Mechanical insights into the physiological functions of claudin mediated adhesion at tight junctions c

Research ArticleProbing effects of pH change on dynamic response of Claudin-2 mediated adhesion using single molecule force spectroscopy aBioinformatics Institute, Agency for Science, Te

Research Article

Probing effects of pH change on dynamic response of Claudin-2 mediated adhesion using single molecule force spectroscopy

aBioinformatics Institute, Agency for Science, Technology and Research (A⁎STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671 b

NUS Graduate School for Integrative Sciences & Engineering (NGS), Centre for Life Sciences (CeLS), #05-01,

28 Medical Drive Singapore 117456

cDivision of Bioengineering & Department of Mechanical Engineering, 9 Engineering Drive 1, National University of Singapore,

Singapore 117576

dInstitute of Molecular and Cell Biology, Agency for Science, Technology and Research (A⁎STAR), 61 Biopolis Drive, Proteos, Singapore 138673

A R T I C L E I N F O R M A T I O N A B S T R A C T

Article Chronology:

Received 7 April 2008

Revised version received

27 May 2008

Accepted 27 May 2008

Available online 3 June 2008

Claudins belong to a large family of transmembrane proteins that localize at tight junctions (TJs) where they play a central role in regulating paracellular transport of solutes and nutrients across epithelial monolayers Their ability to regulate the paracellular pathway is highly influenced by changes in extracellular pH However, the effect of changes in pH on the strength and kinetics of claudin mediated adhesion is poorly understood Using atomic force microscopy, we characterized the kinetic properties of homophilic trans-interactions between full length recombinant GST tagged Claudin-2 (Cldn2) under different pH conditions In measurements covering three orders of magnitude change in force loading rate of 102–104pN/s, the Cldn2/Cldn2 force spectrum (i.e., unbinding force versus loading rate) revealed a fast and a slow loading regime that characterized a steep inner activation barrier and a wide outer activation barrier throughout pH range of 4.5–8 Comparing to the neutral condition (pH 6.9), differences in the inner energy barriers for the dissociation of Cldn2/Cldn2 mediated interactions at acidic and alkaline environments were found to beb0.65 kBT, which

is much lower than the outer dissociation energy barrier (N1.37 kBT) The relatively stable interaction of Cldn2/Cldn2 in neutral environment suggests that electrostatic interactions may contribute to the overall adhesion strength of Cldn2 interactions Our results provide an insight into the changes in the inter-molecular forces and adhesion kinetics of Cldn2 mediated interactions in acidic, neutral and alkaline environments

© 2008 Elsevier Inc All rights reserved

Keywords:

Claudin

Tight junction

Cell–cell adhesion

Molecular force spectroscopy

Atomic force microscopy

pH

Introduction

Tight junctions (TJs) are the apical most constituents of the

intercellular adhesion complex in epithelial monolayers Their

primary function is to regulate the paracellular transport of

ions, solutes and water across epithelia In addition, they interact with a variety of signaling and trafficking molecules to regulate cell differentiation, proliferation and polarity[1,2] The selective permeability of TJs is largely determined by a protein family called claudins (Cldns)[3–5] Although the contribution of

⁎ Corresponding author Division of Bioengineering and Department of Mechanical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576

E-mail address:ctlim@nus.edu.sg(C.T Lim)

0014-4827/$– see front matter © 2008 Elsevier Inc All rights reserved

doi:10.1016/j.yexcr.2008.05.015

a va i l a b l e a t w w w s c i e n c e d i r e c t c o m

w w w e l s e v i e r c o m / l o c a t e / ye x c r

Cldns to the charge selective permeability and ion homeostasis

of epithelia is well established[6–16], details about the strength

and adhesion kinetics of the interactions mediated by Cldns are

being understood only recently[17]

Cldn2 was found to exhibit Ca2+-independent adhesion

activities in cell aggregation assays[18] Expression of Cldn2

has been shown to induce cation-selective channels in TJs of

epithelial cells[6] Also, increased expression of Cldn2 has

been shown to decrease transepithelial electrical resistance

(TER)[19,20]while increasing the density of small TJ pores[21]

Furthermore, the knockdown of endogenous Cldn2 expression

in MDCK cells using siRNA resulted in decreased Na+

permea-tion and loss of capermea-tion selectivity[22] In a more recent study,

Cldn2 was shown to be critical for Vitamin D-dependent Ca2+

absorption between enterocytes[23] Since it has been shown

that the transport of several nutrients can be influenced by

varying the extracellular pH [24–26], understanding the

pH-associated changes of the adhesion kinetics mediated by

Cldns will provide us a better perspective on how it regulates

the paracellular transportation of solutes and intercellular

adhesions To address this question, we used single molecule

force spectroscopy to investigate the molecular interactions

between recombinant N-terminal glutathione S-transferase

(GST) tagged full length human Cldn2 (GST-Cldn2) under

different pH conditions (pH 4.5, 5.1, 6.9 and 8)

