Mol. Cells 2020; 43(11): 909-920
Published online November 9, 2020
https://doi.org/10.14348/molcells.2020.0144
© The Korean Society for Molecular and Cellular Biology
Correspondence to : hjibio@korea.ac.kr
Cytosolic Ca2+ levels ([Ca2+]c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca2+]c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca2+]c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca2+]c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca2+-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca2+]c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca2+]c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca2+]c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca2+]c in single cells and animal models.
Keywords calmodulin, cytosolic Ca2+ sensor, internal ribosome entry site, myosin light chainC kinase 1/2, NanoBiT assay
Intracellular cytosolic calcium (
In general, there are two main classes of
Genetically encoded indicators are produced by translation of a nucleic acid sequence without the addition of any chemical compounds (Mank and Griesbeck, 2008). GECIs include fluorescent protein (FP)-based sensors detected via Förster resonance energy transfer (FRET), and bioluminescent protein (BP)-based sensors detected via a specific chemical reaction within the cell. BP-GECIs require addition of the luciferase substrate luciferin for the detection reaction, although the resulting acquired signal originates from FPs. The major drawback of FP-GECIs is that they require external illumination, which can damage cells or cause artifacts. By contrast, BP-GECIs are superior for light-sensitive applications such as optogenetics or long-term continuous monitoring (Nagai et al., 2014). Both FP-GECIs and BP-GECIs have poorer SNR than synthetic indicators (Perez Koldenkova and Nagai, 2013). A common approach to improve SNR and brightness of GECIs involves indicator overexpression driven by a strong promoter such as the cytomegalovirus (CMV), and CMV early enhancer/chicken beta actin promoter. However, under some conditions, this overexpression strategy can lead to adverse effects such as cell/tissue damage, abnormal brain activity in indicator-expressing transgenic models, nonfunctional indicators, or perturbations in membrane-gated L-type Ca2+ channels and distorted Ca2+ dynamics (Tian et al., 2009; Yang et al., 2018).
The discovery of the 19.1 kDa luciferase enzyme NanoLuc (Nluc), which is derived from the deep-sea shrimp
Specialized microscope instrumentation is required for [Ca2+]c detection using intensiometric or ratiometric sensors. Ratiometric sensor monitoring of [Ca2+]c change requires equipment that detects rapid changes in wavelength emission, such as confocal or two-photon microscopy, which limits their application. Chemically synthesized indicators can be detected with specialized instruments such as fluorescence imaging plate reader (FLIPR) and the FlexStation microplate reader (Luo et al., 2011; Wu et al., 2019), which are feasible for high-throughput assays for drug discovery, but expensive. Therefore, the development of a novel Ca2+ assay system that uses common laboratory equipment would be invaluable for applications ranging from simple signaling mechanism analysis to high-throughput drug screening.
In this study, we developed a novel intensiometric bioluminescent GECI method using split Nluc without a FP. Our calcium sensor consists of two components as fusion constructs with split Nluc fragments: calmodulin (CaM), and the calmodulin-binding peptide. We used these sensors and luminescence detection instruments to monitor real-time changes in [Ca2+]c in response to extracellular stimulation. We also identified a suitable calmodulin-binding peptide and promoter to optimize expression level of the bioluminescent GECI to efficiently detect changes in [Ca2+]c. Although these assay data were collected from cell populations, our method may be applicable for investigating single-cell [Ca2+]c responses, drug development, and
A NanoBiT starter kit containing all plasmids and reagents for the protein interaction assay was purchased from Promega (USA). The pcDNA3.1 expression vector was purchased from Invitrogen (San Diego, CA, USA). The SRE-Luc vector containing four copies of the serum response element (SRE, CCATATTAGG) was purchased from Stratagene (USA). All primers and reagents for gene cloning and PCR were purchased from Cosmo Genetech (Korea). DNA sequencing was conducted by Macrogen (Korea). Unless otherwise stated, all reagents were purchased from Sigma-Aldrich (USA).
HEK293 and HEK293T cells were obtained from the American Type Culture Collection (ATCC, USA). All cell lines in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) were maintained at 37°C with 5% CO2 unless otherwise stated.
Human cytomegalovirus (CMV) promoter sequence in pcDNA3.1 vector was substituted with promoter sequences of
HEK293 or HEK293T cells were seeded in 96-well plates at a cell density of 2 × 104 cells/well. The next day, 30 ng of receptor plasmid, 30 ng of plasmid containing calmodulin tagged with LgBiT or SmBiT, and 30 ng of plasmid containing calmodulin-binding region fused to SmBiT or LgBiT in the N-terminal or C-terminal region were mixed with 0.2 μl Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and added to the plated cells. Subsequent transfection steps were performed according to the manufacturer’s instructions. After 24 h, media was replaced with 100 μl of Opti-MEM and cells were stabilized for 10 min at room temperature before measuring the luminescence. Then, 25 μl of Nano-Glo Live Cell Reagent (furimazine) was added to each well, and basal luminescence was measured using a luminometer (BioTek, Winooski, VT, USA) for the first 10 min. Finally, cells were stimulated by adding 10 μl of appropriate agonists to each well, and the cell plate luminescence was measured for 30 min. The schematics of all NanoBiT constructs and their combinations, and all graphs in dosing experiments were presented in supplementary information (Supplementary Figs. S1-S9). The absolute values of luciferase activities were also shown in it (Supplementary Table S1).
