1Department of Physiology and Biophysics, Confocal Microscopy and Cell Imaging Core, Robert Wood Johnson Medical School, 2Department of Physiology and Biophysics, Robert Wood Johnson Medical School, 3Muscle Biology Research Group-MUBIG Schools of Nursing & Medicine, University of Missouri-Kansas City
Pan, Z., Zhao, X., Brotto, M. Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber. J. Vis. Exp. (60), e3415, doi:10.3791/3415 (2012).
Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.
1. Intracellular Ca2+ measurement for individual cells
The above protocol can be modified for measurement of intracellular Ca2+ in cell suspension system.
2. Mn quenching assay in cultured cells
3. Mn quenching assay in muscle cells
4. Spatially and temporally resolved SOCE in skinned muscle fibers
5. Representative Results
We examined SOCE activity in KYSE-150 cells using intracellular Ca2+ measurement (Fig 2.). Using RFP as reporter, we could select the individual cells transfected with plasmid containing specific shRNA against orai1, a gene encoding the SOCE channel. Compared with cells transfected with plasmids containing scramble shRNA (black trace), the knocking down of Orai1 protein results in decreased SOCE activity (red trace).
SOCE in KYSE-150 cells was also confirmed with the Mn2+ quenching assay (Fig 3.). The excitation wavelength of 360 nm reports the isosbestic point of Fura-2, where the fluorescence is independent of the Ca2+ concentration. After TG completely depleted ER Ca2+ stores, the perfusion of Mn2+ resulted in a significant fluorescence decrease. The overall SOCE activity can be measured by the decrease rate of Fura-2 fluorescence intensity with a steeper slope indicating a more active SOCE, while a shallower slope meaning a less active SOCE. The slope of fluorescence decrease in 2-APB treated cells appeared to be much shallower, which indicated that SOCE activity was blocked by this compound. The Mn2+ quenching rates were determined from the record of the initial 10 seconds, where the quenching was still in linear range without saturation.
A similar Mn2+ quenching assay was performed in the muscle (Fig 4.). In this case Mn2+ was applied together with TG. While TG was depleting the SR Ca2+ stores, the fluorescence quenching slope gradually changed until it reached its maximal rate. The sigmoidal curve indicates the graded activation of SOCE under these experimental conditions.
Healthy intact single muscle fibers in culture showed clear and uniform striation, no signs of contaminations, and no signs of contraction-induced damage. These fibers were able to contract in response to electrical stimulation or depolarizing solutions. Under confocal imaging, skinned fiber trapping of Rhod-5N dye in the TT compartment showed the characteristic mammalian doublet pattern (visualized in the red channel). After loading of the SR with Fluo-5N AM, the typical punctuated SR pattern was visible (in green channel). Upon perfusion of the skinned fiber with TT/SR loading solution, fluorescence intensity of both Rhod-5N and Fluo-5N increased; upon perfusion with SR depletion solution, fluorescence levels in the SR compartment and the TT compartment started to decrease, indicating tight coupling between SR Ca2+ store depletion and SOCE activation, respectively (Fig 5.).
Figure 1. Activation of store-operated Ca2+ entry (SOCE). Orai1, a channel pore forming unit, is located on the plasma membrane (PM) and STIM1, a Ca2+ sensor, is located on the endoplasmic or sarcoplasmic reticulum (ER/SR) membranes. When ER/SR Ca2+ stores are reduced either due to blocking of ER/SR Ca2+ pump (SERCA) or Ca2+ release through IP3 receptor or ryanodine receptor, STIM1 is activated. Activated STIM1 molecules form patches and induce the aggregation of Orai1, which further leads the activation of SOCE.
Figure 2. Measurement of intracellular Ca2+ in KYSE-150 cells. Exchange of extracellular solution from 0 Ca2+ (0.5 mM EGTA) to 2 mM Ca2+ did not induce any change in intracellular Ca2+. 5 μM TG in 0 Ca2+ bath solution induced passive ER Ca2+ release. After ER Ca2+ stores were depleted (>10 min), addition of extracellular Ca2+ (2 mM) activated a sustained intracellular Ca2+ elevation through SOCE, which can be blocked by SOCE inhibitors, e.g. skf-96365 and 2-APB (data not show). Cells transfected with plasmids containing shRNA specifically against orai1 (red) demonstrated significantly reduced SOCE than cells transfected with scramble sequence (black).