Our results show that dissociation of homophilic Cldn2/

Cldn2 complexes follows a two-step energy barriers model

within the pH range of 4.5–8 and loading rates of 102

–104 pN/s

The energy landscape of the dissociations was found to be

dynamically dependent on the changes in environmental pH

Comparison of adhesion kinetics further revealed that Cldn2/

Cldn2 is relatively more stable in neutral solution (pH 6.9)

when compared to acidic or alkaline environments, implying

that electrostatic interactions may contribute to the adhesion

strength of Cldn2 mediated adhesions

Materials and methods

Protein immobilization and cantilever

functionalization

Functionalization of AFM cantilevers was performed using

me-thods described previously[27] Soft silicon nitride tips (Vecco,

Santa Barbara, CA) were UV irradiated for 15 min and incubated

in a mixture of 30% H2O2/70% H2SO4for 30 min After washing

thoroughly in ddH2O, tips were dried and treated with a 4%

solution of APTES (3-aminopropyltriethoxysilane, Sigma) in

acetone for 3 min They were then rinsed thrice in acetone and

incubated in a solution of BS3(Bis (Sulfosuccinimidyl) suberate,

2 mg/ml, Pierce) for 30 min, followed by the incubation of

anti-GST antibody (10 µg/ml, Invitrogen) for 2 h The reaction was

quenched using 1 M Tris buffer, followed by the incubation with

recombinant full length GST-Cldn2 (10 µg/ml, Proteintech

Group, Inc, USA) or GST-Cldn1 (10 µg/ml, Abnova, Taiwan) for

2 h Unbound recombinant proteins were washed off with PBS

Tips were blocked in 1% BSA before experiments[27]

Recombi-nant GST-Cldn2 or GST-Cldn1 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,

pri-mary mouse anti-Cldn2 antibody (Abnova, Taiwan) and Alexa 488-labeled goat anti-mouse secondary antibody (Molecular Probes, Invitrogen) were used to stain the GST-Cldn2-coupled tips For control experiments, all steps were similar except that incubation of recombinant GST-Cldn2 proteins was omitted For blocking experiments, tips and cover slips were incubated with antibody targeting the first extracellular loop of Cldn2 (10 µg/ml, Abnova, Taiwan) for 30 min They were then washed to remove any unbound antibody before the experiments For competition assays, interactions of GST-Cldn2/GST-Cldn2 were probed in the presence of GST-C2E1 (10 µg/ml, Abnova, Taiwan) (C2E1: first extracellular loop of Claudin-2, UUNP_065117UU, 29 a.a.–81 a.a.) or GST-C2E2 (10 µg/ml, Abnova, Taiwan) (C2E2: second extracellular loop of Claudin-2, UUNP_065117UU, 138 a

a.–163 a.a.) in PBS buffer

Molecular force spectroscopy

Force curves were acquired on a MultiMode™ Picoforce™ AFM (Vecco, Santa Barbara, CA) 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 ob-taining force plots Prior to obob-taining force curves, the spring constant was determined using the thermal tune module Target proteins (GST-Cldn2 or GST-Cldn1) immobilized on the glass cover slips were probed with cantilevers functionalized with recombinant proteins (GST-Cldn2 or GST-Cldn1) under different pH conditions (pH 4.5–8) in PBS buffer Force plots were obtained at different reproach velocities (0.1–2 μm/s) and 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.[28]and Panorchan et al.[29], 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 By plotting the mean force as a function of loading rate, the unstressed dissociation rate and reactive compliance characterizing Cldn2/Cldn2 interactions in differ-ent pH conditions were extracted (see Results section)

Results

Measurement of Cldn2/Cldn2 interaction forces

Trans-interactions between full length human Cldn2 (Cldn2/ Cldn2) were characterized at the level of single molecule using atomic force microscopy (AFM) (Fig 1)[28–30] The interaction was established by bringing GST-Cldn2 functionalized canti-lever in close contact to a glass cover slip coated with GST-Cldn2 under different pH conditions (pH 4.5–8) (see Materials and methods) To confirm that GST-Cldn2 was efficiently linked to the silanized tips, primary mouse Cldn2 body and Alexa 488-labeled goat mouse secondary anti-body were used to stain the GST-Cldn2-coupled tips Confocal images showed that GST-Cldn2 was efficiently coupled to the AFM tips and cover slips (Fig 2)

