A genetically encoded ratiometric calcium sensor enables quantitative measurement of the local calcium microdomain in the endoplasmic reticulum
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The local Ca2+ release from the heterogeneously distributed endoplasmic reticulum (ER) calcium store has a critical role in calcium homeostasis and cellular function. However, single fluorescent protein-based ER calcium probes experience challenges in quantifying the ER calcium store in differing live cells, and intensity-based measurements make it difficult to detect local calcium microdomains in the ER. Here, we developed a genetically encoded ratiometric ER calcium indicator (GCEPIA1-SNAPER) that can detect the real-time ER calcium store and local calcium microdomains in live cells. GCEPIA1-SNAPER was located in the lumen of the ER and showed a linear, reversible and rapid response to changes in the ER calcium store. The GCEPIA1-SNAPER probe effectively monitored the depletion of the ER calcium store by TG or starvation treatment, and through its use we identified heterogeneously distributed calcium microdomains in the ER which were correlated with the distribution of STIM1 clusters upon ER calcium store depletion. Lastly, GCEPIA1-SNAPER can be used to detect the ER calcium store by high-throughput flow cytometry and confers the ability to study the function of calcium microdomains of the ER.
KeywordsRatiometric calcium sensor Local calcium microdomain Endoplasmic reticulum
The calcium stores in the endoplasmic/sarcoplasmic reticulum (ER) are heterogeneously distributed in most cells because of the uneven distribution of ER Ca2+-binding proteins and immobile Ca2+-buffered solutions. Numerous protein processes may be affected by the nonuniform distribution of [Ca2+]ER; for example, Ca2+-sensitive chaperones such as the lectin-like proteins calnexin and calreticulin (Carreras-Sureda et al. 2017; Clapham 2007). Ca2+ acts as a vital second messenger responding to a variety of environmental stimuli, and thus the heterogeneous release of Ca2+ from different parts of the ER undoubtedly could affect Ca2+-dependent processes including store-operated Ca2+ entry (SOCE), fusion of autophagosomes, cell motility or calcium transfer from the ER to mitochondria in the mitochondria-associated membrane (Ghislat and Knecht 2012; Rizzuto et al. 1998). Thus, it is important to monitor the dynamic change of calcium stores in these microdomains in living cells.
GCaMP proteins are widely used to image the change in intracellular calcium concentration of cells under different physiological stimuli. Subcellular-located GCaMPs were used to measure changes in the calcium concentration of different organelles. Currently, many probes based on GCaMPs have been developed for the measurement of ER calcium in live cells, and even in animals (Akerboom et al. 2012; Mao et al. 2008; Shigetomi et al. 2010; Tian et al. 2009). Usually, the measurements of ER calcium content are based on the fluorescence intensity of probes. However, the intensity-based measurements of the ER calcium store have several limitations. First, the fluorescence intensity is dependent on the expression levels of ER calcium probes, which differs among cells. Therefore, it is difficult to make cell-to-cell comparisons of the ER calcium store when studying a gene function and enormous amounts of data should be collected to minimize the variation. Second, the fluorescence intensity is also dependent on the local density of the ER calcium probe, which is determined by the local ER structures such as ER sheets. Thus, the intensity-based measurements face difficulty in precisely quantifying the calcium of microdomains. Third, time course quantification of the ER calcium store in live cells is challenging with intensity-based measurements, since the ER is highly dynamic, and the recorded calcium signal could be affected by defocus caused by the movement of the ER. Hence, it is confusing to judge whether the changed fluorescence signal reflects a change of true signal or simply defocus. Finally, imaging methods with intensity-based probes may be incapable of reliably quantifying and monitoring dynamic changes of local calcium in the ER domains of live cells.
All the above limitations could be overcome by normalizing probe expression levels with a ratiometric ER-located calcium sensor. Zhao et al. developed ratiometric calcium probes with excitation and emission at distinct wavelengths in the Ca2+-free and Ca2+-bound states (Zhao et al. 2011). After that, Suzuki et al. optimized the blue-green emission ratiometric GECO and developed GEM-CEPIA1ER with a low Ca2+ affinity and a large dynamic range for imaging ER calcium (Suzuki et al. 2014). However, there is a lethal weakness that the excited wavelength is around 400 nm, which has severe phototoxicity to cells and is not suitable for live cell imaging (Icha et al. 2017; Wäldchen et al. 2015). Furthermore, the cytosolic GCaMP-based ratiometric probes, generally fused with red fluorescent proteins (FPs) such as mCherry (Cho et al. 2017), although avoiding the phototoxic effects of excitation light on cells, experience inherent disadvantages due to the aggregation property and slow maturation rate of mCherry. In addition, Förster resonance energy transfer (FRET) will decrease calcium signal and cannot be entirely avoided because of the spectral overlap between GCaMP and mCherry, despite a series of strategies adopted to reduce FRET (e.g., inserting rigid linkers into GCaMP and mCherry) (Cho et al. 2017).