HEK293 cells in 6-well plates were transfected with 2 μg pcDNA3.1 vector harboring the receptor genes using 4 μl Lipofectamine 2000. The next day, cells were incubated with serum-free media overnight, and then treated with their cognate ligand for the indicated time. After washing with cold phosphate-buffered saline, the cells were harvested with lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM NaF, 1% Triton X-100, and protease inhibitors) and the protein extracts were clarified by centrifugation at 15,000 rpm for 15 min at 4°C. Then, 20 μg of cellular proteins were examined using SDS-PAGE followed by western blotting with anti-pERK or anti-ERK antibodies.
HEK293 cells were seeded in 24-well plates at a density of 8 × 104 cells/well. The next day, a mixture of 400 ng of receptor gene–containing plasmids, 50 ng of SRE-Luc reporter gene plasmids, and 1 μl of Lipofectamine 2000 was added per well according to the manufacturer’s instructions. The next day, cells were maintained in serum-free DMEM overnight. Approximately 36 h after the start of transfection, cells were treated with appropriate ligands for 6 h. Cells were then lysed using 100 µl of lysis buffer, and the luciferase activity of the cell extract was measured using a luciferase assay and the standard protocol for the Synergy 2 Multi-Mode Microplate Reader (BioTek).
Statistical differences among experimental groups were analyzed by unpaired Student’s
Ca2+-bound calmodulin can bind various effector molecules via distinct amino acid sequence motifs. Therefore, a structural complementation assay using NanoBiT technology must be applicable to detect [Ca2+]c increases (Fig. 1A). We developed constructs containing
The calmodulin-binding motifs have different amino acid sequences although they may have similar structures, suggesting that the motifs bind to calmodulin with different affinities. To identify the best calmodulin-binding motif, we further produced vector constructs expressing the NanoBiT fragments fused with the calmodulin-binding motifs of MYLK2 and CAMK2. All assays for this experiment were conducted in HEK293 cells instead of HEK293T cells to reduce receptor expression levels. All NanoBiT constructs were developed in vectors harboring the
To verify the feasibility of this [Ca2+]c assay system, cells expressing calmodulin-SmBiT, LgBiT-MYLK2S, and exogenous receptors that increase [Ca2+]c were treated with cognate ligands of the receptors and then subjected to the luciferase assay. Luciferase activity increased in cells expressing EGFR and GPCRs, which activate the Gαq pathway (e.g., 5-HT2AR, AGTR1, TACR1, and ADRA1A), but not in cells expressing ADRB1, which activates the Gαs pathway (Fig. 3A). These luciferase activities quickly increased in response to their cognate ligands, but the duration of increased activity differed among the receptors. The luciferase activity peaked early and remained elevated for a long time (until 40 min, a maximum detection time) in cells expressing TACR1 and ADAR1A, whereas the activity rapidly declined in cells expressing 5-HT2AR and AGTR1.
These GPCRs also mediate ERK phosphorylation (Gooz et al., 2006; Kanazawa et al., 2015; Rhodes et al., 2009; Wu and O'Connell, 2015). When cells expressing each of the receptors were treated with its cognate ligand for different times, the duration of induced phosphorylation varied depending on the receptors. ADAR1A and TACR1-mediated phosphorylation was detected within 2 min and was sustained for 20 to 30 min. These temporal patterns were similar to the luciferase activity dynamics described above. 5-HT2AR-mediated phosphorylation was detected within 5 min and was sustained for 30 min. By contrast, AGTR1-mediated phosphorylation was detected at 10 min, indicating a slow, weak, short response (Fig. 3B). These results suggest that activation of downstream signaling molecules may depend on receptor activation intensity and individual pathway dynamics.
