doi:10.1016/j.jmb.2008.06.009 J. Mol. Biol. (2008) 381, 681–691 Available online at www.sciencedirect.com Kinetics of Adhesion Mediated by Extracellular Loops of Claudin-2 as Revealed by Single-Molecule Force Spectroscopy Tong Seng Lim 1,2 , Sri Ram Krishna Vedula , Walter Hunziker and Chwee Teck Lim ⁎ Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671 NUS Graduate School for Integrative Sciences and Engineering (NGS), Centre for Life Sciences (CeLS), #05-01, 28 Medical Drive, Singapore 117456 Division of Bioengineering and Department of Mechanical Engineering, Engineering Drive 1, National University of Singapore, Singapore 117576 Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore 138673 Received 22 April 2008; received in revised form 27 May 2008; accepted June 2008 Available online 10 June 2008 Edited by K. Kuwajima Claudins (Cldns) comprise a large family of important transmembrane proteins that localize at tight junctions where they play a central role in regulating paracellular transportation of solutes across epithelia. However, molecular interactions occurring between the extracellular domains of these proteins are poorly understood. Here, using atomic force microscopy, the adhesion strength and kinetic properties of the homophilic interactions between the two extracellular loops of Cldn2 (C2E1or C2E2) and full-length Cldn2 were characterized at the level of single molecule. Results show that while the first extracellular loop is sufficient for Cldn2/Cldn2 transinteraction, the second extracellular loop does not interact with the fulllength Cldn2, with the first extracellular loop, or with itself. Furthermore, within the range of loading rates probed (102–104 pN/s), dissociation of Cldn2/Cldn2 and C2E1/C2E1 complexes follows a two-step energy barrier model. The difference in activation energy for the inner and outer barriers of Cldn2/Cldn2 and C2E1/C2E1 dissociation was found to be 0.26 and 1.66 kBT, respectively. Comparison of adhesion kinetics further revealed that Cldn2/Cldn2 dissociates at a much faster rate than C2E1/C2E1, indicating that the second extracellular loop probably has an antagonistic effect on the kinetic stability of Cldn2-mediated interactions. These results provide an insight into the importance of the first extracellular loop in trans-interaction of Cldn2-mediated adhesion. © 2008 Elsevier Ltd. All rights reserved. Keywords: claudins; tight junctions; cell–cell adhesion; molecular force spectroscopy; atomic force microscopy Introduction Tight junctions (TJs) form a continuous belt of intercellular contacts in the apical region of epithelial monolayers. Their primary function is to regulate the *Corresponding author. E-mail address: ctlim@nus.edu.sg. Abbreviations used: Cldn, claudin; TJ, tight junction; AFM, atomic force microscopy; MC, Monte Carlo. paracellular transport of solutes across epithelia. The selective permeability of TJs largely results from a family of transmembrane proteins called claudins (Cldns).1,2,3 With the use of dual pipette and cell aggregation assays, Cldn1 and Cldn2 were found to exhibit Ca 2+ -independent adhesion activities. 4,5 However, little is known about the strength and kinetics of the interactions mediated by Cldns. Structurally, Cldns consist of four transmembrane helices,2 a short cytoplasmic N-terminal sequence, two extracellular loops, and an intracellular C-terminus 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. 682 that binds to cytoplasmic proteins through a PDZ motif.6 The two extracellular loops of Cldns of adjacent cells trans-interact to form the paracellular TJ strands. It has previously been shown that the first extracellular loop of Cldn2,7,8 Cldn4,8,9,10 Cldn5,11 Cldn7,12 Cldn8,13 Cldn15,9,14 Cldn16,15 and Cldn1916,17 confers charge-selective paracellular permeability to epithelial monolayers while the second extracellular loop acts as a receptor for a bacterial toxin for Cldn318 and Cldn4.19 However, the interaction kinetics of the individual extracellular loops at the molecular level remains unclear. In this study, we have used singlemolecule force spectroscopy to probe the molecular interactions between recombinant N-terminal glutathione S-transferase (GST)-tagged full-length human Cldn2 (GST-Cldn2) and the two extracellular loops of Cldn2 (GST-C2E1 and GST-C2E2) to gain an insight into the contribution of the individual extracellular loops to the overall adhesion kinetics. Our results show that the first extracellular