To measure the de-adhesion forces at single molecular

resolution[30–32], a contact force of 200 pN and a contact time

of 1 ms were used Under such conditions, low frequency of

de-adhesion events (b25%) was achieved, which ensured a

N86% probability of single bond rupture based on Poisson

statistics [33] Upon retraction of the cantilever, force as a

function of pulling distance was recorded (Fig 3a)[34] For

each reproach velocity, hundreds of force–distance curves

(nN500) were collected and analyzed to extract rupture force, F

and loading rate, rf(Fig 3b) Data obtained were subsequently

pooled into histograms to analyze the frequency of adhesion

events for different interactions (Table 1, Fig 4) Results

showed that adhesion frequency was significantly reduced

in control experiments performed using AFM tips

functiona-lized with only anti-GST antibody (Cldn2_Anti-GST, Table1,

Fig 4) On the other hand, the low frequency of interaction

between Cldn1 and Cldn2 (Cldn1_Cldn2 or Cldn2_Cldn1,Table

1,Fig 4) demonstrated that they do not trans-interact which is

consistent with previous findings [35] In addition, it was

found that experiments performed using antibody specifically

targeting the first extracellular loop of Cldn2 (C2E1)

signifi-cantly reduced the binding frequency Competition assays

revealed that only the first but not the second extracellular

loop of Cldn2 can compete for the interactions between Cldn2/

Cldn2 (Cldn2_Cldn2_C2E1, Cldn2_Cldn2_C2E2,Table 1,Fig 4)

These results suggest that C2E1 itself is sufficient to promote

the trans-interactions of Cldn2/Cldn2

Extraction of the kinetic parameters of Cldn2/Cldn2 interactions

Biophysical parameters characterizing the kinetics of Cldn2/

Cldn2 interactions under different pH conditions were

eval-uated using the Bell–Evans model[36,37] 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 other intercellular adhesion molecules, such as nec-tin/nectin [38,39], VE-cadherin/VE-cadherin[40], N-cadherin/ N-cadherin and E-cadherin/E-cadherin interactions[29,38,41]

In this model, the probability density function for the dissociation of a bound complex at force f is given by:

P fð Þ ¼ k0off

rf

exp xbf

kBT

exp k0offkBT

xβrf 1 exp xbf

kBT

ð1Þ where rfis rate of force application (i.e., loading rate), kBis Boltzmann constant, T is the absolute temperature, k0

offis the unstressed dissociation constant and xβ is the reactive compliance Moreover, as shown in Eq (2), the average unbind-ing force of a complex,bfN, increases with rf[27,28,32,42,43], bfN ¼kBT

xβ exp k0offkBT

xβrf

Ei k

0 offkBT

xβrf

ð2Þ Here Eið Þ ¼z Rl

z

t1exp tð Þdt is the exponential integral Eq (2) describes the dynamic properties of a system consisting of a single activation barrier For each pH condition, by fitting the rupture force vs loading rate data points using Eq (2), the

Fig 2– Confocal images of silanized AFM cantilevers functionalized (a) with GST-Cldn2 and (b) without GST-Cldn2 Both images were taken after the cantilevers/tips were incubated with anti-Cldn2 primary antibody and Alexa 488-labeled secondary antibody (see Materials and methods section for details) Images were acquired under the same conditions (pixel dwell time, laser power and gain) Scale bar size: 50μm

Fig 1– Schematic of the atomic force microscopy (AFM)

experimental setup Recombinant GST-Cldn2 was linked to

the AFM tip or immobilized on glass cover slip using the

linker APTES-BS3-anti-GST (see Materials and methods for

details) GST-Cldn2 immobilized on glass cover slip was

probed using these functionalized tips under different pH

condition in PBS buffer The arrow indicates the direction of

pull in the AFM experiment GST: glutathione

S-transferase; Cldn2: Claudin-2; APTES:

3-aminopropyl-triethoxysilane; BS3: Bis (Sulfosuccinimidyl) suberate;

anti-GST: antibody targeting GST; PBS: phosphate buffered

saline

unstressed dissociation constant (k0

off) and the reactive com-pliance (xβ) of the Cldn2/Cldn2 interaction were extracted (Fig 5)

Within the range of loading rates probed (102–104

pN/s), the average unbinding force of Cldn2/Cldn2 complexes was found to

be higher with increasing loading rate Moreover, the Cldn2/

Cldn2 force spectrum (i.e., unbinding force versus loading rate)

revealed a fast and a slow loading regime that characterized a

steep inner activation barrier and a wide outer activation barrier

throughout all pH conditions (pH 4.5, 5.1, 6.9 and 8.0) (Fig 5) A

gradual increase in unbinding force was observed with increas-ing loadincreas-ing rate of up to ~103pN/s Beyond this point, a second loading regime exhibiting a faster increase in the unbinding force was observed Table 2 lists the kinetic parameters (unstressed dissociation off rate k0

offand reactive compliance

xβ) of the two energy barriers that were derived from fitting the experimental data with Eq (2) using non-linear least square method with trust-region algorithm[44] The fitted curves using Bell–Evans model are overlaid on the experimental measure-ments (Fig 5)

Monte Carlo simulation

Monte Carlo (MC) simulations of receptor–ligand bond rupture under constant loading rates were performed to further cor-roborate our experimental results with Bell–Evans model pre-dictions using a previously described procedure [27,29] One thousand rupture forces (Frup= (rf)(nΔt)) were calculated for which the probability of bond rupture Prup:

Prup¼ 1  exp k

0 offKBT

xhrf exp xhrfnDt

kBt

 1

ð3Þ was greater than Pran, a random number between zero and one Here nΔt is the time interval needed to break a bond and Δt is the time step (Δt=10−6 s was used in the simulation) The values of unstressed dissociation rate (k0

off= 6.39 s−1) and reactive compliance (xβ= 0.19 nm) obtained experimentally in neutral pH (pH 6.9) for Cldn2/Cldn2 between loading rate of

103–104pN/s were used in the simulation Results obtained

Fig 3– Force–Displacement curves showing rupture of individual bonds mediated by Cldn2/Cldn2 interactions (a) Typical force–distance curves obtained between tip functionalized with GST-Cldn2 and GST-Cldn2 immobilized on glass cover slips Arrows indicate rupture of homophilic Cldn2/Cldn2 interactions The curves show either no or single bond rupture event Only curves showing a single clear rupture event were used for generating the histograms (b) The slope of the curve just before rupture multiplied by the reproach velocity (Vr, expressed in nm/s) defines the loading rate (rf, expressed in pN/s) The height of the rupture event defines the magnitude of rupture force (F) (expressed in pN) GST: glutathione S-transferase

Table 1– Experiments for studying homophilic

Cldn2/Cldn2 interactions corresponding to histograms

depicted inFig 4

Interaction type AFM tipa Glass substratea

Cldn2_Cldn2_Ab GST-Cldn2 + Antibody GST-Cldn2 + Antibody

Cldn2_Cldn2_C2E1 GST-Cldn2 + GST-C2E1 GST-Cldn2 + GST-C2E1

Cldn2_Cldn2_C2E2 GST-Cldn2 + GST-C2E2 GST-Cldn2 + GST-C2E2

a Anti-GST: antibody targeting GST; Cldn1: recombinant

GST-tagged Claudin-1 protein; GST-Cldn2: recombinant GST-GST-tagged

Claudin-2 protein; GST-C2E1: recombinant GST-tagged first

extra-cellular loop of Claudin-2 protein C2E2: recombinant

GST-tagged second extracellular loop of Claudin-2 protein; antibody:

antibody targeting the first extracellular loop of Claudin-2 protein

(See Materials and methods for details about the immobilization of

proteins onto AFM tip and glass substrate.)

Fig 4– Rupture force histograms obtained at a reproach velocity of 1 µm/s for the different interaction types listed inTable 1 Cldn2: Claudin-2; C2E1: first extracellular loop of Claudin-2; C2E2: second extracellular loop of Claudin-2

Fig 5– Molecular force spectroscopy of homophilic Cldn2/Cldn2 interactions probed under different pH conditions The mean rupture force was plotted as a function of loading rate There was a gradual increase in rupture force along with loading rates up

to ~1000 pN/s This was followed by the faster increase in the unbinding force for loading rates greater than 1000 pN/s By fitting the experimental data from each loading rate regime to Eq (2), the unstressed dissociation rate (k0

off) and reactive compliance (x ) for Cldn2/Cldn2 interactions were extracted (seeTable 2) The error bars are the standard errors of the measurements

from the simulation agreed well with the experimental results

(Fig 6b) The loading rate (logarithmic scale) was assumed to be

normally distributed within the simulation range of loading

(103–104pN/s) (x2test, pb0.05) This assumption is valid and

will not cause significant deviations to the simulation as the

cumulative distribution function of the loading rate agrees well

between experimental data and MC simulation (Fig 6a) 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 Claudins are critical tetra-span proteins localizing at TJs which create the barrier and regulate electrical resistance and size and ionic charge selectivity during paracellular epithelial transport[45] While the role of Cldns in creating charge selec-tivity in the paracellular pathway is well established and has been characterized in some detail[6–16], little is known about the strength of adhesion forces mediated by Cldns at TJs Here,

we used GST tagged full length human Cldn2 to understand kinetic properties and adhesion strength of homophilic Cldn2 interactions in more detail The present work examines the interactions at the level of single molecules instead of descri-bing global cellular adhesion behavior which has been measured previously using dual pipette[17]or cell aggregation