In the current study, we developed a ratiometric ER-located calcium sensor, GCEPIA1-SNAPER, by fusing GCEPIA1ER, a mutant of GGaMP, with a SNAP-tag to dynamically quantify local calcium of ER microdomains in live cells. The SNAP-tag protein undergoes an irreversible self-labeling reaction to form a covalent bond with O6-benzylguanine (BG) derivatives, which can be modified with a variety of reporter molecules such as fluorophores, peptides, or oligonucleotides (Cole 2013). In this study, we used SNAP-Cell® 647-SiR to covalently couple SNAP-tag for two considerations. First, the far-infrared SNAP-647 uses 647 nm for illumination, which has less phototoxic than 405 nm illumination used for GEM-CEPIA1er when imaging live cells. Second, the emission spectrum of SNAP-647 is separated from GCEPIAER and there is no FRET between GEM-CEPIA1ER and SNAP-647 that would affect calcium detection. Because the SNAP-tag is co-expressed in-frame with GCEPIA, and the fluorescence intensity of SNAP-647 does not change in different calcium concentrations, the SNAP-tag can be used for ratiometric measurement to indicate the location and expression level of GCEPIA. This tandem constructed probe facilitates pixel-wise ratiometric measurements of the calcium instead of fluorescence intensity alone. The local ER microdomains could be detected precisely by the ratiometric-based method.
Characteristics of ER-located ratiometric calcium sensor GCEPIA1-SNAPER
Application of GCEPIA1-SNAPER to detect the whole-cell ER calcium store
To prove that GCEPIA1-SNAPER can monitor the dynamic ER calcium store, we treated cells with thapsigargin (TG), the most widely used noncompetitive inhibitor of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), to deplete the ER calcium store; we then measured ER calcium at different time points. Laser scanning confocal microscope images at different time points after TG stimulation showed that the green GCEPIA1 fluorescence was decreased gradually while the red SNAP-dye fluorescence was decreased slightly due to photo-bleaching (Fig. 2A–C).
The heterogeneous distribution of local ER calcium store
Detection of local ER calcium microdomains by GCEPIA1-SNAPER
Relationship between local calcium microdomains and distribution of STIM1 cluster
The content of the ER calcium store decreased under starvation
The ER calcium store is precisely regulated by different types of calcium channels and pumps: inositol trisphosphate receptor (IP3R) binds to IP3 and allows calcium to exit the ER, while the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump transports Ca2+ from the cytosol to refill the ER in an ATP-consuming fashion (Stutzmann 2005). For the reason that the SERCA pump calcium transport requires much energy, we proposed that the activity of the SERCA will be decreased under starvation and lead to the reduction of the ER calcium store.
GCAMPIA1-SNAPER for high-throughput detection of ER calcium content by flow cytometry
In the current study, we developed a new ratiometric ER calcium probe GCEPIA1-SNAPER. Compared to currently used fluorescence intensity-based ER calcium probes, GCEPIA1-SNAPER provides several advantages.
First, GCEPIA1-SNAPER has the ability to measure the ER calcium store more accurately, and it can measure the basal ER calcium store. Previously, the fluorescence intensity was normalized by dividing the average of the total fluorescence values (F0) for each cell at resting state, and the change of ER calcium store was calculated as ∆F/F0 = (Fn − F0)/F0, where Fn is the total fluorescence value for each cell at different time points (Fig. 2D) (Koldenkova and Nagai 2013; Suzuki et al. 2014). However, besides the expression level of the probe, the resting fluorescence F0 is also affected by the dynamic exchange between cellular and extracellular calcium concentrations; thus, normalized fluorescence intensity would also eliminate the information of the initial calcium concentration at resting state. Moreover, the major limitation of the intensity-based method is the dependence on drug stimulation to empty the ER calcium—the method cannot be used to directly monitor the basal ER calcium store at the resting state without TG stimulation. Differently from the currently used fluorescence protein intensity-based ER calcium sensor, our ratiometric probe GCEPIA1-SNAPER utilizes a SNAP-tag for direct calibration of probe expression and can be used for measuring the basal ER calcium store. Additionally, ratiometric GCEPIA1-SNAPER can calibrate the deviations caused by defocusing of the focal plane, transfection/translation efficiency differences, dynamic movement of the ER, and others. Furthermore, compared to two-fluorescence protein dual reporter systems, GCEPIA1-SNAPER minimizes the problem of maturation because the chromophore of the Alexa 647 dye has already been formed before binding to the SNAP-tag.
Second, GCEPIA1-SNAPER enables simultaneous detection of the ER calcium store and other cellular dynamics in one live cell. Due to the influence of the variant expression level of probe, early methods for measuring and comparing ER calcium stores of different cells cannot directly read the calcium store signal, which must be calculated by measuring the reduction of calcium fluorescence signal after completely depleting the ER with TG. Since TG affects the function of cells, it is not possible to simultaneously detect the ER calcium store and other functions in one live cell. In the current study, since GCEPIA1-SNAPER is capable of calibrating the difference in expression amount in real time and reporting the entire ER calcium content, GCEPIA1-SNAPER enables simultaneous detection of the ER calcium store and other cellular dynamics in one live cell. Furthermore, GCEPIA1-SNAPER can be easily labeled with specific BG dyes with separate emission spectra, which enables multicolor imaging of the ER calcium store and other biological processes.
Third, GCEPIA1-SNAPER enables dynamic detection of the local ER calcium microdomains in live cells. Using fluorescence intensity-based ER calcium probes, the local ER calcium cannot be normalized by local F0, as ER is highly dynamic and, accordingly, local probe density changes quickly. Thus, it is impossible that fluorescence intensity-based ER calcium probes can be used for detecting local calcium microdomains. In our study, the tandem fusion of GCEPIA1ER with a SNAP-tag assures two proteins localized in the same pixel, which makes it feasible to perform local calibration and monitor the real-time ER calcium microdomains. Moreover, the formation process of dynamic clusters of STIM1 has been successfully imaged simultaneously with local ER calcium store in live cells in the current study.
The pAAV-H1-GCEPIA1ER plasmid is a gift from Dr. Liangyi Chen at Peking University. SNAP-tag was fused in frame with GCEPIA1 containing the ER retention signal sequence (KDEL) using primers 1–4 (Supplementary Table S2).
For prokaryote expression of GCEPIA1-SNAPER and GCEPIA1ER, we cloned GCEPIA1-SNAPER or GCEPIA1ER into the pRset (A) vector using primers 55 s, 55a, 56 s, and 61 s with the template of pAAV-H1-GCEPIA1-SNAPER or pAAV-H1-GCEPIA1ER.
BL21 (DE3) competent E. coli cells (TransGen Biotech, China) were transfected with plasmids and incubated on LB Agar ampicillin (100 μg/mL) plates for 12 h at 37 °C. Single colonies were separately inoculated in 10 mL of LB broth medium and were left overnight at 37 °C with moderate shaking and then induced with IPTG (0.8 mg/mL) for 16–24 h at 16 °C for target protein expression. The cells were resuspended in binding buffer (50 mmol/L Tris, 500 mmol/L NaCl, 10 mmol/L imidazole). After cell lysis with sonication, recombinant proteins were purified using a Ni–NTA His-Bind resin (Qiagen) followed by a gel filtration step with the Superdex 200 increase 10/300 GL column (GE Healthcare, Sweden). For further analysis, the purified proteins were concentrated via ultrafiltration and then resuspended in KCl/MOPS buffer (130 mmol/L KCl, 50 mmol/L MOPS, pH 7.2).
In vitro spectroscopic determination
Ca2+ titration curves of GCEPIA1ER and GCEPIA1-SNAPER were obtained by adding small aliquots of CaCl2 to probes in a KCl/MOPS buffer containing 0 or 1 mmol/L EGTA at pH 7.2. The free calcium concentration was calculated using MaxChelator Version 1.2. Fluorescence intensity was measured with a spectrophotometer (F7000, HITACHI, Japan) for protein at 488 nm excitation/512 nm emission wavelengths. The obtained Ca2+-fluorescence intensity relationship was fitted by the Hill plot equation: F = Fmin + (Fmax − Fmin) × ([Ca2+]free)n/[([Ca2+]free)n + (Kd)n]. Kd denotes dissociation constant, which also means the calcium concentration with half of the probe binding to Ca2+. The n represents the Hill coefficient. Fmin refers to the value of emission peak of the lowest calcium concentration, and Fmax refers to the value of emission peak of the highest calcium concentration. The dynamic range of the indicator was calculated as the ratio of maximum and minimum fluorescence intensity (Fmax/Fmin). The absorbance spectra were measured by a spectrophotometer (VARIOSKAN FLASH, Thermo Fisher Scientific, USA) for each protein in KCl/MOPS buffer with 1 mmol/L EGTA or with 5 mmol/L CaCl2.
COS7 cells and MEF cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (HyClone, Logan, UT) in an incubator at 37 °C with 5% CO2. For Ca2+ imaging, the cells were plated on 35 mm glass-bottom dishes (D35-10-1-N, Cellvis).
SNAP-Cell® 647-SiR was purchased from New England Biolabs (NEB) and cell labeling was performed according to the manufacturer’s instructions. Briefly, the cells were transfected with GCEPIA1-SNAPER plasmid. After 24 h, the cells were incubated with diluted SNAP-Cell® 647-SiR (25 nmol/L) labeling solution and incubate at 37 °C, 5% CO2 for 30 min.
MEF cells were collected and homogenized using a Dounce Tissue Grinder (Thermo Fisher) in ice-cold homogenized buffer (20 mmol/L HEPES, 250 mmol/L sucrose, 1 mmol/L EDTA, pH 7.4) containing protease inhibitor cocktail (Sigma). The homogenates were centrifuged at 1000 g for 10 min at 4 °C. Then, the supernatant was collected and centrifuged at 8000 g for 20 min at 4 °C. The supernatant was ultracentrifuged at 100,000 g for 1 h at 4 °C. The precipitate was collected and dissolved in homogenization buffer to obtain microsomes.
ATPase activity measurement
ATPase activities of MEF cells were measured using a colorimetric ATPase assay kit according to the manufacturer’s protocol (Innova Biosciences, Cambridge, UK). Protein concentration was analyzed using the BCA assay (Thermo Scientific, Waltham, MA), and the ATPase activity was normalized by protein concentration.
ER calcium store imaging
Cells were incubated with 5 μmol/L Fura-2/AM (DOJINDO, Japan) in KREB buffer (115 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4·7H2O, 1.2 mmol/L KH2PO4, 25 mmol/L NaHCO3, 10 mmol/L HEPES, 2.5 mmol/L Ca2+) with or without 25 mmol/L d-Glucose and 0.1% BSA at room temperature for 30 min. For the starvation group, the cells were treated with Hanks buffer for 2 h followed by Fura-2/AM incubation. The culture medium was replaced with 0 mmol/L Ca2+ KREB buffer followed by 2 μmol/L TG treatment. Images were captured using an inverted microscope (IX81, Olympus, Japan) equipped with a 40× oil objective (numerical aperture (NA) = 1.3, UPlanSApo, Olympus) and Zyla scientific complementary metal-oxide semiconductor (sCMOS) camera (ANDOR, Britain) at a rate of one frame per 800 ms. The calcium concentration was represented by the ratio of 340/380 of Fura-2/AM.
ER Ca2+ dynamic imaging
Cells were transfected with GCEPIA1ER or GCEPIA1-SNAPER using Lipofectamine 2000 (Invitrogen), and images were captured at a rate of one frame per 1 to 2 s using an FV1200 confocal microscope (OLYMPUS, Japan) equipped with a 60× oil objective lens (NA = 1.35, UPlanSApo, Olympus). To display Ca2+ content at different time points or areas of the z-axis, a linear region of interest (ROI) over the time or z-axis area was plotted.
GCEPIA1-SNAPER was transfected into MEF cells with Lipofectamine 2000 (Invitrogen). After 2 days, the cells were cultured in DMEM with or without 2 μmol/L TG treatment for 1 h, followed by incubation with 25 nmol/L SNAP-647 (NEB, USA) in an incubator at 37 °C with 5% CO2 for 30 min. And, finally, sorting by Flow Cytometry (FACSAriaIIIu, BD, USA). Data were acquired and analyzed on the FACSAriaIIIu with DIVA software (BD, USA).
Data analysis was performed using OriginPro 8.1 software (OriginLab, USA). The unpaired t test was used to determine the differences between nonstarvation and starvation groups. The values are presented as the mean ± SD. The significant difference was set at P < 0.05.
This project was supported by the National Key R&D Program of China (2016YFA0501500), the National Natural Science Foundation of China (31421002, 31401174 and 21778069), and the Project of the Chinese Academy of Sciences (XDB08030203). We thank Mr. Junying Jia from the Institute of Biophysics, Chinese Academy of Science for his help in FACS data collection.
Compliance with Ethical Standards
Conflict of interest
Chen Luo, HuiyuWang, Qi Liu, Wenting He, Lin Yuan and Pingyong Xu declare that they have no conflict of interest.
Human and animal rights and informed consent
This article does not contain any studies with human or animal subjects performed by the any of the authors.
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