Received: 5 May 2010, Revised: 8 November 2010, Accepted: 18 November 2010, Published online in Wiley Online Library: 2011

Characterization of N-cadherin unbindingproperties in non-malignant (HCV29) andmalignant (T24) bladder cellsMałgorzata Lekkaa*, Dorota Gilb, Wojciech Dabrosc, Justyna Jaczewskad,Andrzej J. Kulike, Janusz Lekkia, Zbigniew Stachuraa, Jerzy Stachurac

and Piotr Laidlerb

The expression of N-cadherin, characteristic of various cancers, very often leads to changes in the cells’ adhesiveproperties. Thus, we sought to find out if N-cadherin expressed in various, but cancer-related cells, differs in itsfunctional properties that could contribute to variations in cells’ phenotypes. In our work, measurements of anunbinding force of a single N-cadherin molecule, probed with the same antibody both on a surface of livingnon-malignant (HCV29) and malignant cells (T24) of bladder cancer, were carried out with the use of an atomic forcemicroscopy. The results show the enhanced N-cadherin level in T24 malignant cells (8.7% vs. 3.6% obtained fornon-malignant one), confirmed by the Western blot and the immunohistochemical staining. The effect wasaccompanied by changes in unbinding properties of an individual N-cadherin molecule. Lower unbinding forcevalues (26.1W 7.1 pN) in non-malignant cells reveal less stable N-cadherin complexes, as compared to malignant cells(61.7W 14.6 pN). This suggests the cancer-related changes in a structure of the binding site of the antibody, located atthe extracellular domain of N-cadherin. Copyright � 2011 John Wiley & Sons, Ltd.

Keywords: unbinding force; N-cadherin; bladder cancer; single molecule spectroscopy; specific interactions

INTRODUCTION

Cadherins are one of the key molecules that mediate andregulate many cellular processes, such as intercellular adhesion,cell motility, and also cell signaling (Gooding et al., 2004). Onadjacent cells, they are located at the intercellular adhesionjunctions and mediate calcium-dependent adhesion through thedirect interactions between their extracellular domains. Theadhesion also requires anchorage of cadherins to cytoskeletonthrough a complex of proteins associated with their internaldomains, i.e., catenins. These proteins, clustered on thecytoplasmic side of cadherin, act as a switch point for theregulation of adhesive strength (Mege et al., 2006). The structuresof N- and E-cadherins are similar. They are single pass,transmembrane proteins possessing both extracellular andcytoplasmic domains (Gooding et al., 2004). The former oneconsists of five repeats, so-called ectodomains, possessingbinding sites for calcium. The latter one is directly associatedwith b-catenin and/or plakoglobin (g-catenin), which links it toactin cytoskeleton through a-catenin (Stemmler, 2008). Theinteraction of cadherins with cytoplasmic proteins and actinfilaments is thought to reveal many aspects of cell–cell adhesion,such as strengthening of adhesive contacts (Kobielak and Fuchs,2004) or modulation of cell motility (Mege et al., 2006).The alterations in expression and functions of cadherins

correlate with the progression to tumor malignancy (Ramburanand Govender, 2002; Stemmler, 2008; Wheelock et al., 2008). Theloss of expression E-cadherin in epithelial carcinomas has longbeen thought to be the main reason for the disruption of tightepithelial cell–cell contacts, leading to the progression of tumors

to an invasive state (Mareel et al., 1997). Aberrant expression ofcadherins very often involves changes in the catenin expression,i.e., b-catenin and plakoglobin (Ben-Ze’ev and Geiger, 1998;Zhurinsky et al., 2000). Increased expression of N-cadherin hasbeen found to be a potential tumor marker (Hajra and Fearon,

(wileyonlinelibrary.com) DOI:10.1002/jmr.1123

Research Article

* Correspondence to: M. Lekka, Laboratory of Biophysical Microstudies, TheHenryk Niewodniczanski Institute of Nuclear Physics, Polish Academy ofSciences, Radzikowskiego 152, 31–342 Krakow, Poland.E-mail: malgorzata.lekka@ifj.edu.pl; E-mail: www.ifj.edu.pl

a M. Lekka, J. Lekki, Z. Stachura

Laboratory of Biophysical Microstudies, The Henryk Niewodniczanski Institute

of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31–342

Krakow, Poland

b D. Gil, P. Laidler

Chair of Medical Biochemistry, Jagiellonian University Medical College,

Kopernika 7, 31–034 Krakow, Poland

c W. Dabros, J. Stachura

Chair of Patomorphology, Jagiellonian University Medical College, Grzegor-

zecka 16, 31–531 Krakow, Poland

d J. Jaczewska

Smoluchowski Institute of Physics, Jagellonian University, Reymonta 4, 30-059

Krakow, Poland

e A. J. Kulik

Institute of Physics of Biological Systems, Laboratory of Physics of

Living Matter, Ecole Polytechnique Federale de Lausanne, 1015 Lausanne,

Switzerland

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2002; Derycke and Bracke, 2004; Hazan et al., 2004) also in case ofbladder cancers (Gee et al., 1998; Bringuier et al., 1999; Lascombeet al., 2006). In particular, expression of N-cadherin has beenstudied as a function of the stage of tumor development, aimingat evaluation of prognostic value of the molecule. The studieswere performed within a cohort of 101 patients suffering frombladder cancer. The results showed that N-cadherin can be asuccessful prognostic marker of progression for the superficialurothelial tumors (Lascombe et al., 2006).As the changes in adhesive properties very often reflect the

