Bioluminescence Bimodal

Jun 24, 2019 - ... using the BigDye Terminator v1.1 Cycle Sequencing kit (Thermo Fisher Scientific). .... A Ca2+ titration curve was used to calculate...
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Article Cite This: ACS Sens. 2019, 4, 1825−1834

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Genetically Encoded Fluorescence/Bioluminescence Bimodal Indicators for Ca2+ Imaging Israt Farhana,† Md Nadim Hossain,† Kazushi Suzuki,† Tomoki Matsuda,†,‡ and Takeharu Nagai*,†,‡ †

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan Department of Biomolecular Science and Engineering, The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan

‡

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S Supporting Information *

ABSTRACT: Fluorescent and bioluminescent genetically encoded Ca2+ indicators (GECIs) are an indispensable tool for monitoring Ca2+ dynamics in numerous cellular events. Although fluorescent GECIs have a high spatiotemporal resolution, their application is often confined to short-term imaging due to the external illumination that causes phototoxicity and autofluorescence from specimens. Bioluminescent GECIs overcome these pitfalls with enhanced compatibility to optogenetic manipulation and photophysiological processes; however, they are compromised for spatiotemporal resolution. Therefore, there has been a push toward the use of Ca2+ indicators that possess the advantages of both fluorescent and bioluminescent GECI for a wide range of applications. To address this, we developed a high-affinity bimodal GECI, GLICO, using a single fluorescent protein-based GECI combined with a split luciferase. Through this novel design, the fusion protein becomes bimodal and possesses Ca2+ sensing properties similar to those of its fluorescent ancestor and confers bioluminescence-based Ca2+ imaging. GLICO in bioluminescence mode has the highest dynamic range (2200%) of all bioluminescent GECIs. We demonstrated the performance of GLICO in studying cytosolic Ca2+ dynamics in different cultured cells in each mode. With the purpose of Ca2+ imaging in high Ca2+ content organelle, we also created a low-affinity variant, ReBLICO and performed Ca2+ imaging of the ER in both fluorescence and bioluminescence modes. The ability to switch between fluorescence and bioluminescence modes with a single indicator would benefit transgenic applications by presenting an opportunity for a wide range of live Ca2+ imaging in physiological and pathophysiological conditions. KEYWORDS: GECI, bimodal, fluorescence, bioluminescence, high-affinity, low-affinity, dynamic range

A

s a ubiquitous second messenger, the calcium ion (Ca2+) is intricately associated with numerous signaling cascades in living organisms and engaged in the control of diverse physiological events such as fertilization, proliferation, muscle contraction, learning and memory, and gene regulation.1−3 To visualize Ca2+ dynamics in live cells, genetically encoded Ca2+ indicators (GECIs) are preferred over synthetic Ca2+-dyes due to their ability to be continuously synthesized by cellular machinery and targetability to specific cell types or specific subcellular compartments using appropriate gene promotors or localization signal peptides.4−6 Imaging with fluorescent protein (FP)-based GECIs has progressed greatly in visualizing Ca2+, from subcellular levels to cell populations.7−10 FP-based GECIs (FP-GECIs) include resonance energy transfer (RET) type GECIs such as cameleons,11−13 troponin C (TN) Ca2+ indicators,14 and single FP type GECIs such as GCaMPs,15−18 GECOs,19,20 pericams,21,22 and Camgaroo.23 The GCaMP series have been highly optimized by mutagenesis and mammalian cell-based large-scale screening for monitoring in vivo brain activity with high dynamic range and faster kinetics.15,16 © 2019 American Chemical Society

Although FP-GECIs have been widely used as powerful tools for live Ca2+ imaging, they also hold potential difficulties arising from excitation light irradiation, such as signal decrease by photobleaching, cell phototoxicity, and reduced signal-tonoise ratio (SNR) due to autofluorescence from the specimen.24 In particular, imaging in long-term or in multilayer cells could be influenced by these difficulties.25,26 In addition, they are limited to simultaneous application with photomanipulation of physiological functions by optogenetic tools. In order to prevent unwanted actuation of optogenetic tools, only the FPGECIs, whose range of excitation wavelength does not overlap with that for photostimulation, are selected. Difficulties of FP-GECIs can be circumvented by bioluminescent protein based GECIs (BP-GECIs) that generate bioluminescence by substrate oxidation catalysis and are free from excitation light irradiation.27,28 While a major drawback of BPs for practical bioimaging was weak signal intensity, the recent emergence of BPs with improved brightness, such as a Received: March 18, 2019 Accepted: June 24, 2019 Published: June 24, 2019 1825