Enhanced [Ca2+]c triggers SRE-dependent gene transcription through protein kinase C and Ca2+/calmodulin-dependent kinase (CaMK) signaling pathways (Hardingham et al., 1997). ERK phosphorylation also enhances SRE-dependent transcription (Bluthgen et al., 2017; Zhang et al., 2008). We investigated correlations between SRE-dependent transcription and these signaling events (Figs. 3A and 3B) by performing reporter gene assays with cells containing each receptor and SRE-Luc. Stimulation of each receptor with the cognate ligand increased the luciferase activities to varying levels (Fig. 3C). The SRE-dependent transcriptional activities in cells expressing TACR1 and ADAR1A were high enough to reflect strong and sustained luciferase activities in NanoBiT assays and ERK phosphorylation analyses. 5-HT2AR-induced SRE activation appears to be predominantly mediated via the ERK pathway rather than Ca2+-mediated signaling pathway. The weak AGTR1-induced SRE activation correlates with a transient decrease in [Ca2+]c and ERK phosphorylation.
Most assay systems that use heterologous cells expressing exogenous genes utilize CMV promoter-driven gene expression because target gene overexpression results in easily detectable cellular responses. However, the viral CMV promoter potently induces abnormally high gene expression levels, thereby precluding analyses of physiological protein function. Overexpression of G protein-coupled receptors spontaneously stimulates cellular responses without ligands (Wacker et al., 2017). Therefore, optimized expression levels of target genes may be crucial to obtain relevant output from the assay system. To optimize receptor expression levels, cells expressing receptors driven by different promoters and UbiC-driven calmodulin-SmBiT and LgBiT-MYLK2S were treated with their cognate ligands and then subjected to luciferase assay. Transcriptional potency of the promoters was determined by the GFP expression level as follows: CMV >> UbiC > HSV-tk (data not shown). Ligand-stimulated luciferase activities varied depending on the promoter. Ligand-stimulated luciferase activities of all three receptors tested were significantly higher in cells expressing UbiC-driven receptors. Receptors driven by CMV and HSV-tk promoters displayed relatively lower increases in luciferase activity (Figs. 4A and 4B). Basal luciferase activity levels in cells expressing receptor constructs may affect the final ligand-induced fold-increase in luciferase activity, as observed for 5-HT2AR and TACR1 (Fig. 4C). CMV-driven overexpression of 5-HT2AR and TACR1 significantly increased basal luciferase activities. By contrast, the basal activity levels were similar in HEK293 cells expressing AGTR1 under the control of three different promoters. These results suggest that receptor overexpression does not always increase basal luciferase activity. Nevertheless, UbiC-driven receptor expression is likely to produce acceptable levels of basal luciferase activity and efficient cellular responses. Finally, when cells expressing UbiC-driven receptors were treated with different amounts of the ligands, luciferase activities were increased in a dose-dependent manner with a reasonable EC50 value (Fig. 4D). The EC50 did not significantly differ between CMV-driven 5-HT2AR and UbiC-driven 5-HT2AR, but fold-increases in response to different ligand doses were easily distinguished in UbiC-driven 5-HT2AR expression.
As shown above, HEK293 cell transfection efficiency is high enough to detect stimulation-dependent signaling changes even by transfection with three vectors including each of the receptor and two NanoBiT fragment constructs. However, it may be possible to reduce the number of plasmids to develop versatile applications and stable assay systems. Multicistronic expression vectors have been developed using viral gene sequences such as an IRES and a 2A self-cleavage site originated from food-and mouth disease virus (T2A). First, we developed NanoBiT constructs linked by T2A and used them for the [Ca2+]c assay. The SmBiT-fused calmodulin constructs in N-ter or C-ter, which displayed relatively high ligand-induced luciferase activity, were combined with the LgBiT-MYLK2S through T2A linker under control of the UbiC promoter. The basal luciferase activities for both constructs were high compared with that of the three-vector system. The calmodulin-SmBiT construct displayed much higher basal luciferase activities than the SmBiT-Calmodulin (Fig. 5A). When these constructs were expressed along with 5-HT2AR, the slight fold increase of ligand-stimulated luciferase activities appeared due to higher basal luciferase activities (Fig. 5B). Next, the NanoBiT constructs were inserted into a bicistronic expression vector containing IRES. HEK293 cells transfected with the calmodulin-SmBiT-IRES-LgBiT-MYLK2S construct displayed low basal luciferase activities and poor responsiveness for ligand stimulation, which may be due to low expression of IRES-dependent LgBiT-MYLK2S. By contrast, the LgBiT-MYLK2S-IRES-calmodulin-SmBiT construct produced relatively high basal luciferase activities (data not shown). This construct was inserted into the vectors containing different promoters including UbiC, elongation factor (EF), and CMV, and then coexpressed with UbiC-driven GPCRs in HEK293 cells. The resulting basal and ligand-stimulated luciferase activities differed depending on the promoter driving the NanoBiT construct (Figs. 5C and 5D); the EF promoter resulted in the best fold-change in luciferase activity in response to all GPCR ligands compared to other promoter-driven constructs. However, these optimized fold-changes were approximately four times less than those from the three-vector system, although the temporal response patterns were similar. The high basal luciferase activities observed with the EF promoter may explain the lower stimulated fold-change (Fig. 5C), whereas the UbiC-dependent bicistronic construct likely led to very low expression of IRES-dependent calmodulin-SmBiT, which might not be sufficient to represent [Ca2+]c changes. This suggests that EF-driven bicistronic NanoBiT constructs may induce suitable expression in a two-vector system.
