We would like to thank Atsushi Miyawaki for providing us with the ratiometric-pericam-mt expression construct. This work was supported by grant GM081685 from the National Institutes of Health (DB).

1. Pericam-mt Transfection and Cell Preparation.
<ol>
	<li>Plate HeLa cells the night before transfection on sterile glass coverslips placed in a standard 6-well culture plate.</li>
	<li>Prepare DNA/Lipofectamine 2000 complexes as suggested by the manufacturer (Invitrogen). We use 4 &mu;g of ratiometric-pericam-mt expression vector and 10 &mu;L of Lipofectamine 2000 diluted in 0.5 mL of Opti-MEM for each well. The cells should be ~70% confluent before transfection.</li>
	<li>Replace HeLa culture media (Dulbecco's modified eagles medium, 10% FBS, penicillin/streptomycin) in each well with 1.5 mL of the same media without antibiotics.</li>
	<li>Add the DNA/Lipofectamine complexes to each well to be transfected. Mix gently with rocking and incubate for 4 hours in a cell culture incubator at 37oC, 5 % CO2.</li>
	<li>Replace antibiotic-free media with culture media with antibiotics. Allow cells to express ratiometric pericam for 1-2 days before imaging.</li>
</ol>
2. Microscope Setup and Image Acquisition
<ol>
	<li>Place a coverslip with HeLa cells into an appropriate imaging chamber with imaging solution: 107mM NaCl, 7.2mM KCl, 1.2mM MgCl2, 1mM CaCl2,11.5mM glucose, 0.1% bovine serum albumin, and 20mM HEPES 7.2. Our laboratory uses the Attofluor cell chamber from Invitrogen. Mount the imaging chamber in an inverted microscope stage.</li>
	<li>Find an area of interest containing one or more cells expressing ratiometric pericam using a 40x (or higher) oil immersion objective. We have found that ratiometric pericam is less resistant to photobleaching at 380 nm, and thus we use this wavelength to identify suitable cells. For the images acquired in Figure 1, a Nikon Superfluor 40X objective with a 1.3 numerical aperture was used. Higher magnification objectives would also be appropriate (60-100x). Excitation filters used were Chroma Technology filters D380/30x (380 +/- 30 nm) and D495/10 (495 +/- 5 nm), beamsplitter was 505DCXR (505nm longpass), and emission filter was HQ535/50m (535 +/- 25nm). 
	Our microscope used to acquire the images in Figure 1 consists of a Nikon TE2000 inverted microscope equipped with a Roper Scientific Coolsnap HQ cooled CCD camera, Ludl MAC6000 rapid filter wheel and changer, and a MetaFluor data acquisition and analysis software. This protocol can be adapted to any standard wide-field microscope with the appropriate filters and software capable of capturing and processing ratiometric images.</li>
	<li>Set up the software for dual excitation using the 495 and 380 nm filters. Calcium binding to ratiometric pericam increases absorbance at 495 nm, thus the ratio should be set up to be 495 nm/380 nm. 
	Ratiometric pericam has a calcium-free excitation peak at 415 nm4. In this protocol we suggest using a 380 nm filter only because it is ubiquitous in most calcium imaging laboratories. If a 410 nm filter is available, it is preferable to the 380 nm filter.</li>
	<li>Acquire images sequentially by alternatively exciting at 495 and 380 nm. Acquire images of baseline calcium levels for at least 30 s before application of agonist via a perfusion apparatus. Mark the addition time for each agonist. Exposure times and acquisition intervals should be optimized to prevent photobleaching while still allowing sufficient temporal resolution.For example, for semi-rapid calcium dynamics monitored in response to agonist stimulation, images were acquired every two seconds in Figures 1B and C. Much faster acquisition rates are also possible6. Long-term imaging after staurosporine administration was acquired with a longer interval (30s) between acquisitions (Figures 1 D and E). </li>
</ol>
3. Image Processing and Analysis
<ol>
	<li>Once the experiment is complete, obtained images can be analyzed offline. Define regions of interest (ROI) in which you want to monitor changes in calcium levels. Use a ROI selected within an empty area of the field to subtract background if necessary. It should be noted that mitochondria are quite motile and dynamic, and extrapolating events in a single mitochondrion over extended periods is not always possible. Thus, considering the high motility of this organelle in some cell types, selection of ROI should be carefully monitored. Furthermore, as evidenced in Figure 1, mitochondria response heterogeneously to various stimuli. It is therefore important to analyze data offline and monitor RO1 placement on a frame-by-frame basis. A detailed description of measuring mitochondrial calcium in highly motile mitochondria has been described elsewhere7.</li>
	<li>Obtained ratio measurements from the ROI can be imported into Excel or similar graphingsoftware. Data can be presented as a trace representing changes in mitochondrial calcium levels in a single cell or single mitochondrion, or averaged fluorescence signals from several ROI. We have also used this technique to measure average mitochondrial calcium levels in a population of lymphocytes undergoing apoptosis induced by Fas ligand8.</li>
</ol>
4. Representative Results:
<img src="/files/ftp_upload/2579/2579fig1.jpg" alt="Figure 1" /> 
Figure 1. (A) Subcellular localization of ratiometric-pericam-mt. This first image shows live HeLa cell expressing ratiometric-pericam-mt. The second image shows fluorescent staining with mitochondrion-selective dye MitoTracker Red CMXRos. The yellow fluorescence in the merged image demonstrates co-localization of ratiometric-pericam-mt and MitoTracker fluorescence. (B) A series of pseudocolor ratio (495:380 nm) images of 4 HeLa cells expressing mt-ratiometric pericam treated with 10 mM ATP. (C) Quantification of changes in mitochondrial calcium levels in the region of interest (ROI) indicated in (B) treated with 10 mM ATP. (D) Series of pseudocolor ratio (495:380 nm) images of a HeLa cell expressing mt-ratiometric pericam treated with 0.5 &mu;M staurosporine. Heterogeneity in the calcium response in individual mitochondria is evident, as well as significant fragmentation of mitochondria by 60 minutes. Some mitochondria have oscillatory increases in calcium (white arrow head), whereas others do not show significant changes in calcium level (yellow arrow head).(E) Quantification of increases in global mitochondrial calcium levels in a single HeLa cell after induction of apoptosis with 0.5 &mu;M staurosporine.