DOI: 10.1021/acssensors.9b00531 ACS Sens. 2019, 4, 1825−1834

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Terminator v1.1 Cycle Sequencing kit (Thermo Fisher Scientific). The bioluminescent substrate coelenterazine-h was purchased from Wako Chemicals, and furimazine was purchased from Promega. Genetic Construction of GLICO and ReBLICO in Bacterial Expression Vectors. For GLICO construction, the cDNA for the GCaMP6f was amplified by PCR from AAV-EF1a-DIO-GCaMP6fP2A-nls-dTomato (Addgene # 51083), using a sense primer containing 3 amino acid randomized linker (BCTBCTBCT, B = G/ C/T), a 17 bp homology with C-terminal of LgBiT (Promega) and a reverse primer containing 3 amino acid randomized linker (AGVAGVAGV, V = A/G/C) and 16 bp homology with N-terminal of SmBiT (Promega). We used a combination of alanine, serine, proline amino acids to create a mixture of flexible and nonflexible linker sequences.33 We also destroyed the EcoRI site at the 283rd residue by incorporating a silent mutation. The LgBiT was amplified by PCR using a sense primer containing a Kozak sequence following a BamHI site and the reverse primer. The SmBiT was amplified by PCR using a sense and a reverse primer containing stop codon and an EcoRI site. Mixture of PCR products of LgBiT, SmBiT and, GCaMP6f with extended overlapping sequence of LgBiT and SmBiT and 3 amino acid randomized linkers were used as a template for fusion PCR by overlap extension. A primer containing a Kozak sequence following a BamHI site and a primer containing a stop codon after the EcoRI site were used as forward and reverse primers, respectively. Amplified fragments were then restriction digested by BamHI and EcoRI and cloned in-frame between BamHI and EcoRI sites of pRSETB (Invitrogen) for bacterial expression (Supporting Information). For ReBLICO construction, we followed the same construction strategy using the cDNA for the R-CEPIA1er from pCMV RCEPIA1er (Addgene #58216) instead of GCaMP6f (Supporting Information). Construction of GLICO and ReBLICO in Mammalian Expression Vectors. To construct the plasmids for evaluation of the performance of GLICO in mammalian cells, GLICO in pRSETB was digested by BamHI and EcoRI and inserted into a pcDNA3 mammalian expression vector using BamHI and EcoRI sites. For neurons, pAAV2-hSyn-GLICO constructs were made by amplification of GLICO by PCR using a sense primer containing a Kozak sequence following a BamHI site and reverse primer containing a HindIII site. These amplified fragments were then restriction digested by BamHI and HindIIl and then ligated with pAAV2-hSyn.34 For the ER targeted construction of ReBLICO, we used our previously developed low-affinity bioluminescent Ca2+ indicator pcDNA3-CeNL(Ca2+)_110 μ-ER35 vector, which contains a signal peptide from calreticulin (MLLSVPLLLGLLGLAAAD) and a KDEL signal for ER retention located at the N-terminus and C-terminus of CeNL(Ca2+)_110 μ, respectively. We amplified ReBLICO with the sense primer containing a Kozak sequence following the BamHI site and reverse primer containing KpnI site without stop codon by PCR. We digested the PCR products and pcDNA3-CeNL(Ca2+)_110 μ-ER [to remove CeNL(Ca2+)_110 μ] by restriction enzymes BamHI and KpnI. These two fragments were then ligated and cloned. Protein Expression, Purification, and In Vitro Experiments. GLICO and ReBLICO with N-terminal polyhistidine tags were expressed in E. coli strain JM109 (DE3) at 23 °C for 69 h in 20 and 200 mL of LB bacterial growth medium supplemented with 0.1 mg/ mL carbenicillin for small-scale and large-scale purifications, respectively. Harvested cells were suspended in PBS buffer and was lysed with 0.5 μg/μL lysozyme, followed by five freeze−thaw cycles.35 The lysate was clarified by centrifugation (8000 rpm at 4 °C for 20 min). The recombinant proteins were purified from the supernatant using Ni-NTA agarose affinity columns (QIAGEN), followed by buffer-exchange to 50 mM HEPES, at pH 7.2, with the desalting column PD-10 (GE Healthcare).27,36 The protein purification process after lysis was conducted on ice to avoid protein degradation. Protein concentrations were determined by the alkaline-denaturation method using the extinction coefficient 44,000 M−1 cm−1 at 447 nm for denatured GFP chromophore.37 The fluorescence emission intensities of both GLICO and ReBLICO were measured by uisng a fluorescence spectrophotometer (F-7000, Hitachi High-Technologies) using a final

NanoLuc (Nluc) derived from the deep-sea shrimp Oplophorus gracilirostris,29 enabled the attainment of imaging quality close to that with FPs. The Nluc has been utilized to develop BPGECIs intensiometric GeNL(Ca2+)27 and ratiometric CalfluxVTN,30 which were proved to possess satisfactory performance for live cell imaging. However, it is inferior to FP-GECIs in terms of spatiotemporal resolution and kinetics.25,30 Moreover, they also require the continuous supply of luminescent substrates during imaging and face challenges in constitution of 3D images due to the luminescence from the out-of-focal plane, that is the general difficulty of BPs.31 Needless to say, GECIs, which include advantages of both FPand BP-GECIs together with the exclusion of their drawbacks could solve the problem. However, development of such GECIs is quite difficult due to current technological obstacles. Meanwhile, even though development of perfectly ideal GECIs is unrealistic, development of GECIs which simultaneously possess all the advantages in a single molecule is possible with a combination of FP- and BP-GECIs as an alternative option. Recently, Qian et al. developed a Ca2+ sensor by fusing nonsplit Nluc to a topologically altered GCaMP6s, which allows both fluorescence and bioluminescence-based detection of Ca2+ dynamics.32 In this study, we developed a novel designed bimodal (both FP and BP-based) GECI, GLICO (Green Luminescent Indicator for Calcium Observation) possessing high dynamic range (>2,000%) in both fluorescence and bioluminescence modes. GLICO enables Ca2+ imaging in the same specimen in both fluorescence and bioluminescence modes. This bimodal sensor will enable Ca2+ imaging in all cell systems involving light-sensitive and light-insensitive events and during prolonged observation with bioluminescence mode where excitation light gives rise to high background signals. The applicability of GLICO for studying cytosolic Ca2+ dynamics in dual modalities in live cells was demonstrated in HeLa cells, rat pituitary tumor (GH3) cells and dissociated rat hippocampal neurons. GLICO in its bioluminescence mode also made possible concurrent calcium imaging and optogenetic activation that is difficult in the fluorescence mode. Furthermore, we developed bimodal GECI with lower affinity for organelles having higher Ca2+ concentration than the cytosolic level such as ER, Golgi apparatus. Combination of bimodal GECIs with different affinities localized in each subcellular compartment is expected to contribute to investigation of intraorganellar relationship of Ca2+ dynamics.

