We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.
Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.
The spatio-temporal attributes of intracellular calcium (Ca2+) transients activate various biological functions1. These Ca2+ signaling events are triggered extracellularly through different stimuli and controlled intracellularly by the major Ca2+ storage organelle and by numerous Ca2+ pumps, channels, and Ca2+ binding proteins. Ca2+ transients can be significantly altered as a result of defects with signal modulation, leading to different diseases2. Because of the speed and intricacy of the Ca2+ signaling system, with the endo- (ER) and sarcoplasmic reticulum (SR) at the center, genetically-encoded Ca2+ probes that have been optimized for mammalian expression with fast kinetics are needed to observe global and local Ca2+ changes in different cells3.
The ER and the SR, its counterpart in muscle cells, are the major intracellular Ca2+ storage organelles and act as Ca2+ sinks that help to amplify the Ca2+ signal4. The ER/SR is an integral part in Ca2+ signaling with dual roles as a transmitter and receiver of signals5. The ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) are Ca2+ release receptors located on the membranes of the ER/ SR that are regulated by Ca2+ 6. Other agents directly or indirectly stimulate the function of these receptors. 4-chloro-m-cresol (4-cmc) is a potent agonist of the RyR, having a 10 fold higher sensitivity than caffeine for inducing SR Ca2+ release where both are regularly employed to study RyR-mediated Ca2+ release in healthy and diseased cells7. ATP increases IP3-mediated Ca2+ release through the IP3R8. ATP binds to the purinergic receptor P2YR, a G-protein coupled receptor (GPCR), triggering the production of IP3 that binds to the IP3R to release Ca2+ from the ER9,10. The sarco-endoplasmic reticulum calcium ATPase (SERCA) pump is a P-type ATPase pump, also located on the ER/SR membrane that reduces cytosolic Ca2+ and refills the ER/SR by actively pumping the ion into the ER/SR lumen11. Specific inhibitors of the SERCA pump include thapsigargin, from Thapsia garganica, and cyclopiazonic acid (CPA), from Aspergillus and Penicillium. CPA has a low affinity for the pump and reversibly blocks the Ca2+ access point12. Thapsigargin, on the other hand, irreversibly binds to the Ca2+ free pump at residue F256 in the M3 helix with nanomolar affinity11. Analyzing and quantifying the changes involved in Ca2+ stimulated events has been and remains a challenge. Since the ER/SR is the major subcellular Ca2+ containing compartment with a central function in the propagation of the Ca2+ signal, much work has been focused on understanding ER/SR Ca2+ signaling5.
The creation of synthetic Ca2+ dyes helped to advance the field and practice of Ca2+ imaging. Although dyes, such as Mag-Fura-2, have been widely used to measure compartmentalized Ca2+ in different cells,13,14,15 they have limitations such as uneven dye loading, photobleaching, and the inability to be targeted to specific organelles. The discovery of the green fluorescent protein (GFP) and the advancement of fluorescent protein-based Ca2+ probes has propelled the field of Ca2+ imaging forward16. Some of the existing GECIs are Förster resonance energy transfer (FRET) pairs involving yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), calmodulin and the M13 binding peptide17,18. Troponin C-based GECIs are also available as FRET pairs of CFP and Citrine and as single fluorophore probes19,20,21. Others, such as GCaMP2 and R-GECO are single fluorophore sensors involving calmodulin22,23. To overcome the limitations of narrow tuning of Kd's and cooperative binding associated with multiple Ca2+ binding sites found in their Ca2+ binding domains24, a novel class of calcium sensors was created by designing a Ca2+ binding site on the surface of the beta barrel in a chromophore sensitive location of enhanced green fluorescent protein (EGFP)25,26. This highly touted sensor, called CatchER, has a Kd of ~0.18 mM, a kon near the diffusion limit, and a koff of 700 s-1. CatchER has been used to monitor receptor-mediated ER/SR calcium release in different mammalian cell lines such as HeLa, HEK293, and C2C1225. Because of its fast kinetics, CatchER was used in flexor digitorum brevis (FDB) muscle fibers of young and old Friend Virus B NIH Jackson (FVB) mice to reveal that more Ca2+ remains in the SR after 2 s of depolarization in the FDB fibers of old mice compared to that of young mice27. To overcome its low fluorescence at 37 °C, which hinders its applications in calcium imaging of mammalian cells, we have developed an improved version of CatchER called CatchER+. CatchER+ exhibits enhanced fluorescence at 37 °C for better application in mammalian cells. Additional mutations were incorporated into CatchER to improve the thermostability and fluorescence at 37 °C28,29, to create CatchER+. CatchER+ exhibits a six-fold increase in its signal to noise ratio (SNR) over CatchER30.
