Name: an iodide-yellow fluorescent protein-gap junction-intercellular communication assay
Uploaded: 2019-02-01T14:00:00
Description: Watch this Scientific Journal Video about An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay at JoVE.com

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2011-0023701, 2016R1D1A1A02937397, and 2018R1A6A1A03023718).

1. Generation of lentiviruses expressing YFPQL and SLC26A4
<ol>
	<li>Grow HEK293T human embryonic kidney cells to 80% confluency on 100 mm culture plates. Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin is the culture medium used throughout the protocol to maintain HEK293T and other cells mentioned below.</li>
	<li>Coat 6-well culture plates by adding 2 mL of 0.005% of sterile poly-L-lysine (PLL) solution to each well ...

<ol>
	<li>Goodenough, D. a., Goliger, J. a., Paul, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexins,+connexons,+and+Intercellular+Communication.">Connexins, connexons, and Intercellular Communication.</a> Annual Review of Biochemistry. 65, 475-502 (1996).</li><li>Upham, B. L., Weis, L. M., Trosko, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulated+Gap+Junctional+Intercellular+Communication+as+a+Biomarker+of+PAH+Epigenetic+Toxicity:+Structure-Function+Relationship.">Modulated Gap Junctional Intercellular Communication as a Biomarker of PAH Epigenetic Toxicity: Structure-Function Relationship.</a> Environmental Health Perspectives. 106, 975 (1998).</li><li>U, J. E. T., Chang, C., Upham, B., Wilson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+toxicology+as+toxicant-induced+changes+in+intracellular+signalling+leading+to+altered+gap+junctional+intercellular+communication.">Epigenetic toxicology as toxicant-induced changes in intracellular signalling leading to altered gap junctional intercellular communication.</a> Toxicology Letters. , 71-78 (1998).</li><li>Yamasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+disrupted+gap+junctional+intercellular+communications+in+detection+and+characterization+of+carcinogens.">Role of disrupted gap junctional intercellular communications in detection and characterization of carcinogens.</a> Mutation Research - Reviews in Genetic Toxicology. , (1996).</li><li>Yamasaki, H., 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=Gap+junctional+intercellular+communication+and+cell+proliferation+during+rat+liver+carcinogenesis.">Gap junctional intercellular communication and cell proliferation during rat liver carcinogenesis.</a> Environmental Health Perspectives. 101, 191-197 (1993).</li><li>Vinken, 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=Gap+junctional+intercellular+communication+as+a+target+for+liver+toxicity+and+carcinogenicity.">Gap junctional intercellular communication as a target for liver toxicity and carcinogenicity.</a> Critical Reviews in Biochemistry and Molecular Biology. 44, 201-222 (2009).</li><li>Fonseca, C. G., Green, C. R., Nicholson, L. F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+in+astrocytic+connexin+43+gap+junction+levels+may+exacerbate+generalized+seizures+in+mesial+temporal+lobe+epilepsy.">Upregulation in astrocytic connexin 43 gap junction levels may exacerbate generalized seizures in mesial temporal lobe epilepsy.</a> Brain Research. 929 (1), 105-116 (2002).</li><li>Garbelli, R., 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=Expression+of+connexin+43+in+the+human+epileptic+and+drug-resistant+cerebral+cortex.">Expression of connexin 43 in the human epileptic and drug-resistant cerebral cortex.</a> Neurology. 76 (10), 895-902 (2011).</li><li>Schulz, R., 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=Connexin+43+is+an+emerging+therapeutic+target+in+ischemia/reperfusion+injury,+cardioprotection+and+neuroprotection.">Connexin 43 is an emerging therapeutic target in ischemia/reperfusion injury, cardioprotection and neuroprotection.</a> Pharmacology and Therapeutics. 153, 90-106 (2015).</li><li>Sarrouilhe, D., Dejean, C., Mesnil, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+gap+junction+channels+in+the+pathophysiology+of+migraine+with+aura.">Involvement of gap junction channels in the pathophysiology of migraine with aura.</a> Frontiers in Physiology. , (2014).</li><li>Patel, S. 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=Gap+junction+inhibition+prevents+drug-induced+liver+toxicity+and+fulminant+hepatic+failure.">Gap junction inhibition prevents drug-induced liver toxicity and fulminant hepatic failure.</a> Nature Biotechnology. 30 (2), 179-183 (2012).</li><li>Kandyba, E. E., Hodgins, M. B., Martin, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+living+skin+equivalent+amenable+to+live-cell+imaging:+analysis+of+the+roles+of+connexins+in+the+epidermis.">A murine living skin equivalent amenable to live-cell imaging: analysis of the roles of connexins in the epidermis.</a> Journal of Investigative Dermatology. 128 (4), 1039-1049 (2008).</li><li>Upham, B. L., 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=Differential+roles+of+2+,+6+,+and+8+carbon+ceramides+on+the+modulation+of+gap+junctional+communication+and+apoptosis+during+carcinogenesis.">Differential roles of 2 , 6 , and 8 carbon ceramides on the modulation of gap junctional communication and apoptosis during carcinogenesis.</a> Cancer Letters. 191, 27-34 (2003).</li><li>Upham, B. L., 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=Structure-activity-dependent+regulation+of+cell+communication+by+perfluorinated+fatty+acids+using+in+vivo+and+in+vitro+model+systems.">Structure-activity-dependent regulation of cell communication by perfluorinated fatty acids using in vivo and in vitro model systems.</a> Environmental Health Perspectives. 117 (4), 545-551 (2009).</li><li>Weis, L. 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=Bay+or+Baylike+Regions+of+Polycyclic+Aromatic+Hydrocarbons+Were+Potent+Inhibitors+of+Gap+Intercellular+Communication.">Bay or Baylike Regions of Polycyclic Aromatic Hydrocarbons Were Potent Inhibitors of Gap Intercellular Communication.</a> Environmental Health Perspectives. 106 (1), 17-22 (1998).</li><li>el-Fouly, M. H., Trosko, J. E., Chang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrape-Loading+and+Dye+Transfer:+A+Rapid+and+Simple+Technique+to+Study+Gap+Junctional+Intercellular+Communication.">Scrape-Loading and Dye Transfer: A Rapid and Simple Technique to Study Gap Junctional Intercellular Communication.</a> Experimental Cell Research. 168 (2), 422-430 (1987).</li><li>Wade, M. H., Trosko, J. E., Schindler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobleaching+Assay+of+Gap+Junction-+Mediated+Communication+Between+Human+Cells.">Photobleaching Assay of Gap Junction- Mediated Communication Between Human Cells.</a> Advancement of Science. 232 (4749), 525-528 (2010).</li><li>Abbaci, 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=In+vitro+characterization+of+gap+junctional+intercellular+communication+by+gap-FRAP+technique.">In vitro characterization of gap junctional intercellular communication by gap-FRAP technique.</a> Proceedings of SPIE. , 585909 (2005).</li><li>Neyton, J., Trautmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-channel+currents+of+an+intercellular+junction.">Single-channel currents of an intercellular junction.</a> Nature. 317 (6035), 331-335 (1985).</li><li>Wilders, R., Jongsma, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+the+dual+voltage+clamp+method+in+assaying+conductance+and+kinetics+of+gap+junction+channels.">Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels.</a> Biophysical Journal. 63 (4), 942-953 (1992).</li><li>Lee, J. Y., Choi, E. J., Lee, 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+new+high-throughput+screening-compatible+gap+junctional+intercellular+communication+assay.">A new high-throughput screening-compatible gap junctional intercellular communication assay.</a> BMC Biotechnology. 15 (1), 1-9 (2015).</li><li>Galietta, L. J. V., Haggie, P. M., Verkman, A. 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The I-YFP-GJIC assay can be used for HTS because it is robust, rapid, and inexpensive. An HTS assay is considered robust if the Z&#39;-factor is above 0.525. See Zhang et al. for a description of the statistical analysis used to assessing the suitability of HTS assays25. When LN215 cells were used, the Z&#39;-factor was &#62;0.5 without any assay optimization. If other cell types are used in the assay and its Z&#39;-factor is &#60;0.5, the robustness can be improved by exte...

