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
Trang 1Kinetics of Adhesion Mediated by Extracellular
Loops of Claudin-2 as Revealed by Single-Molecule Force Spectroscopy
Tong Seng Lim1,2, Sri Ram Krishna Vedula3, Walter Hunziker4
and Chwee Teck Lim3⁎
1Bioinformatics Institute,
Agency for Science,
Technology and Research
(A*STAR), 30 Biopolis Street,
#07-01 Matrix,
Singapore 138671
2NUS Graduate School for
Integrative Sciences and
Engineering (NGS), Centre for
Life Sciences (CeLS), #05-01,
28 Medical Drive,
Singapore 117456
3Division of Bioengineering and
Department of Mechanical
Engineering, 9 Engineering
Drive 1, National University of
Singapore, Singapore 117576
4Institute 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 4 June 2008
Available online
10 June 2008
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 trans-interaction, the second extracellular loop does not interact with the full-length 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
Edited by K Kuwajima
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
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 Ca2+-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,2a short cytoplasmic N-terminal sequence, two extracellular loops, and an intracellular C-terminus
*Corresponding author E-mail address:ctlim@nus.edu.sg
Abbreviations used: Cldn, claudin; TJ, tight junction;
AFM, atomic force microscopy; MC, Monte Carlo
Available online at www.sciencedirect.com
0022-2836/$ - see front matter © 2008 Elsevier Ltd All rights reserved.
Trang 2that binds to cytoplasmic proteins through a PDZ
motif.6The two extracellular loops of Cldns of adjacent
cells trans-interact to form the paracellular TJ strands
It has previously been shown that the first
extra-cellular loop of Cldn2,7,8Cldn4,8,9,10Cldn5,11Cldn7,12
Cldn8,13 Cldn15,9,14 Cldn16,15 and Cldn1916,17
con-fers charge-selective paracellular permeability to
epithelial monolayers while the second extracellular
loop acts as a receptor for a bacterial toxin for Cldn318
and Cldn4.19However, the interaction kinetics of the
individual extracellular loops at the molecular level
remains unclear In this study, we have used
single-molecule force spectroscopy to probe the molecular
interactions between recombinant N-terminal
glu-tathione 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
extra-cellular loops to the overall adhesion kinetics
Our results show that the first extracellular loop
of Cldn2 is the major determinant of
trans-interac-tions 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
mi-croscopy (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,
Tai-wan) and Alexa-488-labeled goat anti-mouse
sec-ondary 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.21,23,24When a contact force of
200 pN and contact time of 1 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
N86% 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
3a).26For each reproach velocity, hundreds of force– distance curves (nN500) were collected and ana-lyzed 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 functiona-lized 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 do not trans-interact, which is consistent with previous findings.27
The first extracellular loop of Cldn2 is sufficient for promotingtrans-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 interac-tions in the presence of an antibody targeting the first extracellular loop indicated that C2E2/C2E2 do not trans-interact (C2E2_C2E2 and Cldn2_Cldn2_Ab,
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, clau-din-2; C2E1, first extracellular loop of Cldn2; APTES, 3-aminopropyltriethoxysilane; BS3, bis(sulfosuccinimidyl) suberate; AntiGST, antibody targeting GST
Trang 3Table 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 do 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 interac-tion kinetics of Cldn2/Cldn2 and C2E1/C2E1 inter-actions 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 inter-actions between intercellular adhesion molecules, such as nectin/nectin,30,31 VE-cadherin/VE-cadhe-rin,32N-cadherin/N-cadherin, and
E-cadherin/E-cad-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 50μm
Trang 4herin interactions.22,30,33In the model, the probability
density function for the dissociation of a bound
complex at force f is given by:
P fð Þ¼ koff0
rf
exp xhf
kBT
exp k0offkBT
xhrf 1 exp xhf
kBT
ð1Þ where rfis the rate of force application (i.e., loading
rate), kBis the Boltzmann constant, T is the absolute
temperature, koff0 is the unstressed dissociation
con-stant, and xβis 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)
h f i ¼kBT
xh exp koff0 kBT
xhrf
Ei k
0 offkBT
xhrf
ð2Þ
Here, EiðzÞ ¼Rzlt1expðtÞdt 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
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
Table 1 Experiments for studying homophilic Cldn2/Cldn2, C2E1/C2E1, C2E1/C2E2, and C2E2/C2E2 interactions corresponding to histograms depicted inFig 4
Interaction type AFM tip a Glass substrate a
4 Cldn2_Cldn2_Ab GST-Cldn2 + Antibody GST-Cldn2 + Antibody
5 Cldn2_Cldn2_C2E1 GST-Cldn2 + GST-C2E1 GST-Cldn2 + GST-C2E1
7 Cldn2_Cldn2_C2E2 GST-Cldn2 + GST-C2E2 GST-Cldn2 + GST-C2E2
9 C2E1_Cldn2_Ab GST-C2E1 GST-Cldn2 + Antibody
10 C2E1_C2E1_Ab GST-C2E1 + Antibody GST-C2E1 + Antibody
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).
