Name: monitoring er/sr calcium release with the targeted ca2+ sensor catcher+
Uploaded: 2017-05-19T15:00:00
Description: Watch this Scientific Journal Video about Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+ at JoVE.com

This work was funded by NIH GM62999, NIH EB007268, NIH AG15820, B&amp;B Seed Grant, and a NIH Supplemental Grant to FR, BB fellowship to CM, CDT fellowship to RG.

1. Slide Preparation
<ol>
	<li>Place 22 mm x 40 mm glass microscope slides in 6 cm cell culture dishes, 1 slide per dish.</li>
	<li>Expose each side of each slide as well as the 6 cm dish to ultraviolet (UV) light for 15-20 min to sterilize in a sterile hood.</li>
	<li>Cover the dishes with slides inside with parafilm and store at 4 &#176;C until ready to use.</li>
</ol>
2. Preparation of Media, Buffers, Solutions, and Reagents
<ol>
	<li>Prepare 1 L of Hank&#39;s balanced salt solution (HBSS) ...

<ol>
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Biol. 27, 90-97 (2015).</li><li>Rizzuto, R., 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=Microdomains+of+intracellular+Ca2+:+Molecular+determinants+and+functional+consequences.">Microdomains of intracellular Ca2+: Molecular determinants and functional consequences.</a> Physiol. Rev. 86 (1), 369-408 (2006).</li><li>Berridge, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endoplasmic+reticulum:+a+multifunctional+signaling+organelle.">The endoplasmic reticulum: a multifunctional signaling organelle.</a> Cell Calcium. 32 (5-6), 235-249 (2002).</li><li>Clapham, D. 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Chem. 279 (14), 14280-14286 (2004).</li><li>Mank, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+FRET-based+calcium+biosensor+with+fast+signal+kinetics+and+high+fluorescence+change.">A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.</a> Biophys. J. 90 (5), 1790-1796 (2006).</li><li>Garaschuk, O., Griesbeck, O., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troponin+C-based+biosensors:+A+new+family+of+genetically+encoded+indicators+for+in+vivo+calcium+imaging+in+the+nervous+system.">Troponin C-based biosensors: A new family of genetically encoded indicators for in vivo calcium imaging in the nervous system.</a> Cell Calcium. 42 (4-5), 351-361 (2007).</li><li>Carlson, H. J., Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+Analysis+of+a+Red+Fluorescent+Protein-Based+Calcium+Ion+Indicator.">Mutational Analysis of a Red Fluorescent Protein-Based Calcium Ion Indicator.</a> Sensors. 13 (9), 11507-11521 (2013).</li><li>Wang, Q., Shui, B., Kotlikoff, M. I., Sondermann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Basis+for+Calcium+Sensing+by+GCaMP2.">Structural Basis for Calcium Sensing by GCaMP2.</a> Structure. 16 (12), 1817-1827 (2008).</li><li>Koldenkova, V. P., Nagai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+Ca2++indicators:+Properties+and+evaluation.">Genetically encoded Ca2+ indicators: Properties and evaluation.</a> Biochim. Biophys. Acta-Mol. Cell Res. 1833 (7), 1787-1797 (2013).</li><li>Tang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+application+of+a+class+of+sensors+to+monitor+Ca2++dynamics+in+high+Ca2++concentration+cellular+compartments.">Design and application of a class of sensors to monitor Ca2+ dynamics in high Ca2+ concentration cellular compartments.</a> Proc. Natl. Acad. Sci. U. S. A. 108 (39), 16265-16270 (2011).</li><li>Zou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+sensors+for+real-time+measurement+of+high+Ca2++concentrations.">Developing sensors for real-time measurement of high Ca2+ concentrations.</a> Biochemistry. 46 (43), 12275-12288 (2007).</li><li>Wang, Z. -. M., Tang, S., Messi, M. L., Yang, J. 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R., Golbik, R., Sever, R., Haseloff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+that+suppress+the+thermosensitivity+of+green+fluorescent+protein.">Mutations that suppress the thermosensitivity of green fluorescent protein.</a> Current Biology. 6 (12), 1653-1663 (1996).</li><li>Reddish, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Design Of Genetically-Encoded Ca2+ Probes With Rapid Kinetics for Sub-Cellular Application [dissertation]. , (2016).</li><li>Seo, M. D., Enomoto, M., Ishiyama, N., Stathopulos, P. 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Live single-cell imaging of fluorescent probes, such as CatchER+, is an effective technique to analyze intricate ER/SR Ca2+ signaling processes in each cell in response to receptor agonists or antagonists. This technique is also useful for imaging using multiple wavelengths concurrently, such as needed for Fura-2 or to image CatchER+ and Rhod-2 together to monitor both ER and cytosolic calcium changes, respectively. There are several critical steps in this protocol; cell transfection can ...

The spatio-temporal attributes of intracellular calcium (Ca2+) transients activate various biological functions1. These Ca2+ signaling events are triggered extracellularly through different stimuli and controlled intracellularly by the major Ca2+ storage organelle and by numerous Ca2+ pumps, channels, and Ca2+ binding proteins. Ca2+ transients can be significantly altered as a result of defects with signal modulation, leading to different diseases2. Because of the speed and intricacy of the Ca2+ signaling system, with the endo- (ER) and sarcoplasmic reticulum (SR) at the center, genetically-encoded Ca2+ probes that have been optimized for mammalian expression with fast kinetics are needed to observe global and local Ca2+ changes in different cells3.
The ER and the SR, its counterpart in muscle cells, are the major intracellular Ca2+ storage organelles and act as Ca2+ sinks that help to amplify the Ca2+ signal4. The ER/SR is an integral part in Ca2+ signaling with dual roles as a transmitter and receiver of signals5. The ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) are Ca2+ release receptors located on the membranes of the ER/ SR that are regulated by Ca2+ 6. Other agents directly or indirectly stimulate the function of these receptors. 4-chloro-m-cresol (4-cmc) is a potent agonist of the RyR, having a 10 fold higher sensitivity than caffeine for inducing SR Ca2+ release where both are regularly employed to study RyR-mediated Ca2+ release in healthy and diseased cells7. ATP increases IP3-mediated Ca2+ release through the IP3R8. ATP binds to the purinergic receptor P2YR, a G-protein coupled receptor (GPCR), triggering the production of IP3 that binds to the IP3R to release Ca2+ from the ER9,10. The sarco-endoplasmic reticulum calcium ATPase (SERCA) pump is a P-type ATPase pump, also located on the ER/SR membrane that reduces cytosolic Ca2+ and refills the ER/SR by actively pumping the ion into the ER/SR lumen11. Specific inhibitors of the SERCA pump include thapsigargin, from Thapsia garganica, and cyclopiazonic acid (CPA), from Aspergillus and Penicillium. CPA has a low affinity for the pump and reversibly blocks the Ca2+ access point12. Thapsigargin, on the other hand, irreversibly binds to the Ca2+ free pump at residue F256 in the M3 helix with nanomolar affinity11. Analyzing and quantifying the changes involved in Ca2+ stimulated events has been and remains a challenge. Since the ER/SR is the major subcellular Ca2+ containing compartment with a central function in the propagation of the Ca2+ signal, much work has been focused on understanding ER/SR Ca2+ signaling5.
The creation of synthetic Ca2+ dyes helped to advance the field and practice of Ca2+ imaging. Although dyes, such as Mag-Fura-2, have been widely used to measure compartmentalized Ca2+ in different cells,13,14,15 they have limitations such as uneven dye loading, photobleaching, and the inability to be targeted to specific organelles. The discovery of the green fluorescent protein (GFP) and the advancement of fluorescent protein-based Ca2+ probes has propelled the field of Ca2+ imaging forward16. Some of the existing GECIs are F&#246;rster resonance energy transfer (FRET) pairs involving yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), calmodulin and the M13 binding peptide17,18. Troponin C-based GECIs are also available as FRET pairs of CFP and Citrine and as single fluorophore probes19,20,21. Others, such as GCaMP2 and R-GECO are single fluorophore sensors involving calmodulin22,23. To overcome the limitations of narrow tuning of Kd&#39;s and cooperative binding associated with multiple Ca2+ binding sites found in their Ca2+ binding domains24, a novel class of calcium sensors was created by designing a Ca2+ binding site on the surface of the beta barrel in a chromophore sensitive location of enhanced green fluorescent protein (EGFP)25,26. This highly touted sensor, called CatchER, has a Kd of ~0.18 mM, a kon near the diffusion limit, and a koff of 700 s-1. CatchER has been used to monitor receptor-mediated ER/SR calcium release in different mammalian cell lines such as HeLa, HEK293, and C2C1225. Because of its fast kinetics, CatchER was used in flexor digitorum brevis (FDB) muscle fibers of young and old Friend Virus B NIH Jackson (FVB) mice to reveal that more Ca2+ remains in the SR after 2 s of depolarization in the FDB fibers of old mice compared to that of young mice27. To overcome its low fluorescence at 37 &#176;C, which hinders its applications in calcium imaging of mammalian cells, we have developed an improved version of CatchER called CatchER+. CatchER+ exhibits enhanced fluorescence at 37 &#176;C for better application in mammalian cells. Additional mutations were incorporated into CatchER to improve the thermostability and fluorescence at 37 &#176;C28,29, to create CatchER+. CatchER+ exhibits a six-fold increase in its signal to noise ratio (SNR) over CatchER30.
Here, the protocols for the culture and transfection of HEK293 and C2C12 cells with CatchER+ and its application for monitoring ER/SR receptor-mediated calcium transients are presented. Representative results are shown for CatchER+ expressed in HEK293 cells treated with 4-cmc, CPA and ATP. We also provide a protocol for determining the in situ Kd of CatchER+ in C2C12 myoblast cells and quantification of basal [Ca2+].

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-Chloro-3-methylphenol (4-CmC)</td>
			<td>Sigma-Aldrich</td>
			<td>C55402</td>
		</tr>
		<tr>
			<td>515DCXR dichroic mirror</td>
			<td>Chroma Technology Corp.</td>
			<td>NC338059</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A26209</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>EMD Millipore</td>
			<td>102382</td>
		</tr>
		<tr>
			<td>Corning tissue-culture treated culture dishes (100 mm)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430167</td>
		</tr>
		<tr>
			<td>Corning tissue-culture treated culture dishes (60 mm)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430166</td>
		</tr>
		<tr>
			<td>Cyclopiazonic Acid (CPA)</td>
			<td>EMD Millipore</td>
			<td>239805</td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>ACROS Organics</td>
			<td>41095-0010</td>
		</tr>
		<tr>
			<td>Dow Corning 111 Valve Lubricant &#38; Sealant</td>
			<td>Warner Instruments</td>
			<td>64-0275</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D7777</td>
		</tr>
		<tr>
			<td>Ethylenebis(oxyethylenenitrilo) 
			tetraacetic Acid (EGTA)</td>
			<td>ACROS Organics</td>
			<td>409911000&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFisher</td>
			<td>26140087</td>
		</tr>
		<tr>
			<td>Fisherbrand Cover Glasses 22x40 mm</td>
			<td>Fisher Scientific</td>
			<td>&#160;12-544B</td>
		</tr>
		<tr>
			<td>Hanks&#8217; Balanced Salts (HBSS)</td>
			<td>Sigma-Aldrich</td>
			<td>H4891</td>
		</tr>
		<tr>
			<td>HEPES, Free Acid, Molecular Biology Grade</td>
			<td>EMD Millipore</td>
			<td>391340</td>
		</tr>
		<tr>
			<td>Immersion Oil without autofluorescence</td>
			<td>Leica</td>
			<td>11513859</td>
		</tr>
		<tr>
			<td>Ionomycin, Free Acid</td>
			<td>Fisher Scientific</td>
			<td>50-230-5804</td>
		</tr>
		<tr>
			<td>Leica DM6100B inverted microscope with a cooled EM-CCD camera</td>
			<td>Hamamatsu</td>
			<td>C9100-13</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>ThermoFisher</td>
			<td>11668019</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>ThermoFisher</td>
			<td>L3000015</td>
		</tr>
		<tr>
			<td>Low Profile Open Diamond Bath Imaging Chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26GLP</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate</td>
			<td>Fisher Scientific</td>
			<td>M33-500</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>ThermoFisher</td>
			<td>51985034</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>EMD Millipore</td>
			<td>PX1405</td>
		</tr>
		<tr>
			<td>Potassium Phosphate, Dibasic</td>
			<td>EMD Millipore</td>
			<td>PX1570</td>
		</tr>
		<tr>
			<td>Potassium Phosphate, Monobasic</td>
			<td>EMD Millipore</td>
			<td>PX1565</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>47036</td>
		</tr>
		<tr>
			<td>SimplePCI Image Analysis Software</td>
			<td>Hamamatsu</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher Scientific</td>
			<td>S233-3</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
		</tr>
		<tr>
			<td>Sterivex-GV 0.22 &#181;m filter</td>
			<td>EMD Millipore</td>
			<td>SVGVB1010</td>
		</tr>
		<tr>
			<td>Till Polychrome V Xenon lamp</td>
			<td>Till Photonics</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Trypsin (2.5%), no phenol red (10X)</td>
			<td>ThermoFisher</td>
			<td>15090046</td>
		</tr>
	</tbody>
</table>

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.

This section will illustrate the results that were achieved using the previously described methods using the optimized ER/SR-targeted GECI CatchER+ to monitor changes in ER/SR Ca2+ through different receptor mediated pathways.
Figure 1 illustrates ER emptying through the RyR stimulated with 200 &#181;M 4-cmc. 4-cmc is an agonist of the RyR. Addition of the drug induces a decrea...

Watch this Scientific Journal Video about Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+ at JoVE.com

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.

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 ...

The overall goal of this experiment is to display our protocols for the culture and transfection of HEK293 and C2C12 cells with CatchER plus and its application for monitoring ERSR receptor mediated calcium transients in the measurement of the NC2KD. This method can help answer key questions in calcium signaling and the imaging such as the basal calcium concentration and its rapid changes in the ERSR of definite cells and how to apply biosensor CatchER plus for these measurements. The main advantage of this technique is that the fluorescence response of single cells can be recorded in real time with rapid kinetics.The implications of this technique extend toward diagnosis of ERSR calcium related diseases because CatchER plus can monitor abnormal calcium transients in these organelles that arise from dysfunctional calcium signaling elements. The visual demonstration for this method is critical as the cell transfection and fluorescence imaging steps are difficult to learn because cell culture and microscopy have multiple areas for error. Demonstrating the procedure will be Florence Reddish, a post-doc from our laboratory.To begin this experiment, seed HEK293 cells onto the sterilized glass microscope slides in the six centimeter dishes and culture for one to two days until they are about 70%confluent on the day of transfection. Next, empty DMEM from the dish and add three milliliters of the reduced serum medium. Then, add DNA transfection reagent mixture to the dish and incubate for four to six hours at 37 degrees Celsius.Transfection of HEK293 cells is generally easier than other cell lines, but the cells are less resilient to harsh conditions. Therefore, it is critical to only expose them to transfection reagents for four to six hours to avoid apoptosis. After four to six hours, wash the cells with five to six milliliters of HBSS.Discard the HBSS from the dish and replace with three milliliters of fresh DMEM. Subsequently, incubate the cells for 48 hours at 37 degrees Celsius to allow the expression of GECI. For C2C12 myoblast cells, trypsinize the cells by adding one to two milliliters of trypsin to cover the bottom of the dish.Next, place the dish in a 37 degrees Celsius incubator for two to six minutes to allow the trypsin to loosen the cells from the bottom of the dish. Once the incubation is complete, remove the trypsin and aspirate the cells in eight milliliters of DMEM. Pipette the cells thoroughly with DMEM to evenly disperse and re-suspend the cells.Then, seed them onto the sterile coverslips in six centimeter culture dishes and then add three milliliters of DMEM to achieve a final confluence of 60%Following that, transfect the cells on the same day. Incubate them for 24 hours at 37 degrees Celsius. After incubation, wash the cells with five to six milliliters of HBSS.Discard the HBSS from the dish and replace with three milliliters of fresh DMEM. Then, incubate them at 37 degrees Celsius for 48 hours to allow expression of the GECI. In this procedure, turn on the microscope and light source, then open the simple PCI program.Using a cotton swab, cover the bottom of the low-profile open-diamond bath imaging chamber with a thin layer of sealant. Then, take a slide containing the transfected cells out of the incubator and gently rinse it three times with around one milliliter of Ringer's buffer with 10 millimolar glucose and 1.8 millimolar calcium preheated to 37 degrees Celsius. Leave roughly one milliliter of Ringer's buffer in the dish to prevent the cells from drying and use forceps to take the slide out of the dish.Absorb excess solution by touching the edge of the slide to a laboratory tissue. Then, place it onto the side of the chamber with the cells facing towards the grease. Subsequently, secure the chamber onto the stage mount using a screwdriver.Add around one milliliter of Ringer's buffer to the chamber to prevent the cells from drying while mounting it onto the stage and completing the microscope setup. Switch the microscope objective to the 40 times oil immersion objective. Add one drop of the immersion oil to the objective.Afterward, place the mount onto the microscope stage. Then, place the vacuum tip in the chamber to maintain the solution leveled and prevent it from overfilling. Next, raise the objective using the course adjustment until the oil on the objective touches the bottom of the slide.Using the bright field mode, adjust the gain to 175 and the exposure time to 0.03 seconds to focus the cells. Once the cells are focused, switch to fluorescence mode using 488 nanometer excitation and change the exposure time to 0.07 seconds. Locate a field of view that has enough cells that are healthy with adequate fluorescence for imaging.Then, take pictures in fluorescence and bright field mode. Circle a region of interest in the cells or circle the whole cell to record the intensity from the chosen area. Now, start the intensity measurement with the frame rate set at one every five seconds.Allow the baseline intensity to stabilize for 20 to 30 frames. Next, calculate the amount of 4-CMC needed from a 20 millimolar stock based on the final chamber volume of 450 microliters to make a final concentration of 200 micromolar. Then, carefully transfer 60 microliters of Ringer's buffer from the chamber to a microcentrifuge tube.Dilute the calculated amount of 4-CMC with this solution and add back to the chamber to evoke the intended response from the cells being imaged. Allow time for the drug to bind to the receptor and subsequent signal decrease. Then, wash the cells with three to six milliliters of Ringer's buffer while simultaneously adding the event marker.Repeat the procedures for 100 micromolar ATP and 15 micromolar CPA using a new slide of the transfected cells for each assay. Once the measurement is complete, end the data collection in the intensity measurement window in the simple PCI program. Subsequently, take the fluorescence and bright field pictures of the cells.Then, open the data file for the experiment. Re-circle the cells or regions of interest to recalculate the intensity values if necessary. To determine the NC2KD and C2C12 myoblast cells, repeat the transfection procedure and add one milliliter of calcium-free Ringer's buffer in the chamber.Permeabilize the cells with 0.002 to 0.005%saponin and intracellular buffer for 15 to 30 seconds to empty the cells of any CatchER plus that may be in the cytosol. Then, wash the cells with three to six milliliters of KCL buffer containing EGTA and 10 micromolar ionomycin. Allow the intensity to decrease until a plateau is reached.Following that, rinse the cells with three to six milliliters of KCL buffer with 10 micromolar ionomycin and allow the intensity to stabilize. Afterward, add different calcium solutions to allow the intensity to reach a plateau between each addition. To calibrate the sensor for basal calcium concentration quantification, use EGTA and 100 millimolar calcium buffer to get the minimum and maximum fluorescence intensity.Shown here is the representative traits for the normalized intensity collected from the permeabilized C2C12 myoblast cells transfected with CatchER plus. Cells were permeabilized with 0.005%saponin in the intracellular buffer for 15 to 30 seconds. EGTA and calcium solutions were prepared in KCL buffer and N refers to the number of cells imaged.These inset fluorescence images are the representatives of cells before and after treatment. 0.2, 0.6, two, five, 10, 20, 50, and 100 millimolar calcium was added to the permeabilized C2C12 myoblast cells in the presence of 10 micromolar ionomycin to get a KD of 3.1 plus or minus 1.4 millimolar. This figure shows a representative intensity measurement of CatchER plus transfected cells at minimum and maximum values after the addition of one millimolar EGTA and 100 millimolar calcium with 10 micromolar ionomycin each respectively.Once mastered, this technique can be done in 30 to 60 minutes for the imaging or three days if including the transfection. While attempting this procedure, it's important to remember to always handle the cells in a sterile environment, get reagents ready before the experiment, to apply an even layer of grease to the chamber, and to have your vacuum attached before beginning. Following this procedure, other methods like western blot can be performed in order to answer additional questions like the expression level of CatchER plus.After its development, this technique paved the way for researchers in the field of calcium signaling and imaging to explore ERSR calcium dynamics in mammalian cells. After watching this video you should have a good understanding of how to transfect cells and image them using CatchER plus.

monitoring-ersr-calcium-release-with-the-targeted-ca2-sensor-catcher

Research

JoVE Journal

Biology

Monitoring Actin Disassembly with Time-lapse Microscopy

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

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, ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

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 ...