Here, the protocols for the culture and transfection of HEK293 and C2C12 cells with CatchER+ and its application for monitoring ER/SR receptor-mediated calcium transients are presented. Representative results are shown for CatchER+ expressed in HEK293 cells treated with 4-cmc, CPA and ATP. We also provide a protocol for determining the in situ Kd of CatchER+ in C2C12 myoblast cells and quantification of basal [Ca2+].
1. Slide Preparation
2. Preparation of Media, Buffers, Solutions, and Reagents
3. Cell Culture
4. Transfection of HEK293 Cells
5. Preparation of Slide and Fluorescence Microscope
6. Imaging Drug-induced Ca2+ Transients and In Situ Kd Calibration
7. Data Processing
This section will illustrate the results that were achieved using the previously described methods using the optimized ER/SR-targeted GECI CatchER+ to monitor changes in ER/SR Ca2+ through different receptor mediated pathways.
Figure 1 illustrates ER emptying through the RyR stimulated with 200 µM 4-cmc. 4-cmc is an agonist of the RyR. Addition of the drug induces a decrease in ER calcium as measured by the decrease in the CatchER+ fluorescence intensity. As marked, the protocol outlined here allows the visualization and measurement of receptor-mediated Ca2+ transients using CatchER+ which provides information about the changes in ER Ca2+.
As depicted in Figure 2, the addition of 15 µM CPA, to reversibly block the SERCA pump, produces a large decrease in the fluorescence intensity that forms a plateau. As the function of the SERCA pump is inhibited, the ER Ca2+ release channels are still actively releasing Ca2+ into the cytosol causing the fluorescence intensity to decrease. The cells recover after washing CPA away with Ringer's buffer containing 1.8 mM Ca2+ and 10 mM glucose. These results confirm the ability of CatchER+ to monitor Ca2+ transients specific to the SERCA pump.
Figure 3 shows representative data for the indirect activation of the IP3R with 200 µM ATP in HEK293 cells transfected with CatchER+. ATP binds to the P2Y receptor, a G-protein coupled receptor, which generates IP3 that activates Ca2+ release through the IP3R. Although the change is small, the addition of 200 µM ATP produces a visible decrease in signal intensity. Washing the cells with Ringer's buffer containing 1.8 mM Ca2+ and 10 mM glucose allows the ER to refill and the signal to return to the baseline. These results highlight the ability of CatchER+ to monitor IP3R-mediated calcium release through indirect stimulation.
In Figure 4, CatchER+ was transfected into C2C12 myoblast cells to determine the in situ Kd. The data was collected as outlined in the protocol. C2C12 myoblast cells were permeabilized with 0.005% saponin for 15-30 s then washed with 1 mM EGTA in KCl buffer containing 10 µM ionomycin. The various Ca2+ concentrations were prepared in KCl buffer containing 10 µM ionomycin and were added in a stepwise manner. The data was normalized and fitted using the 1:1 binding equation. The average Kd was 3.1 ± 1.4 mM for 7 cells. The weak affinity of CatchER+ allows it to sense Ca2+ in high Ca2+ organelles like the ER or SR. To determine the basal [Ca2+] in the SR, C2C12 cells were permeabilized with 0.005% saponin for 15-30 s, washed with KCl buffer, and then washed with 1 mM EGTA in KCl buffer containing 10 µM ionomycin to obtain Fmin. After washing the EGTA away with KCl buffer a maximum, Fmax, of 100 mM Ca2+ with 10 µM ionomycin was added. The basal Ca2+ was calculated to be 0.4 ± 0.2 mM.
Figure 1: Stimulation of RyR using 4-cmc in HEK293 cells. (A) Depiction of the activation of RyR with 4-cmc. (B) Intensity recording of HEK293 cells transfected with CatchER+, localized in the ER, treated with 200 µM 4-cmc. Stimulation of the RyR with 4-cmc causes a decrease in the normalized fluorescence intensity that directly correlates to a decrease in [Ca2+]ER. n refers to the number of cells imaged. The average signal decrease was 12.0 ± 2.2%. The presence of 1.8 mM Ca2+ in the Ringer's buffer facilitated refilling of the ER reflected in the intensity recovery to baseline. Inset pictures show the cells before and after treatment with 4-cmc. SNR was calculated to be 4.3 ± 0.8. Please click here to view a larger version of this figure.
Figure 2: Decrease in [Ca2+]ER using the reversible SERCA pump inhibitor CPA in HEK293 cells. (A) The inhibition of the SERCA pump by CPA. (B) Live imaging of HEK293 cells transfected with CatchER+, localized in the ER, treated with 15 µM CPA. Because Ca2+ can still flow through the IP3R and RyR, blocking the SERCA pump with CPA causes a decrease in the normalized fluorescence intensity that directly correlates to a decrease in [Ca2+]ER. n refers to the number of cells imaged. Inset pictures are HEK293 cells transfected with CatchER+ before and after treatment. The average signal decrease was 21.0 ± 0.3%. The presence of 1.8 mM Ca2+ in the Ringer's buffer facilitated refilling of the ER reflected in the intensity recovery to baseline. SNR was calculated to be 5.5 ± 0.8. Please click here to view a larger version of this figure.
Figure 3: ATP decreases [Ca2+]ER in HEK293 cells. (A) Schematic of the indirect activation of IP3R with ATP through the purinergic receptor P2YR. P2YR is a GPCR that generates IP3 upon ATP binding. The IP3 activates the IP3R, stimulating calcium release from the ER. (B) Real time imaging of HEK293 cells transfected with CatchER+, localized in the ER, treated with 200 µM ATP. Indirect stimulation of the IP3R via the P2Y receptor with ATP causes a decrease in the normalized fluorescence intensity that directly correlates to a decrease in [Ca2+]ER. n refers to the number of cells imaged. The average signal decrease was 6.0 ± 3.0%. SNR was calculated to be 6.7 ± 2.7. Please click here to view a larger version of this figure.
Figure 4: In situ Kd of CatchER+ in C2C12 myoblast cells. Representative trace for normalized intensity collected from permeabilized C2C12 myoblast cells transfected with CatchER+. Cells were permeabilized with 0.005% saponin in intracellular buffer for 15-30 s. EGTA and Ca2+ solutions were prepared in KCl buffer, n refers to the number of cells imaged. (A) Inset fluorescence images are representative of cells before and after treatment. 0.3, 0.6, 2, 5, 10, 20, 50, and 100 mM Ca2+ was added to permeabilized C2C12 myoblast cells in the presence of 10 µM ionomycin to get a Kd of 3.1 ± 1.4 mM. (B) Inset fluorescence images are representative of cells at minimum and maximum values, after addition of 1 mM EGTA and 100 mM Ca2+ with 10 µM ionomycin each, respectively. The basal Ca2+ was calculated to be 0.4 ± 0.2 mM. Please click here to view a larger version of this figure.
Live single-cell imaging of fluorescent probes, such as CatchER+, is an effective technique to analyze intricate ER/SR Ca2+ signaling processes in each cell in response to receptor agonists or antagonists. This technique is also useful for imaging using multiple wavelengths concurrently, such as needed for Fura-2 or to image CatchER+ and Rhod-2 together to monitor both ER and cytosolic calcium changes, respectively. There are several critical steps in this protocol; cell transfection can have a great effect on the viability of imaging. Low transfection rates can result in insufficient expression of the GECI for monitoring calcium responses. On the other hand, overexpression of CatchER+ will not have a buffering effect on ER/ER Ca2+, a problem associated with synthetic calcium dyes. Buffering effects of Ca2+ probes are influenced by the [Ca2+] in the organelle, the Kd of the probe and the concentration of the probe required for measurement. Cytosolic [Ca2+] is typically in the low nanomolar range, however, the required probe concentration is in a comparable range. In this case, the amount of dye concentration and its capability in buffering cytosolic calcium was, and is, a major concern for cellular imaging. In contrast, ER/SR [Ca2+] is in the 100s of micromolar to millimolar range31 as we determined for different mammalian cell lines25. Since 0.1-1 µM expression of CatchER+ is sufficient to enable detection of ER calcium, the buffering effect of CatchER is negligible. Thus, CatchER+ can monitor Ca2+ flux in high [Ca2+] environments without any buffering effect on luminal ER/SR Ca2+32.
Several factors can affect transfection efficiency of the GECI such as the incubation time, the transfection reagent, the incubation temperature, and the cell confluency. The cell confluency, when seeding the cells on the slide, can significantly affect the imaging quality. Low confluency can result in a low number of cells (n) and less statistical accuracy, while high levels of confluency lead to overlapping layers of cells producing large variables in imaging. The time and temperature for transfection should be optimized based on the cell type. Additionally, adding DMEM with reduced serum media is optimal for longer transfection times to prevent apoptosis. The original GECI CatchER fluorescence in mammalian cells, when cultured and transfected, was only 30 °C. The fluorescence of CatchER was successfully optimized and improved for expression at 37 °C resulting in the new, improved variant CatchER+. A western blot using an antibody against EGFP can be performed to confirm the expression of Ca2+ probe.
Moreover, the method and consistency of reagent delivery is critical. Reagents can be added to the chamber by perfusion, small volume diffusion, or by mechanical pumps, all of which can elicit differing responses. It is imperative that the solution chamber is properly sealed by applying a layer of sealant grease on the bottom of the chamber that will be placed onto the slide. The correct wavelengths must be selected for the probe. Excitation and emission wavelengths for CatchER+ are 395/488 nm and 510 nm, respectively. For cell imaging, only 488 nm is used for excitation to avoid the detrimental effect of UV light on cells. Therefore, using any optical filters which excite at 488 nm and collect the emission at 510 nm would be acceptable to use to image with CatchER+. To prevent photobleaching, efforts may be focused to optimize light intensity and light exposure such as frames/s33.