malignant transformation, a natural direction for the studies isdetermination of these alterations. At the molecular level, it canbe accomplished throughmeasurements of the unbinding forcesbetween particular molecules that are recognized as potentialmarkers of the disease. Expression of different types of moleculesat the protein (functional) level can be studied with severalmethods such as immunodetection of blotted proteins (i.e., theWestern blot technique), fluorescence microscopy or flowcytometry. Recently, novel methods have appeared. They canoperate with high resolution at a single molecule level. One ofthem is the atomic force microscopy (AFM; Binnig et al., 1986).Using this technique, the forces involved in specific interactionscan be measured as a force needed for detaching the AFM tip,modified with ligands, from receptors either immobilized on thesubstrate, or present on a surface of the cell plasma membrane.So far, the AFM technique has beenmostly applied to the study ofthe interaction of different molecular complexes for isolatedmolecules in buffer conditions (Lekenkari and Horton, 1999;Zlatanova and vanHolde, 2006). However, there is a number ofstudies showing the use of AFM for probing them directly on asurface of living cells (Zhang et al., 2004; Taubenberger et al.,2007). Several studies have been focused on differences betweennormal and cancerous cells, where the AFM technique has beenshown to be complementary to other biochemical methods(Laidler et al., 2005; Lekka et al., 2006; Winiarska et al., 2008). In ourprevious studies (Laidler et al., 2005), we were able to detect andcompare the expression of the membrane form of prostate-specific antigen (PSMA) in prostate cancer cells of various origins.The results proved the identity of the PSMA binding potentialregardless of whether it was expressed by the cells naturally orupon simulation with either fibroblast growth factor (FGF) and/orestradiol. This could be material to the search of membranetumor markers potentially useful for immunotherapy. In aseparate study, the differences of single-molecule interactionsfor carbohydrates recognition (Lekka et al., 2006) clearlydemonstrated the AFM as a method to investigate the bindingsite identity of the complex carbohydrates.A number of AFM studies has also been devoted to the

molecular aspects of cadherins, aiming at exploring mechanismsof their molecular interactions (Baumgartner et al., 2000; duRoure et al., 2006; Praksam et al., 2006; Pittet et al., 2007; Shi et al.,2008; Sivasankar et al., 2009; Zhang et al., 2009). Although thecadherin–cadherin interactions have already been studied byAFM, the technique has not yet been applied to measure theinteraction forces for a complex, which is composed of anantibody and a cadherin. For this reason, we used a monoclonalantibody in our work to detect N-cadherin and to analyze itsunbinding properties. The studies were addressed to the issue ofwhether the altered expression of N-cadherin in cancerous cells isaccompanied by distinct unbinding properties of the singleN-cadherin molecule as compared to that present in referencecells.

MATERIAL AND METHODS

Cell lines

The studies were performed on two human cell lines: referenceHCV29 (non-malignant transitional epithelial cells of the urether,ATCC) and T24 (transitional cancer cells of the urine bladder,ATCC). Cells were grown in culture flasks (Sarstedt) in a RPMI 1640medium (Sigma) supplemented with 10% fetal calf serum (Alab,pH7.4) at 378C in 95% air/5%CO2 atmosphere. After few passages,cells were trypsinized (0.05% in EDTA-trypsin solution, Sigma),transferred to Petri dishes with a coverslip inside, and cultureduntil semi-confluent layer was formed. Afterwards, they wereused either for AFM measurements or in immunohistochemicalstaining.

Antibodies

Primary antibodies were used for: N-cadherin against cytoplasmicdomain (clone BTA7, R&D System), N-cadherin against extra-cellular part (clone GC-4, Sigma), E-cadherin (clone 34/E-Cadherin, BD Transduction LaboratoriesTM), b-catenin (clone14/Beta-Catenin, BD Transduction LaboratoriesTM), g-catenin(clone 11B6, Novocastra), and b-actin (clone AC-15, Sigma).