<ol>
	<li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat Rev Mol Cell Biol. 1, 11-21 (2000).</li><li>Hajnoczky, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+calcium+signalling+and+cell+death:+approaches+for+assessing+the+role+of+mitochondrial+Ca2++uptake+in+apoptosis.">Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis.</a> Cell Calcium. 40, 553-560 (2006).</li><li>Nagai, T., Sawano, A., Park, E. S., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circularly+permuted+green+fluorescent+proteins+engineered+to+sense+Ca2.">Circularly permuted green fluorescent proteins engineered to sense Ca2.</a> Proc Natl Acad Sci U S A. 98, 3197-3202 (2001).</li><li>Rizzuto, R., Simpson, A. W., Brini, M., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+changes+of+mitochondrial+Ca2++revealed+by+specifically+targeted+recombinant+aequorin.">Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin.</a> Nature. 358, 325-327 (1992).</li><li>Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+Ca2++based+on+green+fluorescent+proteins+and+calmodulin.">Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.</a> Nature. 388, 882-887 (1997).</li><li>Shimozono, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+of+subcellular+Ca2++concentrations+using+a+dual-excitation+ratiometric+indicator+based+on+green+fluorescent+protein.">Confocal imaging of subcellular Ca2+ concentrations using a dual-excitation ratiometric indicator based on green fluorescent protein.</a> Sci STKE. , pl4-pl4 (2002).</li><li>Wozniak, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+biphasic+calcium+release+from+the+endoplasmic+reticulum+for+Fas-mediated+apoptosis.">Requirement of biphasic calcium release from the endoplasmic reticulum for Fas-mediated apoptosis.</a> J Cell Biol. 175, 709-714 (2006).</li><li>Boehning, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+c+binds+to+inositol+(1,4,5)+trisphosphate+receptors,+amplifying+calcium-dependent+apoptosis.">Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis.</a> Nat Cell Biol. 5, 1051-1061 (2003).</li><li>Csordas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+interorganelle+contacts+and+local+calcium+dynamics+at+the+ER-mitochondrial+interface.">Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface.</a> Mol Cell. 39, 121-132 (2010).</li><li>Matsuyama, S., Llopis, J., Deveraux, Q. L., Tsien, R. Y., Reed, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+intramitochondrial+and+cytosolic+pH:+early+events+that+modulate+caspase+activation+during+apoptosis.">Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis.</a> Nat Cell Biol. 2, 318-325 (2000).</li><li>Jiang, D., Zhao, L., Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screen+identifies+Letm1+as+a+mitochondrial+Ca2+/H++antiporter.">Genome-wide RNAi screen identifies Letm1 as a mitochondrial Ca2+/H+ antiporter.</a> Science. 326, 144-147 (2009).</li></ol>

