Abstract
Epithelial junction formation and maintenance are multistep processes that rely on the clustering of macromolecular complexes. These events are highly regulated by signalling pathways that involve Rho small GTPases. Usually, when analysing the contribution of different components of Rho-dependent pathways to cell–cell adhesion, the localisation of adhesion receptors at junctions is evaluated by immunofluorescence. However, we find that this method has limitations on the quantification (dynamic range), ability to detect partial phenotypes and to differentiate between the participation of a given regulatory protein in assembly and/or maintenance of cell–cell contacts.
In this chapter, we describe a suitable method, the aggregation assay, in which we adapted a quantitative strategy to allow objective and reproducible detection of partial phenotypes. Importantly, this methodology estimates the ability of cells to form junctions and their resistance to mechanical shearing forces (stabilisation).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fukata, M., and Kaibuchi, K. (2001) Rho-family GTPases in cadherin-mediated cell-cell adhesion. Nat Rev Mol Cell Biol 2, 887–897
Braga, V.M. (2002) Cell-cell adhesion and signalling. Curr Opin Cell Biol 14, 546–56
Braga, V.M., and Yap, A.S. (2005) The challenges of abundance: epithelial junctions and small GTPase signalling. Curr Opin Cell Biol 17, 466–74
Takeichi, M. (1977) Functional correlation between cell adhesive properties and some cell surface proteins. J Cell Biol 75, 464–474
Shoshani, L, Contreras, R.G., Roldán, M.L., Moreno, J., Lázaro, A., Balda, M.S., Matter, K., and Cereijido, M. (2005) The polarized expression of Na+,K + −ATPase in epithelia depends on the association between β-subunits located in neighboring cells. Mol Biol Cell 16, 1071–1081
Katsamba, P, Carroll, K., Ahlsen, G., Bahna, F., Vendome, J., Posy, S., Rajebhosale, M., Price, S., Jessell, T.M., Ben-Shaul, A., Shapiro, L.,and Honig, B.H. (2009) Linking molecular affinity and cellular specificity in cadherin-mediated adhesion. Proc Natl Acad Sci USA 106, 11594–11599
Wendeler, MW, Praus, M., Jung, R., Hecking, M., Metzig, C., and Gessner, R. (2004) Ksp-cadherin is a functional cell-cell adhesion molecule related to LI-cadherin. Exp Cell Res 294, 345–355
Tzima, E. (2006) Role of small GTPases in endothelial cytoskeletal dynamics and the shear stress response. Circ Res 98, 176–185
Kim, J-B, Islam, S., Kim, Y.J., Prudoff, R.S., Sass, K.M., Wheelock, M.J., and Johnson, K.R. (2000) N-Cadherin extracellular repeat 4 mediates epithelial to mesenchymal transition and increased motility. J Cell Biol 151, 1193–1206
Bardella, C, Costa, B., Maggiora, P., Patane’, S., Olivero, M., Ranzani, G.N., De Bortoli, M., Comoglio, P.M., and Di Renzo, M.F. (2004) Truncated RON tyrosine kinase drives tumor cell progression and abrogates cell-cell adhesion through E-cadherin transcriptional repression. Cancer Res 64, 5154–5161
Carrozzino, F, Soulié, P., Huber, D., Mensi, N., Orci, L., Cano, A., Féraille, E., and Montesano, R. (2005) Inducible expression of Snail selectively increases paracellular ion permeability and differentially modulates tight junction proteins. Am J Physiol Cell Physiol 289, C1002–C1014
Yanagisawa, M., and Anastasiadis, P.Z. (2006) p120 catenin is essential for mesenchymal cadherin–mediated regulation of cell motility and invasiveness. J Cell Biol 174, 1087–1096