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MATERIALS AND METHODS

General Molecular Biology. DNA oligonucleotides used in this study were purchased from Hokkaido System Science. KOD-Plus (Toyobo Life Science) was used for conventional PCR amplification and PCR products were purified using phenol-chloroform extraction followed by ethanol precipitation. Restriction digestion was performed by using endonucleases (Takara Bio or New England Biolabs) following the manufacturer’s recommended protocol, and digested products were purified from agarose electrophoresis gel using the QIAEX II gel extraction kit (QIAGEN). For ligation, T4 ligase and 2× Rapid Ligation Buffer (Promega) were used. Small-scale plasmid DNA was prepared by alkaline lysis method from the bacterial pellet harvested from 1.5 mL of LB liquid culture followed by ethanol precipitation. Large-scale plasmid DNA was obtained from bacterial pellets from 200 mL of LB-liquid culture by alkaline lysis, PEG-8000 precipitation, two rounds of phenol/chloroform extraction, and isopropanol precipitation. cDNA sequences for all constructs were confirmed by dye terminator cycle sequencing using the BigDye 1826

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ACS Sensors protein concentration 0.3 μM. Bioluminescence emission intensities were measured using a microplate reader (SH-9000, Corona Electric) using a final concentration of 2 nM of protein and 5 μM of the bioluminescent substrate coelenterazine-h (Wako chemicals) for these measurements. Experiments were performed at least in triplicate, and the averaged data were used for further analysis. In both fluorescence and bioluminescence readouts, Ca2+ titrations for high-affinity GLICO were performed by reciprocal dilution of Ca2+-free and Ca2+-saturated buffers prepared by using O,O′-bis(2-aminoethyl)ethylene glycol-N,N,N′,N′-tetraacetic acid (EGTA) and N-(2 hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid(HEEDTA).13 Ca2+ was added as CaCO3, at pH 7.2, at 25 °C. Ca2+ titrations for ReBLICO were performed by the dilution of small aliquots of concentrated CaCl2. A Ca2+ titration curve was used to calculate the apparent Kd value by nonlinear regression analysis. Sigmoidal binding curves were fitted to the data to extract the single Hill equation using Origin7 software (OriginLab). pH titration was performed using a series of 20 mM buffers with 100 mM KCl in pH 5.7 and 6.2 (MES), 6.6 and 7.0 (MOPS), 7.4 (Tris), 7.8 and 8.0 (HEPES), and 8.6 (Glycin) in dual readout.38 Measurements of Ca2+ dissociation kinetics of GLICO were performed by using stopped-flow photometry system consisting of SFS-853 stopped-flow unit (JASCO, Japan) and FP-8300 spectrophotometer (JASCO, Japan). In fluorescence mode, koff was determined from a single exponential fit to the fluorescence decay following rapid mixing of Ca2+-saturated protein samples with a Ca2+-free buffer containing EGTA at room temperature, both buffered with 10 mM MOPS, 100 mM KCl at pH 7.2. For fluorescence measurements, 480 nm excitation wavelength was used and emission at 520 nm was monitored at 1 kHz rate. HeLa Cell Culture, Transfection, and Ca2+ Imaging. HeLa cells (RIKEN BRC) were cultured on homemade 35 mm glassbottom dishes in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS). The next day, HeLa cells (at ∼70−80% confluency) were transfected with 5.0 μg of plasmid DNA using the calcium phosphate transfection protocol.35 The DMEM (with 10% FBS) medium was changed after 12 h, and the cells were grown for an additional 18 h in a CO2 incubator (Sanyo) at 37 °C in 5% CO2. Phenol-red-free DMEM/F12 (ThermoFisher Scientific) supplemented with 1% penicillin/streptomycin (ThermoFisher Scientific) was used as imaging medium. To observe Ca2+ signals in living HeLa cells, an Eclipse Ti-E inverted microscope (Nikon) equipped with 40× oil-immersion objectives (Nikon Plan Fluor, NA 1.3) was used. The signal was recorded by an iXon Ultra EMCCD camera (Andor Technology) with MetaMorph software (Molecular Devices) with 1 × 1 binning. The optical filter set for Ca2+ imaging by GLICO included excitation filter 472/30 (Semrock), dichroic mirror 502/950 (Semrock), and emission filter 520/35 (Semrock). For the Ca2+ imaging with ReBLICO, we used a similar microscopy setup with a 60× oil-immersion objective (Nikon Plan Fluor, NA 1.4) and optical filter set including excitation filters FF01-562/40 (Semrock), FF01-624/40 (Semrock), and dichroic mirror 601/950 (Semrock). GH3 Cell Culture, Transfection, and Ca2+ Imaging. GH3 (rat pituitary tumor, ATCC CCL-82.1) cells were cultured on homemade 0.1% poly-D-lysine-coated 35 mm glass-bottom dishes in DMEM/F12 supplemented with 15% horse serum and 2.5% FBS. For Ca2+ imaging with GLICO, GCaMP6f or GeNL(Ca2+)_480, GH3 cells (at ∼70% confluency) were transfected with 3.0 μg plasmid DNA using Lipofectamine 2000 Transfection Reagent (ThermoFisher Scientific) according to the manufacturer’s recommended protocol. The medium was replaced after 5 h, and the cells were grown for an additional 16 h in a CO2 incubator (Sanyo) at 37 °C in 5% CO2. The GH3 cells were washed with phenol-red-free DMEM/F12 and imaged in phenol-redfree DMEM/F12. At 3 to 4 min prior to bioluminescence imaging, 20 μM furimazine was added to the imaging medium. The same microscope and optical filter set up were used as in HeLa cell and imaging was performed at 5 Hz recording rate. Binnings were 1 × 1 and 8 × 8 for fluorescence and bioluminescence imaging, respectively. Primary Rat Hippocampal Neuron Culture, Transfection, and Imaging. Primary cultures of hippocampal neurons were