The expression levels of all tested receptors affected ligand-induced luciferase activities in the three-vector system. To examine whether this occurs in the two-vector system, the bicistronic NanoBiT construct was expressed along with different promoter-driven GPCRs in HEK293 cells. As observed in the three-vector system, when all receptors were expressed under control of the UbiC promoter, the fold-increase in luciferase activities was much higher than those for other promoters (Figs. 6A and 6B). Basal luciferase activities in the two-vector system were much higher than those in three-vector system, as the EF promoter is stronger than the UbiC promoter (Figs. 4C and 6C). CMV-driven receptor overexpression significantly increased basal luciferase activities, which ultimately may generate a lower ligand-stimulated fold-increase in luciferase activities. When the ligand dose dependency of luciferase activities was determined in cells expressing UbiC-driven GPCRs along with the EF-driven IRES-linked NanoBiT construct, the EC50 values were quite similar to those from three-vector system, although their fold-increases were lower (Fig. 6D). These results suggest that the two-vector system may be useful for developing a stable
Bioluminescence-based
In this work, we introduced a novel design for intensiometric bioluminescent GECI based on NanoBiT technology with greatly improved properties. Nluc can be split into two fragments, LgBiT and SmBiT, and subsequent interaction of their fusion proteins to acquire the enzymatic activity. Increased
Many cell stimulators transduce signals through dynamic [Ca2+]c changes; therefore a
Traditionally, drug studies targeting GPCRs focused on efficacy, potency, and selectivity, whereas analysis of ligand-binding kinetics (e.g., residence time between ligand-receptor interactions) is challenging due to complex signaling transduction pathways (Hothersall et al., 2016; Strasser et al., 2017). It is becoming increasingly apparent that measuring residence time would be useful for drug development (Grundmann and Kostenis, 2017; Hothersall et al., 2016). In the current study, we used four different GPCRs (5-HT2A, AGTR1, TACR1, and ADRA1A) to evaluate the assay system kinetics in reflecting the transient [Ca2+]c increases. Although these GPCRs couple to Gαq, we observed differences in peak magnitudes and signal durations. Upon stimulation, 5-HT2AR and AGTR1 induced a transient rise in [Ca2+]c followed by a quick return to approximate basal, whereas TACR1 and ADRA1A peaked and then attained long-term plateaus that were much higher than basal. The differences in signal duration among the four receptors was confirmed by other assays (i.e., SRE-dependent transcription, ERK1/2 phosphorylation). These differences depend on factors such as the degree of receptor activation, inducer, and receptor preference for specific downstream responses (Charlton and Vauquelin, 2010); however, these data suggest that our
We were not able to demonstrate comparable sensitivity between
Although performing transient transfections is relatively simple and effortless, creating stably expressing clonal cell lines may be more suitable for high-throughput applications (Farhana et al., 2019; Wu et al., 2019). These cell lines would continuously manufacture specific indicators, thereby reducing the number of steps before the assay. However, it may be difficult to establish stable cell lines for the exogenous multigene expression system, also due to transfection efficiency. Here, we used IRES and 2A self-cleavage peptide to construct multicistronic expression vectors containing both LgBiT-MYLK2S and Calmodulin-SmBiT, which yielded promising results for establishing a stable
Although this PPI-based
Usually, luciferase assays have been performed with general luminometer to measure the luciferase activity in cell population. Recent development of new equipment enables to detect the luminescence at single cell level (Yasunaga et al., 2015). Our assay method may apply to detect cytosolic Ca2+ change in individual cell using the equipment.
Taken together, we used NanoBiT technology to develop an innovative
This work was supported by the Korea Research Foundation Grant (NRF-2019R1A2C1090051, NRF-2020M3E5D 9080792) which is funded by the Ministry of Science and ICT, and partly supported by a Korea University Grant.
L.P.N. performed experiments and wrote the manuscript. H.T.N., H.J.Y., A.R.A., and Y.H.N. constructed plasmids. H.K.P. and Y.N.L. performed reporter gene assay. B.J.H., C.S.L., and J.Y.S. reviewed and edited the manuscript. J.I.H. supervised the experiments and wrote the manuscript.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2020; 43(11): 909-920
Published online November 30, 2020 https://doi.org/10.14348/molcells.2020.0144
Copyright © The Korean Society for Molecular and Cellular Biology.
Lan Phuong Nguyen1 , Huong Thi Nguyen1
, Hyo Jeong Yong1
, Arfaxad Reyes-Alcaraz2
, Yoo-Na Lee1
, Hee-Kyung Park1
, Yun Hee Na1
, Cheol Soon Lee1
, Byung-Joo Ham3
, Jae Young Seong1
, and Jong-Ik Hwang1,*
1Department of Biomedical Sciences, Korea University College of Medicine, Seoul 02841, Korea, 2College of Pharmacy, University of Houston, Houston, TX 77204, USA, 3Department of Psychiatry, Korea University College of Medicine, Seoul 02841, Korea
Correspondence to:hjibio@korea.ac.kr
Cytosolic Ca2+ levels ([Ca2+]c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca2+]c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca2+]c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca2+]c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca2+-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca2+]c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca2+]c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca2+]c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca2+]c in single cells and animal models.
Keywords: calmodulin, cytosolic Ca2+ sensor, internal ribosome entry site, myosin light chainC kinase 1/2, NanoBiT assay
Intracellular cytosolic calcium (
In general, there are two main classes of
Genetically encoded indicators are produced by translation of a nucleic acid sequence without the addition of any chemical compounds (Mank and Griesbeck, 2008). GECIs include fluorescent protein (FP)-based sensors detected via Förster resonance energy transfer (FRET), and bioluminescent protein (BP)-based sensors detected via a specific chemical reaction within the cell. BP-GECIs require addition of the luciferase substrate luciferin for the detection reaction, although the resulting acquired signal originates from FPs. The major drawback of FP-GECIs is that they require external illumination, which can damage cells or cause artifacts. By contrast, BP-GECIs are superior for light-sensitive applications such as optogenetics or long-term continuous monitoring (Nagai et al., 2014). Both FP-GECIs and BP-GECIs have poorer SNR than synthetic indicators (Perez Koldenkova and Nagai, 2013). A common approach to improve SNR and brightness of GECIs involves indicator overexpression driven by a strong promoter such as the cytomegalovirus (CMV), and CMV early enhancer/chicken beta actin promoter. However, under some conditions, this overexpression strategy can lead to adverse effects such as cell/tissue damage, abnormal brain activity in indicator-expressing transgenic models, nonfunctional indicators, or perturbations in membrane-gated L-type Ca2+ channels and distorted Ca2+ dynamics (Tian et al., 2009; Yang et al., 2018).
The discovery of the 19.1 kDa luciferase enzyme NanoLuc (Nluc), which is derived from the deep-sea shrimp
Specialized microscope instrumentation is required for [Ca2+]c detection using intensiometric or ratiometric sensors. Ratiometric sensor monitoring of [Ca2+]c change requires equipment that detects rapid changes in wavelength emission, such as confocal or two-photon microscopy, which limits their application. Chemically synthesized indicators can be detected with specialized instruments such as fluorescence imaging plate reader (FLIPR) and the FlexStation microplate reader (Luo et al., 2011; Wu et al., 2019), which are feasible for high-throughput assays for drug discovery, but expensive. Therefore, the development of a novel Ca2+ assay system that uses common laboratory equipment would be invaluable for applications ranging from simple signaling mechanism analysis to high-throughput drug screening.
In this study, we developed a novel intensiometric bioluminescent GECI method using split Nluc without a FP. Our calcium sensor consists of two components as fusion constructs with split Nluc fragments: calmodulin (CaM), and the calmodulin-binding peptide. We used these sensors and luminescence detection instruments to monitor real-time changes in [Ca2+]c in response to extracellular stimulation. We also identified a suitable calmodulin-binding peptide and promoter to optimize expression level of the bioluminescent GECI to efficiently detect changes in [Ca2+]c. Although these assay data were collected from cell populations, our method may be applicable for investigating single-cell [Ca2+]c responses, drug development, and
A NanoBiT starter kit containing all plasmids and reagents for the protein interaction assay was purchased from Promega (USA). The pcDNA3.1 expression vector was purchased from Invitrogen (San Diego, CA, USA). The SRE-Luc vector containing four copies of the serum response element (SRE, CCATATTAGG) was purchased from Stratagene (USA). All primers and reagents for gene cloning and PCR were purchased from Cosmo Genetech (Korea). DNA sequencing was conducted by Macrogen (Korea). Unless otherwise stated, all reagents were purchased from Sigma-Aldrich (USA).
HEK293 and HEK293T cells were obtained from the American Type Culture Collection (ATCC, USA). All cell lines in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) were maintained at 37°C with 5% CO2 unless otherwise stated.
Human cytomegalovirus (CMV) promoter sequence in pcDNA3.1 vector was substituted with promoter sequences of
HEK293 or HEK293T cells were seeded in 96-well plates at a cell density of 2 × 104 cells/well. The next day, 30 ng of receptor plasmid, 30 ng of plasmid containing calmodulin tagged with LgBiT or SmBiT, and 30 ng of plasmid containing calmodulin-binding region fused to SmBiT or LgBiT in the N-terminal or C-terminal region were mixed with 0.2 μl Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and added to the plated cells. Subsequent transfection steps were performed according to the manufacturer’s instructions. After 24 h, media was replaced with 100 μl of Opti-MEM and cells were stabilized for 10 min at room temperature before measuring the luminescence. Then, 25 μl of Nano-Glo Live Cell Reagent (furimazine) was added to each well, and basal luminescence was measured using a luminometer (BioTek, Winooski, VT, USA) for the first 10 min. Finally, cells were stimulated by adding 10 μl of appropriate agonists to each well, and the cell plate luminescence was measured for 30 min. The schematics of all NanoBiT constructs and their combinations, and all graphs in dosing experiments were presented in supplementary information (Supplementary Figs. S1-S9). The absolute values of luciferase activities were also shown in it (Supplementary Table S1).
HEK293 cells in 6-well plates were transfected with 2 μg pcDNA3.1 vector harboring the receptor genes using 4 μl Lipofectamine 2000. The next day, cells were incubated with serum-free media overnight, and then treated with their cognate ligand for the indicated time. After washing with cold phosphate-buffered saline, the cells were harvested with lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM NaF, 1% Triton X-100, and protease inhibitors) and the protein extracts were clarified by centrifugation at 15,000 rpm for 15 min at 4°C. Then, 20 μg of cellular proteins were examined using SDS-PAGE followed by western blotting with anti-pERK or anti-ERK antibodies.
HEK293 cells were seeded in 24-well plates at a density of 8 × 104 cells/well. The next day, a mixture of 400 ng of receptor gene–containing plasmids, 50 ng of SRE-Luc reporter gene plasmids, and 1 μl of Lipofectamine 2000 was added per well according to the manufacturer’s instructions. The next day, cells were maintained in serum-free DMEM overnight. Approximately 36 h after the start of transfection, cells were treated with appropriate ligands for 6 h. Cells were then lysed using 100 µl of lysis buffer, and the luciferase activity of the cell extract was measured using a luciferase assay and the standard protocol for the Synergy 2 Multi-Mode Microplate Reader (BioTek).
Statistical differences among experimental groups were analyzed by unpaired Student’s
Ca2+-bound calmodulin can bind various effector molecules via distinct amino acid sequence motifs. Therefore, a structural complementation assay using NanoBiT technology must be applicable to detect [Ca2+]c increases (Fig. 1A). We developed constructs containing
The calmodulin-binding motifs have different amino acid sequences although they may have similar structures, suggesting that the motifs bind to calmodulin with different affinities. To identify the best calmodulin-binding motif, we further produced vector constructs expressing the NanoBiT fragments fused with the calmodulin-binding motifs of MYLK2 and CAMK2. All assays for this experiment were conducted in HEK293 cells instead of HEK293T cells to reduce receptor expression levels. All NanoBiT constructs were developed in vectors harboring the
To verify the feasibility of this [Ca2+]c assay system, cells expressing calmodulin-SmBiT, LgBiT-MYLK2S, and exogenous receptors that increase [Ca2+]c were treated with cognate ligands of the receptors and then subjected to the luciferase assay. Luciferase activity increased in cells expressing EGFR and GPCRs, which activate the Gαq pathway (e.g., 5-HT2AR, AGTR1, TACR1, and ADRA1A), but not in cells expressing ADRB1, which activates the Gαs pathway (Fig. 3A). These luciferase activities quickly increased in response to their cognate ligands, but the duration of increased activity differed among the receptors. The luciferase activity peaked early and remained elevated for a long time (until 40 min, a maximum detection time) in cells expressing TACR1 and ADAR1A, whereas the activity rapidly declined in cells expressing 5-HT2AR and AGTR1.
These GPCRs also mediate ERK phosphorylation (Gooz et al., 2006; Kanazawa et al., 2015; Rhodes et al., 2009; Wu and O'Connell, 2015). When cells expressing each of the receptors were treated with its cognate ligand for different times, the duration of induced phosphorylation varied depending on the receptors. ADAR1A and TACR1-mediated phosphorylation was detected within 2 min and was sustained for 20 to 30 min. These temporal patterns were similar to the luciferase activity dynamics described above. 5-HT2AR-mediated phosphorylation was detected within 5 min and was sustained for 30 min. By contrast, AGTR1-mediated phosphorylation was detected at 10 min, indicating a slow, weak, short response (Fig. 3B). These results suggest that activation of downstream signaling molecules may depend on receptor activation intensity and individual pathway dynamics.
Enhanced [Ca2+]c triggers SRE-dependent gene transcription through protein kinase C and Ca2+/calmodulin-dependent kinase (CaMK) signaling pathways (Hardingham et al., 1997). ERK phosphorylation also enhances SRE-dependent transcription (Bluthgen et al., 2017; Zhang et al., 2008). We investigated correlations between SRE-dependent transcription and these signaling events (Figs. 3A and 3B) by performing reporter gene assays with cells containing each receptor and SRE-Luc. Stimulation of each receptor with the cognate ligand increased the luciferase activities to varying levels (Fig. 3C). The SRE-dependent transcriptional activities in cells expressing TACR1 and ADAR1A were high enough to reflect strong and sustained luciferase activities in NanoBiT assays and ERK phosphorylation analyses. 5-HT2AR-induced SRE activation appears to be predominantly mediated via the ERK pathway rather than Ca2+-mediated signaling pathway. The weak AGTR1-induced SRE activation correlates with a transient decrease in [Ca2+]c and ERK phosphorylation.
Most assay systems that use heterologous cells expressing exogenous genes utilize CMV promoter-driven gene expression because target gene overexpression results in easily detectable cellular responses. However, the viral CMV promoter potently induces abnormally high gene expression levels, thereby precluding analyses of physiological protein function. Overexpression of G protein-coupled receptors spontaneously stimulates cellular responses without ligands (Wacker et al., 2017). Therefore, optimized expression levels of target genes may be crucial to obtain relevant output from the assay system. To optimize receptor expression levels, cells expressing receptors driven by different promoters and UbiC-driven calmodulin-SmBiT and LgBiT-MYLK2S were treated with their cognate ligands and then subjected to luciferase assay. Transcriptional potency of the promoters was determined by the GFP expression level as follows: CMV >> UbiC > HSV-tk (data not shown). Ligand-stimulated luciferase activities varied depending on the promoter. Ligand-stimulated luciferase activities of all three receptors tested were significantly higher in cells expressing UbiC-driven receptors. Receptors driven by CMV and HSV-tk promoters displayed relatively lower increases in luciferase activity (Figs. 4A and 4B). Basal luciferase activity levels in cells expressing receptor constructs may affect the final ligand-induced fold-increase in luciferase activity, as observed for 5-HT2AR and TACR1 (Fig. 4C). CMV-driven overexpression of 5-HT2AR and TACR1 significantly increased basal luciferase activities. By contrast, the basal activity levels were similar in HEK293 cells expressing AGTR1 under the control of three different promoters. These results suggest that receptor overexpression does not always increase basal luciferase activity. Nevertheless, UbiC-driven receptor expression is likely to produce acceptable levels of basal luciferase activity and efficient cellular responses. Finally, when cells expressing UbiC-driven receptors were treated with different amounts of the ligands, luciferase activities were increased in a dose-dependent manner with a reasonable EC50 value (Fig. 4D). The EC50 did not significantly differ between CMV-driven 5-HT2AR and UbiC-driven 5-HT2AR, but fold-increases in response to different ligand doses were easily distinguished in UbiC-driven 5-HT2AR expression.
As shown above, HEK293 cell transfection efficiency is high enough to detect stimulation-dependent signaling changes even by transfection with three vectors including each of the receptor and two NanoBiT fragment constructs. However, it may be possible to reduce the number of plasmids to develop versatile applications and stable assay systems. Multicistronic expression vectors have been developed using viral gene sequences such as an IRES and a 2A self-cleavage site originated from food-and mouth disease virus (T2A). First, we developed NanoBiT constructs linked by T2A and used them for the [Ca2+]c assay. The SmBiT-fused calmodulin constructs in N-ter or C-ter, which displayed relatively high ligand-induced luciferase activity, were combined with the LgBiT-MYLK2S through T2A linker under control of the UbiC promoter. The basal luciferase activities for both constructs were high compared with that of the three-vector system. The calmodulin-SmBiT construct displayed much higher basal luciferase activities than the SmBiT-Calmodulin (Fig. 5A). When these constructs were expressed along with 5-HT2AR, the slight fold increase of ligand-stimulated luciferase activities appeared due to higher basal luciferase activities (Fig. 5B). Next, the NanoBiT constructs were inserted into a bicistronic expression vector containing IRES. HEK293 cells transfected with the calmodulin-SmBiT-IRES-LgBiT-MYLK2S construct displayed low basal luciferase activities and poor responsiveness for ligand stimulation, which may be due to low expression of IRES-dependent LgBiT-MYLK2S. By contrast, the LgBiT-MYLK2S-IRES-calmodulin-SmBiT construct produced relatively high basal luciferase activities (data not shown). This construct was inserted into the vectors containing different promoters including UbiC, elongation factor (EF), and CMV, and then coexpressed with UbiC-driven GPCRs in HEK293 cells. The resulting basal and ligand-stimulated luciferase activities differed depending on the promoter driving the NanoBiT construct (Figs. 5C and 5D); the EF promoter resulted in the best fold-change in luciferase activity in response to all GPCR ligands compared to other promoter-driven constructs. However, these optimized fold-changes were approximately four times less than those from the three-vector system, although the temporal response patterns were similar. The high basal luciferase activities observed with the EF promoter may explain the lower stimulated fold-change (Fig. 5C), whereas the UbiC-dependent bicistronic construct likely led to very low expression of IRES-dependent calmodulin-SmBiT, which might not be sufficient to represent [Ca2+]c changes. This suggests that EF-driven bicistronic NanoBiT constructs may induce suitable expression in a two-vector system.
The expression levels of all tested receptors affected ligand-induced luciferase activities in the three-vector system. To examine whether this occurs in the two-vector system, the bicistronic NanoBiT construct was expressed along with different promoter-driven GPCRs in HEK293 cells. As observed in the three-vector system, when all receptors were expressed under control of the UbiC promoter, the fold-increase in luciferase activities was much higher than those for other promoters (Figs. 6A and 6B). Basal luciferase activities in the two-vector system were much higher than those in three-vector system, as the EF promoter is stronger than the UbiC promoter (Figs. 4C and 6C). CMV-driven receptor overexpression significantly increased basal luciferase activities, which ultimately may generate a lower ligand-stimulated fold-increase in luciferase activities. When the ligand dose dependency of luciferase activities was determined in cells expressing UbiC-driven GPCRs along with the EF-driven IRES-linked NanoBiT construct, the EC50 values were quite similar to those from three-vector system, although their fold-increases were lower (Fig. 6D). These results suggest that the two-vector system may be useful for developing a stable
Bioluminescence-based
In this work, we introduced a novel design for intensiometric bioluminescent GECI based on NanoBiT technology with greatly improved properties. Nluc can be split into two fragments, LgBiT and SmBiT, and subsequent interaction of their fusion proteins to acquire the enzymatic activity. Increased
Many cell stimulators transduce signals through dynamic [Ca2+]c changes; therefore a
Traditionally, drug studies targeting GPCRs focused on efficacy, potency, and selectivity, whereas analysis of ligand-binding kinetics (e.g., residence time between ligand-receptor interactions) is challenging due to complex signaling transduction pathways (Hothersall et al., 2016; Strasser et al., 2017). It is becoming increasingly apparent that measuring residence time would be useful for drug development (Grundmann and Kostenis, 2017; Hothersall et al., 2016). In the current study, we used four different GPCRs (5-HT2A, AGTR1, TACR1, and ADRA1A) to evaluate the assay system kinetics in reflecting the transient [Ca2+]c increases. Although these GPCRs couple to Gαq, we observed differences in peak magnitudes and signal durations. Upon stimulation, 5-HT2AR and AGTR1 induced a transient rise in [Ca2+]c followed by a quick return to approximate basal, whereas TACR1 and ADRA1A peaked and then attained long-term plateaus that were much higher than basal. The differences in signal duration among the four receptors was confirmed by other assays (i.e., SRE-dependent transcription, ERK1/2 phosphorylation). These differences depend on factors such as the degree of receptor activation, inducer, and receptor preference for specific downstream responses (Charlton and Vauquelin, 2010); however, these data suggest that our
We were not able to demonstrate comparable sensitivity between
Although performing transient transfections is relatively simple and effortless, creating stably expressing clonal cell lines may be more suitable for high-throughput applications (Farhana et al., 2019; Wu et al., 2019). These cell lines would continuously manufacture specific indicators, thereby reducing the number of steps before the assay. However, it may be difficult to establish stable cell lines for the exogenous multigene expression system, also due to transfection efficiency. Here, we used IRES and 2A self-cleavage peptide to construct multicistronic expression vectors containing both LgBiT-MYLK2S and Calmodulin-SmBiT, which yielded promising results for establishing a stable
Although this PPI-based
Usually, luciferase assays have been performed with general luminometer to measure the luciferase activity in cell population. Recent development of new equipment enables to detect the luminescence at single cell level (Yasunaga et al., 2015). Our assay method may apply to detect cytosolic Ca2+ change in individual cell using the equipment.
Taken together, we used NanoBiT technology to develop an innovative
This work was supported by the Korea Research Foundation Grant (NRF-2019R1A2C1090051, NRF-2020M3E5D 9080792) which is funded by the Ministry of Science and ICT, and partly supported by a Korea University Grant.
L.P.N. performed experiments and wrote the manuscript. H.T.N., H.J.Y., A.R.A., and Y.H.N. constructed plasmids. H.K.P. and Y.N.L. performed reporter gene assay. B.J.H., C.S.L., and J.Y.S. reviewed and edited the manuscript. J.I.H. supervised the experiments and wrote the manuscript.
The authors have no potential conflicts of interest to disclose.
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