Immunohistochemical staining

Immunohistochemical examinations were performed with theuse of immunoperoxidase methods. The coverslip with cells waswashed with phosphate-buffered saline (PBS; pH 7.4) and fixedwith a cold acetone for 10min. Then, coverslips were washedwith deionized water. Endogenous peroxidase activity wasblocked by 3% hydrogen peroxidase (H2O2) in deionized waterfor 8min, followed by rinsing in pure water. Afterwards, coverslipswere immediately immersed in PBS buffer for 5min andincubated with the following antibodies either against cyto-plasmic domain of N-cadherin (HCV29 cells: 1:50, 60min; T24cells: 1:50, 30min) or b-catenin (HCV29 cells: 1:100, 60min; T24cells: 1:100, 30min) or plakoglobin (HCV29 cells: 1:50, overnight;T24 cells: 1:50, 30min). Finally, coverslips were washed with PBSbuffer, and EnVisionTM Systems (Dako) was applied for 30min,washed again with PBS buffer. Visualization was performed bytheir incubation for 10min in a solution of carbazol (Dako). Afterwashing with deionized water, coverslips with cells werecounter-stained with hematoxylin (�8 s) to visualize a cellnucleus. Such prepared samples were examined using opticalmicroscope (Olympus type BX51, magnification 640�).

Western blot analysis

The Western blotting analysis was performed for cells lysed in asample buffer composed of 62.5mM Tris–HCl (pH 6.8, Sigma),2%w/v sodium dodecylsulfate (SDS; Sigma), 10% glycerol, and50mM b-mercaptoethanol (Sigma). Next, cells were sonicated for15 s and heated to 95–1008C for 5min. Protein concentration wasassayed using Total Protein Kit (Micro Lowry, Peterson’sModification, Sigma TP0300). Sample of cells extracts containing40mg of total protein was loaded on 10% gels for SDS–PAGE andafterwards transferred to a polyvinylidene difluoride membrane(Roche) and blocked for 1 h with casein solution 1� in distilledwater (SP-5020, Vector Laboratories). Membranes were thenincubated overnight with the appropriate primary antibodyagainst: E-cadherin (1:2000, 0.125mg/ml), N-cadherin (1:1500,

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3mg/ml), g-catenin (1:50), b-catenin (1:2000, 0.125mg/ml), andb-actin (1:4000, 0.5mg/ml).

Fluorescence microscopy

Fluorescent microscopy was carried out on cells (both HCV29 andT24 ones) cultured on glass coverslips for 72 h in a RPMI 1640medium supplemented with 10% fetal calf serum at 378C in 95%air/5%CO2 atmosphere (same concentrations of 1� 104 cells wasused for both cell lines). Next, cells were washed with PBS bufferand fixed with 2% of paraformaldehyde (30min at roomtemperature). After removing the fixative, cells were incubatedwith the 0.2% solution of Triton X-100 in 48C for 5min, and nextrinsed with PBS. Afterwards, coverslips were incubated withAlexa-Fluor 488 conjugated with phalloidin (1:100; PBS solution,Molecular Probes) for 30min, washed again with PBS buffer andclosed.The fluorescence images were recorded using Olympus

BX51 microscope equipped with a 100W mercury lamp(uniformly illuminating the whole sample area), a filter U-MWIG2(lexit¼ 530–550 nm, lemit> 580 nm), and an objective (UPlanApo)at magnification 4�. For image recording, the QICAM 12 bit digitalcamera was used. This camera provides a 1392� 1040 pixel(1.4 million) image with a 1/2-inch optical format.

AFM measurements

Topography and elasticity

The topography of the living cells and their elastic propertieswere measured using a commercially available device (PSIAXE120, Park Systems, Korea) equipped with the ‘‘liquid cell’’ setup.Standard, non-sharpened, gold coated, and silicon nitridecantilevers (MLCT-AUHW, Veeco), with the nominal spring

constant of 0.01 N/m were used in all measurements. Thecoverslips with cells were immersed in 50mM Tris-buffered saline(pH7.4, Sigma), supplemented with 1mM of Ca2þ, Mg2þ, andMn2þ), and placed into the ‘‘liquid cell’’ laid on the AFM scanner.All measurements were performed at room temperature. Surfacetopography was recorded in contact mode with scan rate of 2 Hz,set point of 2 nN, for scan size of 70� 70mm (256� 256 points).The elasticity measurements were performed on 24 cells

chosen randomly for each cell line. First, a cell was localized withthe use of optical microscope and the cantilever was placedaround cell center, i.e., cell nucleus. Then a map consisting of4� 4 points corresponding to a scan size of 10� 10mm waschosen. Thus, 16 force curves were recorded for each cell (thesenumber was sufficient to notice the differences in the cellelasticity between both studied cell lines; see Figure 1A). TheYoung’s modulus was calculated with the approximation of theAFM four-sided pyramidal tip by a cone with a¼ 358, andassuming the Poisson coefficient to be 0.5 (Lekka et al., 1999). TheYoung’s modulus values were used to calculate the contact areabetween the antibody-modified AFM tip and cell surface usingthe Hertz model (Sneddon, 1965).