[18]assays

It has been shown previously that the transportation of several nutrients as well as changes to TJ permeability (for example induced by copper) are influenced by variations of the extracellular pH [24–26] To understand the dynamic response of individual Cldn2/Cldn2 interactions to the change

in pH, we used AFM to probe the trans-interaction of Cldn2/

Table 2– pH dependence of adhesion kinetics mediated

by Claudin-2/Claudin-2 interactions

pH Loading rate

(pN/s)

Rate of dissociationa

k0 off(s−1)

Reactive compliancea

xβ(nm)

a Reactive compliance xβ, and the unstressed bond dissociation

rate k0

off, were fitted from the loading rate curve (Fig 5) using Eq (2)

using non-linear least square method with trust-region algorithm

(Gilles et al., 2002)

Fig 6– Comparison of the experimental and theoretical rupture force distribution of Cldn2/Cldn2 interaction (a) Empirical cumulative distribution function of loading rate for experimental data (dotted line) and Monte Carlo (MC) simulations (continuous solid line) (b) Experimental (black) and theoretical (white) histograms of rupture forces to break a single

Cldn2/Cldn2 bond at loading rate between 103and 104pN/s MC simulations, which were conducted using the Bell-Evans model unstressed off rate (k0

off= 6.39 s−1) and reactive compliance (xβ= 0.19 nm) obtained from force spectroscopy experiments, were consistent with experimental data and further indicate that only one single type of bond was analyzed

Cldn2 pairs at different pH conditions In the range of pH 4.5–8,

dissociations of Cldn2/Cldn2 complexes were found to follow a

two-step energy activation barrier process within the probed

loading rate of 102–104pN/s (Figs 5 and 7) The dissociation

rate of the bound complex in N barrier model is given by

multiple Bell's model arranged in series[36,46,47]:

k1¼XN

i¼1

k0

iexp x βiF=kBT 1

ð4Þ

where kBis the Boltzmann constant, T is the absolute

tem-perature, and xβ i and ki0 (i = 1, 2, , N) are parameters

corresponding to reactive compliance and unstressed

dis-sociation rate for ith activation barrier along disdis-sociation of

bounded complex These kinetic parameters can be further

used to construct the geometry of the conceptual energy

landscape for the dissociation pathway To compare the

topography of the energy landscapes of the dissociation of

Cldn2/Cldn2 complexes at different pH conditions, the

geometric locations of their bound states were plotted on

the same reactive coordinates (Fig 7) In general,

dissocia-tions of Cldn2/Cldn2 may involve different reactive

coordi-nates Compared to acidic (pH ~ 4.5) or alkaline (pH 8.0) conditions, the energy barriers for the dissociation of Cldn2/ Cldn2 are highest at pH 6.9, indicating that the Cldn2/Cldn2 bond is more stable in neutral solution This may explain why the structural integrity of the tight junctions in epithelia

is affected by low pH[48] The dissociation rate constants were used to estimate the energy differences (ΔG) among transition state energies of Cldn2/Cldn2 complexes at different pH conditions:

DG ¼ kBTln k0

A=k0

ð5Þ where k0

Aand k0

Bare dissociation rate constants of the Cldn2/ Cldn2 interaction at different pH conditions of A and B, respec-tively The analysis reveals that the outer activation barriers of the Cldn2/Cldn2 complex at acidic (pH 4.5) and alkaline con-ditions (pH 8.0) are 1.37 and 4.9 kBT greater than at neutral condition (pH 6.9) (Fig 7) Moreover, the energy difference of the inner barrier is small (b0.65 kBT), which implies that the diffe-rence in equilibrium dissociation constant between Cldn2/Cldn2 complexes at acidic or alkaline conditions arises from differ-ences in the activation energies of the outer barrier

Fig 7– Comparison of conceptual energy landscapes of dissociation pathway between homophilic Cldn2/Cldn2 interactions probed under different pH conditions The dissociation of Cldn2/Cldn2 involves two energy activation barriers They were constructed using the kinetic parameters obtained from the molecular force spectroscopy (Table 2,Fig 4) Activation energy difference for inner and outer barriers was sketched in the figure, respectively In general, dissociation pathways for

Cldn2/Cldn2 interactions may take different reactive coordinates Here, the geometric locations for their bound states in different pH conditions were plotted on the same reactive coordinates for the purpose of comparison