While this protocol is ideal to analyze the effect of ER/SR changes using CatchER+ Ca2+ ER/SR fluorescent probe, there are limitations as expected in any experiment. Since single-cell live imaging only analyzes a small frame of cells, the number of cells (n) can vary from 1-100 cells, on average, but can be lower for larger cell lines. This leads to lower cell numbers and statistically varied results without multiple trials. An n>6 is required for statistical accuracy. To get a larger n, many dishes must be imaged or other techniques must be used, concurrently. Additionally, CatchER+ is a single wavelength ER/SR Ca2+ probe, this leads to the issues of other single wavelength probes and dyes with not being able to quantify the signal, not knowing if signal is true as opposed to disruption of the cells artifact, and having larger noise as compared to ratiometric systems34.
This work shows that these optimized Ca2+ probes can be applied in different cell types or tissue types to monitor receptor-mediated ER/SR Ca2+ release. CatchER+ is a single wavelength ER/SR Ca2+ probe. Therefore, it is important that the experimental parameters and settings are consistent for quantitative measurement. A detailed calibration of calcium concentration and Kd of calcium sensor in the cell lines are additional important measurements for quantitative analysis.
These developed calcium sensors can also be targeted to specific locations within the ER/SR to monitor the vastly different Ca2+ transients that exist compared to global Ca2+ changes in various biological and pathological conditions. These findings will continue to push the fields of Ca2+ imaging and probe design forward to provide future tools for diagnosing Ca2+-related diseases. The reported protocols and developed sensor can also be adapted for drug discoveries against diseases associated with calcium signaling.
The authors have nothing to disclose.
This work was funded by NIH GM62999, NIH EB007268, NIH AG15820, B&B Seed Grant, and a NIH Supplemental Grant to FR, BB fellowship to CM, CDT fellowship to RG.
4-Chloro-3-methylphenol (4-CmC) | Sigma-Aldrich | C55402 |
515DCXR dichroic mirror | Chroma Technology Corp. | NC338059 |
Adenosine 5′-triphosphate disodium salt hydrate | Sigma-Aldrich | A26209 |
Calcium chloride dihydrate | EMD Millipore | 102382 |
Corning tissue-culture treated culture dishes (100 mm) | Sigma-Aldrich | CLS430167 |
Corning tissue-culture treated culture dishes (60 mm) | Sigma-Aldrich | CLS430166 |
Cyclopiazonic Acid (CPA) | EMD Millipore | 239805 |
D(+)-Glucose | ACROS Organics | 41095-0010 |
Dow Corning 111 Valve Lubricant & Sealant | Warner Instruments | 64-0275 |
Dulbecco’s Modified Eagle’s Medium (DMEM) | Sigma-Aldrich | D7777 |
Ethylenebis(oxyethylenenitrilo) tetraacetic Acid (EGTA) |
ACROS Organics | 409911000 |
Fetal Bovine Serum (FBS) | ThermoFisher | 26140087 |
Fisherbrand Cover Glasses 22×40 mm | Fisher Scientific | 12-544B |
Hanks’ Balanced Salts (HBSS) | Sigma-Aldrich | H4891 |
HEPES, Free Acid, Molecular Biology Grade | EMD Millipore | 391340 |
Immersion Oil without autofluorescence | Leica | 11513859 |
Ionomycin, Free Acid | Fisher Scientific | 50-230-5804 |
Leica DM6100B inverted microscope with a cooled EM-CCD camera | Hamamatsu | C9100-13 |
Lipofectamine 2000 Transfection Reagent | ThermoFisher | 11668019 |
Lipofectamine 3000 Transfection Reagent | ThermoFisher | L3000015 |
Low Profile Open Diamond Bath Imaging Chamber | Warner Instruments | RC-26GLP |
Magnesium Chloride Hexahydrate | Fisher Scientific | M33-500 |
Opti-MEM | ThermoFisher | 51985034 |
Potassium Chloride | EMD Millipore | PX1405 |
Potassium Phosphate, Dibasic | EMD Millipore | PX1570 |
Potassium Phosphate, Monobasic | EMD Millipore | PX1565 |
Saponin | Sigma-Aldrich | 47036 |
SimplePCI Image Analysis Software | Hamamatsu | N/A |
Sodium Bicarbonate | Fisher Scientific | S233-3 |
Sodium Chloride | Fisher Scientific | S271-500 |
Sterivex-GV 0.22 µm filter | EMD Millipore | SVGVB1010 |
Till Polychrome V Xenon lamp | Till Photonics | N/A |
Trypsin (2.5%), no phenol red (10X) | ThermoFisher | 15090046 |