Cantilever functionalization

Before functionalization, cantilevers (0.01N/m, MLCT, Veeco)were cleaned in acetone, and then irradiated with UV light for30min. Afterwards, they were immersed in 4% solution of3-amino-propyltriethoxysilne (APTES, Sigma), in toluene for 2 handwashed in pure water (0.08mS, Cobrabid purification system).Next, they were functionalized in a 2.5% glutaraldehyde (Sigma)aqueous solution for 20min. Finally, such pre-treated cantileverswere immersed in the antibody solution (N-cadherin, 1:42) for 1 h,and then rinsed with the PBS buffer. They were immediately used

Figure 1. (A) The Young’s modulus determined for two studied bladder cell lines. The mean value was 3.09� 0.33 and 0.78� 0.20 kPa for HCV29 and

T24 cells, respectively. Insets show the AFM image in deflection mode of the corresponding cell type after 24 h of growing after trypsinization. (B and C)Fluorescent images of HCV29 (B) and T24 (C) bladder cells cultured on glass coverslips 72 h after trypsinization. The actin filaments were stained with

phalloidin-Alexa-Fluor 488 conjugate.

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in the AFMmeasurements. The spring constant of cantilevers wascontrolled by monitoring thermally excited resonant frequencyof cantilever (Sader et al., 1995) before applying the functiona-lization protocol. The determined spring constant varied from0.008 to 0.009 N/m.

Unbinding force

Measurements were performed with the use of a home-builtatomic force microscope (Lekka et al., 1999) equipped with a‘‘liquid cell’’ setup, in 50mM Tris-buffered saline (pH7.4, Sigma),supplemented with 1mM of Ca2þ, Mg2þ, and Mn2þ, at roomtemperature. Each measured cell was localized with the use of anoptical microscope, and then force curves were recorded with theloading rates in the range between 2500 and 5000 pN/s forHCV29 cells, and between 3600 and 6600 pN/s for T24 cells.For the measurements, a map of 9� 9 spots was chosen giving

81 sampling points per one cell. In each spot, so-called forcecurve was recorded. It represents the relationship between thecantilever deflection and the relative cantilever displacement,which is, afterwards, converted into force–distance curve. Such away of recording was dictated by the aim of the studies, whichwas to quantify the expression of N-cadherin–antibody inter-action in a given cell population. Thus, to collect informationabout the alterations present in the entire cell population, ca 30randomly chosen cells were measured. Measurements wererepeated three times, each time with a fresh AFM probe and anew sample (fresh-cultured cells). The recorded number ofunbinding events was similar to those applied for oligosaccharidedetection on a living cell surface reported earlier (Lekka et al.,2006).

Specificity of the measured interaction

To assure specificity of the interaction with N-cadherin, both celltypes were incubated at two concentrations of free antibody: 0.1and 0.4mg/ml, and added to the buffer for 30min in cultureconditions (378C, 5% CO2/95% air). Afterwards, cells were washedwith RPMI medium, only to remove excess of unboundmolecules, and immediately taken for the measurements withthe antibody-modified AFM cantilever. In this manner, freelydissolved antibody molecules bind to some of the N-cadherinbinding sites, thereby competing with antibody moleculesattached to the AFM probe.

RESULTS

Elastic properties of living cells

Two cell lines were chosen: (i) HCV29 cells (non-malignanttransitional epithelial cells of urether) and (ii) T24 cells (malignantcells of urine bladder cancer) to carry out the characterization ofN-cadherin molecule.Determining the Young’s modulus during the measurements

where cells were pressed by the probing AFM tip was the firststep. This was dictated by the fact that the chosen cells possessdistinct elastic properties (Lekka et al., 1999) resulting in variouscontact areas. The elasticity measurement data are presented inFigure 1A. The non-malignant HCV29 cells were around fourtimes stiffer as compared to malignant ones. These values arequantitatively different from those reported previously (Lekkaet al., 1999) due to different culture conditions (no poly-L-lysine

modified glass coverslips, different buffer used during themeasurements). Also, previously larger indentation depths(�1000 nm) were applied while, for the present study, indenta-tion depth of 400 nm has been chosen. In spite of differentconditions, similar tendency clearly persists: HCV29 cells arestiffer than T24 ones. The differences in cell elasticity are usuallyattributed to distinct organization of cytoskeleton filaments. Thiscan be observed in the deflection mode images (insets, AFMimages were recorded after 24 h of cell growth).The Young’s modulus values were used to adjust the size of the

contact area between the probing tip and the cell surface, whichshould be constant during the unbinding force measurements.The elasticity measurements were typically performed on cells

cultured between 24 and 72 h after trypsinization. The cell’sdistribution in the culture is presented in Figure 1B and C. Bothcell types were stained against actin filaments using phalloidinconjugated with Alexa-Fluor 488, and observed using afluorescent microscope. Since the initial concentration of cellsfor both lines was the same (1� 104 cells), one can clearlyobserve the increased number of T24 cells after the same growthtime.

