Gap junctional intercellular communication (GJIC) is a vital cellular process required for maintenance of tissue homeostasis. In vitro assessment of GJIC represents valuable phenotypic endpoint that could be effectively utilized as an integral component in modern toxicity testing, drug screening or biomedical in vitro research. However, currently available methods for quantifying GJIC with higher-throughputs typically require specialized equipment, proprietary software and/or genetically engineered cell models. To overcome these limitations, we present here an innovative adaptation of traditional, fluorescence microscopy-based scrape loading-dye transfer (SL-DT) assay, which has been optimized to simultaneously evaluate GJIC, cell density and viability. This multiparametric method was demonstrated to be suitable for various multiwell microplate formats, which facilitates an automatized image acquisition. The assay workflow is further assisted by an open source-based software tools for batch image processing, analysis and evaluation of GJIC, cell density and viability. Our results suggest that this approach provides a simple, fast, versatile and cost effective way for in vitro high-throughput assessment of GJIC and other related phenotypic cellular events, which could be included into in vitro screening and assessment of pharmacologically and toxicologically relevant compounds.

Masaryk University Faculty of Science RECETOX Kamenice 5 CZ 62500 Brno Czech Republic

Zobrazit více v PubMed

Zhou JZ, Jiang JX. Gap junction and hemichannel-independent actions of connexins on cell and tissue functions–an update. FEBS Lett. 2014;588:1186–1192. doi: 10.1016/j.febslet.2014.01.001. PubMed DOI PMC

Guiza J, Barria I, Saez JC, Vega JL. Innexins: expression, regulation, and functions. Front. Physiol. 2018;9:Article 1414. doi: 10.3389/fphys.2018.01414. PubMed DOI PMC

Nielsen MS, et al. Gap junctions. Compr. Physiol. 2012;2:1981–2035. PubMed PMC

Aasen T, Mesnil M, Naus CC, Lampe PD, Laird DW. Gap junctions and cancer: communicating for 50 years. Nat. Rev. Cancer. 2016;16:775–788. doi: 10.1038/nrc.2016.105. PubMed DOI PMC

Kurtenbach S, Kurtenbach S, Zoidl G. Gap junction modulation and its implications for heart function. Front. Physiol. 2014;5:Article 82. doi: 10.3389/fphys.2014.00082. PubMed DOI PMC

Freund-Michel V, Muller B, Marthan R, Savineau J-P, Guibert C. Expression and role of connexin-based gap junctions in pulmonary inflammatory diseases. Pharmacol. Ther. 2016;164:105–119. doi: 10.1016/j.pharmthera.2016.04.004. PubMed DOI

Kidder GM, Cyr DG. Roles of connexins in testis development and spermatogenesis. Semin. Cell Dev. Biol. 2016;50:22–30. doi: 10.1016/j.semcdb.2015.12.019. PubMed DOI

Winterhager E, Kidder GM. Gap junction connexins in female reproductive organs: implications for women’s reproductive health. Hum. Reprod. Update. 2015;21:340–352. doi: 10.1093/humupd/dmv007. PubMed DOI

Mesnil M, et al. An update on minding the gap in cancer. Biochim. Biophys. Acta. Biomembr. 2018;1860:237–243. doi: 10.1016/j.bbamem.2017.06.015. PubMed DOI

Tschernig T. Connexins and gap junctions in cancer of the urinary tract. Cancers (Basel). 2019;11:704. doi: 10.3390/cancers11050704. PubMed DOI PMC

Willebrords J, Maes M, Crespo Yanguas S, Vinken M. Inhibitors of connexin and pannexin channels as potential therapeutics. Pharmacol. Ther. 2017;180:144–160. doi: 10.1016/j.pharmthera.2017.07.001. PubMed DOI PMC

Laird DW, Lampe PD. Therapeutic strategies targeting connexins. Nat. Rev. Drug Discov. 2018;17:905–921. doi: 10.1038/nrd.2018.138. PubMed DOI PMC

Zheng W, Thorne N, McKew JC. Phenotypic screens as a renewed approach for drug discovery. Drug Discov. Today. 2013;18:1067–1073. doi: 10.1016/j.drudis.2013.07.001. PubMed DOI PMC

Upham, B. L., Sovadinova, I. & Babica, P. Gap junctional intercellular communication: A functional biomarker to assess adverse effects of toxicants and toxins, and health benefits of natural products. J. Vis. Exp. 2016, 10.3791/54281 (2016). PubMed PMC

Babica, P., Sovadinová, I. & Upham, B. L. Scrape loading/dye transfer assay. In Gap Junction Protocols (eds. Vinken, M. & Johnstone, S. R.) 1437, 133–144 (Springer New York, 2016).

Elfouly MH, et al. Scrape-loading and dye transfer: A rapid and simple technique to study gap junctional intercellular communication. Exp. Cell Res. 1987;168:422–430. doi: 10.1016/0014-4827(87)90014-0. PubMed DOI

Upham, B. L. Role of integrative signaling through gap junctions in toxicology. In Current Protocols in Toxicology (ed. Maines, M. D.) (2011). PubMed PMC

Picoli C, et al. Human connexin channel specificity of classical and new gap junction inhibitors. J. Biomol. Screen. 2012;17:1339–1347. doi: 10.1177/1087057112452594. PubMed DOI

Abbaci M, Barberi-Heyob M, Blondel W, Guillemin F, Didelon J. Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication. Biotechniques. 2008;45:33–62. doi: 10.2144/000112810. PubMed DOI

Opsahl H, Rivedal E. Quantitative determination of gap junction intercellular communication by scrape loading and image analysis. Cell Adhes. Commun. 2000;7:367–375. doi: 10.3109/15419060009109019. PubMed DOI

Hofgaard JP, Mollerup S, Holstein-Rathlou N-H, Nielsen MS. Quantification of gap junctional intercellular communication based on digital image analysis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2009;297:R243–R247. doi: 10.1152/ajpregu.00089.2009. PubMed DOI

Begandt D, et al. Gold nanoparticle-mediated (GNOME) laser perforation: a new method for a high-throughput analysis of gap junction intercellular coupling. J. Bioenerg. Biomembr. 2015;47:441–449. doi: 10.1007/s10863-015-9623-y. PubMed DOI

Liu J, et al. High-throughput measurement of gap junctional intercellular communication. Am. J. Physiol. Heart Circ. Physiol. 2014;306:H1708–13. doi: 10.1152/ajpheart.00110.2014. PubMed DOI

Ye N, Bathany C, Hua SZ. Assay for molecular transport across gap junction channels in one-dimensional cell arrays. Lab Chip. 2011;11:1096–1101. doi: 10.1039/c0lc00350f. PubMed DOI

Chen S, Lee LP. Non-invasive microfluidic gap junction assay. Integr. Biol. (Camb). 2010;2:130–138. doi: 10.1039/b919392h. PubMed DOI

Roemer E, Lammerich HP, Conroy LL, Weisensee D. Characterization of a gap-junctional intercellular communication (GJIC) assay using cigarette smoke. Toxicol. Lett. 2013;219:248–253. doi: 10.1016/j.toxlet.2013.03.028. PubMed DOI

Dukic AR, McClymont DW, Tasken K. A cell-based high-throughput assay for gap junction communication suitable for assessing connexin 43-Ezrin interaction disruptors using IncuCyte ZOOM. SLAS Discov. Adv. life Sci. R D. 2017;22:77–85. PubMed

Li Z, et al. Identification of gap junction blockers using automated fluorescence microscopy imaging. J. Biomol. Screen. 2003;8:489–499. doi: 10.1177/1087057103257309. PubMed DOI

Haq N, et al. A high-throughput assay for connexin 43 (Cx43, GJA1) gap junctions using codon-optimized aequorin. Assay Drug Dev. Technol. 2013;11:93–100. doi: 10.1089/adt.2012.469. PubMed DOI

Lee JY, Choi EJ, Lee J. A new high-throughput screening-compatible gap junctional intercellular communication assay. BMC Biotechnol. 2015;15:90. doi: 10.1186/s12896-015-0211-3. PubMed DOI PMC

Goldenberg RCS, et al. Modulation of gap junction mediated intercellular communication in TM3 Leydig cells. J. Endocrinol. 2003;177:327–335. doi: 10.1677/joe.0.1770327. PubMed DOI