prepared from embryonic day 17 Sprague−Dawley rats. Cells were dissociated in plating medium consisting of Hanks’ Balanced Salt solution (HBSS; Wako), which was supplemented with 1 mM HEPES (pH 7.2) and 100 U/ml penicillin/streptomycin. The cells were then plated on a homemade polyethylenimine (Sigma-Aldrich) and poly-Dlysine (Sigma-Aldrich) coated 35 mm dish with a coverslip bottom at a density of 3.5 × 105 cells/12 mm diameter coverslip. The medium was changed to culture medium constituting Neuro Basal (ThermoFisher Scientific), supplemented with 2% B27 (ThermoFisher Scientific) and L-glutamine, 5 h after plating, and the cultures were grown in 5% CO2 at 37 °C. On the seventh day in vitro (DIV-7), the cells were transfected with pAAV2-hSyn-GLICO (for imaging spontaneous oscillations and glutamate stimulation) by calcium phosphate precipitation. For optical stimulation with ChR2(H134R), the cultured neurons were cotransfected with pAAV2-hSyn-GLICO and pcDNA3-ChR2(H134R)-mCherry. For imaging spontaneous Ca2+ spiking, the neuronal cells were imaged at DIV-10 on a wide-field epi-fluorescence inverted microscope as described above for HeLa cells. For the bioluminescence imaging, a solvent of furimazine solution was evaporated with a VDR20G vacuum desiccator (Jeio Tech) and a BSW-50N belt drive rotary vane vacuum pump (Sato Vac Inc.) overnight under dark conditions. The precipitate was eventually dissolved in propylene glycol (up to 5 mM). The furimazine precipitate was kept at −30 °C as a stock solution and used for further imaging experiments. Immediately prior to imaging, 5 μM furimazine dissolved in propylene glycol was added to the culture medium. Imaging of spontaneous Ca2+ oscillations was performed at 1 × 1 and 4 × 4 binning for fluorescence and bioluminescence modes, respectively. To image the Ca2+ transient induced by high glutamate stimulation, 20 μL of 2 mM D-glutamate solution was added to neurons at DIV-11 at binning 1 × 1, and time-lapse images were taken in fluorescence mode. Several minutes later, in the same dish, 20 μM furimazine was added for the demonstration of Ca2+ response in neurons in bioluminescence mode. Four minutes after furimazine addition, 20 μL of 2 mM glutamate solution was added, and then imaging was done at binning 2 × 2. Ca2+ Imaging with Optogenetic Actuator. To activate ChR2(H134R) during Ca2+ imaging, stimulating light (47 mW/ cm2) from a LightEngine SPECTRA (Lumencor) was applied within the dead time of the charge-coupled device camera, as reported previously.39 Briefly, the exposure time-out signals from the EMCCD camera were used as the trigger for a WF1973 multifunction generator (NF Corporation) to generate the pulsed signals for turning on the stimulation light. The entire illumination duration was modulated by a function of the multifunction generator. Camera binning was set at 4. Light was passed through a 438/24 nm filter from above the culture dish to activate ChR2(H134R). Irradiation from above the dish was applied by the LightEngine liquid light guide, connected using a homemade adaptor.40

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RESULTS AND DISCUSSION Development of Bimodal GECI, GLICO. We designed a bimodal GECI composed of an FP and a BP, and each mechanism for Ca2+ dependent emission light intensity change is conferred here. Its core Ca2+ sensing mechanism was based on that of FP-GECI, GCaMP6f, which is composed of a circularly permuted GFP (cpGFP), a calcium-binding protein (CaM) and a Ca2+-CaM interacting peptide (RS20).16 In the GCaMP6f, CaM and RS20 are fused to the C- and N-terminus of cpGFP, respectively. Ca2+ binding to CaM induces interaction between Ca2+-CaM and RS20. That modulates solvent access of the chromophore of cpGFP, resulting in the increase in fluorescence intensity of GCaMP6f relative to the unbound form of Ca2+. To provide the Ca2+ dependent light intensity change to a BP moiety, we attempted to incorporate the interaction between Ca2+-CaM and RS20 moieties also into 1827