Expression of N-cadherin in HCV29 and T24 cells

The transition from epithelial to mesenchymal phenotypereported for cancer progression (Huber et al., 2005) is associatedwith the increased expression of N-cadherin. Therefore, proteinexpression for both studied cell lines was investigated using theWestern blot performed on cell extracts. The obtained resultsshowed the expression of N-cadherin in cancerous T24 cells to behigher than in non-malignant ones (Figure 2A). Such behaviorwas accompanied by an opposite effect observed for E-cadherin,which was detected in trace amounts only in HCV29 cells

Figure 2. (A) The Western blot analysis clearly shows the over-expression of N-cadherin (129 kDa) in cancerous T24 cells. The transition

from non-malignant (HCV29) to malignant (T24) cell phenotype is accom-

panied by the switch between E-cadherin (120 kDa) and N-cadherin. (B)

b-catenin (92 kDa) and plakoglobin (82 kDa), constituting the cadherin–catenin complex linked with actin cytoskeleton, are present in both cell

lines at similar levels. (C) Level of b-actin (42 kDa) in both cell types.

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(Figure 2A, a very weak band almost invisible in all assays). Thelack of E-cadherin expression in the non-malignant epithelialurothelium cell line HCV29 has already been observed (Laidleret al., 2000; Guan et al., 2009). It was recently confirmed by Guanet al. (2009) indicating that deficiency of E-cadherin is aphenomenon characteristic of in vitro cultures of bladder celllines. Two catenins, b-catenin and g-catenin (plakoglobin), wereexpressed in both cell types, at a similar level (Figure 2B). Also, thelevel of b-actin was comparable (Figure 2C).Since the Western blot provides results for a whole cell extract,

the immunohistochemical staining with respective antibodieswas performed in order to localize the N-cadherin, b-catenin, andplakoglobin in an individual cell. Proteins were indentified at thesites of the cell–cell adhesive contacts (white arrows in Figure 3).These results indirectly estimate the expression level of thestudied molecules in malignant T24 cells since the same antibodyconcentrations were applied. However, for N-cadherin andb-catenin, the incubation time for T24 cells was two timesshorter (30min). In the reference, non-malignant HCV29 cells,plakoglobin was visualized after 24 h of incubation, while forcancerous cells 30min incubation was sufficient to observemembrane expression of this protein.

N-cadherin expression using AFM

The expression of N-cadherin in HCV29 and T24 cells wasinvestigated with the use of an AFM. The unbinding forcemeasurements were accomplished by anchoring the monoclonalantibody against extracellular domain of N-cadherin (clone GC4)to the AFM probe surface through covalent binding. Suchmodified AFM probe was brought to contact with the surface ofliving cells, and then withdrawn (Figure 4A). At themoment whenthe tip detaches from the cell surface, a characteristic jump isobserved (Figure 4B and C). Such a jump corresponds to anunbinding event, i.e., to the unbinding of two interactingmolecules. In our work, force curves were recorded for individualN-cadherin–antibody complexes directly on the surface of livingbladder cells. They show both (i) a rupture of a single complex(Figure 4B and C, upper curves) and (ii) ruptures of severalcomplexes (Figure 4B and C, lower curves). Their presence wasindependent of the cell type.Based on AFM unbinding force measurements, the expression

of N-cadherin can be quantified with an unbinding probability – aparameter, which can be related to the number of receptors oncell surface. It is usually defined as the ratio of the number of

Figure 3. Immunolocalization of N-cadherin, b-catenin, and plakoglobin in bladder cells which was carried out using corresponding monoclonalantibodies against cytoplasmic domains of these proteins. White arrows indicate that the reactions with the corresponding antibodies take place at cell

membrane, i.e., at the border between cells, where adherens junctions occur (for visualization, cell nuclei were stained with hematoxylin). Bar size – 8mm.

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unbinding events to the recorded total number of force curves(i.e., the total number of sampling). For HCV29 cells, the recordedtotal amount of curves was 10 166while for T24 ones its valuewaslower (of 2873), which stemmed from different efficiencies ofdetecting the unbinding events in both cells. The correspondingnumbers of unbinding events were 366 for HCV29 and 250 forT24 cells, resulting with the unbinding probability values of 3.6%and 8.7%, respectively. It includes all measured cells and spots.These probabilities were reproduced in a separate experimentwhere these values were 4.0% and 7.8%, respectively. This clearlyshows larger expression of N-cadherins in malignant T24 cells.The origin of the observed unbinding events was verified by

blocking binding sites of N-cadherin. It was realized by addingfree antibodymolecules to the solution. As a result, the number ofunbinding events decreased, confirming specificity of the studiedinteraction (Figure 5A and B). For HCV29 cells, a drop from 3.6%(no inhibition) to 2.7% (antibody concentration¼ 0.1mg/ml), andto 0.4% (antibody concentration¼ 0.4mg/ml) was observed. ForT24 cells the analogous decrease was noticed. The unbindingprobability dropped from 8.7% to 4.6% and to 2.1% for 0.1 and0.4mg/ml antibody concentrations, respectively.Since the binding site for the GC4 antibody is placed in the