loop of Cldn2 is the major determinant of trans-interactions involving Cldn2. Dissociation of homophilic Cldn2/Cldn2 and C2E1/C2E1 complexes follow a two-energy-barrier model within the range of loading rates probed (102–104 pN/s). Comparison of interaction kinetics further revealed that Cldn2/ Cldn2 dissociates at a much faster rate than C2E1/ C2E1, implying that the second extracellular loop has an antagonistic effect on the kinetic stability of Cldn2-mediated adhesions. Results Measurement of Cldn2/Cldn2 and C2E1/C2E1 interaction forces Trans-interactions between full-length human Cldn2 (Cldn2/Cldn2) or between first extracellular loops of Cldn2 (C2E1/C2E1) were characterized at the level of single molecule using atomic force microscopy (AFM) (Fig. 1).20,21,22 The interaction was established by bringing GST-Cldn2 (or GST-C2E1) functionalized cantilever in close contact to a glass cover slip coated with GST-Cldn2 (or GST-C2E1) (see Materials and Methods). The functionalization of the tips and cover slips was confirmed using mouse anti-Cldn2 primary antibody (Abnova, Taiwan) and Alexa-488-labeled goat anti-mouse secondary antibody (Molecular Probes, Invitrogen). Confocal images showed that GST-Cldn2 was efficiently coupled to the AFM tips and cover slips (Fig. 2). In single-molecule force spectroscopy experiments, contact force and contact time are crucial for measuring discrete de-adhesion forces at molecular resolution.21,23,24 When a contact force of 200 pN and contact time of ms were used, b25% of the force–distance curves showed bond rupture events. On the basis of Poisson statistics,25 the low frequency of these de-adhesion events ensured a N 86% probability of the rupture being due to a single bond. Upon retraction of the cantilever, force as a function of pulling distance was recorded (Fig. Adhesion Kinetics of Claudin-2 Extracellular Loops Fig. 1. Schematic of the AFM experimental setup. Recombinant GST-Cldn2 or GST-C2E1 was linked to the AFM tip or immobilized on glass cover slip using the linker APTES-BS3-AntiGST (see Materials and Methods for details). GST-Cldn2 or GST-C2E1 immobilized on glass cover slip was probed using these functionalized tips. The arrow indicates the direction of pulling in the AFM experiment. GST, glutathione S-transferase; Cldn2, claudin-2; C2E1, first extracellular loop of Cldn2; APTES, 3aminopropyltriethoxysilane; BS3, bis(sulfosuccinimidyl) suberate; AntiGST, antibody targeting GST. 3a).26 For each reproach velocity, hundreds of force– distance curves (n N 500) were collected and analyzed to extract rupture force, F, and loading rate, rf (Fig. 3b). The data obtained were subsequently pooled into histograms to analyze the frequency of adhesion events for different interactions (Table 1; Fig. 4). Binding was specific because adhesion frequency was significantly reduced in control experiments performed using AFM tips functionalized with only anti-GST antibody. Furthermore, blocking experiments performed using antibody specifically targeting the first extracellular loop of Cldn2 significantly reduced the binding frequency (Table 1; Fig. 4). The low frequency of interaction between Cldn1 and Cldn2 (Cldn1/Cldn2 or Cldn2/ Cldn1, Table 1; Fig. 4) demonstrated that they not trans-interact, which is consistent with previous findings.27 The first extracellular loop of Cldn2 is sufficient for promoting trans-interactions Since Cldn2 consists of two extracellular loops (C2E1 and C2E2), the interactions observed between full-length Cldn2/Cldn2 could have resulted from interactions between two first extracellular loops (C2E1/C2E1), two second extracellular loops (C2E2/C2E2), or one first extracellular loop and another second extracellular loop (C2E1/C2E2) of apposing Cldn2 molecules. Histogram depicting the distribution of C2E1/C2E1 interaction forces demonstrated that the first extracellular loop can trans-interact with itself (C2E1_C2E1, Table 1; Fig. 4). Low adhesion frequency of C2E2/C2E2 interactions and significant reduction in Cldn2/Cldn2 interactions in the presence of an antibody targeting the first extracellular loop indicated that C2E2/C2E2 not trans-interact (C2E2_C2E2 and Cldn2_Cldn2_Ab, 683 Adhesion Kinetics of Claudin-2 Extracellular Loops Fig. 2. Confocal images of silanized AFM cantilevers functionalized (a) with GST-Cldn2 and (b) without GST-Cldn2. Confocal images of silanized glass cover slip functionalized (c) with GST-Cldn2 and (d) without GST-Cldn2. All images were