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increase by Ca2+ binding (Figure 1B). Based on this design, we made constructs in which GCaMP6f and fragments of NBiT moieties were connected with random three-residue linkers (linker 1 and linker 2). We made a cDNA expression library with randomized three amino acid linkers between NBiT and GCaMP6f. E. coli cells were transformed using this library and cultured on LB plates. After the colonies size grew to about 1 mm in diameter, brighter colonies were selected by visual inspection upon blue light excitation. Then, the proteins were purified and evaluated in terms of increased luminescence intensity and dynamic range, in both fluorescence and bioluminescence modes. Finally, six gene constructs were short-listed (Table 1). Variant 1 showed the best properties and is hereafter referred to as GLICO (Green Luminescent Indicator for Calcium Observation). In Vitro Characterization of GLICO. Fluorescence spectra and Ca2+-induced fluorescence change of GLICO are basically very similar to those of GCaMP, and the dynamic range is also comparable to that of GCaMP6f (GLICO and GCaMP6f are 2000% and 2600%, respectively) (Figure 2A and C). GLICO retained almost 65% fluorescence intensity of GCaMP6f even after the fusion of NBiT. Although the bioluminescence intensity of GLICO in the absence of Ca2+ was one-tenth of GeNL(Ca2+) due to the higher RET efficiency in GeNL(Ca2+), it exhibited a large signal change in the presence of Ca2+ (Table 1). GLICO in bioluminescence mode possessed the highest dynamic range (2200%, calculated from intensiometric measurements) among the currently available BP-GECIs (ratiometric GECI: CalfluxVTN 900%; and intensiometric GECI: GeNL(Ca2+) 450%) (Figure 2D and F).27,30 Combination of the mechanics, reconstitution of NBiT subunits, and RET to the GCaMP6f enhanced the signal intensity upon Ca2+-binding, owing to the high dynamic range in bioluminescence modality. GLICO also showed a dynamic range of ∼200% calculated from the RET ratio (520 nm/450 nm) in the bioluminescence mode. Although the dynamic range from ratiometric measurement is smaller than that of the intensiometric measurements, it is helpful when its advantages are rather important than the dynamic range, like in the situation where the signal difference due to the local concentration of the bioluminescent protein/substrate or time dependent focal drift of the observation object exists. We checked that there was no Ca2+-dependent intensity change on Nluc and NBiT (Figure S1). We also compared GLICO with another GCaMP6s-based bimodal Ca2+ indicator, LUCI-GECO1.32 LUCI-GECO1 exhibited a dynamic range of

its mechanism. To this end, a binary complementation reporter system NanoBiT (NBiT)41 that is derived from Nluc was selected as a BP moiety of the bimodal GECI. Components of the NBiT, large BiT (LgBiT; 18 kDa), and small BiT (SmBiT; 1.3 kDa)41,42 were fused to the N-terminal of RS20 and Cterminal of CaM in GCaMP6f, respectively, to cause Ca2+ dependent reconstitution of LgBiT-SmBiT complex to increase Ca2+ dependent bioluminescence (Figure 1). Along with this,

Figure 1. Engineering of GCaMP6f-based bimodal Ca2+ indicator. (A) This bimodal GECI consisted of N-terminal large BiT (LgBiT), smooth muscle myosin light chain kinase derived calmodulin binding peptide (RS20), a circularly permutated GFP (cpGFP), calcium binding domain, calmodulin (CaM) and C-terminal small BiT (SmBiT). The start and end amino acid residue numbers are shown. Both linker 1 and linker 2 are randomized three amino acid linkers. (B) Schematic representation of Ca2+ sensing mechanism of GLICO in fluorescence mode (top) and bioluminescence mode (bottom). Ca2+ and substrate are indicated as red and blue circles, respectively.

RET from LgBiT-SmBiT complex to GCaMP6f is expected because of the adjacency of these moieties. Moreover, increase in RET efficiency by Ca2+ binding is also expected owing to the increase in molar extinction coefficient of the GCaMP6f moiety. Overall, signal intensities of both the fluorescence and bioluminescence modes in the bimodal GECI are intended to Table 1. Property of the Variants Selected by Screeninga construct variant 1 (GLICO) variant 2 variant 3 variant 4 variant 5 variant 6 GCaMP6f GeNL(Ca2+)

linker 1 linker 2

relative fluorescence at Ca2+ saturation

intensity change (fluorescence)b

relative bioluminescence at Ca2+ saturation

intensity change (bioluminescence)b

ASS

SPA

1

21.5

1

23

SPS APP SSA AAS PSA

PAP SAP PSP PPA SSS

0.73 0.92 0.72 0.84 0.45 1.66

11 8 17 14 7 27

0.87 0.69 0.5 0.54 0.58

22 17 14 15 16

11

5

a

Bold fonts indicate the best variant. bIntensity changes were measured in presence and absence of Ca2+ and calculated using the following formula, (ΣI+Ca2+ − ΣI−Ca2+)/ΣI−Ca2+ where ΣI+Ca2+ and ΣI−Ca2+ are fluorescence or bioluminescence intensities in the presence and absence of Ca2+, respectively. 1828

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Figure 2. Characteristics of GLICO in vitro and in HeLa cells. In vitro characteristics in fluorescence mode (A−C) and bioluminescence mode (D−F). (A, D) Normalized emission spectra of GLICO at Ca2+ saturated (black solid line) and Ca2+ free (dashed gray line) states. (B, E) Ca2+ titration curves. In drawing the Ca2+ dependent curve, the summation of the intensity of the whole wavelength (490−600 nm and 400−600 nm in fluorescence and bioluminescence mode, respectively) was used. Fitted Hill plot curves are shown. (C) Comparison of dynamic ranges of GCaMP6f, YC 3.60, LUCI-GECO1, and GLICO. (F) Comparison of dynamic ranges of GeNL(Ca2+)_480, CalfluxVTN, LUCI-GECO1, and GLICO. Dynamic range was calculated as [R − R0]/R0 (for YC 3.60, CalfluxVTN and LUCI-GECO1) or as [ΣI − ΣI0]/ΣI0 (for intensiometric Ca2+ sensors), where R and I are the ratio and intensity, respectively. Bioluminescence and fluorescence spectra are normalized at 450 and 514 nm, respectively. The averaged data and standard deviations are shown for n = 3. Characteristics in HeLa cells expressing GLICO in fluorescence mode (G, H) and bioluminescence mode (I, J). (G, I) Images of cells. Scale bar 20 μm. (H, J) Signal changes depend on the Ca2+ oscillations evoked by histamine. Regions of interest for measurements are shown as white circles in (G) and (I). Arrow indicates the time point of 20 μM histamine addition.