extracellular domain of N-cadherin, the observed jumps couldindicate unfolding of cadherin repeats – ectodomains (Harrisonet al., 2005). This was however excluded. If the force needed forunfolding a single cadherin ectodomain was smaller than theunbinding force recorded for N-cadherin–antibody interaction,one could expect that such a pulling will cause unfolding of these

ectodomains. In such a case, one can observe a characteristicsaw-tooth pattern composed of a few jumps corresponding toparallel unfolded single ectodomains (Carrion-Vazquez et al.,2000). There were no such curves recorded by AFM in ourexperiments (Figure 4B and C). On the contrary, the recordedretrace paths showed jumps characteristic of unbinding of asingle molecular complex typical for many antibody–antigencomplexes (Hinterdorfer and Dufrene, 2006). The moment whenthe tip detaches from the cell surface, i.e., the interactingmolecules unbind, corresponds to the most probable unbindingforce F. Its value should be dependent on a loading rate (Evans1999). For this reason such dependence was also considered(Figure 6). It should be pointed out here that the loading ratecannot be controlled directly in the AFM experiment. Its value isusually derived from the product of the retraction velocity (thisparameter can be set in AFM) and the system spring constant(taking into account the spring constants of the cantilever andthe studied complex). For the same retraction velocity range(1.5–2.9mm/s) used during the measurements in both cell lines,the loading rates varied from 2500 to 5000 and from 3600 to6600 pN/s for HCV29 and T24 cells, respectively. The unbindingforce values, calculated for these limits, varied between 25.3� 8.2and 27.9� 7.5 pN (reference HCV29, force variation�5–6 pN) andof 59.7� 13.0 and 64.1� 10.6 pN (malignant T24, force variation�4–5 pN). The obtained force variations are comparable with theexperimental noise level (7 pN). They are much lower than theerror of estimation of the unbinding force. This allows of aconclusion that there is almost no (or a weak) dependency on the

Figure 4. (A) The mechanical force detection of the complex of monoclonal antibody (GC4) and N-cadherin on a living cell surface using a designedAFM tip. When it is pulled off, the rupture of a single complex occurs. It may be also associated with cell membrane bending and stretching of the linkage

with actin cytoskeleton (arrows indicate two possible linkages of N-cadherin to actin cytoskeleton). (B and C) Force curves recorded for individual

N-cadherin–GC4 antibody complexes, probed directly on a surface of living bladder cells: reference, non-malignant HCV29 (B), and malignant T24 (C).

Independently of the cell type, both (i) a rupture of a single complex (upper curves) and (ii) simultaneous rupture of a few complexes (lower curves) wereobserved. For clarity, only retraction curve is shown.

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applied loading rates. Therefore N-cadherin interactions in bothcell types can be compared directly. If strong loading ratedependence existed, the direct comparison would be difficult.

Properties of a single N-cadherin–antibody complex

Apart from counting how many unbinding events are observed,the AFM enables characterization of properties of a ruptured

single molecular complex through the determination of theunbinding force.All collected unbinding events were used to create corre-

sponding histograms (Figure 7A and B). The size of its bin was setto 7 pN, reflecting the noise level. Then, the force value neededfor breaking a single N-cadherin–antibody complex wasdetermined through analyzing them. The shape of the histogramwas distinct for each cell type (HCV29 or T24 ones). It can beexplained as follows: depending on the number of moleculespresent within the contact area, i.e., receptor density on a cellsurface, such histograms can show either only one peakattributed to a single bond rupture, or multiple peakscorresponding to the case when simultaneous rupture of oneor more bonds takes place (Lekka et al., 2006). The first peakcorresponds to the unbinding event involving the rupture of onebond, the second is related to the simultaneous unbinding of twobonds (the force values at the secondmaximum are doubled), thethird is related to the unbinding of three bonds, etc. For HCV29cells, the histogram of the unbinding force showed fourdistinguishable maxima (Figure 7A). For malignant T24 cells onlytwo maxima were visible (Figure 7B). Next, histograms werefitted with the superposition of the Gauss functions to obtainpeak positions. To compare the properties of a single N-cadherin–antibody complex studied, only first maximum wasconsidered. It denotes the rupture of a simplest, single complex.The other maxima denoting multiple ruptures were omitted onaccount of them requiring a more profound analysis. Thus, forreference cells, the position of the first peak was at 26.1� 7.1 pN,whereas for cancerous cells, the rupture of a single complex wascentered at 61.7� 14.6 pN. The errors are the half of the width atthe half height of the peak.

DISCUSSION

The alterations in the expression and function of cell–celladhesion molecules correlate with the cancer progression(Mialhe et al., 2000; Van Aken et al., 2001; Christofori, 2006).The switch between N- and E-cadherin in cancerous cells,reported for many cancer types (Christofori, 2006; Stemmler,2008), was also observed for the studied human bladder cell lines(HCV29 and T24). The Western blot and the immunohistochem-ical staining detected higher expression of N-cadherin incancerous cells. These techniques have sufficient resolution todetect molecules in the cell extracts and/or to show theirlocalization in a cell. One should also note that the fullcompatibility between AFM and these techniques is not possiblesince AFMdetects molecules only on a cell surface while the lattermethods deliver information on a protein concentration of awhole cell extract. The quantitative results of 3.6% and 8.7% ofN-cadherin expression in non-malignant HCV29 and malignantT24 cells respectively agree very well with the qualitative resultsobtained from the Western blot and immunohistochemicalstaining.Taking advantage of the AFM measurements at a single-

molecule level, characterization of an individual complexcomposed of N-cadherin and its monoclonal antibody (GC4)was performed. The interaction force between these molecules isexpected to have the same value if the structure of thecadherin-binding site remains unaffected upon cancer pro-gression. Surprisingly, the obtained experimental results clearlysuggest otherwise. The unbinding force of the individual

Figure 6. Loading rate dependence for N-cadherin–antibody com-

plexes, determined for HCV29 and T24 cells in the range of

2500–6600pN/s. The observed force variations were within the exper-imental errors, thus, the increasing tendency (line) cannot be relied upon

or estimation of interaction kinetics.

Figure 5. Inhibition of the N-cadherin–antibody binding sites showing

the decrease in the number of unbinding events. Black columns: controlmeasurements; White columns: events after the inhibition of the inter-

action with N-cadherin–antibody at concentration of 0.1mg/ml; Light

gray columns: after the inhibition with the antibody concentration of0.4mg/ml. All histograms were normalized by the total number of force

curve recorded.

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N-cadherin–antibody complex present in T24 cells was approxi-mately 2.5 times higher than in the case when these moleculeswere probed on the surface of HCV29 cells (61.7� 14.6 vs.26.1� 7.1 pN, respectively). The lower unbinding force in bladdernon-malignant cells denotes an easier dissociation (unbinding) ofthe individual complex as compared to cancerous cells. Thisindicates smaller binding stability of the antibody–N-cadherincomplex in non-malignant cells. Since the same antibody wasused to detect the N-cadherin in both cell types, the distinctunbinding force reveals alterations in the structure and/orproperties of N-cadherin epitope recognized by the GC4antibody. These results are partially confirmed by the resultsreported by Bajpai et al. (2009) in terms of detection of alterationsin cadherin–cadherin unbinding force depending on differentcell types. They have reported weaker interaction strengths ofsingle E-cadherin/E-cadherin bonds between parental cells. Theirresults were referred to the absence of a-catenin.The possible explanations may involve the following

approaches focused on various aspects, such as: the presenceof Trp2 in the GC4 epitope (Volk and Geiger, 1984; Harrison et al.,2005); differences in glycosylation patterns of N-cadherin in thestudied bladder cell lines (Przybyło et al., 2002; Hoja-Łukowiczet al., 2007); differences in membrane corrugations of bothnormal and cancerous cells, recently reported by Iyer et al. (2009);or interactions of N-cadherin with other molecules.In the first reasoning, the binding of GC4 to N-cadherin requires

Trp2 to be docked into the domain structure, regardless of bothpresence as well as absence of calcium ions. Since Trp2 is locatedon the opposite site of the binding domain, 30 A away from theGC4 epitope, the reactivity with GC4 antibody requires Trp2 to belocated in the hydrophobic cavity in the ectodomain 1. Theseconditions impose structural constrains in the GC4 epitope.The second explanation takes into account the degree of

glycosylation of cadherins (Gahmberg and Tolvenen, 1996). InN-cadherin, the glycosylation sites are located on the extracellularparts in the ectodomains 2–5 (Hatta et al., 1988). It is alsowell-known that tumorigenesis and metastasis are frequentlyassociated with altered structure and expression of oligosac-charides on cell surface (Fukuda, 1996). Therefore, we suspectedthat N-cadherins from T24 cells might represent differentglycosylation patterns in comparison with those from HCV29cell line. The studies reported by Przybyło et al. (2002) confirmedthis hypothesis. N-cadherin from non-malignant HCV29 cell line

possessed bi- and/or 2,4-branched triantennary complex typeglycans and, in addition, some of them were a2,6-sialylated.On the contrary, the oligasaccharides of N-cadherin malignantT24 cells were high mannose type as well as 2,6-branched tri-and/or tetrantennary poly-N-acetyllactosamine complex typeoligosaccharides, which are a2,3- or a2,6-sialylated. Additionally,N-cadherin from highly invasive tumor T24 possesses also afucosylated core. Thus, the altered unbinding force can stem fromthe different structure of N-glycans presenting the proximity ofthe GC4 binding site.The third interpretation considers the degree of a membrane