Figure 3. Mn2+ quenching assay of SOCE in KYSE-150 cells. The quenching of Fura-2 fluorescence by Mn2+ (0.5 mM) was measured at the Ca2+-independent excitation wavelength of Fura-2 (360 nm). Cells were treated with 5 μM TG for 10 min to completely deplete ER Ca2+ stores. The decay slope of Fura-2 fluorescence upon Mn2+ addition (dash lines, within the first 10 sec) represented the activation of SOCE, which was expressed as percent decrease in fluorescence per unit time (the initial value as 100%). The maximally quenched fluorescence signal was established at the end of the experiment by lysing the cells with 0.1% Triton X-100 (as 0%). Cells treated with 2-APB (75 μM) demonstrated a much shallower slope, suggesting SOCE is blocked by this compound in KYSE-150 cells.
Figure 4. Graded activation of SOCE in muscle cells revealed by Mn2+ quenching assay. Activation of SOCE could be registered while TG was depleting SR Ca2+ stores. Simultaneous application of Mn2+ (0.5mM) and TG (20 μM) induced a graded- and sigmoidal decrease of intracellular Fura-2 fluorescence (cyan dash line to show the fastest quenching point), which was distinct from initial Mn2+ quenching rate (blue dash line). Mn2+ quenching slope remained almost the same as basal level in cells treated 2-APB (75 μM), suggesting that SOCE was inhibited.
Figure 5. Spatially and temporally resolved SOCE in skinned muscle fiber. (A) Rhod-5N salt was loaded into TT and Fluo-5N AM was loaded into SR. (B) After applying Ca2+ depletion solution, the fluorescence in both TT and SR compartments decreased. The loss of Fluo-5N fluorescence indicated rapidly released SR Ca2+ content and the loss of Rhod-5N fluorescence suggested activation of SOCE.
Although the excitation wavelengths for Ca2+-binding and Ca2+ free Fura-2 are 340 nm and 380 nm, respectively, the best ratio dynamic range of Fura-2 for Ca2+ concentration measurement may occur at other wavelengths in a particular microscope system. Such shifts in wavelength are usually due to changes in the optical path with the addition of various optical components. In this study, the excitation wavelengths for Fura-2 were determined as 350 nm and 390 nm by performing spectral analysis of Fura-2 fluorescence.
The intracellular Ca2+ level in the cytosol results from a balance of several sources, including intracellular Ca2+ release, extracellular Ca2+ entry, as well as Ca2+ exclusion mechanism at the ER and plasma membrane. To isolate the unidirectional SOC-mediated Ca2+ influx from intracellular Ca2+ release and Ca2+ extrusion, the Mn2+ quenching assay can be used. Mn2+ is known to be able to permeate into cells via SOCE but is impervious to surface membrane extrusion or ER uptake by Ca2+ pumps. Therefore, fluorescence quenching represents a measurement of unidirectional Mn2+ flux into cells that estimates the degree of activation of SOCE. Mn2+ quenching assay is performed at the isosbestic point, at such wavelength Fura-2 fluorescence intensity is independent of Ca2+ concentration. The isosbestic point for each system should be determined by spectrum scanning. Alternatively, Mn2+ quenching can be recorded by adjusted fluorescence at any two wavelengths in such a way that the final value is independent of Ca2+ concentration13.
To gain the spatial and temporal information of activity of SOCE in skeletal muscles, we employed a dual-dye method, which allows simultaneous measurement of SOCE activity and SR Ca2+ content in skinned adult mammalian EDL muscle fibers. The correlation between changes in SR Ca2+ content and SOCE activity can be plotted to indicate the threshold and sensitivity of SOCE activation to depletion of the SR Ca2+ storage. The TT-loading solution is optimized to additionally load the T-tubules with Ca2+ and for priming of the T-tubules and the SR-loading solution is designed to promote maximal SR Ca2+ loading and to additionally load the T-tubules with ~500 μM Ca2+. FCCP is included in the TT/SR-loading solution and the SR-depleting solution to eliminate the effects from the mitochondria.
No conflicts of interest declared.
We thank Dr. Noah Weisleder for reading and editing of this manuscript. This work was supported by Research Grants UMDNJ Foundation 62-09 to ZP, American Heart Association SDG2630086 to XZ, 0535555N to MB, and National Institutes of Health RC2AR058962-01 to MB.
|Glass Bottom Dish||MatTek||P35G-1.5-14-C|
|Fura-2 AM||Invitrogen (Molecular Probes)||F1221||-20 °C, seal tight, protected from light, dissolved into DMSO|
|Fluo-5N AM||Invitrogen (Molecular Probes)||F14204||-20 °C, seal tight, protected from light|
|Rhod-5N||Invitrogen (Molecular Probes)||R14207||-20 °C, seal tight, protected from light|
|Quartz Cuvette||Starna Cells||3-Q-10|
|N-benzyl-p-toluene sulphonamide (BTS)||Sigma-Aldrich||435600|
|Collagenase Type I||Sigma||C0130|