In this study, we have elucidated the dynamic response of

Cldn2 mediated interactions in various acidic, neutral, and

alkaline environments The relative stability of the

trans-actions at neutral conditions suggests that electrostatic

inter-actions may contribute to the overall adhesion strength

Decreased stability of Cldn2/Cldn2 interactions in acidic and

alkaline environments could be attributed either to

conforma-tional changes in Cldn2 or changes in the charge distribution of

critical amino acids mediating trans-interactions It has

pre-viously been shown that the first extracellular loop of Cldn2[6,7]

confers charge selective paracellular permeability to epithelial

monolayers In the acidic environment (pH ~ 4.5), most of the

amide groups in the side chain of positively charged residues

such as tyrosine (NP_065117, a.a 35 and 67), lysine (a.a 31 and

48) and histidine (a.a 57) in the first extracellular loop of Cldn2

are protonated (pK valueN4.5) whereas the carboxylic acids in

the side chain of aspartic (a.a 65 and 76) and glutamic acids

(a.a 53) are uncharged Under such conditions, though the

trans-interactions could still occur via other means, such as the

hy-drophobic interactions of nonpolar residues, the electrostatic

repulsion caused by the positive charged residues would

desta-bilize the overall adhesion strength When the pH is changed

from neutral to 5.1, only the charge of a single histidine residue

(a.a 57) is affected, which could explain the small effect on the

adhesion kinetics of Cldn2/Cldn2 interactions under this

condi-tion (Table 2,Fig 7)

Given that the charged residues of Cldns influence the

paracellular ion selectivity in the TJs pore, it will be interesting

to compare the kinetics of interactions by mutating the

charged residues in future experiments Understanding

inter-actions 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 receptors for bacterial toxin

[49,50]and co-receptors for the entry of hepatitis C virus[51]

Acknowledgments

This work was supported by the Biomedical Research Council

(BMRC) from the Agency for Science, Technology & Research

(A⁎STAR), Singapore Their funding support is gratefully

acknowledged

R E F E R E N C E S

[1] K Matter, M.S Balda, Signalling to and from tight junctions,

Nat Rev Mol Cell Biol 4 (2003) 225–236

[2] S Tsukita, M Furuse, M Itoh, Multifunctional strands in tight

junctions, Nat Rev Mol Cell Biol 2 (2001) 285–293

[3] M Furuse, K Fujita, T Hiiragi, K Fujimoto, S Tsukita,

Claudin-1 and -2: novel integral membrane proteins

localizing at tight junctions with no sequence similarity to

occludin, J Cell Biol 141 (1998) 1539–1550

[4] K Morita, M Furuse, K Fujimoto, S Tsukita, Claudin

multigene family encoding four-transmembrane domain

protein components of tight junction strands, Proc Natl

Acad Sci U S A 96 (1999) 511–516

[5] Y.H Loh, A Christoffels, S Brenner, W Hunziker, B Venkatesh,

Extensive expansion of the claudin gene family in the

teleost fish, Fugu rubripes, Genome Res 14 (2004) 1248–1257

[6] S Amasheh, N Meiri, A.H Gitter, T Schoneberg, J Mankertz, J.D Schulzke, M Fromm, Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells,

J Cell Sci 115 (2002) 4969–4976

[7] O.R Colegio, C Van Itallie, C Rahner, J.M Anderson, Claudin extracellular domains determine paracellular charge selectivity and resistance but not tight junction fibril architecture, Am J Physiol Cell Physiol 284 (2003) C1346–C1354

[8] O.R Colegio, C.M Van Itallie, H.J McCrea, C Rahner, J.M Anderson, Claudins create charge-selective channels in the paracellular pathway between epithelial cells, Am J Physiol Cell Physiol 283 (2002) C142–C147

[9] C Van Itallie, C Rahner, J.M Anderson, Regulated expression

of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability, J Clin Invest 107 (2001) 1319–1327

[10] H Wen, D.D Watry, M.C Marcondes, H.S Fox, Selective decrease in paracellular conductance of tight junctions: role

of the first extracellular domain of claudin-5, Mol Cell Biol

24 (2004) 8408–8417

[11] M.D Alexandre, B.G Jeansonne, R.H Renegar, R Tatum, Y.H Chen, The first extracellular domain of claudin-7 affects paracellular Cl− permeability, Biochem Biophys Res Commun 357 (2007) 87–91

[12] A.S Yu, A.H Enck, W.I Lencer, E.E Schneeberger, Claudin-8 expression in Madin-Darby canine kidney cells augments the paracellular barrier to cation permeation, J Biol Chem 278 (2003) 17350–17359

[13] C.M Van Itallie, A.S Fanning, J.M Anderson, Reversal of charge selectivity in cation or anion-selective epithelial lines

by expression of different claudins, Am J Physiol Renal Physiol 285 (2003) F1078–F1084

[14] J Hou, D.L Paul, D.A Goodenough, Paracellin-1 and the modulation of ion selectivity of tight junctions, J Cell Sci

118 (2005) 5109–5118

[15] S Angelow, R El-Husseini, S.A Kanzawa, A.S Yu, Renal localization and function of the tight junction protein, claudin-19, Am J Physiol Renal Physiol 293 (2007) F166–F177

[16] M Konrad, A Schaller, D Seelow, A.V Pandey, S Waldegger,

A Lesslauer, H Vitzthum, Y Suzuki, J.M Luk, C Becker, K.P Schlingmann, M Schmid, J Rodriguez-Soriano, G Ariceta,

F Cano, R Enriquez, H Juppner, S.A Bakkaloglu, M.A Hediger,

S Gallati, S.C Neuhauss, P Nurnberg, S Weber, Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular involvement, Am J Hum Genet 79 (2006) 949–957 [17] S.R.K Vedula, T.S Lim, P.J Kausalya, B Lane, G Rajagopal,

W Hunziker, C.T Lim, Quantifying forces mediated by integral tight junction proteins in cell-cell adhesion, Exp Mech (in press),doi:10.1007/s11340-007-9113-1

[18] K Kubota, M Furuse, H Sasaki, N Sonoda, K Fujita, A Nagafuchi, S Tsukita, Ca(2+)-independent cell-adhesion activity of claudins, a family of integral membrane proteins localized at tight junctions, Curr Biol 9 (1999) 1035–1038

[19] M Furuse, K Furuse, H Sasaki, S Tsukita, Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells,

J Cell Biol 153 (2001) 263–272

[20] J.H Lipschutz, S Li, A Arisco, D.F Balkovetz, Extracellular signal-regulated kinases 1/2 control claudin-2 expression in Madin-Darby canine kidney strain I and II cells, J Biol Chem

280 (2005) 3780–3788

[21] C.M Van Itallie, J Holmes, A Bridges, J.L Gookin, M.R Coccaro,

W Proctor, O.R Colegio, J.M Anderson, The density of small

tight junction pores varies among cell types and is increased

by expression of claudin-2, J Cell Sci 121 (2008) 298–305

[22] J Hou, A.S Gomes, D.L Paul, D.A Goodenough, Study of

claudin function by RNA interference, J Biol Chem 281 (2006)

36117–36123

[23] H Fujita, K Sugimoto, S Inatomi, T Maeda, M Osanai, Y

Uchiyama, Y Yamamoto, T Wada, T Kojima, H Yokozaki,

T Yamashita, S Kato, N Sawada, H Chiba, Tight junction

proteins claudin-2 and -12 are critical for vitamin D-dependent

Ca2+ absorption between enterocytes, Mol Biol Cell 19 (2008)

1912–1921

[24] D.T Thwaites, G.T McEwan, N.L Simmons, The role of the

proton electrochemical gradient in the transepithelial

absorption of amino acids by human intestinal Caco-2 cell

monolayers, J Membr Biol 145 (1995) 245–256

[25] F.H Leibach, V Ganapathy, Peptide transporters in the

intestine and the kidney, Annu Rev Nutr 16 (1996) 99–119

[26] S Ferruzza, M.L Scarino, G Rotilio, M.R Ciriolo, P Santaroni,

A.O Muda, Y Sambuy, Copper treatment alters the

permeability of tight junctions in cultured human intestinal

Caco-2 cells, Am J Physiol 277 (1999) G1138–G1148

[27] W Hanley, O McCarty, S Jadhav, Y Tseng, D Wirtz, K

Konstantopoulos, Single molecule characterization of

P-selectin/ligand binding, J Biol Chem 278 (2003) 10556–10561

[28] W.D Hanley, D Wirtz, K Konstantopoulos, Distinct kinetic

and mechanical properties govern selectin–leukocyte

interactions, J Cell Sci 117 (2004) 2503–2511

[29] P Panorchan, M.S Thompson, K.J Davis, Y Tseng, K

Konstantopoulos, D Wirtz, Single-molecule analysis of

cadherin-mediated cell–cell adhesion, J Cell Sci 119 (2006)

66–74

[30] M Benoit, D Gabriel, G Gerisch, H.E Gaub, Discrete

interactions in cell adhesion measured by single-molecule

force spectroscopy, Nat Cell Biol 2 (2000) 313–317

[31] C.M Franz, A Taubenberger, P.H Puech, D.J Muller, Studying

integrin-mediated cell adhesion at the single-molecule level

using AFM force spectroscopy, Sci STKE 2007 (2007) pl5

[32] D.F Tees, R.E Waugh, D.A Hammer, A microcantilever device

to assess the effect of force on the lifetime of selectin–

carbohydrate bonds, Biophys J 80 (2001) 668–682

[33] S.E Chesla, P Selvaraj, C Zhu, Measuring two-dimensional

receptor–ligand binding kinetics by micropipette, Biophys J

75 (1998) 1553–1572

[34] S.R Vedula, T.S Lim, P.J Kausalya, W Hunziker, G Rajagopal,

C.T Lim, Biophysical approaches for studying the integrity

and function of tight junctions, Mol Cell Biomech 2 (2005)

105–123

[35] M Furuse, H Sasaki, S Tsukita, Manner of interaction of

heterogeneous claudin species within and between tight

junction strands, J Cell Biol 147 (1999) 891–903

[36] G.I Bell, Models for the specific adhesion of cells to cells,

Science 200 (1978) 618–627

[37] E Evans, K Ritchie, Dynamic strength of molecular adhesion

bonds, Biophys J 72 (1997) 1541–1555

[38] Y Tsukasaki, K Kitamura, K Shimizu, A.H Iwane, Y Takai,

T Yanagida, Role of multiple bonds between the single

cell adhesion molecules, nectin and cadherin, revealed

by high sensitive force measurements, J Mol Biol 367 (2007) 996–1006

[39] S.R Vedula, T.S Lim, S Hui, P.J Kausalya, E.B Lane, G Rajagopal, W Hunziker, C.T Lim, Molecular force spectroscopy of homophilic nectin-1 interactions, Biochem Biophys Res Commun 362 (2007) 886–892

[40] W Baumgartner, P Hinterdorfer, W Ness, A Raab, D Vestweber, H Schindler, D Drenckhahn, Cadherin interaction probed by atomic force microscopy, Proc Natl Acad Sci U S A 97 (2000) 4005–4010

[41] E Perret, A Leung, H Feracci, E Evans, Trans-bonded pairs of E-cadherin exhibit a remarkable hierarchy of mechanical strengths, Proc Natl Acad Sci U S A 101 (2004) 16472–16477

[42] C Gergely, J Voegel, P Schaaf, B Senger, M Maaloum, J.K Horber, J Hemmerle, Unbinding process of adsorbed proteins under external stress studied by atomic force microscopy spectroscopy, Proc Natl Acad Sci U S A 97 (2000) 10802–10807

[43] X Zhang, E Wojcikiewicz, V.T Moy, Force spectroscopy of the leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction, Biophys J 83 (2002) 2270–2279

[44] L Gilles, C.R Vogel, B.L Ellerbroek, Multigrid preconditioned conjugate-gradient method for large-scale wave-front reconstruction, J Opt Soc Am A Opt Image Sci Vis 19 (2002) 1817–1822

[45] C.M Van Itallie, J.M Anderson, Claudins and epithelial paracellular transport, Annu Rev Physiol 68 (2006) 403–429

[46] R Merkel, P Nassoy, A Leung, K Ritchie, E Evans, Energy landscapes of receptor–ligand bonds explored with dynamic force spectroscopy, Nature 397 (1999) 50–53

[47] E Evans, Probing the relation between force–lifetime–and chemistry in single molecular bonds, Annu Rev Biophys Biomol Struct 30 (2001) 105–128

[48] K.T Ferreira, B.S Hill, The effect of low external pH on properties of the paracellular pathway and junctional structure in isolated frog skin, J Physiol 332 (1982) 59–67 [49] K Fujita, J Katahira, Y Horiguchi, N Sonoda, M Furuse, S Tsukita, Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein, FEBS Lett 476 (2000) 258–261 [50] B Litkouhi, J Kwong, C.M Lo, J.G Smedley III, B.A McClane,

M Aponte, Z Gao, J.L Sarno, J Hinners, W.R Welch, R.S Berkowitz, S.C Mok, E.I Garner, Claudin-4 overexpression in epithelial ovarian cancer is associated with hypomethylation and is a potential target for modulation of tight junction barrier function using a C-terminal fragment of Clostridium perfringens enterotoxin, Neoplasia (New York, N.Y.) 9 (2007) 304–314

[51] M.J Evans, T von Hahn, D.M Tscherne, A.J Syder, M Panis,

B Wolk, T Hatziioannou, J.A McKeating, P.D Bieniasz, C.M Rice, Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry, Nature 446 (2007) 801–805