corrugation in malignant cells compared to normal ones (Iyeret al., 2009). The effect was found for cervical cells but it can betrue also for other types of epithelial cells. If N-cadherin moleculesare attached to a highly corrugated, easy to deform membrane,then the stretching of N-cadherin molecules attached to thesofter surface is slower than in case of a more rigid surface. As aresult, lower unbinding force should be expected. To verifythis, the extension lengths were delivered from the analysis of20 randomly chosen force curves (Figure 4B and C). In the caseof cancerous T24 cells, the unbinding event takes placeimmediately after the probe starts to disconnect from the cellsurface. The extension length equaled 17� 8 nm. On thecontrary, for the HCV29 cells, the unbinding happens somewhatlater at the extension length of 56� 16 nm. Hence, the unbindingforce should be higher for molecules attached to a rigid surface.However, this is in conflict with the elastic properties reported forthese cells. The Young’s modulus for reference cells was aroundfour times larger than for T24 cells. Thus, one could expect thesmaller unbinding force for softer cells (i.e., malignant ones).It is known that N-cadherin interacts with other molecules,

namely, with the FGF (Suyama et al., 2002) or glutamate (Sagliettiet al., 2007) receptors family. The association of fibroblast growthfactor receptors (FGFRs) isoforms with N-cadherin has beencorrelated with cancer state of the cell. This has been reported,e.g., breast or prostate cancers. Bladder carcinoma cell linesgenerally express no FGFR2IIIc isoform and very little FGFR2IIIbcompared to normal urothelium. Moreover, introduction ofFGFR2IIIb into the human T24 bladder carcinoma cell line leads toa decrease in proliferation and decreased tumor growth aftersubcutaneous inoculation in nude mice (Ricol et al., 1999; Chafferet al., 2007). With the FGFR receptors, the N-cadherin can formdirect interaction. The functional cooperativity between the FGFR

Figure 7. (A) Force histogram for non-malignant HCV29 cells shows four maxima indicating that during AFM measurements simultaneous rupture ofmore than one complex occurred. (B) Analogous distribution of the unbinding force for N-cadherin–GC4 complex in malignant T24 cells. In most cases,

only a rupture of a single, individual complex is observed. For all histograms, lines are Gaussian fits used for the determination of the mean unbinding

force (i.e., a center of the maximum) and the standard deviation (i.e., a half width at half maximum height).

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with N-cadherin in neurite outgrowth was postulated to involvethe HAV motif on FGFR and the fourth extracellular domain (EC4)on N-cadherin, based on the inhibitory effect of peptides derivedfrom the HAV domain, as well as function-blocking anti-EC4antibodies on neuronal growth (Williams et al., 2001). Glutamatereceptor (GluR) also interacts with N-cadherin. Saglietti et al.(2007) used the protein-docking algorithm to model the way ofbinding between the extracellular domain of N-cadherin (usingthe crystallographic structure of C-cadherin ectodomain) andGluR2 isoform of GluR family (using GluR2-NTD structureobtained by homology modeling). The predicted interactionsites lay on the internal side of EC3 and EC2.The antibody used in our studies reacts with the N-terminal

portion of the extracellular domain of N-cadherin. The bindingsite of the antibody is placed between 1 and 2 ectodomains, asreported by Harrison et al. (2005). Based on the differencesbetween FGFR and GluR expression in cancerous bladder andnormal urothelium and assuming different bindingmechanism inmalignant cells, one might also conclude with such origin ofdistinct unbinding forces in non-malignant and malignantbladder cells.

CONCLUSIONS

In spite of the origin of the unbinding force changes observed forthe individual antibody–N-cadherin complexes, the possibility todetect conformational or structural changes within the bindingsite of a singlemolecule is an indispensable value of the AFM. Thiscan be applied to study either the antigenic identity of receptors,or their dissimilarities, or even alterations of the binding site

within the same receptor type. The results obtained from therecognition of N-cadherin by monoclonal antibody demonstratecancer-related changes in the structure of the binding site ofthe antibody, located at the extracellular domain of thecadherin. The enhanced N-cadherin level in T24 malignantcells was accompanied by changes in unbinding properties ofan individual N-cadherin molecule. Lower unbinding force(26.1� 7.1 pN) in non-malignant HCV29 cells reveals less stableN-cadherin complexes as compared to malignant T24 cells(61.7� 14.6 pN). The possible explanations for the observedunbinding force difference may involve few approaches focusedon various elements such as the presence of Trp2 in the GC4epitope; differences in glycosylation patterns of N-cadherin; theinfluence of membrane mechanical properties on unbindingforce; or the interactions either with FGF and Glu receptors.The obtained difference between the interaction forces ofN-cadherin–antibody complexes could partially explain themalignant nature of these cells. It also demonstrates thepossibility of detection of N-cadherin in cancerous cells withoutexpensive isolation and purification methodology.

Acknowledgements

We thank Mrs Grazyna Igielska for technical assistance duringimmunochemical staining procedure. This work was partiallysupported by the project K/ZDS/000455 Jagiellonian UniversityMedical College (JUMC) and by the project SMW (SingleMolecule Workstation), grant agreement no. 213717 (NMP4-SE-2008–213717).

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