We thank National Institute of Plant Genome Research - Phytotron Facility for plant growth, Bombay Locale for the video shoot, and the Department of Biotechnology- eLibrary Consortium for providing access to e-resources. This work was supported by the Department of Biotechnology, India through the National Institute of Plant Genome Research Core Grant, Max Planck Gesellschaft-India Partner Group program; and CSIR-Junior Research Fellowship (to D.M and S.M) and Department of Biotechnology-Junior Research Fellowship (to R.P).

1. EMS mutagenesis and single pedigree-based seed collection (1-3 months)
<ol>
	<li>Weigh 150 mg of seeds (~7500) of aequorin for EMS mutagenesis (M0 seeds). Weigh another 150 mg of seeds to be used as a control.</li>
	<li>Transfer the seeds to a 50 mL tube and add 25 mL of 0.2% EMS (v/v) (CAUTION) (for treatment) or 25 mL of autoclaved water (for control). 
	NOTE: Ethyl-methane sulfonate is a chemical agent for mutagenizing plant material.</li>
	<li>Seal the tube with parafilm and wrap it in aluminum foil. Rotate the tube end-over-end for 18 h at room temperature.</li>
	<li>Allow the seeds to settle. Remove the EMS solution carefully and discard in a waste container containing 1 M NaOH in equal volumes (NaOH helps to neutralize/inactivate EMS making it safe to discard). Discard the used plasticware in a 1 M NaOH solution. 
	NOTE:&#160;After 24 h, dispose the discards according to hazardous material laboratory practices.</li>
	<li>Wash the mutagenized seeds thoroughly with 40 mL of autoclaved water at least 8 times. Discard EMS containing water in a NaOH waste container as mentioned above.</li>
	<li>For the final wash, add 40 mL of 100 mM sodium thiosulfate and rinse 3 times to remove traces of EMS.</li>
	<li>Soak the seeds in 40 mL of autoclaved water for ~1 h to diffuse EMS out of seeds and then place them on filter paper until completely dry.</li>
	<li>Transfer the seeds to a microcentrifuge tube and stratify the seeds at 4 &#176;C in 40 mL of autoclaved water for 2-4 days. This helps in breaking seed dormancy and ensures homogenous growth.</li>
	<li>Transfer both the mutagenized seeds (M0) and the control seeds on to soil (soil composition: agropet: soilrite, 1:1) and transfer them to growth rooms with a 16 h light/8 h dark photoperiod, a light intensity of 150 &#956;mol&#8729;m&#8722;2&#8729;s&#8722;1 and ~70% relative humidity.</li>
	<li>To determine if mutagenesis was successful, look for reduced germination speed and seedling growth, and chlorophyll sectoring18 (Figure 1A). Compare the mutagenized plants to the control plants to identify these physiological and developmental differences. 
	NOTE: Different methods can be used for harvesting seeds from M1 plants. In this protocol, we have used the single pedigree-based seed collection method. Each M1 plant is given a unique number, starting from A1 to A3500.</li>
	<li>Maintain individually numbered plants as discrete plant lines (Figure 1B).</li>
	<li>Upon maturation, harvest seeds from these individual mutant plants and store as individual M1 lines (Figure 2). From the single pedigree-based seed collection, we obtained around 5000 M1 lines out of which 3500 M1 lines were screened.</li>
</ol>
2. High-throughput screening to select mutants (8 months)
<ol>
	<li>Identify novel mutants based on the Ca2+ response to a selected stimulus. Here, we used H2O2 as an example.</li>
	<li>For identifying mutants, screen the M2 generation. Since recessive mutants segregate at 1/8 frequency in M2 generation upon EMS mutagenesis14, screening of 8-12 M2-segregating plants covers one M1 line and identifies a homozygous recessive mutant (using 12 seedlings increases the probability of finding a mutant). From each independent M1 line, test 12 M2 seedlings for Ca2+ response to H2O2 (12 M2 seedlings per M1 line).</li>
	<li>Use a high throughput seed sterilization and hydroponic plant growth protocol19. Place nearly 12-15 M2 seeds per M1 line in individual wells of a 24-well tissue culture plate and sterilize using chlorine gas (40 mL of 12% sodium hypochlorite and hydrochloric acid, 3:1, v/v) in a desiccator for 4 h in a fume hood. After the procedure, open the desiccator and leave overnight for chlorine gas to evaporate.</li>
	<li>After sterilization, bring plates outside and add liquid 1/2 MS media (half-strength MS without agar) to individual wells. Seal the plates with parafilm and stratify the seeds for 2-4 days at 4 &#176;C and then move seeds to a growth chamber with 10 h light/ 14 h dark photoperiod at 20-22 &#176;C with 70% relative humidity.</li>
	<li>Once the seedlings are 8-12 days old, place 12 M2 seedling from each line individually in a 96-well luminometer plate. For measuring Ca2+ response to H2O2, use a luminometer plate reader.</li>
	<li>After seedling transfer, add 150 &#181;L of 5-10 &#181;M coelentrazine solution (diluted in autoclaved water from a 5 mM stock in methanol) (CAUTION) in individual wells in a dark/low-light area and store in dark at 21 &#176;C for 8 h. 
	NOTE: Coelentrazine is a prosthetic group that binds to apo-aequorin and reconstitutes it to functional aequorin. Coelentrazine is light sensitive and is hence stored in dark colored bottles, protected from light.</li>
	<li>The next day, perform mutant screen using 10 mM H2O2 as a stimulus and measure the subsequent Ca2+ response.</li>
	<li>For simultaneous measurement of 24 wells, create an automated kinetic program that measures the background for 1 min, followed by stimuli addition (40 &#956;L) and measurement for 10 min, followed by total aequorin discharge (2 M CaCl2 in 150 &#181;L 20% ethanol) for 1-2 min. 
	NOTE: An end discharge for the total aequorin is needed to quantify the measured Ca2+ and as additional control for functional aequorin. The end discharge is a short run of 1-2 min and does not cause significant plant death. Alternatively, if the plants die after discharge step, then re-screen the specific M1 line and rescue without discharge. Such mutants can be confirmed in M3 and M4 generations using the discharge step.</li>
	<li>Use a 24-well format scanning method that measures each row in 7 s interval with 300 ms integration time per well per measurement point. Use a wild-type seedling as control in each row for comparison and evaluation of the mutant. 
	NOTE: A single 96 well plate containing M2 seedlings will cover 8 individual M1 line (8 M1*12= 96 M2) and can be screened in 2.5 h and each day 32 M1 lines would be screened, 640 M1 plants per month. The whole screening procedure after the plants are ready would take around 8 months.</li>
	<li>Identify mutants based on loss of or reduced Ca2+ response with H2O2. Rescue the selected mutants through an antibiotic-based washing process. Wash the seedling with 25 mg/L cefotaxime solution twice and then transfer to rescue medium that contains 25 mg/L cefotaxime in 1/2 MS agar.</li>
	<li>Grow the plates at a 10 h light/ 14 h dark photoperiod to obtain a mutant plant. The cefotaxime wash helps to remove any harmful micro-organisms on the seedling. Since the seedlings after reconstitution are kept un-sealed, minimize contamination when transferring back to sterile condition.</li>
	<li>Transfer the mutant plant to soil for obtaining the homozygous M3 population.</li>
</ol>
3. Data analysis and mutant identification (1-3 months)
NOTE: The readings provided from the above method are in relative light units (RLU).
<ol>
	<li>For calculating [Ca2+]cyt concentration, use the following concentration equation20. 
	pCa = 0.332588(&#8722;log (L/Lmax)) + 5.5593 
	Luminescence counts () and total remaining counts (max).</li>
	<li>Convert the obtained pCa values to [Ca2+]cyt values in &#956;M by multiplying by 106. Then graphically plot against an aequorin (transgenic Col-0 aequorin) control for identifying putative H2O2 mutants to Ca2+ response.</li>
	<li>Rescue seedlings showing a loss of or reduced response to H2O2 application rescued.</li>
	<li>Upon maturation, harvest seeds from the rescued mutant and re-screen for response to H2O2 in M3 seedlings. If all 12 M3 seedlings show a Ca2+ response similar to the mother plant, consider the population to be a homozygous M3 mutant population. If all 12 seedlings do not show such a response, then take them to M4 generation and re-screen to obtain homozygous population.</li>
	<li>Once a homozygous plant line has been obtained, identify the gene leading to altered phenotype using next generation sequencing.</li>
</ol>

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None of the authors have any conflicts of interest to declare.

EMS mutagenesis is a powerful tool to generate mutations in population. The classical forward genetic screens using EMS has been an effective tool to identify novel genes for two major reasons: firstly, they do not require any prior assumptions on gene identity and secondly, they do not introduce any bias. There are several methods to generate a screening populations like EMS, T-DNA insertions, radiations etc. Out of all the methods, EMS-based mutagenesis has few advantages over the other methods. First, it is easier to ...

A change in cytosolic calcium (Ca2+) concentration upon perception of biotic or abiotic stimulus is a well-studied early signaling event that activates many signaling pathways1,2,3,4. A cell in its basal resting state maintains a lower Ca2+ concentration in the cytosol and sequesters excess Ca2+ in various intracellular organelles and extracellular apoplast leading to a steep Ca2+ gradient5,6. Upon signal perception, Ca2+ levels rise in the cytosol due to an influx of Ca2+ from extracellular and/or intracellular sources and generate a stimuli specific calcium signature7,8,9. Ca2+ elevations in the cytosol are activated by many stimuli, but specificity is maintained by distinct stores releasing Ca2+, a unique Ca2+ signature and appropriate sensor proteins10,11.
The use of alkylating agent, ethyl-methane sulfonate (EMS) for mutagenesis is a powerful tool in classical forward genetic screens to identify multiple independent genes involved in a process. EMS is a chemical mutagen predominantly inducing C to T and G to A transitions randomly throughout the genome and produces a 1 bp change in every 125 kb of the genome. EMS mutagenesis will induce &#8776;1000 single base pair changes, either insertion/deletions (InDel) or single nucleotide polymorphism (SNP) per genome12. EMS-induced mutations are multiple point mutations with a mutation frequency ranging from 1/300 to 1/30000 per locus. This reduces the number of M1 plants needed to find a mutation in a given gene. A M1 seed population range of 2000-3000 is typically used to obtain mutations of interest in Arabidopsis thaliana13,14.
Aequorin transgenics are Arabidopsis Columbia-0 (Col-0) ecotype plants expressing p35S-apoaequorin (pMAQ2) in the cytosol15. Aequorin is a Ca2+ binding protein composed of apoprotein and a prosthetic group consisting of luciferin molecule, coelenterazine.&#160;The binding of Ca2+ to aequorin, which has three Ca2+ binding EF-hands sites, results in coelenterazine being oxidized and cyclized to give the dioxetanone intermediate, followed by a conformational change of the protein accompanied by the release of carbon dioxide and singlet-excited coelenteramide16. The coelenteramide so produced emits a blue light (&#955;max, 470 nm) that can be detected by the luminometer17. The extremely fast Ca2+ elevations can thus be measured in real time, and exploited for rapid forward genetic screens. This protocol aims to use the specificity of calcium response to identify novel key players that are involved in the Ca2+ signature. To achieve this task, we use EMS mutagenesis in transgenic aequorin and identify the SNPs associated with altered Ca2+ signaling. The protocol identifies mutants that show no or reduced Ca2+ elevations upon stimuli addition. These mutants can then be mapped to identify the genes responsible for the Ca2+ response. The method is applicable to any kind of liquid stimuli in plants that results in a Ca2+ elevation. Since Ca2+ elevation is one of the first responses in the plant defense signaling pathway, the identification of upstream response components can provide candidates for genetic engineering to develop resilient plants.

<table border="0" cellpadding="0" cellspacing="0" style="width:695px;" width="694">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">24 well tissue culture plate</td>
			<td style="width:125px;">Jetbiofil</td>
			<td style="width:116px;">11024</td>
			<td style="width:241px;">for growing seedlings</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well white cliniplate</td>
			<td>Thermo Scientific</td>
			<td>9502887</td>
			<td>for luminometer measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aequorin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agropet</td>
			<td>Lab Chem India</td>
			<td></td>
			<td>for plant growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>12135</td>
			<td>for discharge solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coelenterazine</td>
			<td>PJK</td>
			<td>55779-48-1</td>
			<td>prosthetic group for aequorin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ehtylmethane sulfonate</td>
			<td>Sigma Aldrich</td>
			<td>M0880-5G</td>
			<td>for seed mutagenesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Analytical reagent</td>
			<td>1170</td>
			<td>for discharge solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td>Merck Life Sciences</td>
			<td>1.93001.0521</td>
			<td>sterlization solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrogen peroxide</td>
			<td>Fisher Scientific</td>
			<td>15465</td>
			<td>as stimulus for Calcium elevation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luminoskan ascent</td>
			<td>Thermo Scientific</td>
			<td>5300172</td>
			<td>aequorin luminescence measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MES buffer</td>
			<td>Himedia</td>
			<td>RM1128-100G</td>
			<td>plant growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Murashige and skoog media</td>
			<td>Himedia</td>
			<td>PT021-25L</td>
			<td>plant growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>27805</td>
			<td>for neutralizing EMS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hypochlorite</td>
			<td>Merck Life Sciences</td>
			<td>1.93607.5021</td>
			<td>sterlization solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium thiosulfate</td>
			<td>Fisher Scientific</td>
			<td>28005</td>
			<td>for seed washing in step 1.6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">soilrite</td>
			<td>Lab Chem India</td>
			<td></td>
			<td>for plant growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Square pots</td>
			<td>Lab Chem India</td>
			<td></td>
			<td>for plant growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389</td>
			<td>plant growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taxim</td>
			<td>Alkem</td>
			<td>7180720</td>
			<td>for seedling rescue</td>
		</tr>
	</tbody>
</table>

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.

The EMS population was screened for H2O2 induced Ca2+ elevation. As discussed earlier, 12 individual M2 seedlings were screened from each M1 line. In Figure 3, one such M1 line is plotted with each panel showing 12 individual M2 seedlings. A wild-type aequorin is used as control for comparing and evaluating the mutant response. A recessive mutant segregates in the ratio of 1:7 (mutant: non mutant). When screening 12...

Watch this Scientific Journal Video about Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling at JoVE.com

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

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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.1K Views.  National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India. 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.

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

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 ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

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 ...

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.

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, ...

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

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 ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

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 ...

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

Summary

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Chapters in this video

Calcium Imaging of Cortical Neurons using Fura-2 AM

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

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

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

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

Measuring Fast Calcium Fluxes in Cardiomyocytes

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP