Name: measurement of calcium fluctuations within the sarcoplasmic reticulum of cultured smooth muscle cells using fret-based confocal imaging
Uploaded: 2016-06-20T13:00:00
Description: Watch this Scientific Journal Video about Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging at JoVE.com

This work was supported by funding from the Canadian Institutes of Health Research [MOP-1112666 to CVB &#38; ME].

Ethics statement: All experiments and procedures were conducted in agreement with the Laboratory Biosafety guidelines of the University of British Columbia, Vancouver, Canada.
1. Preparation of 35 mm Glass-bottom Culture Dishes for FRET-based Confocal Microscopy
<ol>
	<li>Prepare plates using the following procedure 36-48 hr prior to experiments.</li>
	<li>Retrieve chosen gelatinous protein mixture and 0.25% trypsin-EDTA from -20 &#176;C storage and keep them in mobile ice bath until use. 
	NOTE: The coatin...

<ol>
	<li>Hennings, H., Michael, D., Cheng, C., Steinert, P., Holbrook, K., Yuspa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+regulation+of+growth+and+differentiation+of+mouse+epidermal+cells+in+culture.">Calcium regulation of growth and differentiation of mouse epidermal cells in culture.</a> Cell. 19 (1), 245-254 (1980).</li><li>Whitfield, J. F., 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=The+roles+of+calcium+and+cyclic+AMP+in+cell+proliferation.">The roles of calcium and cyclic AMP in cell proliferation.</a> Ann N Y Acad Sci. 339 (1), 216-240 (1980).</li><li>Nicotera, P., Orrenius, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+calcium+in+apoptosis.">The role of calcium in apoptosis.</a> Cell Calcium. 23 (2-3), 173-180 (1998).</li><li>Wei, C., Wang, X., Chen, M., Ouyang, K., Song, L. S., Cheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+flickers+steer+cell+migration.">Calcium flickers steer cell migration.</a> Nature. 457 (7231), 901-905 (2009).</li><li>Ashby, M. C., Tepikin, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+calcium+and+the+functions+of+intracellular+organelles.">ER calcium and the functions of intracellular organelles.</a> Sem Cell Dev Biol. 12 (1), 11-17 (2001).</li><li>Mattson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+calcium+and+Alzheimer's+disease:+in+a+state+of+flux.">ER calcium and Alzheimer's disease: in a state of flux.</a> Sci signal. 3 (114), (2010).</li><li>Rutkowski, D. T., Kaufman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+trip+to+the+ER:+coping+with+stress.">A trip to the ER: coping with stress.</a> Trends Cell Biol. 14 (1), (2004).</li><li>Schroder, M., Kaufman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+stress+and+the+unfolded+protein+response.">ER stress and the unfolded protein response.</a> Mutat Res. 569 (1-2), 29-63 (2005).</li><li>Palmer, A. E., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+calcium+signaling+using+genetically+targetable+fluorescent+indicators.">Measuring calcium signaling using genetically targetable fluorescent indicators.</a> Nat Protoc. 1 (3), 1057-1065 (2006).</li><li>Poburko, D., Liao, C. H., van Breemen, C., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+regulation+of+sarcoplasmic+reticulum+Ca2++content+in+vascular+smooth+muscle+cells.">Mitochondrial regulation of sarcoplasmic reticulum Ca2+ content in vascular smooth muscle cells.</a> Circ Res. 104 (1), 104-112 (2009).</li><li>Esfandiarei, M., Fameli, N., Choi, Y. Y., Tehrani, A. Y., Hoskins, J. G., van Breemen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Waves+of+calcium+depletion+in+the+sarcoplasmic+reticulum+of+vascular+smooth+muscle+cells:+an+inside+view+of+spatiotemporal+Ca2++regulation.">Waves of calcium depletion in the sarcoplasmic reticulum of vascular smooth muscle cells: an inside view of spatiotemporal Ca2+ regulation.</a> PloS one. 8 (2), e55333 (2013).</li><li>Cobbold, P. H., Rink, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+and+bioluminescence+measurement+of+cytoplasmic+free+calcium.">Fluorescence and bioluminescence measurement of cytoplasmic free calcium.</a> Biochem J. 248 (2), 313-328 (1987).</li><li>Park, M. K., Ashby, M. C., Erdemli, G., Petersen, O. H., Tepikin, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perinuclear,+perigranular+and+sub-plasmalemmal+mitochondria+have+distinct+functions+in+the+regulation+of+cellular+calcium+transport.">Perinuclear, perigranular and sub-plasmalemmal mitochondria have distinct functions in the regulation of cellular calcium transport.</a> The EMBO journal. 20 (8), 1863-1874 (2001).</li><li>Lovett, J. L., Sibley, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+stores+in+Toxoplasma+gondii+govern+invasion+of+host+cells.">Intracellular calcium stores in Toxoplasma gondii govern invasion of host cells.</a> J Cell Sci. 116 (14), 3009-3016 (2003).</li><li>Kleinman, H. K., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+basement+membrane+matrix+with+biological+activity.">Matrigel: basement membrane matrix with biological activity.</a> Semin Cancer Biol. 15 (5), 378-386 (2005).</li><li>Palmer, A. E., 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=Ca2++indicators+based+on+computationally+redesigned+calmodulin-peptide+pairs.">Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs.</a> Chem Biol. 13 (5), 521-530 (2006).</li></ol>

This protocol describes the transfection of VSMCs with the D1SR adenovirus to study Ca2+ concentrations within the lumen of the SR. This method allows for the direct measurement of Ca2+ within this organelle as well as its movement into/out of the SR as a result of the application of different calcium-moving agents. This method has many advantages for investigators working towards a comprehensive understanding of how changes to SR and cytoplasmic Ca2+ levels affect overall cellular health...

Ca2+ is a very important ion found in abundance within every single cell in the body. It has significant roles in many different cellular functions, including growth, proliferation, migration, and apoptosis1-4. It is well established that Ca2+ can affect these processes through both direct and indirect actions, and therefore, changes to the normal intracellular concentrations of this ion can easily result in negative outcomes for affected cells. The SR is considered as the largest intracellular Ca2+ store within the cell5. Steady state levels of Ca2+ in both the SR and cytoplasm are normally maintained through constant flux into and out of the Ca2+-transporting channels of this organelle. The stressful conditions imposed upon cells due to any form of injury or disease are commonly associated with significant changes in SR Ca2+ that go on to have long-lasting effects on the health of the cell. In the most severe of cases, the inability of a stressed cell to recover and maintain steady state SR Ca2+ levels may even culminate in death by apoptosis6-8.
Current research into cellular Ca2+ dynamics is limited by the fact that few studies test organelle Ca2+ store content directly9-11. The most common practice instead involves measurement of cytoplasmic Ca2+ levels as indirect measurements of changes in SR Ca2+ content 12-14. In these experiments, Ca2+ is commonly induced to be released from the SR through the use of pharmacological agents causing the organelle&#39;s depletion (e.g. thapsigargin). Conclusions are then drawn with regard to changes to SR Ca2+ based on fluctuations in the cytoplasmic Ca2+ concentrations. Despite the ability remaining for investigators to draw such conclusions in a roundabout manner, this method of SR Ca2+ measurement is clearly an indirect way to glean such information, with many limitations concerning the interpretation of collected data. In order to bypass this clear restriction, it is necessary to measure the amount of Ca2+ found directly within the SR luminal network.
Vital to the final outcome of being able to directly record intraluminal SR Ca2+ levels are the cell culture tools and the Ca 2+ indicator used. For the data referenced in the current manuscript, it is important to note that VSMCs used came from a frozen cell line. Cells were cultured in Dulbecco&#8217;s Modified Eagle&#8217;s medium (DMEM) + 10% newborn calf serum (NCS)&#160;over passages 22-26 for the outlined experiments, incubated at a constant 37 &#176;C with 5% CO2 supply. Using the current method, for example, cells have been very successfully grown on a protein mixture that simulates the extracellular environment of many different types of tissue15. Important for success along the vein of SR Ca2+ research is the type of protein mixture used; in this case, a low growth factor variety was necessary to avoid components of this tool from affecting the regular Ca2+ signaling and movements constantly occurring within the tested cells. Following successful growth of test cells, the Ca2+ indicator must also be effectively introduced to these cultured cells. This has become possible by using an adenoviral vector that carries the SR-residing Ca2+ indicator D1SR. To achieve a high transfection efficiency, cells must be incubated with viral vectors for at least 36-48 hr prior to imaging. This preparation provides a reliable tool to measure Ca2+ transients within the SR lumen with high accuracy and reproducibility.
D1SR indicator used in this protocol is a modified variant of the D1ER Ca2+ indicator that was originally created by Dr. Roger Y. Tsien&#39;s laboratory at the University of California, San Diego, USA9,16. The original D1ER belongs to a second generation of genetically encoded Ca2+ indicators called cameleons that display Ca2+ sensitivities over a much broader range (0.5-160 &#181;M) as compared to previous Ca2+ indicators9,16. The new variant D1SR, a kind gift from Dr. Wayne Chen (University of Alberta, Canada), however, carries a mutant calsequestrin sequence (instead of a calreticulin sequence as in the original D1ER). The mutant calsequestrin has a reduced binding to Ca2+ that eliminates the issue of competing with endogenous calsequestrin in binding to Ca2+ within the SR lumen11. The D1SR indicator carries a truncated enhanced cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) that bind by a linker protein containing modified calmodulin (CaM) and M13 (the 26-residue peptide of myosin light-chain kinase that binds to CaM) sequences. The CaM-M13 sequence has been modified to prevent M13 from binding to endogenous calmodulin. Also, to ensure SR retention, a calsequestrin sequence has been added on the 5&#180; end of CFP11. When bound to Ca2+, the CaM-M13 domain goes through conformational changes that result in an increase in the energy transfer between the flanking CFP and YFP, which is recorded as an increase in the FRET signal intensity. On the other hand, when the concentration of SR luminal Ca2+ drops, the CaM-M13 domain goes through reverse conformational changes, resulting in a decrease in the energy transfer between the flanking CFP and YFP, and a significant drop in FRET signal intensity.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (high glucose, pyruvate)</td>
			<td>Life technologies</td>
			<td>11995-065</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix Growth Factor Reduced, 5mL</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Keep at -20&#176;C until right before use</td>
		</tr>
		<tr>
			<td>Newborn Calf Serum</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Keep at -20&#176;C until right before use</td>
		</tr>
		<tr>
			<td>35mm glass-bottomed plates</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL 0.2 &#956;M vented blue plug seal cap flask</td>
			<td>BD Falcon</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA Solution</td>
			<td>Life technologies</td>
			<td>25200056</td>
			<td>Keep at -20&#176;C until right before use</td>
		</tr>
		<tr>
			<td>50 mL Polypropylene Conical Tube</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>D1SR</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Gift</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8779</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo 4-AM</td>
			<td>Life technologies</td>
			<td>F-23917</td>
			<td>Keep at -20&#176;C until right before use</td>
		</tr>
		<tr>
			<td>1.5 mL Black Microcentrifuge Tubes</td>
			<td>Argos Technologies</td>
			<td>T7100BK</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP5 confocal microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite 2.6.3 (with FRET SE Wizard application)</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 5.0</td>
			<td>GraphPad Software, Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of calcium fluctuations within the sarcoplasmic reticulum of cultured smooth muscle cells using fret-based confocal imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

This section provides examples of the results that can be obtained using the described method to capture SR luminal Ca2+ concentration data, compared to the commonly employed manner of indirectly measuring changes to organelle Ca2+ store concentrations by observing fluctuations in cytoplasmic Ca2+.
Figure 1A shows a snapshot of a region of interest (ROI) of the SMC culture transf...

Watch this Scientific Journal Video about Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging at JoVE.com

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

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