Fang, WB, Ireton, R.C., Zhuang, G., Takahashi, T., Reynolds, A., and Chen, J. (2008) Overexpression of EPHA2 receptor destabilizes adherens junctions via a RhoA-dependent mechanism. J Cell Sci 121, 358–368
Ehrlich, J.S., Hansen, M.D.H., and Nelson, W.J. (2002) Spatio-temporal regulation of Rac1 localization and lamellipodia dynamics during epithelial cell-cell adhesion. Dev Cell 3: 259–270
Katuri, V, Tang, Y., Marshall, B., Rashid, A., Jogunoori, W., Volpe, E.A., Sidawy, A.N., Evans, S., Blay, J., Gallicano, G.I., Premkumar Reddy, E., Mishra, L., and Mishra, B. (2005) Inactivation of ELF//TGFβ signaling in human gastrointestinal cancer. Oncogene 24, 8012–8024
Chakraborty, S, Mitra, S., Falk,M.M., Caplan, S.H., Wheelock, M.J., Johnson, K.R., and Mehta, P.P.. (2010) E-cadherin differentially regulates the assembly of connexin43 and connexin32 into gap junctions in human squamous carcinoma cells. J Biol Chem 285, 10761–10776
Crooke, C.E, Pozzi, A., and Carpenter, G.F. (2009) PLC-γ1 regulates fibronectin assembly and cell aggregation. Exp Cell Res 315, 2207–2214
Govindarajan, R, Chakraborty, S., Johnson, K.E., Falk, M.M., Wheelock, M.J., Johnson, K.R., and Mehta, P.P. (2010) Assembly of Connexin43 into gap junctions is regulated differentially by E-cadherin and N-cadherin in rat liver epithelial cells. Mol Biol Cell 21, 4089–4107
Qin, Y, Capaldo, C., Gumbiner,B.M., and Macara, I.G. (2005) The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin. J Cell Biol 171, 1061–1071
Pappas, D.J. and Rimm, D.L. (2006) Direct interaction of the C-terminal domain of α-catenin and F-actin is necessary for stabilized cell-cell adhesion. Cell Comm Adh 13, 151–170
Sarrio, D, Palacios, J., Hergueta-Redondo, M., Gómez-López, G., Cano, A., and Moreno-Bueno, G. (2009) Functional characterization of E- and P-cadherin in invasive breast cancer cells. BMC Cancer 9, 74
De Corte, V, Bruyneel, E., Boucherie, C., Mareel, M., Vandekerckhove,J., and Gettemans, J. (2002) Gelsolin-induced epithelial cell invasion is dependent on Ras-Rac signaling. EMBO J 21, 6781–6790
Hatta, K., Okada, T.S., and Takeichi, M. (1985) A monoclonal antibody disrupting calcium-dependent cell-cell adhesion of brain tissues: possible role of its target antigen in animal pattern formation. Proc Natl Acad Sci USA 82, 2789–2793
Watt, F.M. (1994) Cultivation of human epidermal keratinocytes with a 3 T3 feeder layer. In: Celis, J.E. (ed) Cell biology: a laboratory handbook. Academic Press, London
Hodivala, K.J. and Watt, F.M. (1994) Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation. J Cell Biol 124, 589–600
Neelamegham, S., and Zygourakis, K. (1997) A Quantitative assay for intercellular aggregation. Ann Biomed Engineer 25, 180–189
Acknowledgements
We would like to thank the Wellcome Trust (VIP award and GR081357MA) and Cancer Research UK (C1282/A11980) for the generous support to the lab. We also thank Dr. A. Wheeler for images shown in Fig. 1.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Nola, S., Erasmus, J.C., Braga, V.M.M. (2012). Quantitative and Robust Assay to Measure Cell–Cell Contact Assembly and Maintenance. In: Rivero, F. (eds) Rho GTPases. Methods in Molecular Biology, vol 827. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-442-1_10
Download citation
DOI: https://doi.org/10.1007/978-1-61779-442-1_10
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-61779-441-4
Online ISBN: 978-1-61779-442-1
eBook Packages: Springer Protocols