Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Podsumowanie

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 K_d measurement and calibration is also discussed.

Streszczenie

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 K_d 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.

Wprowadzenie

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 Ca^{2+ 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 ER^9,10. 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 peptide^17,18. Troponin C-based GECIs are also available as FRET pairs of CFP and Citrine and as single fluorophore probes^19,20,21. Others, such as GCaMP2 and R-GECO are single fluorophore sensors involving calmodulin^22,23. To overcome the limitations of narrow tuning of K_d'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 K_d of ~0.18 mM, a k_on near the diffusion limit, and a k_off 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 °C^28,29, 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 K_d of CatchER⁺ in C2C12 myoblast cells and quantification of basal [Ca²⁺].

Protokół

1. Slide Preparation

1. Place 22 mm x 40 mm glass microscope slides in 6 cm cell culture dishes, 1 slide per dish.
2. Expose each side of each slide as well as the 6 cm dish to ultraviolet (UV) light for 15-20 min to sterilize in a sterile hood.
3. Cover the dishes with slides inside with parafilm and store at 4 °C until ready to use.

2. Preparation of Media, Buffers, Solutions, and Reagents

1. Prepare 1 L of Hank's balanced salt solution (HBSS) in deionized water and add 10 mM HEPES, 5 mM NaHCO₃, and 1 mM EGTA. Adjust to pH 7.2-7.3 with NaOH. Filter buffer with a 0.22 µm filter into an autoclaved bottle.
2. Prepare 900 mL of high glucose Dulbecco's Modified Eagle's Medium (DMEM) h in deionized water. Add 44 mM NaHCO₃ and adjust the pH to 7.2-7.3 with HCl. Filter media with a 0.22 µm filter into an autoclaved bottle. Add 100 mL of fetal bovine serum (FBS) to the media to make the FBS concentration 10%. Store at 4 °C.
3. Prepare 1 L of intracellular buffer (125 mM KCl, 25 mM NaCl, 10 mM HEPES, 0.2 mM CaCl₂, 0.5 mM EGTA, 0.2 mM MgCl₂) in deionized water, and adjust the pH to 7.25. The final [Ca²⁺] will be ~100 nM. Before use, add 0.5 mM ATP. Store at room temperature.
4. Prepare 1 L of Ringer's buffer (121 mM NaCl, 2.4 mM K₂HPO₄, 0.4 mM KH₂PO₄, 10 mM HEPES, and 1.2 mM MgCl₂) in deionized water, and adjust the pH to 7.2. Store at room temperature. To use, aliquot 50 mL into a 50 mL tube and add 1.8 mM Ca²⁺ and 10 mM glucose.
5. Prepare 1 L of KCl rinse solution (125 mM KCl, 25 mM NaCl, 10 mM HEPES, 0.2 mM MgCl₂) in deionized water, and adjust the pH to 7.25.
6. Dissolve 5 mg of ionomycin in 1 mL of dimethyl sulfoxide (DMSO). Aliquot 30 µL into microtubes and store at -20 °C.
7. Prepare the cyclopiazonic acid (CPA) stock by dissolving CPA into DMSO. Make sure the final percent of DMSO is ≤ 1% for the CPA added to the cells to prevent apoptosis. Prepare 20 mM 4-chloro-m-cresol (4-cmc) by dissolving into filtered H₂O and letting it dissolve overnight by shaking at 37 °C. Prepare the ATP stock by dissolving in filtered H₂O to 100 mM.
8. To determine the in situ K_d, prepare 15 mL each of 1 mM EGTA, 0.2 mM, 0.6 mM, 2 mM, 5 mM, 10 mM, 20 mM, 50 mM, and 100 mM Ca²⁺ in KCl buffer using a 100 mM EGTA stock and a 1 M CaCl₂ stock dissolved in deionized water.

3. Cell Culture

1. Culture C2C12 myoblast and HEK293 cells according to ATCC protocols for each using 10 cm cell culture dishes in a sterile UV hood.
   NOTE: C2C12 cells should not be allowed to grow higher than ~70% confluence since the myoblasts will begin to differentiate into myotubes. Both HEK293 and C2C12 cells grow readily and should not be allowed to grow past 100% confluency to maintain cell integrity. Additionally, cells should not be passaged more than 10 times before using them for an experiment to preserve cell health.

4. Transfection of HEK293 Cells

1. Seed HEK293 cells onto sterilized 22 mm x 40 mm glass microscope slides in 6 cm dishes so they are ~70% confluent the day of transfection.
2. The next day, follow the manufacturer's protocol for the transfection reagent to transfect cells using 2 µg of CatchER⁺ cDNA and a 1:3 (weight/volume) DNA:transfection reagent ratio in the reduced serum media (e.g. Opti-MEM).
3. Empty the DMEM media from the dish, and add 3 mL of reduced serum media. Add DNA:transfection reagent mixture to the dish and incubate for 4-6 h at 37 °C.
4. After incubation, wash the cells with 5-6 mL of HBSS. Discard the HBSS from the dish and replace with 3 mL of fresh DMEM and incubate them at 37 °C for 48 h to allow expression of the GECI.
5. Transfection of C2C12 myoblast cells
   1. For C2C12 myoblast cells, trypsinize cells by adding enough trypsin to cover the bottom of dish (1-2 mL). Place the dish in a 37 °C incubator for 2-6 min to allow the trypsin to loosen the cells from the bottom of the dish.
   2. Once the incubation is complete, remove the trypsin and aspirate the cells in 8 mL of DMEM. Pipette the cells thoroughly with the DMEM to evenly disperse and re-suspend te cells and seed them onto sterile coverslips in 6 cm cell culture dishes containing 3 mL of DMEM so that the final confluence is 60%.
   3. Transfect the cells the same day as outlined in step 4.2-4.3. Incubate for 24 h at 37 °C.
   4. Repeat step 4.4.

5. Preparation of Slide and Fluorescence Microscope

1. Turn on microscope and light source, then open the Simple PCI program. Allow the microscope to warm up for 15-20 min or until the charge-coupled device (CCD) camera cools to -65 °C. Prepare the chamber and slide with cells while waiting.
2. Cover the bottom of the low profile open diamond bath imaging chamber with a thin layer of sealant, using a cotton swab.
3. Take the slide containing transfected cells out of the incubator and gently rinse three times with 1 mL of Ringer's buffer with 10 mM glucose and 1.8 mM Ca²⁺ preheated to 37 °C.
4. Leave 1 mL of Ringer's buffer in the dish, to prevent cells from drying, and use forceps to take the slide out of the dish. Allow excess solution to absorb onto a laboratory tissue by touching the edge of the slide to a laboratory tissue. Then place onto the side of the chamber containing the grease with cells facing up towards grease and secure chamber onto the stage mount using a screwdriver.
5. Add 1 mL of Ringer's buffer to the chamber to prevent cells from drying while mounting the chamber onto the stage and completing the rest of the microscope setup. Once the vacuum suction hose is attached to the chamber, the final volume the chamber holds is ~450 µL.
6. Switch the microscope objective to the 40X oil immersion objective.
7. Use lens paper and lens cleaning solution to clean and dry the objective. Do not rub the objective. Dab gently until the surface is clean.
8. Add one drop of the immersion oil to the objective.
9. Place the mount onto the microscope stage and add the vacuum tip to the chamber. Once the vacuum suction hose is attached to the chamber and turned on, the final volume the chamber holds is ~450 µL. This final volume is level with the sides of the chamber. It is imperative for the chamber solution to be level, during the experiment, to prevent the solution from spilling down into the objective due to overfilling.
10. Raise the objective and use the coarse adjustment until the oil on the objective touches the bottom of the slide.
11. Using the bright field mode, adjust the gain to 175 and the exposure time to 0.03 s to focus the cells.
12. Once cells are focused, switch to fluorescence mode using 488 nm excitation. Change the exposure time to 0.07 s. Locate a field of view that has enough cells that are healthy with adequate fluorescence to image.
13. Once a field of view has been chosen, take pictures in fluorescence and bright field mode.
14. Circle a region of interest (ROI) in the cells or circle the whole cell to record the intensity from the chosen area.

6. Imaging Drug-induced Ca²⁺ Transients and In Situ K_d Calibration

1. Start intensity measurement with frame rate set at one every 5 s.
2. Allow the baseline intensity to stabilize for 20-30 frames (100-150 s). If needed, decrease the frames/s during this step to prevent photobleaching.
3. Calculate the amount of 4-cmc needed from a 20 mM stock based on the final chamber volume of ~450 µL to make a final concentration of 200 µM. Make dilutions of the stock as necessary.
4. Carefully take 60 µL of Ringer's buffer from the chamber and place in a microcentrifuge tube. Dilute the calculated amount of 4-cmc with this solution and add back to the chamber to evoke the intended response from the cells being imaged.
5. Add the calculated amount of 4-cmc to the microcentrifuge tube and mix by pipetting.
6. Gently add the solution over the field of view while simultaneously adding the event marker in the intensity measurement.
7. Allow time for the drug to bind to the receptor and subsequent signal decrease, then wash with 3-6 mL of Ringer's buffer with 1.8 mM Ca²⁺ and 10 mM glucose while simultaneously adding the event marker. Since the chamber volume is 450 µL and is connected to a vacuum, adding 3-6 mL of buffer ensures that the former solution containing the drug has been washed away and replaced with the newly added solution.
8. Repeat 6.1-6.7 for 100 µM ATP and 15 µM CPA, using a new slide of transfected cells for each assay.
9. Once the measurement is complete, end the data collection in the intensity measurement window in the Simple PCI program.
10. Take fluorescence and brightfield pictures of the cells.
11. Open the data file for the experiment. Here, re-circle the cells or regions of interest to re-calculate the intensity values, if needed.
12. To determine the in situ K_d in C2C12 myoblast cells, follow the protocol as outlined in section 4.2 and section 5 and put 1 mL of 0 mM Ca²⁺ Ringer's buffer in the chamber.
    1. Permeabilize the cells with 0.002-0.005% saponin in intracellular buffer for 15-30 s to empty the cells of any CatchER⁺ that may be in the cytosol. Wash the cells with 3-6 mL of 1 mL EGTA in KCl buffer with 10 µM ionomycin. Allow the intensity to decrease until a plateau is reached.
    2. Rinse with 3-6 mL of KCl buffer with 10 µM ionomycin and allow the intensity to stabilize, then add each Ca²⁺ solution detailed in step 2.7. Allow the intensity to reach a plateau between each addition.
13. To calibrate the sensor for basal [Ca²⁺] quantification, follow step 6.12. Use the EGTA and the 100 mM Ca²⁺ buffer from step 2.7 to get the minimum and maximum fluorescence intensity.

7. Data Processing

1. Download the spreadsheet file of the intensity measurement data.
2. Normalize the data for each cell to the baseline intensity (F/F₀). Plot the normalized data versus time in any graphing program.
3. To calculate the % change, subtract the normalized peak value from 1 and multiply by 100. Calculate the average and standard deviation if multiple cells were imaged.
4. To calculate the signal to noise ratio, SNR, open up the image file saved from the experiment in an image processing software, such as ImageJ, and then measure the signal, transfected cells, and noise, background. Calculate the SNR using the equation SNR = signal/noise.
5. To calculate the in situ K_d from the collected fluorescence imaging data, average the intensity data, comprising the plateaus, after the addition of each Ca²⁺ concentration and EGTA (0 mM Ca²⁺). Calculate the fractional saturation (relative intensity) using θ = (F – F_min)/(F_max- F_min), where θ is the relative intensity, F is the fluorescence at any point, F_min is the minimum fluorescence intensity, and F_max is the maximum fluorescence intensity, to see the change in the intensity of the probe with the addition of Ca²⁺ compared to the minimum intensity. Plot the relative intensity data against the [Ca²⁺] in any graphing and curve fitting program. Use the 1:1 binding equation θ = [M_T]/(K_d + [M_T]) translated for any graphing and curve fitting program to calculate the K_d. M_T is the total metal concentration.
6. To quantify the basal calcium from the calibration outlined in 6.13, average the intensity data, comprising the plateaus, after the addition of 1 mM EGTA (F_min) and 100 mM Ca²⁺ (F_max). Use the calibration equation [Ca²⁺] = K_d([F – F_min)/(F_max – F)] to calculate the basal calcium.

Wyniki

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 decrea...

Dyskusje

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 ...

Materiały

Name	Company	Catalog Number	Comments
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 22x40 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