No conflicts of interest declared.

Here we present a very simple method for measuring mitochondrial calcium using mitochondrial-targeted ratiometric pericam. As shown in Figure 1A, using standard widefield optics with no deconvolution it is possible to easily view individual mitochondria in HeLa cells with acceptable signal-to-noise ratio. This is because HeLa cells, like most cells in culture, flatten out significantly when adherent obviating the need for confocal microscopy or other specialized equipment. We have found similar results in Jurkat cells adhered to poly-lysine coated coverslips8. In contrast to methodologies using dyes, it is also possible to non-invasively monitor mitochondrial calcium levels for hours using genetically encoded calcium indicators such as ratiometric pericam (Figure 1E). This is especially important when analyzing mitochondrial calcium during cell death. Furthermore, it is also possible to visualize fission/fragmentation of mitochondria during the apoptotic process. As shown in Figures 1D and E, staurosporine treatment causes a slow increase in calcium in select subpopulations of mitochondria which peaks at 20 minutes. Calcium levels go down again concomitant with mitochondrial fragmentation before going up again 2 hours after treatment. This is consistent with the slow waves in cytosolic calcium induced by staurosporine treatment measured using Fura-29. Thus, important kinetic information can be obtained which is not possible with methods employing static measurements. Although we have presented ratios and not calcium concentrations in Figure 1, it is possible to calibrate the sensor in situ to calculate absolute calcium levels4, 10. One important caveat to consider is that ratiometric pericam is sensitive to pH4, 10. As both cytosolic and mitochondrial pH levels can change dramatically during apoptosis11, this is an important consideration. Titration of pH in isolated mitochondria expressing pericam demonstrate that increasing [H+] increases emission of pericam when excited at 495 nm, with little effect on emission stimulated by excitation at 410 nm, a property which has been exploited to simultaneously measure both mitochondrial calcium and pH12. Thus, selectively monitoring emission with 410 nm excitation provides a means to non-ratiometrically monitor mitochondrial calcium with reduced concern for pH. Finally, the protocol presented here only requires access to a standard epifluorescent microscope with widely available filters, thus making this technique accessible to most laboratories.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Inverted wide-field fluorescent calcium imaging microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>MEA53310</td>
<td>Any wide-field inverted microscope equipped with 380 nm/495 nm excitation filters and appropriate longpass and emission filter can be easily adapted to use this protocol.</td>
</tr>
<tr>
<td>Imaging Software</td>
<td>&nbsp;</td>
<td>Molecular Devices, Inc</td>
<td>Metafluor</td>
<td>&nbsp;</td>
<tr>
<td>Attofluor cell chamber</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>A-7816</td>
<td>Any imaging chamber for an inverted microscope which allows perfusion would suffice.</td>
</tr>
<tr>
<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>11668-019</td>
<td>Most transfection reagents/protocols would be appropriate for these studies </td>
</tr>
<tr>
<td>MitoTracker Red CMXRos</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>M7512</td>
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monitoring dynamic changes in mitochondrial calcium levels during apoptosis using a genetically encoded calcium sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

Watch this Scientific Journal Video about Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor at JoVE.com

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

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23.8K Views.  University of Texas Medical Branch. This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria. 

Video: Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases 1, 2 . Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago 3, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca2+ or Na+ currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells 4-8.

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

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

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.

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

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 ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca2+) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca2+ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca2+ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca2+ reporter background and identify novel Ca2+ regulated targets.

A forward genetic screen based on Ca2+ elevation as a read-out leads to identification of genetic components involved in calcium dependent signaling pathways in plants.

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis ...