Single-Molecular-Level Study of Claudin-1-Mediated Adhesion Tong Seng Lim, ²,‡ Sri Ram Krishna Vedula, § P. Jaya Kausalya, | Walter Hunziker, | and Chwee Teck Lim* ,§ Bioinformatics Institute, A * STAR (Agency for Science, Technology and Research), 30 Biopolis Street, Singapore 138671, NUS Graduate School for IntegratiVe Sciences and Engineering, 28 Medical DriVe, Singapore 117456, DiVision of Bioengineering and Department of Mechanical Engineering, National UniVersity of Singapore, 9 Engineering DriVe 1, Singapore 117576, and Institute of Molecular and Cell Biology, A * STAR (Agency for Science, Technology and Research), 61 Biopolis DriVe, Singapore 138673 ReceiVed August 7, 2007. In Final Form: September 28, 2007 Claudins are proteins that are selectively expressed at tight junctions (TJs) of epithelial cells where they play a central role in regulating paracellular permeability of solutes across epithelia. However, the role of claudins in intercellular adhesion and the mechanism by which they regulate the diffusion of solutes are poorly understood. Here, using single molecule force spectroscopy, the kinetic properties and adhesion strength of homophilic claudin-1 interactions were probed at the single-molecule level. Within the range of tested loading rates (10 3 -10 5 pN/s), our results showed that homophilic claudin-1 interactions have a reactive compliance of 0.363 ( 0.061 nm and an unstressed dissociation rate of 1.351 ( 1.312 s -1 . This is more than 100-fold greater than that of E-cadherin. The weak and short-lived interactions between claudin-1 molecules make them highly unstable and dynamic in nature. Such a dynamic interaction is consistent with a model where breaking and resealing of TJ strands regulate the paracellular diffusion of solutes. Introduction Intercellular junctions play an extremely important role in maintaining homeostasis in multicellular organisms. The epithelial intercellular adhesion complex consists of several components that include the adherens junctions (AJs), tight junctions (TJs), gap junctions, and desmosomes. TJs constitute the most apical junctional complex in epithelial cells. 1 Apart from acting as barriers to paracellular diffusion of extracellular solutes, 2 they also restrict the apical proteins from diffusing to the basolateral membrane, 3,4 regulate cell proliferation and differentiation, 5 and have been recently identified as coreceptors for hepatitis C virus. 6 In recent years, the adhesion behavior of E-cadherins (localizing at AJs) has been extensively investigated at the levels of both the cell and single molecule using flow chamber assay, 7 dual pipet assay, 8,9 cell aggregation assay, 10 atomic force microscopy (AFM), 11,12 and surface force analysis. 13 While the role of E-cadherins at AJs is well established and has been characterized in some detail, little is known about the strength of adhesion forces mediated by TJ proteins. Claudins (Cldns) comprise a protein family of 24 members in mammals that have been identified as major tetraspan transmembrane proteins localized at TJs. 14-16 Structurally, Cldns contain two extracellular loops and four transmembrane regions. The two extracellular loops of Cldns belonging to adjacent cells interact to form the paracellular TJ strands. Using cell aggregation assays, claudin-1 (Cldn1), claudin-2, and claudin-3 were found to exhibit Ca 2+ -independent adhesion activities. 17 However, the strength and kinetics of the interactions mediated by Cldns have not been characterized. In this study we have used single-molecule force spectroscopy to gain insight into the kinetics of Cldn- mediated interactions using full-length human Cldn1, tagged with GST (glutathione S-transferase) on the N-terminal end (GST-Cldn1), as a representative model. Our results show that dissociation of the homophilic Cldn1/ Cldn1 bond involves a single energy barrier in the range of loading rates between 10 3 and 10 5 pN/s. Comparison of interaction kinetics revealed that Cldn1 dissociates at a much faster rate than E-cadherins. This supports the fact that E-cadherin is more important in providing mechanical stability to epithelial cell junctions. The weak and short-lived interactions between claudin-1 molecules are highly unstable and dynamic in nature. The dynamic nature of these interactions is consistent with the model in which breaking and resealing of TJ strands regulate the paracellular diffusion of solutes across epithelia. 18,19 * To whom correspondence should be addressed. Phone: +65 6516 7801. E-mail: ctlim@nus.edu.sg. ² Bioinformatics Institute. ‡ NUS Graduate School for Integrative Sciences and Engineering. § National University of Singapore. | Institute of Molecular and Cell Biology. (1) Tsukita, S.; Furuse, M.; Itoh, M. Nat. ReV. 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Cell Sci. 2004, 117, 1247. 490 Langmuir 2008, 24, 490-495 10.1021/la702436x CCC: $40.75 © 2008 American Chemical Society Published on Web 12/21/2007