1900% and 400% in fluorescence and bioluminescence modes, respectively (Figure 2C and F). GLICO exhibited a dissociation constant (Kd) of 590 and 230 nM in fluorescence and bioluminescence modes, respectively. The Kd is determined by the summation of the whole emission wavelength (490−600 nm and 400−600 nm in fluorescence and bioluminescence modes, respectively) (Figure 2B and E). The bioluminescence emission from 400 to 600 nm includes the Ca2+ response range of both blue and green emission. We also calculated the Kd’s for the peak intensity at 450 and 520 nm, which corresponded to the emission from the NBiT and GCaMP6f moiety in GLICO, as 190 and 450 nM, respectively (Figure S2A and B). The Kd calculated from the RET ratio (520 nm/450 nm) is 590 nM (Figure S2C). For the apparent smaller Kd in bioluminescence mode, we presume that smaller structural change of CaM-RS20 interaction is required for the reconstitution of NBiT than that necessary for the influence on the fluorescence state of the chromophore in GCaMP6f. LgBiT and SmBiT in NBiT were

originally designed for detection of interaction between two separate molecules that were linked to each of them.41 In order to deal with detection of weak interaction, LgBiT and SmBiT had lower affinity (Kd = 190 μM) for suppressing the influence of the LgBiT−SmBiT interaction. However, in the case of GLICO, where LgBiT and SmBiT are tethered to the GCaMP6f, their effective concentration for interaction tends to be higher. Therefore, intrinsic LgBiT-SmBiT interaction influenced the Ca2+-dependent conformational change of CaM-RS20 and suggested an apparent higher affinity for Ca2+ in bioluminescence mode. Since a higher affinity version of the NBiT system was developed as HiBiT41,43 by introducing mutations into the smaller peptide subunit corresponding to the SmBiT, a lower affinity might also be achieved by engineering the SmBiT to develop a bimodal indicator with similar affinity in dual modes. Ca2+ dissociation rate constant (koff) for GLICO in fluorescence mode determined by stopped-flow photometry system was 2.48 s−1 (τ = 0.40 s), that is comparable to the koff for GCaMP6f 3.19 s−1 (τ = 0.31 s). By considering the Kd’s of 1829

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ACS Sensors GLICO (590 nM) and GCaMP6f (375 nM),16 estimated that the association rate constant (kon) calculated by koff/Kd is also comparable between GLICO and GCaMP6f. We also attempted to measure the kinetic parameters for the bioluminescence mode to compare with that of the fluorescence mode. However, time dependent bioluminescence intensity decay due to the consumption of the bioluminescent substrate complicated the analysis. Time dependent reconstitution or separation of LgBiT and SmBiT in the NBiT for association or dissociation kinetics measurements, which causes time dependent change of substrate consumption rate, also adds further complexity to the analysis. In such a situation, where different time-dependent factors are convoluted, data analysis to determine the parameters related to each factor becomes difficult. We evaluated pH sensitivity of GLICO and the results indicated that the signal in both modes were stable in physiological pH range between 6.5 and 8.0 (Figure S3A).38 In addition, response of GLICO is also specific for Ca2+ and showed negligible sensitivity to changes in K+, Na+ or Mg2+ within the range of concentration change that might be expected to occur under physiological condition (Figure S3B-). Characterization of GLICO in Cultured Cells. To test the applicability of GLICO for studying Ca2+ dynamics in living cells, we expressed it in HeLa cells, rat pituitary tumor (GH3) cells, and primary cultured neurons. First, we demonstrated cytosolic Ca2+ imaging in HeLa cells expressing GLICO. Histamine-induced Ca2+ oscillations were successfully observed by GLICO sequentially in both fluorescence and bioluminescence modes (Figure 2G−J). After the histamine stimulation, an acute intensity spike followed by oscillations, that corresponded to the general Ca2+ response in HeLa cells, was observed in both fluorescence and bioluminescence modes. In the fluorescence readout, shape of the peaks during the following oscillations was as sharp as that of GCaMP6f (Figure 2G and H). On the other hand, shallow spikes and slow response were observed in bioluminescence mode (Figure 2I and J). This discrepancy in bioluminescence mode might be due to the higher affinity and slower kinetics of Ca2+. Reconstitution of spatially decoupled segments of NBiT requiring larger structural change for a signal change than cpGFP in the GCaMP6f is presumably the principal reason for that. These results also comply with the previous findings that GECI with low Kd value usually cause an initial peak after histamine stimulation followed by sustained high intensity between spikes due to saturation of the GECI in excessive Ca2+ concentration.44 We also compared the performance of GLICO with the reported Ca2+ indicators, LUCI-GECO1,32 GCaMP6f,16 and GeNL(Ca2+)_480,27 for fluorescence and bioluminescence modes in GH3 cells (rat pituitary tumor) which spontaneously oscillate Ca2+ level45 (Figure 3). Contrast between minimum and maximum fluorescence intensities of GLICO (3.9 ± 1.1fold, n = 6) is higher than that of LUCI-GECO132 (1.7 fold ±0.4, n = 6) and comparable to that of GCaMP6f (4.9 ± 1.9fold, n = 6) in GH3 cells (Figure 3A, C, and E). The contrast of GLICO (2.5 ± 1.2-fold, n = 4) in bioluminescence mode is almost similar to those of LUCI-GECO1 (1.9 fold ±0.4, n = 6) and the intensiometric Ca2+ indicator, GeNL(Ca2+)_48027 (1.8 ± 0.6-fold, n = 5) (Figure 3B, D, and F). The similar spiking pattern of GLICO in fluorescence mode and GCaMP6f suggested similar fast kinetics. However, the spike pattern of GLICO between fluorescence and bioluminescence modes

Figure 3. Comparison of performance of GLICO in rat pituitary tumor cells (GH3). Spontaneous Ca2+ spiking of (A) GLICO_Fluorescence, (B) GLICO_Bioluminescence, (C) LUCI-GECO1_Fluorescence, (D) LUCI-GECO1_Bioluminescence, (E) GCaMP6f, and (F) GeNL (Ca2+)_480.

suggests that the kon might be similar in both modes and slower decay (smaller koff) in bioluminescence mode. The results also correspond to the smaller Kd value of the bioluminescence mode than the fluorescence mode. Spikes with relatively wider width detected in bioluminescence mode of GLICO might also be related to the slower kinetics than of GeNL(Ca2+)_480.27 Next, we tested the performance of GLICO in dissociated rat hippocampal neurons. Increase in Ca2+ levels induced by stimulation of glutamate receptors (GluRs) by a major excitatory neurotransmitter glutamate was measured.46,47 After imaging of the glutamate stimulation dependent Ca2+ change in the fluorescence mode for a few minutes (Figure 4A and B), the Ca2+ level in the neuron was found to return to the basal level. The next round of the glutamate-induced Ca2+ response was recorded in bioluminescence mode (Figure 4C and D). GLICO underwent a 3- and 2.8-fold increase in fluorescence and bioluminescence intensity upon treatment with glutamate, respectively (Figure 4B and D). We also demonstrated the capability of GLICO to detect spontaneous Ca2+ oscillations in cultured hippocampal neuron at DIV-10, and an almost 3-fold intensity change with cytosolic Ca2+ spiking was exhibited in both fluorescence and bioluminescence modes (Figure S4). To validate the compatibility of GLICO in the bioluminescence mode with an optogenetic actuator, we coexpressed GLICO and channel rhodopsin, ChR2(H134R),48 in cultured rat hippocampal neurons. Imaging was performed at a 2 Hz recording rate, and ChR2(H134R) was activated with a 2 ms pulse of blue light during the dead time of the CCD chip for data transfer.39 The blue light pulses provoked a transient increase in bioluminescence intensity indicating that light stimulation of ChR2(H134R) elicited membrane depolarization and subsequent Ca2+ flux was detected by GLICO (Figure 4E). Neurons expressing GLICO without ChR2(H134R) did 1830

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value of ReBLICO. We expect a transient Ca2+ depletion, followed by a recovery with Ca2+ oscillation to be observed with variants having a suitable lower Kd (submicromolar range)49 after further improvement. Mutation in the CaM region in the parental R-CEPIAer and/or using high-affinity variant of NBiT might increase the Kd to a more appropriate range to image ER Ca2+ dynamics. Although we could not obtain a red bioluminescent variant, ReBLICO showed a Ca2+ dependent signal increase for both fluorescence and bioluminescence modes with different affinity range and fluorescence color, by replacing the parental fluorescent Ca2+ indicator. This suggests that GLICO design can provide a platform for further modifications for various applications. Development of the red color variant with similar affinity can also be expected by optimization of the linker length and type between NanoBiT and R-CEPIAer. Ca2+ imaging with a bimodal indicator would enable us to obtain information on both photodependent events such as circadian rhythms, and photosynthesis, as well as photoindependent phenomena simultaneously in a single model, rather than using different monomodal FP-GECI or BP-GECI. In plant biology, due to the strong autofluorescence and intrinsic photosensitivity of chloroplast, a solution for simultaneous monitoring of Ca2+ dynamics in different subcellular compartments in the green and nongreen plant part would be by using bimodal indicators with affinity variants. The characteristics of these bimodal indicators would help to address queries of Ca2+ signaling in whole plant system in response to biotic and abiotic stresses that were previously difficult to address.

Figure 4. Performance of GLICO reports in dissociated rat hippocampal neuron. Series of pseudocolored fluorescence (A) and bioluminescence images of cultured neurons expressing GLICO in cytosol showing Ca2+ dynamics upon glutamate stimulation (C). Time course of fluorescence change (F/F0) (B) and bioluminescence change (B/B0) (D). Scale bar 20 μm. (E) Time course of the B/B0 ratio change in neuron expressing GLICO with ChR2 (H134R) (black line) and without ChR2 (red line). ChR2 was repeatedly photoactivated (450 nm) in illumination sessions (∼5 s, transparent blue bands), consisting of camera exposure (500 ms) and pulses of blue light (∼2 ms) that were delivered during periods (∼8 ms) when camera exposure was off.

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not exhibit any signal change in response to blue light irradiation (Figure 4E). Development of Low-Affinity Bimodal Ca2+ Indicator. Following the strategy of GLICO’s construction, we developed its low-affinity variant. To reduce the affinity, a red variant of the low-affinity FP-GECI family CEPIA, R-CEPIA1er (Kd = 565 μM),49 which was developed from R-GECO1, was used instead of GCaMP6f. LgBiT and SmBiT were fused to the Nand C-terminus of R-CEPIA1er by three amino acid linkers (Figure 5A). The fusion protein, which we named ReBLICO (Red and Blue Luminescent Indicator for Calcium Observation), retained the Ca2+ sensing potency in both fluorescence and bioluminescence modes with dynamic ranges of 1100% and 240%, respectively (Figure 5B and D). Ca2+ dependent bioluminescence signal was only emitted from the RET donor, NBiT, rather than the RET acceptor, cpmApple in RCEPIA1er, because of the poor RET efficiency. The dynamic range of ReBLICO in the fluorescence mode is slightly larger than that of R-CEPIA1er.49 In the bioluminescence mode, the dynamic range is 2-fold higher than that of the low-affinity bioluminescent indicator CeNL(Ca2+)_110 μ, recently developed by our group.35 ReBLICO showed a Kd of 1576 and 1526 μM in fluorescence and bioluminescence modes, respectively (Figure 5C and E), that is slightly above the submillimolar Ca2+ range in the ER. We performed Ca2+ imaging of the ER in HeLa cells using ReBLICO (Figure S5). A transient Ca2+ decrease following recovery in the ER of HeLa cells stimulated by histamine is expected to be visualized by an ER localized Ca2+ indicator.35 However, after histamine stimulation, a signal depletion of ReBLICO expressed in the ER was observed with a small signal change in both fluorescence and bioluminescence modes, and a slight recovery of the signal was observed in the bioluminescence mode (Figure S5) due to the higher Kd

CONCLUSION We developed color variant of bimodal Ca2+ indicators, GLICO and ReBLICO, with high and low affinity for Ca2+, respectively, and demonstrated their performance in cultured cells and in both fluorescence and bioluminescence modes. Due to the low background signal in the bioluminescence mode, these sensors can be used in primary drug screening, and pharmacodynamics of Ca2+ in live animal models can be monitored with minimal invasiveness.50 Development of a color palette or affinity variants of new bimodal Ca2+ indicators might be possible by applying this design strategy to either GECO or GCaMP type single fluorescent protein-based Ca2+ indicators. This is a simple design for changing monomodal FP-GECI to bimodal GECI, without requiring extensive modifications of the prototype FP-GECI. This bimodal Ca2+ sensor retains the already optimized properties of the parental fluorescent moiety and enables additional bioluminescencebased detection including compatibility to optogenetics and luminescence microplate reader assays. As the size of this bimodal indicator is equivalent to or smaller than FRET-based GECI, no allosteric hindrance in sensor activities is expected. As an application to assimilate the bimodality of the GLICO, imaging across the spatial and temporal scale can be suggested. To find out the previously unidentified locus and timing related to the Ca2+ change in the three-dimensional cell tissue, imaging in the wider area during a contentious longer period is required. For this purpose, the bioluminescence mode is useful by which signals from the whole population of cells within tissues or organs can be obtained with high signal-to-noise ratio (SNR) and minimal phototoxicity for an extended period.25 More detailed information can be obtained by the fluorescence mode of the same indicator in combination with 1831

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Figure 5. Development and in vitro properties of R-CEPIA-based low-affinity bimodal Ca2+ indicator ReBLICO. (A) Schematic domain structure of ReBLICO. ReBLICO consists of the N-terminal LgBiT, RS20, circularly permutated red fluorescent protein (cpmApple), CaM, and C-terminal SmBiT. The start and end amino acid residue numbers are shown. Both linker 1 (AAS) and linker 2 (PAA) are three amino acid linkers. Normalized emission spectra of ReBLICO at Ca2+ saturated (solid black line) and Ca2+ free (dashed gray line) state in fluorescence (B) and bioluminescence (D) modes. Ca2+ titration curve in fluorescence (C) and bioluminescence mode (E). Fitted Hill plot curves are shown. The averaged data and SDs are shown for n = 3.

Author Contributions

two-photon microscopy in single cells or at subcellular resolution. Owing to these characteristics, this bimodal indicator would allow trans-scale Ca2+ imaging and open frontiers for in vivo imaging in healthy and diseased conditions by finding a rare event that is difficult to be found by fluorescence imaging with a narrow observation field.

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I.F. developed and performed imaging of GLICO. T.N. and K.S. designed GLICO construction. H.N. developed ReBLICO and performed imaging. Writing of original draft: I.F. and T.M. Review and editing: I.F, T.M., and T.N. Supervision: T.N. Notes

The authors declare no competing financial interest. The nucleotide sequences of the GLICO construct has been deposited in DDBJ under accession numbers LC466954.

ASSOCIATED CONTENT

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.9b00531. Dependence of NBiT and Nluc on Ca2+; Ca2+ titration curves of GLICO; pH dependency and insensitivity of GLICO to other ions; Imaging of spontaneous Ca2+ spiking by GLICOin cultured rat hippocampal neuron; Ca2+ imaging with ReBLICO in HeLa cells and time course of fluorescence and bioluminescence intensity; Nucleotide sequences of bimodal Ca2+ indicators (PDF)

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ACKNOWLEDGMENTS We would like to thank Dr. Kenji Osabe for proofreading of the manuscript. We are also very grateful to the Japan Government and the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) for supporting I.F. and M.N.H. through the scholarships during this study. This work was supported by the MEXT “Grant-in-Aid for Scientific Research on Innovative Areas” “Singularity biology” (No. 18H05410) to T.N., and “Interplay of developmental clock and extracellular environment in brain formation” (No. JP16H06487) to T.M.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-6-6879-8480. E-mail: [email protected].

REFERENCES

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ORCID

Md Nadim Hossain: 0000-0002-8086-8939 1832

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