Trang 5Fig 4 Rupture force histograms obtained at a reproach velocity of 1 μm s− 1for 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 (koff0 ) 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
Trang 6dissociation constant (koff0 ) and the reactive compliance
(xβ) of Cldn2/Cldn2 and C2E1/C2E1 As shown inFig
5, the average unbinding force of Cldn2/Cldn2 and
C2E1/C2E1 complexes increases with increasing
load-ing rate in the range of loadload-ing 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∼103pN/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
force spectroscopy of C2E1/C2E1 (Fig 5).Table 2lists the kinetic parameters (unstressed dissociation off-rate,
koff0, and reactive compliance, xβ) of the two energy 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
To corroborate our experimental results with Bell-Evans model predictions, Monte Carlo (MC) simu-lations of the dissociation of Cldn2/Cldn2 and C2E1/C2E1 under constant loading rate were per-formed using a previously described procedure.22,36 Thousands of rupture forces [Frup= (rf)(nΔt)] were calculated, for which the probability of bond rupture Prup
Prup ¼ 1 exp k0offkBT
xhrf exp xhrfnDt
kBT
1
ð3Þ was greater than Pran, a random number between 0 and 1 Here, nΔt is the time interval needed to break
a bond andΔt is the time step (Δt=10− 6s was used
in the simulation) MC simulations were conducted
by using unstressed dissociation rate (koff0 = 6.39 s–1 for Cldn2/Cldn2 and koff0 = 8.29 s–1for C2E1/C2E1) and reactive compliance (xβ=0.19 nm for Cldn2/ Cldn2 and xβ= 0.1 nm for C2E1/C2E1) obtained
Table 2 Comparison of adhesion kinetics of homophilic
interactions mediated by Cldn2/Cldn2 and C2E1/C2E1
Molecular
pairsa
Loading rate
(pN/s)
Rate of dissociation a ,
k0off (s− 1)
Reactive compliance a ,
x β (nm) C2E1/C2E1 102–10 3
8.4 × 10− 4 0.90
10 3 –10 4 8.29 0.10 Cldn2/Cldn2 102–10 3
4.4 × 10− 3 0.94
10 3 –10 4 6.39 0.19
a Reactive compliance, x β , and the unstressed bond
dissocia-tion rate, k off0 , 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.
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 (koff0 = 6.39 s–1 for Cldn2/Cldn2 and koff0 = 8.29 s–1for 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
Trang 7experimentally for Cldn2/Cldn2 between loading
rates of 103and 104(seeTable 2) As shown inFig 6,
there is a good agreement between the MC
simula-tion and experimental results Loading rate
(loga-rithm scale) was assumed to be normally distributed
within the simulation range of loading (103–104pN/s)
(χ2test, pb0.05) 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
bet-ween 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-interactions27similar 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,40,41or cell aggregation assays.42
Cldns have been predicted to possess four
transmembrane helices,2a short intracellular
N-ter-minal sequence, two extracellular loops, and an
internal C-terminus that binds to cytoplasmic pro-teins through a PDZ binding motif.6It 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 Cldn1916,17 determines paracellular charge selectivity, while the second loop of Cldn3 and Cldn4 acts as a receptor for a bacterial toxin.18Here, 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-interac-tions between Cldn2 (Fig 4) Decrease in the frequency of interactions in the presence of recom-binant C2E1, C2E2, or antibody against C2E1 further confirmed that trans-interactions between either C2E2/C2E2 or C2E1/C2E2 do not occur (Fig 4) More recently, it has been demonstrated that the second extracellular loop of Cldn5 (C5E2) is in-volved in TJ strand formation via trans-interac-tions.43 The exact reasons for why the trans-interaction 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 C5E2.43Thus, 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 cis-interaction with other parts of the Cldn2 protein, for example, C2E1, possibly through an involvement of E159.43,44Thus, 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 concep-tual energy landscapes of dissocia-tion pathway between homophilic Cldn2/Cldn2 and C2E1/C2E1 inter-actions 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 gen-eral, dissociation pathways for Cldn2/Cldn2 and C2E1/C2E1 inter-actions 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
Trang 8activation barrier process (Figs 5 and 7) The
dis-sociation rate of the bound complex in N barrier
model is given by multiple Bell's model arranged in
series28,45,46:
k1¼XN
i¼1
k0iexpðxhiF=kBTÞ
ð4Þ
where kBis 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
con-stants were used to estimate the energy differences
(ΔG) between transition state energies Cldn2/Cldn2
and C2E1/C2E1 complexes:
DG ¼ kBTlnðk0
Cldn2=k0
where kCldn20 and kC2E10 are dissociation rate
con-stants of the Cldn2/Cldn2 and C2E1/C2E1,
respec-tively 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 kBT), 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 forcesN∼50 pN The dissocia-tion 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 5 and 8)
A critical concern in force spectroscopy is whether the recombinant protein under investigation main-tains its functional structure or not It has been shown previously that isolated and purified recom-binant 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 force-dependent 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 inTable 2 Cldn2, claudin-2; C2E1, first extracellular loop of Cldn2
Trang 9Cldn2-transfected L-cells (which do not express any
endogenous Cldns4,48) were incubated with
GST-Cldn2, 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
Colocali-zation 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
Under-standing interactions mediated by Cldns is
impor-tant 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,
single-molecule 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
des-cribed previously.36Soft silicon nitride tips (Veeco, 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-aminopropyl-triethoxysilane, Sigma) in acetone for 3 min They were
then rinsed thrice in acetone and then incubated in a
solution of BS3[bis(sulfosuccinimidyl) suberate, 2 mg/ml,
Pierce] for 30 min, followed by the incubation of anti-GST
antibody (10μg/ml, Invitrogen) for 2 h After quenching
the reaction using 1 M Tris buffer, the tips were incubated
in recombinant full-length GST-Cldn2 (10μg/ml,
Protein-tech Group, Inc, USA), GST-Cldn1, GST-C2E1 (10μg/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 2 h Unbound recombinant proteins were
washed off with phosphate-buffered saline Tips were
blocked in 1% bovine serum albumin before
expe-riments.36 Recombinant Cldn2, Cldn1,
GST-C2E1, 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 Cldn2
anti-body (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-488-labeled 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 (10μg/ml, Abnova) for 30 min They were then washed to remove any unbound antibody before the experiments For competition assays, tips func-tionalized with GST-Cldn2 were probed in the presence of GST-C2E1 or GST-C2E2 (10μg/ml) in phosphate-buffered saline buffer
Molecular force spectroscopy Force curves were acquired on a MultiMode™ Pico-force™ 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 (Cldn1, Cldn2, GST-C2E1, or GST-C2E2) immobilized on the glass cover slips were probed with cantilevers functionalized with recom-binant 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 1 ms Under such condition, the adhesion frequency was b25%, which would give a N86% probability of the rupture forces being due to single-bond rupture according to Poisson statistics.35Force 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.20and Panorchan et al.,22rupture 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 for the mole-cular interactions were extracted (see Results) These parameters characterize the binding interactions between homophilic Cldn2/Cldn2 and C2E1/C2E1 proteins at the single-molecule level
Acknowledgements
This work was supported by the Biomedical Research Council from the Agency for Science, Technology and Research, Singapore Their funding support is gratefully acknowledged
Supplementary Data
Supplementary data associated with this article can be found, in the online version, atdoi:10.1016/ j.jmb.2008.06.009
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