taken after the cantilevers/tips were incubated with anti-Cldn2 primary antibody and Alexa-488-labeled secondary antibody (see Materials and Methods for details). Images were acquired under similar conditions (pixel dwell time, laser power, and gain). The scale bar represents 50 μm. Table 1; Fig. 4). The extremely low adhesion frequency of C2E1 with C2E2 and with Cldn2 incubated with antibody against the first extracellular loop suggested that first and second extracellular loops not trans-interact (C2E1_C2E2, C2E2_C2E1, and C2E1_Cldn2_Ab, Table 1; Fig. 4). Furthermore, it was observed that only C2E1 but not C2E2 can compete for the interactions between Cldn2/Cldn2 (Cldn2_Cldn2_C2E1, Cldn2_Cldn2_C2E2, Table 1; Fig. 4), suggesting that the first extracellular loop is sufficient to promote the trans-interactions of Cldn2/Cldn2. Extraction of the kinetic parameters of Cldn2/Cldn2 and C2E1/C2E1 interactions Biophysical parameters characterizing the interaction kinetics of Cldn2/Cldn2 and C2E1/C2E1 interactions were evaluated using the Bell–Evans model.28,29 This model relates the bond rupture force to the loading rate applied to the bond. It has previously been used to characterize binding interactions between intercellular adhesion molecules, such as nectin/nectin,30,31 VE-cadherin/VE-cadherin,32 N-cadherin/N-cadherin, and E-cadherin/E-cad- 684 Adhesion Kinetics of Claudin-2 Extracellular Loops Fig. 3. Force–displacement curves showing the rupture of single molecular bond of Cldn2/Cldn2 interactions. (a) Typical force–displacement curves from the force spectroscopy experiment obtained between GST-Cldn2-functionalized tip and GST-Cldn2-immobilized glass cover slips. (b) Loading rate (rf, expressed in piconewtons per second) is obtained by multiplying the reproach speed (Vr, expressed in nanometers per second) and the slope of the force-displacement curve just before bond rupture (expressed in piconewtons per nanometer). Magnitude of rupture force (F, expressed in piconewtons) is determined from the height of rupture event. herin interactions.22,30,33 In the model, the probability density function for the dissociation of a bound complex at force f is given by:     &  !' k k kB T xh f xh f À exp Pð f Þ¼ off exp exp off kB T kB T rf xh rf ð1Þ where rf is the rate of force application (i.e., loading rate), kB is the Boltzmann constant, T is the absolute is the unstressed dissociation contemperature, koff stant, and xβ is the reactive compliance. Moreover, it can be shown that the average unbinding force of a complex, 〈f〉, increases with rf,20,24,34–36 as shown in Eq. (2).     k kB T k kB T kB T exp off hfi ¼ Ei off ð2Þ xh xh r f x h rf Rl Here, Ei ðzÞ ¼ z tÀ1 expðÀtÞdt is the exponential integral. Equation (2) describes the dynamic properties of a system consisting of a single activation barrier. Fitting the rupture force versus loading rate data points using Eq. (2) (Fig. 5), we extracted the unstressed Table 1. Experiments for studying homophilic Cldn2/Cldn2, C2E1/C2E1, C2E1/C2E2, and C2E2/C2E2 interactions corresponding to histograms depicted in Fig. Interaction type 1. Cldn2_AntiGST 2. Cldn1_Cldn2 3. Cldn2_Cldn1 4. Cldn2_Cldn2_Ab 5. Cldn2_Cldn2_C2E1 6. Cldn2_Cldn2 7. Cldn2_Cldn2_C2E2 8. C2E1_AntiGST 9. C2E1_Cldn2_Ab 10. C2E1_C2E1_Ab 11. C2E1_C2E1 12. C2E2_AntiGST 13. C2E1_C2E2 14. C2E2_C2E1 15. C2E2_Cldn2 16. Cldn2_C2E2 17. C2E2_C2E2 AFM tipa Glass substratea Anti-GST GST-Cldn1 GST-Cldn2 GST-Cldn2 + Antibody GST-Cldn2 + GST-C2E1 GST-Cldn2 GST-Cldn2 + GST-C2E2 Anti-GST GST-C2E1 GST-C2E1 + Antibody GST-C2E1 Anti-GST GST-C2E1 GST-C2E2 GST-C2E2 GST-Cldn2 GST-C2E2 GST-Cldn2 GST-Cldn2 GST-Cldn1 GST-Cldn2 + Antibody GST-Cldn2 + GST-C2E1 GST-Cldn2 GST-Cldn2 + GST-C2E2 GST-C2E1 GST-Cldn2 + Antibody GST-C2E1 + Antibody GST-C2E1 GST-C2E2 GST-C2E2 GST-C2E1 GST-Cldn2 GST-C2E2 GST-C2E2 a Anti-GST, antibody targeting GST; GST-Cldn1, recombinant GST-tagged Cldn1 protein; GST-Cldn2, recombinant GST-tagged Cldn2 protein; GST-C2E1, recombinant GST-tagged first extracellular loop of Cldn2 protein; GST-C2E2, recombinant GST-tagged second extracellular loop of Cldn2 protein; Antibody, antibody targeting the first extracellular loop of Cldn2 (see Materials and Methods for details about the immobilization of proteins onto AFM tip and glass substrate). Adhesion Kinetics of Claudin-2 Extracellular Loops 685 Fig. 4. Rupture force histograms obtained at a reproach velocity of μm s− for the different interaction types listed in Table 