Gap junctions (GJs) act as intercellular channels to allow the diffusion of small molecules of &#60;1 kDa such as nutrients, metabolites, and signaling molecules between adjacent cells. The junctional elements include a hemichannel or connexon in each cell, and each connexon constitutes six connexins (Cxs)1. GJs and Cxs have been used in toxicology assays of carcinogens such as polycyclic aromatic hydrocarbons (PAH), which are GJ inhibitors2,3,4. Disrupted GJIC has been associated with nongenotoxic carcinogenesis5,6. As a potential therapeutic target, GJ involvement has been reported in particular subtypes of seizures7,8, protection from cardiac and brain ischemia/reperfusion injury9, migraine with aura10, drug-induced liver injury6,11, and wound healing12. High-throughput screening (HTS) assays are required to identify GJ-modulating chemicals or antibodies for drug discovery, for toxicology assays, and to identify novel cellular regulators of GJ activity. HTS assays can also be used to investigate structure-activity relationships of GJ modulators2,13,14,15.
Some GJIC assays include dye transfer or dual patch clamp techniques. Lucifer yellow CH (LY) and calcein acetoxymethyl ester (calcein-AM) have been used in dye-transfer assays. Cells are not permeable to LY, which is introduced by microinjection, scrape loading, or electroporation. Once inside the cell, LY spreads into neighboring cells via GJs and GJ activity is assayed by the extent of the LY migration16. Calcein-AM assays usually involve gap-fluorescence recovery after photobleaching17,18. Calcein-AM is a cell-permeant dye that is converted intracellularly into impermeable calcein by an intrinsic esterase. The assay requires a confocal microscope to observe the transfer of calcein-AM into a cell from those surrounding it following laser photobleaching. If functional GJs are present, calcein-AM in adjacent cells enters the photobleached cells and the fluorescence is recovered. GJ activity is assayed by the extent of fluorescence recovery of the photobleached cells. Dye-transfer assays are laborious and time consuming or have low sensitivities. Dual patch clamping is an electrophysiological method that measures junctional conductance. It is relatively sensitive, with a direct dependence of conductance on the number of open GJs19; however, it is technically demanding, time consuming, and expensive20. The I-YFP-GJIC assay was developed for use in HTS.
Figure 1 illustrates the components and steps of the I-YFP GJIC assay, which utilizes acceptor cells expressing an iodide-sensitive YFP variant bearing H148Q and I152L (YFPQL) and donor cells expressing an iodide transporter (SLC26A4)21. The two mutations carried by YFPQL allow quenching of fluorescence by iodide22. Iodides are added to co-cultured acceptor and donor cells; they do not enter the acceptor cells, but are taken up by the SLC26A4 transporters present on the donor cells. Iodides in the donor cells diffuse through functioning GJs into adjacent acceptor cells where they quench the YFPQL fluorescence. If GJs are closed or blocked by inhibitors, iodide cannot enter the acceptor cells to quench the fluorescence. The YFPQL quenching rate reflects GJ activity. The I-YFP GJIC assay procedure is neither complicated nor time consuming. It is compatible with HTS and can be used to test the effects of a large number of compounds on GJ activity in a relatively short period. It requires only acceptor and donor cells, and two balanced salt solutions. The protocol described below is based on LN215 cells whose major Cx is Cx4321. The LN215-YFPQL receptor and LN215-I&#8722; donor cells were generated by transduction with lentiviruses expressing YFPQL or SLC26A421,23.

<table border="0" cellpadding="0" cellspacing="0" style="width:629px;" width="630">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">96-well plate</td>
			<td style="width:72px;">SPL</td>
			<td style="width:123px;">30096</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Calcium chloride (CaCl2)</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">C5670</td>
			<td>I-solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">D-(+)-Glucose</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">G7021</td>
			<td>C-solution, I-solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:72px;">sigma</td>
			<td style="width:123px;">276855</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">HEPES</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">RES6003H-B7</td>
			<td>C-solution, I-solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Lipofectamine 2000</td>
			<td style="width:72px;">Invitrogen</td>
			<td style="width:123px;">11668-027</td>
			<td>transfection reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">Magnesium chloride hexahydrate (MgCl2 6H2O)</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">M2393</td>
			<td>C-solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">Microplate reader&#160;</td>
			<td style="width:72px;">BMG LabTech&#160;</td>
			<td style="width:123px;">POLARstar Omega 415-1618</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMD2.G&#160;</td>
			<td style="width:72px;">Addgene</td>
			<td>#12259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Polybrene</td>
			<td style="width:72px;">sigma</td>
			<td style="width:123px;">H9268</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Poly-L-lysine solution</td>
			<td style="width:72px;">sigma</td>
			<td style="width:123px;">P4707</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Potassium chloride (KCl)</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">P5405</td>
			<td>C-solution, I-solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">psPAX2&#160;</td>
			<td style="width:72px;">Addgene</td>
			<td style="width:123px;">&#160;#12260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Puromycin Dihydrochloride</td>
			<td style="width:72px;">sigma</td>
			<td style="width:123px;">P8833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Sodium chloride (NaCl)</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">S5886</td>
			<td>C-solution, I-solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Sodium hyroxide (NaOH)</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">S2770</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Sodium Iodide (NaI)</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">383112</td>
			<td>I-solution</td>
		</tr>
	</tbody>
</table>

an iodide-yellow fluorescent protein-gap junction-intercellular communication assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Twenty-nine 96-well culture plates were used to screen 2,320 chemicals to identify novel GJIC modulators by I-YFP GJIC assay using the LN215-YFPQL and LN215-I&#8722; cells. The results obtained with a representative plate are shown in Figure 2. The percentage of YFP fluorescence in each well is shown as a line graph in Figure 2A and the percentage of GJIC activity in each well is shown in th...

Watch this Scientific Journal Video about An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay at JoVE.com

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

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Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...