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

Summary

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.

Abstract

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.

Introduction

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 sarcopla....

Protocol

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 filte....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

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