1. Cldn2, claudin-2; C2E1, first extracellular loop of Cldn2; C2E2, second extracellular loop of Cldn2. Fig. 5. Molecular force spectroscopy of homophilic Cldn2/Cldn2 and C2E1/C2E1 interactions. The mean rupture force was plotted as a function of loading rate. There was a gradual increase in rupture force along with loading rate up to ∼ 1000 pN/s. This was followed by the faster increase in the unbinding force for loading rate greater than 1000 pN/s. By fitting the experimental data from each loading rate regime to Eq. (2), the unstressed dissociation rate (k0off) and reactive compliance (xβ) for Cldn2/Cldn2 and C2E1/C2E1 interactions were extracted (see Table 2). The error bars are the standard errors of the measurements. Cldn2, claudin-2; C2E1, first extracellular loop of Cldn2. 686 Adhesion Kinetics of Claudin-2 Extracellular Loops Table 2. Comparison of adhesion kinetics of homophilic interactions mediated by Cldn2/Cldn2 and C2E1/C2E1 Molecular pairsa C2E1/C2E1 Cldn2/Cldn2 Loading rate (pN/s) Rate of dissociationa, k0off (s− 1) Reactive compliancea, xβ (nm) 102–103 103–104 102–103 103–104 8.4 × 10− 8.29 4.4 × 10− 6.39 0.90 0.10 0.94 0.19 force spectroscopy of C2E1/C2E1 (Fig. 5). Table lists the kinetic parameters (unstressed dissociation off-rate, , and reactive compliance, xβ) of the two energy koff barriers that were derived from fitting the experimental data with Eq. (2) using nonlinear least-squares method with trust-region algorithm.37 The fitted curves are overlaid on the experimental measurements (Fig. 5). Monte Carlo simulation a Reactive compliance, xβ, and the unstressed bond dissociation rate, k0off, were fitted from the loading rate curve (Fig. 5) using Eq. (2) and nonlinear least-squares method with trust-region algorithm.36 C2E1, first extracellular loop of Cldn2. dissociation constant (koff ) and the reactive compliance (xβ) of Cldn2/Cldn2 and C2E1/C2E1. As shown in Fig. 5, the average unbinding force of Cldn2/Cldn2 and C2E1/C2E1 complexes increases with increasing loading rate in the range of loading rates probed. Moreover, both Cldn2/Cldn2 and C2E1/C2E1 interactions showed two distinct loading regimes in the force spectrum. A gradual increase in unbinding force was observed with increasing loading rate up to a loading rate of ∼103 pN/s. Beyond this point, a second loading regime exhibiting a faster increase in the unbinding force was observed. Interestingly, the dynamic response of the Cldn2/Cldn2 complex was found to be sensitive to the presence of the second extracellular loop. The unbinding force acquired in both low and fast loading regime for Cldn2/Cldn2 was amplified in the To corroborate our experimental results with BellEvans model predictions, Monte Carlo (MC) simulations of the dissociation of Cldn2/Cldn2 and C2E1/C2E1 under constant loading rate were performed using a previously described procedure.22,36 Thousands of rupture forces [Frup = (rf)(nΔt)] were calculated, for which the probability of bond rupture Prup    ! xh rf nDt koff kB T Prup ¼ À exp À exp À1 kB T xh r f ð3Þ was greater than Pran, a random number between and 1. Here, nΔt is the time interval needed to break a bond and Δt is the time step (Δt = 10− s was used in the simulation). MC simulations were conducted = 6.39 s–1 by using unstressed dissociation rate (koff –1 for Cldn2/Cldn2 and koff = 8.29 s for C2E1/C2E1) and reactive compliance (xβ =0.19 nm for Cldn2/ Cldn2 and xβ = 0.1 nm for C2E1/C2E1) obtained Fig. 6. Comparison of rupture force distribution of Cldn2/Cldn2 and C2E1/C2E1 interactions in experimental and theoretical study. (a) Empirical cumulative distribution function of loading rate for MC simulations (continuous line) and experimental data (dotted line) for Cldn2/Cldn2 (black) and C2E1/C2E1 (blue) interactions. (b and c) Bond strength histograms of Cldn2/Cldn2 and C2E1/C2E1 interactions at loading rate between 103 and 104 pN/s from single molecular force spectroscopy experiments (black bar) and MC simulations (white bar). Unstressed off-rate (k0off = 6.39 s–1 for Cldn2/Cldn2 and k0off = 8.29 s–1 for C2E1/C2E1) and reactive compliance (xβ = 0.19 nm for Cldn2/Cldn2 and xβ = 0.1 nm for C2E1/C2E1) obtained from force spectroscopy experiments were used in the MC simulations. Cldn2, claudin-2; C2E1, first extracellular loop of Cldn2. Adhesion Kinetics of Claudin-2 Extracellular Loops experimentally for Cldn2/Cldn2 between loading rates of 103 and 104 (see Table 2). As shown in Fig. 6, there is a good agreement between the MC simulation and experimental results. Loading rate (logarithm scale) was assumed to be normally distributed within the simulation range of loading (103–104 pN/s) (χ2 test, p b 0.05). This assumption is valid and will not cause significant bias to the simulation as the cumulative distribution function of the loading rate agrees well between experimental data and MC simulation (Fig. 6a). Since single dissociation rate and reactive compliance were used to simulate the bond strength distributions of Cldn2/Cldn2 and C2E1/C2E1 interactions, the good agreement between computational and experimental distributions indicates that the rupture event is mainly caused by breaking of a single bond and not from the breaking of multiple bonds. Discussion Cldns are critical tetraspan proteins localizing at TJs, which control solute movement through the paracellular pathway across epithelia. Though Cldns undergo both cis- and trans-interactions27 similar to E-cadherins,38 little is known about how Cldns interact at the molecular level to seal the paracellular cleft. Here, we used GST-tagged, full-length, first extracellular loop of human Cldn2 to understand homophilic Cldn2 interactions in more detail. The analysis presented here examines the interactions at the level of single molecules instead of describing global cellular adhesion behavior, which has been measured previously using flow chambers,39 dual pipette assay,40,41 or cell aggregation assays.42 Cldns have been predicted to possess four transmembrane helices,2 a short intracellular N-terminal sequence, two extracellular loops, and an 687 internal C-terminus that binds to cytoplasmic proteins through a PDZ binding motif.6 It is known that the first extracellular loop of Cldn2,7,8 Cldn4,8,9,10 Cldn5,11 Cldn7,12 Cldn8,13 Cldn15,9,14 Cldn16,15 and Cldn19 16,17 determines paracellular charge selectivity, while the second loop of Cldn3 and Cldn4 acts as a receptor for a bacterial toxin.18 Here, using recombinant GST-tagged full-length Cldn2 and the two extracellular loops of Cldn2 (C2E1 or C2E2) in a series of force spectroscopy experiments, we have demonstrated that the first extracellular loop (C2E1) is sufficient to promote trans-interactions between Cldn2 (Fig. 4). Decrease in the frequency of interactions in the presence of recombinant C2E1, C2E2, or antibody against C2E1 further confirmed that trans-interactions between either C2E2/C2E2 or C2E1/C2E2 not occur (Fig. 4). More recently, it has been demonstrated that the second extracellular loop of Cldn5 (C5E2) is involved in TJ strand formation via trans-interactions. 43 The exact reasons for why the transinteraction occurs in C5E2 but not in C2E2 remain unclear. For C5E2, it was found that five residues (NP_003268; F147, Y148, Q156, Y158, and E159) are important to form the proper binding interface of the trans-interaction of C5E2.43 Thus, it is likely that C2E2 lacks the ability to trans-interact due to the changes of two critical residues, with respect to C5E2 (Q156M and Y158F). Alternatively, the ability of C2E2 to interact in trans may depend on its cisinteraction with other parts of the Cldn2 protein, for example, C2E1, possibly through an involvement of E159.43,44 Thus, trans-interaction between C2E2 will not occur in the case of the truncated C2E2 used in our experiments. Future structural information based on the crystal structure of Cldns will be needed to resolve the issue. Dissociations of Cldn2/Cldn2 and C2E1/C2E1 complexes were found to follow a two-step energy Fig. 7. Comparison of conceptual energy landscapes of dissociation pathway between homophilic Cldn2/Cldn2 and C2E1/C2E1 interactions. The dissociation of Cldn2/ Cldn2 and C2E1/C2E1 involves two energy activation barriers. They were constructed using the kinetic parameters obtained from the molecular force spectroscopy (Table 2; Fig. 5). Activation energy differences for inner and outer barriers between Cldn2/Cldn2 and C2E1/C2E1 were found to be 0.26 and 1.66 kBT, respectively. In general, dissociation pathways for Cldn2/Cldn2 and C2E1/C2E1 interactions may take different reactive coordinates. Here, the geometric locations for their bound states were plotted on the same reactive coordinates for the purpose of comparison. Cldn2, claudin-2; C2E1, first extracellular loop of Cldn2. 688 Adhesion Kinetics of Claudin-2 Extracellular Loops activation barrier process (Figs. and 7). The dissociation rate of the bound complex in N barrier model is given by multiple Bell's model arranged in series28,45,46: kÀ1 ¼ N  X ÃÀ1 ki0 expðxhi F=kB TÞ ð4Þ i¼1 where kB is the Boltzmann constant, T is the absolute temperature, and xβi and ki0 (i = 1, 2, …, N) are parameters corresponding to reactive compliance and unstressed dissociation rate for ith activation barrier along dissociation of bounded complex. The geometry of the conceptual energy landscape for the dissociation pathway can be constructed based on these kinetic parameters. The geometric locations of their bound states were plotted on the same reactive coordinates to compare the topography of the energy landscapes of the dissociation of Cldn2/ Cldn2 and C2E1/C2E1 complexes (Fig. 8). However, it is possible that Cldn2/Cldn2 and C2E1/C2E1 interactions may dissociate along different reactive coordinates in general. The dissociation rate constants were used to estimate the energy differences (ΔG) between transition state energies Cldn2/Cldn2 and C2E1/C2E1 complexes: 0 =kC2E1 Þ DG ¼ ÀkB TlnðkCldn2 ð5Þ 0 and kC2E1 are dissociation rate conwhere kCldn2 stants of the Cldn2/Cldn2 and C2E1/C2E1, respectively. The analysis reveals that the outer activation barrier of the Cldn2/Cldn2 complex is 1.66 kBT lower than that of the C2E1/C2E1 complex (Fig. 6). Moreover, the energy difference of the inner barrier is small (∼ 0.26 k BT), which implies that the difference in equilibrium dissociation constant between Cldn2/Cldn2 and C2E1/C2E1 complexes arises from energy difference of the outer barrier. The effect of different activation energy barriers on the dynamic properties of Cldn2/Cldn2 and C2E1/ C2E1 complexes is best illustrated in the comparison of their kinetic profiles (Fig. 8). Based on Bell's model [Eq. (4)], dissociation rates for Cldn2/Cldn2 and C2E1/C2E1 complexes increase exponentially with pulling force. In both interactions, there is initially a fast exponential rise in dissociation rate with increased applied force, followed by a more gradual exponential increase at forces N∼50 pN. The dissociation rate of Cldn2/Cldn2 was found to be faster than that of the C2E1/C2E1 complex in both absence and presence of the applied pulling force (Fig. 8). A similar trend was observed in the comparison of dissociation kinetics between Cldn2/Cldn2 and Cldn2/C2E1 (data not shown). Taken together, this suggests that the second extracellular loop has an inhibitory or antagonizing effect on the adhesion strength and kinetics of the interactions (Figs. and 8). A critical concern in force spectroscopy is whether the recombinant protein under investigation maintains its functional structure or not. It has been shown previously that isolated and purified recombinant GST-Cldn1 proteins retain their functions and are suitable for in vitro experiments.47 When GFP- Fig. 8. Comparison of dissociation rates for homophilic Cldn2/Cldn2 and C2E1/C2E1 interactions. The forcedependent dissociation rates of Cldn2/Cldn2 and C2E1/C2E1 interactions were plotted using multiple Bell's model arranged in series [Eq. (4), two energy barriers, n = 2] with the kinetic parameters tabulated in Table 2. Cldn2, claudin-2; C2E1, first extracellular loop of Cldn2. 689 Adhesion Kinetics of Claudin-2 Extracellular Loops Cldn2-transfected L-cells (which not express any endogenous Cldns4,48 ) were incubated with GSTCldn2, it was observed that GST-Cldn2 colocalizes with GFP-Cldn2 partners on cell surface. This strongly suggests that GST-Cldn2 can still fold properly and retains its ability to trans-interact with GFP-Cldn2 partners on cell surface. Colocalization of GST-Cldn2 with GFP-Cldn2 was specific since blocking experiments using antibody targeting Cldn2 significantly suppressed the colocalization (Supplementary Material). Given that the charged residues on the first extracellular loop of Cldns influence the paracellular ion selectivity in the TJs' pore,7–17,49 it will be interesting to compare the kinetics of interactions by mutating the charged residues of the first extracellular loop in the future experiments. Understanding interactions mediated by Cldns is important not only because of the role that they play in regulating paracellular transport of solutes and intercellular adhesion but also because of their pathological role in acting as coreceptors for the entry of hepatitis C virus.50 In the future, singlemolecule analysis could be performed on other structural components of TJs,51 such as occludin52 and junctional adhesion molecules,53,54 in order to gain a better perspective of how the interaction kinetics of different adhesion molecules affect the organization and functioning of TJs. Materials and Methods Protein immobilization and cantilever functionalization Functionalization of AFM cantilever was done as described previously.36 Soft silicon nitride tips (Veeco, Santa Barbara, CA) were UV irradiated for 15 and incubated in a mixture of 30% H2O2/70% H2SO4 for 30 min. After washing thoroughly in ddH2O, tips were dried and treated with a 4% solution of APTES (3-aminopropyltriethoxysilane, Sigma) in acetone for min. They were then rinsed thrice in acetone and then incubated in a solution of BS3 [bis(sulfosuccinimidyl) suberate, mg/ml, Pierce] for 30 min, followed by the incubation of anti-GST antibody (10 μg/ml, Invitrogen) for h. After quenching the reaction using M Tris buffer, the tips were incubated in recombinant full-length GST-Cldn2 (10 μg/ml, Proteintech Group, Inc, USA), GST-Cldn1, GST-C2E1 (10 μg/ml, Abnova) (C2E1: first extracellular loop of Cldn2, NP_065117, 29–81 aa), or GST-C2E2 (10 μg/ml, Abnova) (C2E2: second extracellular loop of Cldn2, NP_065117, 138–163 aa) for h. Unbound recombinant proteins were washed off with phosphate-buffered saline. Tips were blocked in 1% bovine serum albumin before experiments.36 Recombinant GST-Cldn2, GST-Cldn1, GSTC2E1, or GST-C2E2 was immobilized on glass cover slips using the same procedure as described above. To confirm that GST-Cldn2 was efficiently linked to the silanized tips, we used primary mouse anti-Cldn2 antibody (Abnova) and Alexa-488-labeled goat anti-mouse secondary antibody (Molecular Probes, Invitrogen) to stain the GST-Cldn2-coupled tips. It was found that primary mouse anti-Cldn2 antibody and Alexa-488labeled goat anti-mouse antibody (Molecular Probes, Invitrogen) bound efficiently to the GST-Cldn2-coupled silanized tips but not to silanized tips incubated with only anti-GST. For control experiments, all steps were similar except that incubation with recombinant proteins was omitted. For blocking experiments, functionalized tips and cover slips were incubated with antibody to the first extracellular loop of Cldn2 (10 μg/ml, Abnova) for 30 min. They were then washed to remove any unbound antibody before the experiments. For competition assays, tips functionalized with GST-Cldn2 were probed in the presence of GST-C2E1 or GST-C2E2 (10 μg/ml) in phosphate-buffered saline buffer. Molecular force spectroscopy Force curves were acquired on a MultiMode™ Picoforce™ AFM (Veeco) coupled to an upright microscope at room temperature using a fluid cell. Cantilevers with a nominal spring constant of 0.01–0.03 N/m were used for obtaining force plots. Prior to obtaining force curves, the spring constant was determined using the thermal tune module. Target proteins (GST-Cldn1, GST-Cldn2, GSTC2E1, or GST-C2E2) immobilized on the glass cover slips were probed with cantilevers functionalized with recombinant proteins (GST-Cldn1, GST-Cldn2, GST-C2E1, or GST-C2E2). Force plots were obtained at different reproach velocities (0.1–2 μm/s). To minimize the number of adhesion events and maximize the probability of obtaining single-bond adhesion events, we used a contact force of 200 pN and a contact time of ms. Under such condition, the adhesion frequency was b 25%, which would give a N 86% probability of the rupture forces being due to single-bond rupture according to Poisson statistics.35 Force curves were analyzed for the magnitude of the rupture events and the apparent loading rate (defined as the slope of the retrace curve prior to the rupture event multiplied by the reproach velocity) using MATLAB version 7.1 (The MathWorks, Natick, MA). Following Hanley et al.20 and Panorchan et al.,22 rupture force measurements were partitioned by using binning windows of 50 pN/s for loading rates between 100 and 1000 pN/s and by binning windows of 500 pN/s for loading rates between 1000 and 10,000 pN/s. Each bin yields a mean force by Gaussian fitting. 