 2+ addition, suggesting that the 91/92 site can be used to obtain CSL-based Ca 2+ indicators. On the other hand, CaM-M13@91_Nano-lantern, in which the CaM-M13 was inserted into the 91/92 position of RLuc8_S257G moiety in Nano-lantern, showed almost the same signal change (90%), but its intensity level was 3 times higher than that of CaM-M13@91_RLuc8_S257G. The peak emission ratio was not changed by Ca 2+ addition (530/480 nm ratio was 8.0 for both the Ca 2+ -free and -saturating conditions), indicating that the Nano-lantern-based Ca 2+ indicator indeed used a Ca 2+ -dependent CSL mechanism whose emission was enhanced by BRET, but which was insensitive to Ca 2+ . We next inserted CaM-M13 into the 228/229 position of RLuc8_S257G moiety in Nano-lantern (CaM-M13@228_Nano-lantern), which displayed a much larger signal change (300%) at a higher intensity level (max. 35% of Nano-lantern's brightness, Fig. 3b) than CaM-M13@91_Nano-lantern, demonstrating that the complementation of split RLuc moiety was more efficient when the CaM-M13 domain was inserted at the 228/229 position. We nicknamed the CaM-M13@228_Nano-lantern "Nano-lantern (Ca 2+ )".
Since previously reported CSL-based Ca 2+ indicators suffered from irreversible complementation, due to strong affinity of the split luciferase fragments or problems in the target molecule-sensing domain, we examined the reversibility of Nano-lantern (Ca 2+ ). We first determined the Ca 2+ affinity of Nano-lantern (Ca 2+ ) compared with that of the original recombinant calcium indicator Cameleon, YC3.60. If the reconstitution affinity of split-Nano-lantern @228 was too high, we would obtain an abnormally small apparent K d . However, the Ca 2+ affinity of Nano-lantern (Ca 2+ ) was determined to be K d = 620 nM which was comparable to that of the parental YC3.60 (K d = 780 nM) 15 . This was also true for the other 5 affinity variants of Nano-lantern (Ca 2+ )s, whose K d ranged from 17 nM to 980 nM, suggesting that the reconstitution affinity of split-Nano-lantern@228 would not be high enough to affect its reversible conformational change. The reversibility was further confirmed by in-cell-analysis as described in the main text.

Supplementary Note 3 Design and properties of Nano-lantern (cAMP)
To screen for a cAMP-recognition domain yielding the largest luminescence signal change, we cloned the cAMP-recognition motif of human EPAC1 with different flanking sequences. We choose G170, P183 and A327, S334 for the N-and C-terminal end, respectively. All 4 constructs, G170/S334, G170/A327, P183/S334, and P183/A327 displayed a change in luminescence intensity upon stimulation with 100 M cAMP; the G170 series showed a larger response than the P183 series, and that of G170/A327 was the largest (130%). Next, we performed affinity optimization. The K d of EPAC1 G170/A327 was determined to be 2.2 M (data not shown), a detection range slightly different from the estimated concentration range of cellular cAMP (0.5-3 M) 24,25 . To increase its affinity, we introduced a Q270E mutation into the cAMP-binding pocket, and confirmed that the K d value of EPAC1 G170/A327_Q270E was decreased to 1.6 M, as reported previously 41 . To obtain a low-affinity variant, we inserted 12 amino acids of a polypeptide linker, which was reported to decrease the affinity of a firefly luciferase (FLuc)-PKA-based cAMP indicator 42 . As expected, the K d value of EPAC1 G170/A327_12 amino acids linker was decreased to 3.3 M. To generate a cAMP indicator with increased affinity, we cloned part of the cAMP-recognition motif from the human PKA regulatory subunit Ior II which are reported to have a higher cAMP affinity than EPAC1 41,43 . Among the tested constructs, Nano-lantern with PKA RI_M245/V381 inserted at position 228 in Rluc-S257G moiety displayed the highest affinity for cAMP (K d = 400 nM).
To compare the signal change of a Nano-lantern-based indicator with its related FRET-based indicator, we generated 6xHis-tagged FRET constructs by fusing EPAC1 G170/A327 domain with donor and acceptor pairs 15 . Among those tested, we selected ECFP_EPAC1 G170/A327_cp173Venus for further analysis, because it displayed slightly larger FRET ratio change (+11.7%) than the previously reported EPAC-camp sensor (-9.1% in our measurement) 43 and Fig. S8e).

Supplementary Note 4 Design and properties of Nano-lantern (ATP)
We developed a Nano-lantern-based ATP indicator by using the  subunit of the bacterial F o F 1 -ATP synthase, which was inserted into the 228-229 position of RLuc8-S257G in Nano-lantern. The resulting protein, Nano-lantern (ATP0), exhibited a 360% increase in light output upon the addition of ATP (Fig. 5b and Supplementary Fig. S12a); however, its relative brightness was only 10% that of Nano-lantern, even under ATP-saturating conditions. To increase the maximum brightness, we performed a simple modification of the construct. We developed another Nano-lantern (ATP) variant from VRL10.0_S257G instead of Nano-lantern (Nano-lantern (ATP1)), which has an intact RLuc8_S257G moiety compared to the parental Nano-lantern (ATP0) with its three amino acid N-terminal deletion (Fig. 5a). Although they differ by only three N-terminal amino acidsin the RLuc8-S257G moiety, their dynamic range and brightness were vastly different. Nano-lantern (ATP1) exhibited enough dynamic range (200%) for functional imaging, and was 1.5-fold brighter than Nano-lantern (ATP0) at ATP-saturating condition ( Fig. 5b and Supplementary Fig. S12a). They had similar affinities for ATP ( Supplementary Fig. S12b and Supplementary Table S4). Therefore we used Nano-lantern (ATP1) in further experiments. Chisso. ATP-Na 2 and cAMP-Na were purchased from Wako and Sigma-Aldrich, respectively.
For mammalian expression, pRSET B _eBAF-Y and pRSET B _Nano-lantern were digested with BamHI and EcoRI, and the resulting fragments were cloned into the BamHI/EcoRI site of the pcDNA3 vector (Life Technologies) to yield pcDNA3_eBAF-Y and pcDNA3_Nano-lantern. The N-terminal 12 amino acid sequence of the cytochrome c oxidase subunit IV sequence (CoxIV) was fused to the N-terminus of Nano-lantern to obtain Nano-lantern-mt, which specifically localized to the mitochondria matrix. Nano-lantern-er was generated by extending the N-terminus of Nano-lantern with the signal peptide sequence of calreticulin and the C-terminus of Nano-lantern with an endoplasmic reticulum retention signal sequence KDEL, which specifically localized to the endoplasmic reticulum. The Golgi body-localizing signal sequence of -N-acetylglucosaminyl-glycopeptide-1,4-galactosyltransferase was fused to the N-terminus of Nano-lantern to obtain Nano-lantern-go, which specifically localized to the Golgi body. A peroxisome localization sequence, SKL, was fused to the C-terminus of Nano-lantern to obtain Nano-lantern-pe. Nano-lantern-pm was constructed by adding the myristoylation and palmitoylation sequence from lyn kinase (MGCIKSKRKDNLNDDGVDMKT) to the N-terminus of Nano-lantern, which localized to the plasma membrane. Histone2B (H2B) was fused to the C-terminus of Nano-lantern to obtain Nano-lantern-H2B. Nano-lantern, Nano-lantern-mt, -er, -go, -pe, -pm, and -H2B were cloned into the pcDNA3 vector. The EYFP in pEYFP-Tub and pEYFP-Actin (Takara-bio) was replaced with Nano-lantern to obtain Nano-lantern-Tub, and -Actin. The EGFP in pEGFP-EB3 was replaced with Nano-lantern to obtain Nano-lantern-EB3.

Construction of Nano-lantern (Ca 2+ )
To insert a Ca 2+ -sensing domain into the Rluc8 moiety of Nano-lantern, a cDNA fragment encoding the CaM-M13 peptide of YC3.60 15 was amplified by PCR to attach NcoI and SacI sites to its 5'and To generate affinity variants of Nano-lantern (Ca 2+ ), the cDNA fragments of CaM-M13 were amplified from YC2.60 10 and YC-Nano series 15 , each of which has a different affinity for Ca 2+ .
For mammalian expression experiments, Nano-lantern (Ca 2+ ) cDNA digested with BamHI/EcoRI was cloned into the BamHI/EcoRI site of the pcDNA3 vector.

Construction of Nano-lantern (cAMP)
The cDNA clone encoding the human PKA regulatory subunit I was purchased from DNAFORM (Japan). cDNA fragments corresponding to the cAMP-binding domain and its flanking region were amplified by PCR to attach the oligonucleotide linkers for XhoI and SphI at its 5' and 3' end, respectively (Supplementary Table S5). XhoI-and SphI-tags were introduced at the 228/229 split site of the Nano-lantern cDNA using PCR. The obtained fragments were ligated to yield pRSET B _Nano-lantern (cAMP). The Q270E mutation in EPAC1 was introduced by site-directed mutagenesis 41 . The fragment encoding the 12-amino acid linker PWNWDSGCSREG was attached to the junction between 228 of Nano-lantern and the N-terminus of the cAMP-binding motif, by inserting an oligonucleotide cassette 42 .
To construct the expression vectors for Dictyostelium cells, Nano-lantern (cAMP) cDNA was cloned into the pDM304 expression vector 44 by attaching Dictyostelium's Kozak sequence (aaaaATG) to its 5'-end. To assure the robust expression of Nano-lantern and Nano-lantern (cAMP) in Dictyostelium cells, the Venus moiety of Nano-lantern, whose codon usage was optimized for mammalian but not for Dictyostelium, was replaced with Aequorea victoria's GFP sequences, in which the following mutations were introduced into the encoded Venus protein: F46L, F64L, S65G, M153T, V163A, S175G, T203Y, and A206K.

Construction of Nano-lantern (ATP)
The cDNA fragment encoding the ATP-recognition domain ( subunit of the bacterial F o F 1 -ATP synthase) was PCR-amplified to attach NcoI and SacI sites using ATeam1.03 cDNA as a template 28 .
To construct expression vectors for the chloroplast stroma of Arabidopsis plant cells, we prepared RNA from Arabidopsis leaves and isolated cDNA encoding the RECA transit peptide 29 followed by a 15-amino acid sequence derived from mature RECA and the adaptor sequence RDM, by RT-PCR with Arabidopsis RECA-specific primers (Supplementary Table S5). The cDNA fragment corresponding to CT was amplified by PCR to attach oligonucleotide linkers for BamHI and SalI sites at its 5' and 3' end, respectively (Supplementary Table S5 pENTR1A. The cloned Nano-lantern (ATP1) was transferred from the entry vector to the destination vector pH2GW7 by an LR recombination reaction, to produce pH2GW7_Nano-lantern (ATP1).

Protein purification and in vitro spectroscopy
Nano-lantern, Nano-lantern (Ca 2+ ), and Nano-lantern (ATP) E. coli strain JM109(DE3) was transformed with a pRSET B vector carrying Nano-lantern and its derivative cDNA and grown at 23°C. Recombinant proteins were purified using Ni-NTA agarose  Supplementary Table S6.
Colon26 cell lines stably expressing RLuc8 and Nano-lantern were established.
For tumor cell imaging, BALB/c male 5-week-old nude mice (nu/nu) were anesthetized.
At each of 6 different sites on the back, 1  10 1 -10 7 colon26 cells expressing RLuc8 or Nano-lantern were subcutaneously transplanted, as illustrated in Fig. 2a. binning setting. For spectroscopic analysis, emission intensity at 530 nm was measured for 5 × 10 6 of Nano-lantern (cAMP) expressing wild-type cell or ACA-KO cells every 1 s.

FRET-based cAMP imaging of Dictyostelium discoideum
Cells expressing ECFP_EPAC1 G170/A327_cp173Venus were imaged as described previously 27 .

Preparation and luminescence imaging of plants
Agrobacterium-mediated gene transfer was performed essentially as described previously 46 .
Agrobacterium tumefaciens (strain GV3101) transformed with Nano-lantern (ATP) was pre-cultured overnight at 28 °C in LB medium containing 50 g/mL hygromycin B (Wako) and 25 g/mL gentamicin (Wako), then subjected to an additional overnight culture at 28 °C in fresh LB medium containing 10 mM MES, pH 5.7, 20 M acetosyringone (Sigma-Aldrich), 50 g/mL hygromycin B, and 25 g/mL gentamicin. After centrifugation at 3,000 g for 15 min, the bacterial pellet was collected and then re-suspended in 10 mM MES, pH 5.7, 10 mM MgCl 2 , and 200 M acetosyringone, and incubated at room temperature for 3-4 h. Agrobacterium was inoculated into N.