Ziambaras K, Lecanda F, Steinberg TH, Civitelli R. Cyclic stretch enhances gap junctional communication between osteoblastic cells. J. Bone Miner. Res. 1998;13:218–228. doi: 10.1359/jbmr.1998.13.2.218. PubMed DOI

Meda P. Assaying the molecular permeability of connexin channels. Methods Mol. Biol. 2001;154:201–224. PubMed

Elfgang C, et al. Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J. Cell Biol. 1995;129:805–817. doi: 10.1083/jcb.129.3.805. PubMed DOI PMC

Krutovskikh VA, Piccoli C, Yamasaki H. Gap junction intercellular communication propagates cell death in cancerous cells. Oncogene. 2002;21:1989–1999. doi: 10.1038/sj.onc.1205187. PubMed DOI

Ramirez CN, Antczak C, Djaballah H. Cell viability assessment: toward content-rich platforms. Expert Opin. Drug Discov. 2010;5:223–233. doi: 10.1517/17460441003596685. PubMed DOI PMC

King MA. Detection of dead cells and measurement of cell killing by flow cytometry. J. Immunol. Methods. 2000;243:155–166. doi: 10.1016/S0022-1759(00)00232-5. PubMed DOI

Esseltine JL, Laird DW. Next-generation connexin and pannexin cell biology. Trends Cell Biol. 2016;26:944–955. doi: 10.1016/j.tcb.2016.06.003. PubMed DOI

Saez JC, Leybaert L. Hunting for connexin hemichannels. FEBS Lett. 2014;588:1205–1211. doi: 10.1016/j.febslet.2014.03.004. PubMed DOI

Saez JC, et al. Cell membrane permeabilization via connexin hemichannels in living and dying cells. Exp. Cell Res. 2010;316:2377–2389. doi: 10.1016/j.yexcr.2010.05.026. PubMed DOI

Schalper KA, Palacios-Prado N, Orellana JA, Saez JC. Currently used methods for identification and characterization of hemichannels. Cell Commun. Adhes. 2008;15:207–218. doi: 10.1080/15419060802014198. PubMed DOI

Le HT, et al. Gap junction intercellular communication mediated by connexin43 in astrocytes is essential for their resistance to oxidative stress. J. Biol. Chem. 2014;289:1345–1354. doi: 10.1074/jbc.M113.508390. PubMed DOI PMC

Tarzemany R, Jiang G, Jiang JX, Larjava H, Hakkinen L. Connexin 43 hemichannels regulate the expression of wound healing-associated genes in human gingival fibroblasts. Sci. Rep. 2017;7:14157. doi: 10.1038/s41598-017-12672-1. PubMed DOI PMC

Pollok S, et al. Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non-diabetic cells. J. Cell. Mol. Med. 2011;15:861–873. doi: 10.1111/j.1582-4934.2010.01057.x. PubMed DOI PMC

Retamal MA, et al. Cx43 hemichannels and gap junction channels in astrocytes are regulated oppositely by proinflammatory cytokines released from activated microglia. J. Neurosci. 2007;27:13781–13792. doi: 10.1523/JNEUROSCI.2042-07.2007. PubMed DOI PMC

Abudara V, et al. The connexin43 mimetic peptide Gap19 inhibits hemichannels without altering gap junctional communication in astrocytes. Front. Cell. Neurosci. 2014;8:306. doi: 10.3389/fncel.2014.00306. PubMed DOI PMC

Zanella F, Lorens JB, Link W. High content screening: seeing is believing. Trends Biotechnol. 2010;28:237–245. doi: 10.1016/j.tibtech.2010.02.005. PubMed DOI

Novakova K, Babica P, Adamovsky O, Blaha L. Modulation of gap-junctional intercellular communication by a series of cyanobacterial samples from nature and laboratory cultures. Toxicon. 2011;58:76–84. doi: 10.1016/j.toxicon.2011.05.006. PubMed DOI

Begandt D, Bintig W, Oberheide K, Schlie S, Ngezahayo A. Dipyridamole increases gap junction coupling in bovine GM-7373 aortic endothelial cells by a cAMP-protein kinase A dependent pathway. J. Bioenerg. Biomembr. 2010;42:79–84. doi: 10.1007/s10863-009-9262-2. PubMed DOI

Liu L, et al. The combination of three natural compounds effectively prevented lung carcinogenesis by optimal wound healing. PLoS One. 2015;10:e0143438. doi: 10.1371/journal.pone.0143438. PubMed DOI PMC

Kubincova P, et al. PAHs and endocrine disruption: Role of testicular gap junctional intercellular communication and connexins. Toxicol. Sci. 2019;169:70–83. PubMed

Sovadinova I, et al. Phosphatidylcholine specific PLC-induced dysregulation of gap junctions, a robust cellular response to environmental toxicants, and prevention by Resveratrol in a rat liver cell model. PLoS One. 2015;10:e0124454. doi: 10.1371/journal.pone.0124454. PubMed DOI PMC

Babica P, et al. Methoxychlor and vinclozolin induce rapid changes in intercellular and intracellular sSignaling in liver progenitor cells. Toxicol. Sci. 2016;153:174–185. doi: 10.1093/toxsci/kfw114. PubMed DOI PMC

Siegrist KJ, et al. Early mechanistic events induced by low molecular weight polycyclic aromatic hydrocarbons in mouselung epithelial cells: A role for eicosanoid signaling. Toxicol. Sci. 2019;169:180–193. doi: 10.1093/toxsci/kfz030. PubMed DOI PMC

Steuer A, Schmidt A, Laboha P, Babica P, Kolb JF. Transient suppression of gap junctional intercellular communication after exposure to 100-nanosecond pulsed electric fields. Bioelectrochemistry. 2016;112:33–46. doi: 10.1016/j.bioelechem.2016.07.003. PubMed DOI

Yuan D, et al. Propofol attenuated liver transplantation-induced acute lung injury via connexin43 gap junction inhibition. J. Transl. Med. 2016;14:194. doi: 10.1186/s12967-016-0954-1. PubMed DOI PMC

Rosenberg E, et al. Correlation of expression of connexin mRNA isoforms with degree of cellular differentiation. Cell Adhes. Commun. 1996;4:223–235. doi: 10.3109/15419069609010768. PubMed DOI

Rackauskas M, Verselis VK, Bukauskas FF. Permeability of homotypic and heterotypic gap junction channels formed of cardiac connexins mCx30.2, Cx40, Cx43, and Cx45. Am. J. Physiol. Heart Circ. Physiol. 2007;293:H1729–H1736. doi: 10.1152/ajpheart.00234.2007. PubMed DOI PMC

Beltramello M, et al. Permeability and gating properties of human connexins 26 and 30 expressed in HeLa cells. Biochem. Biophys. Res. Commun. 2003;305:1024–1033. doi: 10.1016/S0006-291X(03)00868-4. PubMed DOI

Cao F, et al. A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes. J. Cell Sci. 1998;111:31–43. PubMed

Eckert R. Gap-junctional single-channel permeability for fluorescent tracers in mammalian cell cultures. Biophys. J. 2006;91:565–579. doi: 10.1529/biophysj.105.072306. PubMed DOI PMC

Teubner B, et al. Functional expression of the new gap junction gene connexin47 transcribed in mouse brain and spinal cord neurons. J. Neurosci. 2001;21:1117–1126. doi: 10.1523/JNEUROSCI.21-04-01117.2001. PubMed DOI PMC

Nielsen PA, et al. Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1. J. Biol. Chem. 2002;277:38272–38283. doi: 10.1074/jbc.M205348200. PubMed DOI

Manthey D, et al. Intracellular domains of mouse connexin26 and -30 affect diffusional and electrical properties of gap junction channels. J. Membr. Biol. 2001;181:137–148. doi: 10.1007/s00232-001-0017-1. PubMed DOI

Teubner B, et al. Functional expression of the murine connexin 36 gene coding for a neuron-specific gap junctional protein. J. Membr. Biol. 2000;176:249–262. doi: 10.1007/s002320001094. PubMed DOI PMC

Manthey D, Bukauskas F, Lee CG, Kozak CA, Willecke K. Molecular cloning and functional expression of the mouse gap junction gene connexin-57 in human HeLa cells. J. Biol. Chem. 1999;274:14716–14723. doi: 10.1074/jbc.274.21.14716. PubMed DOI

Hu J, Cotgreave IA. Glutathione depletion potentiates 12-O-tetradecanoyl phorbol-13-acetate(TPA)-induced inhibition of gap junctional intercellular communication in WB-F344 rat liver epithelial cells: relationship to intracellular oxidative stress. Chem. Biol. Interact. 1995;95:291–307. doi: 10.1016/0009-2797(94)03568-S. PubMed DOI

Upham BL, Weis LM, Rummel AM, Masten SJ, Trosko JE. The effects of anthracene and methylated anthracenes on gap junctional intercellular communication in rat liver epithelial cells. Fundam. Appl. Toxicol. 1996;34:260–264. doi: 10.1006/faat.1996.0195. PubMed DOI

Lee KW, et al. Inhibition of cyclooxygenase-2 expression and restoration of gap junction intercellular communication in H-ras-transformed rat liver epithelial cells by caffeic acid phenethyl ester. Ann. N. Y. Acad. Sci. 2004;1030:501–507. doi: 10.1196/annals.1329.062. PubMed DOI

Jung J-W, et al. Effects of the histone deacetylases inhibitors sodium butyrate and trichostatin A on the inhibition of gap junctional intercellular communication by H2O2- and 12-O-tetradecanoylphorbol-13-acetate in rat liver epithelial cells. Cancer Lett. 2006;241:301–308. doi: 10.1016/j.canlet.2005.10.029. PubMed DOI

Leone A, Longo C, Trosko JE. The chemopreventive role of dietary phytochemicals through gap junctional intercellular communication. Phytochem. Rev. 2012;11:285–307. doi: 10.1007/s11101-012-9235-7. DOI

Babica P, Ctverackova L, Lencesova Z, Trosko JE, Upham BL. Chemopreventive agents attenuate rapid inhibition of gap junctional intercellularcommunication induced by environmental toxicants. Nutr. Cancer. 2016;68:827–837. doi: 10.1080/01635581.2016.1180409. PubMed DOI PMC

Roukos V, Pegoraro G, Voss TC, Misteli T. Cell cycle staging of individual cells by fluorescence microscopy. Nat. Protoc. 2015;10:334–348. doi: 10.1038/nprot.2015.016. PubMed DOI PMC

Reddel RR, et al. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res. 1988;48:1904–1909. PubMed

Yankaskas JR, et al. Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype. Am. J. Physiol. 1993;264:C1219–30. doi: 10.1152/ajpcell.1993.264.5.C1219. PubMed DOI

Mather JP. Establishment and characterization of 2 distinct mouse testicular epithelial-cell lines. Biol. Reprod. 1980;23:243–252. doi: 10.1095/biolreprod23.1.243. PubMed DOI

Mather JP, Rich KA, Perezinfante V, Haour F. The role of insulin in testicular cell-function in serum-free culture. Vitr. Tissue Cult. Assoc. 1982;18:274.

Tsao MS, Smith JD, Nelson KG, Grisham JW. A diploid epithelial cell line from normal adult rat liver with phenotypic properties of ‘oval’ cells. Exp. Cell Res. 1984;154:38–52. doi: 10.1016/0014-4827(84)90666-9. PubMed DOI

Coleman WB, et al. Evaluation of the differentiation potential of WB-F344 rat liver epithelial stem-like cells in vivo. Differentiation to hepatocytes after transplantation into dipeptidylpeptidase-IV-deficient rat liver. Am. J. Pathol. 1997;151:353–359. PubMed PMC

Hayashi T, Nomata K, Chang CC, Ruch RJ, Trosko JE. Cooperative effects of v-myc and c-Ha-ras oncogenes on gap junctional intercellular communication and tumorigenicity in rat liver epithelial cells. Cancer Lett. 1998;128:145–154. doi: 10.1016/S0304-3835(98)00060-3. PubMed DOI

De Feijter AW, et al. Infection of rat liver epithelial cells with v-Ha-ras: correlation between oncogene expression, gap junctional communication, and tumorigenicity. Mol. Carcinog. 1990;3:54–67. doi: 10.1002/mc.2940030203. PubMed DOI

Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods. 2012;9:671. doi: 10.1038/nmeth.2089. PubMed DOI PMC

Dibenzocyclooctadiene Lignans from Schisandra chinensis with Anti-Inflammatory Effects

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project

Applicability of Scrape Loading-Dye Transfer Assay for Non-Genotoxic Carcinogen Testing

Structure-Dependent Effects of Phthalates on Intercellular and Intracellular Communication in Liver Oval Cells