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Energy landscapes of < /b> receptor–ligand bonds explored with dynamic force spectroscopy Nature, 397, 50–53 46 Evans, E (2001) Probing the < /b> relation between force— lifetime—and chemistry in single molecular bonds Annu Rev Biophys Biomol Struct 30, 105–128 47 Tanaka, M., Kamata, R & Sakai, R (2005) Phosphorylation of < /b> ephrin -B1 via the < /b> interaction with claudin < /b> following cell–cell contact formation EMBO J 24, 3700–3711... qualitative and quantitative determinants Dev Biol 253, 309–323 691 43 Piontek, J., Winkler, L., Wolburg, H., Muller, S L., Zuleger, N., Piehl, C et al (2008) Formation of < /b> tight < /b> junction: determinants of < /b> homophilic interaction between classic claudins FASEB J 22, 146–158 44 Krause, G., Winkler, L., Mueller, S L., Haseloff, R F., Piontek, J & Blasig, I E (2008) Structure and function of < /b> claudins Biochim Biophys.. .Adhesion < /b> Kinetics of < /b> Claudin-< /b> 2 Extracellular Loops 33 Perret, E., Leung, A., Feracci, H & Evans, E (2004) Trans-bonded pairs of < /b> E-cadherin exhibit a remarkable hierarchy of < /b> mechanical < /b> strengths Proc Natl Acad Sci USA, 101, 16472–16477 34 Gergely, C., Voegel, J., Schaaf, P., Senger, B. , Maaloum, M., Horber, J K & Hemmerle, J (2000) Unbinding process of < /b> adsorbed proteins under external... Fruscella, P et al (1998) Junctional adhesion < /b> molecule, a novel member of < /b> the < /b> immunoglobulin superfamily that distributes at < /b> intercellular junctions < /b> and modulates monocyte transmigration J Cell Biol 142, 117–127 54 Williams, L A., Martin-Padura, I., Dejana, E., Hogg, N & Simmons, D L (1999) Identification and characterisation of < /b> human junctional adhesion < /b> molecule (JAM) Mol Immunol 36, 1175–1188 ... domains J Biol Chem 281, 2901–2910 41 Chu, Y S., Thomas, W A., Eder, O., Pincet, F., Perez, E., Thiery, J P & Dufour, S (2004) Force measurements in E-cadherin -mediated < /b> cell doublets reveal rapid adhesion < /b> strengthened by actin cytoskeleton remodeling through Rac and Cdc42 J Cell Biol 167, 1183–1194 42 Duguay, D., Foty, R A & Steinberg, M S (2003) Cadherin -mediated < /b> cell adhesion < /b> and tissue segregation:... c-Jun/c-Fos dimerization domains J Biol Chem 277, 19455–19460 39 Niessen, C M & Gumbiner, B M (2002) Cadherinmediated cell sorting not determined by binding or adhesion < /b> specificity J Cell Biol 156, 389–399 40 Chu, Y S., Eder, O., Thomas, W A., Simcha, I., Pincet, F., Ben-Ze'ev, A et al (2006) Prototypical type I E-cadherin and type II cadherin-7 mediate very distinct adhesiveness through their extracellular... Jaramillo, B E (2003) Tight < /b> junction proteins Prog Biophys Mol Biol 81, 1–44 52 Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S & Tsukita, S (1993) Occludin: a novel integral membrane protein localizing at < /b> tight < /b> junctions < /b> J Cell Biol 123, 1777–1788 53 Martin-Padura, I., Lostaglio, S., Schneemann, M., Williams, L., Romano, M., Fruscella, P et al (1998) Junctional adhesion < /b> molecule, a novel member... studied by atomic force microscopy spectroscopy Proc Natl Acad Sci USA, 97, 10802–10807 35 Zhang, X., Wojcikiewicz, E & Moy, V T (2002) Force spectroscopy of < /b> the < /b> leukocyte function-associated antigen-1/intercellular adhesion < /b> molecule-1 interaction Biophys J 83, 2270–2279 36 Hanley, W., McCarty, O., Jadhav, S., Tseng, Y., Wirtz, D & Konstantopoulos, K (2003) Single molecule characterization of < /b> P-selectin/ligand... when expressed in fibroblasts J Cell Sci 110, 1113–1121 49 Van Itallie, C M & Anderson, J M (2006) Claudins and epithelial paracellular transport Annu Rev Physiol 68, 403–429 50 Evans, M J., von Hahn, T., Tscherne, D M., Syder, A J., Panis, M & Wolk, B (2007) Claudin-< /b> 1 is a hepatitis C virus co-receptor required for a late step in entry Nature, 446, 801–805 51 Gonzalez-Mariscal, L., Betanzos, A., Nava,... P-selectin/ligand binding J Biol Chem 278, 10556–10561 37 Gilles, L., Vogel, C R & Ellerbroek, B L (2002) Multigrid preconditioned conjugate-gradient method for large-scale wave-front reconstruction J Opt Soc Am A, Opt Image Sci Vis 19, 1817–1822 38 Ahrens, T., Pertz, O., Haussinger, D., Fauser, C., Schulthess, T & Engel, J (2002) Analysis of < /b> heterophilic and homophilic interactions of < /b> cadherins using the < /b> c-Jun/c-Fos . dissociation rate for ith activation barrier along dissociation of bounded complex. The geometry of the conceptual energy landscape for the dissociation pathway can be constructed based on these. kinetic parameters. The geometric locations of their bound states were plotted on the same reactive coordinates to c ompare the topography of the energy landscapes of the dissociation of Cldn2/ Cldn2. rupture events. On the basis of Poisson statistics, 25 the low frequency of these de -adhesion events ensured a N 86% probability of the rupture being due to a single bond. Upon retraction of the cantilever,