Investigating biomolecular recognition at the cell surface using atomic force microscopy

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nvestigating biomolecular recognition at the cell surfacesing atomic force microscopy

ongzhou Wang, Vamsi K. Yadavalli ∗

epartment of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA 23284, USA

r t i c l e i n f o

rticle history:eceived 28 October 2013eceived in revised form 7 January 2014ccepted 7 January 2014vailable online 17 January 2014

eywords:tomic force microscopy

a b s t r a c t

Probing the interaction forces that drive biomolecular recognition on cell surfaces is essential for under-standing diverse biological processes. Force spectroscopy has been a widely used dynamic analyticaltechnique, allowing measurement of such interactions at the molecular and cellular level. The capa-bilities of working under near physiological environments, combined with excellent force and lateralresolution make atomic force microscopy (AFM)-based force spectroscopy a powerful approach to mea-sure biomolecular interaction forces not only on non-biological substrates, but also on soft, dynamic cellsurfaces. Over the last few years, AFM-based force spectroscopy has provided biophysical insight into

orce spectroscopyiomolecular recognitionell surface

how biomolecules on cell surfaces interact with each other and induce relevant biological processes. Inthis review, we focus on describing the technique of force spectroscopy using the AFM, specifically inthe context of probing cell surfaces. We summarize recent progress in understanding the recognitionand interactions between macromolecules that may be found at cell surfaces from a force spectroscopyperspective. We further discuss the challenges and future prospects of the application of this versatiletechnique.

© 2014 Elsevier Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62. Basic principles of force spectroscopy via AFM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2.1. Force spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.2. Experimental strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3. AFM-FS of cell surface biomolecules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.1. Membrane receptor proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

3.1.1. Vascular endothelial growth factor receptor (VEGFR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93.1.2. Protein tyrosine kinase 7(PTK7) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.1.3. Transforming growth factor �1 (TGF-�1) receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.1.4. Invariant natural killer T cell receptor (iNKTCR). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.1.5. Fission yeast pheromone receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.1.6. Adhesins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.1.7. Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.1.8. Vascular endothelial cadherin (VE-cadherin) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.1.9. Fibrinogen adhesion receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

3.2. Cell surface glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.2.1. Glycans detected using lectin-probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.2.2. Other glycans on bacterial and plant cell surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

4. Applications of cell-surface AFM-FS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
4.1. Probing cancer related biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.1. Prostate specific membrane antigen (PSMA) . . . . . . . . . . . . .4.1.2. Tenascin-C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +1 804 828 0587.E-mail address: [email protected] (V.K. Yadavalli).

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4.1.3. P-selectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134.1.4. Glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4.2. Drug design and detection of action mechanisms at the molecular scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134.2.1. Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134.2.2. Rituximab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144.2.3. Design of a drug delivery system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

4.3. Evaluating the effects of therapeutic agents at cell surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144.3.1. Atorvastatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144.3.2. Effects of cigarette smoke extract (CSE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144.3.3. Trastuzumab and Pertuzumab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

5. Challenges and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Biomolecular interactions play important roles in many bio-ogical and physiological processes (Hinterdorfer and Dufrene,006). Specific recognition processes between macromolecules onell surfaces are essential for diverse cellular functions includingmbryonic development, signal transduction, immune response,ell adhesion, and tissue assembly (Bertozzi and Kiessling, 2001;ustamante et al., 2004; Sheetz, 2001; Vogel and Sheetz, 2006).ecognition events on cell surfaces typically involve complex inter-ctions between molecules such as membrane receptor proteinsnd ligands, carbohydrates and lectins, antigens and antibodies,nd cell adhesion molecules (CAMs) and the extracellular matrixECM) (Fig. 1) (Kienberger et al., 2006; Mrksich, 2002; Wehrle-aller and Imhof, 2002). For instance, membrane receptor proteins

erve as mediators to transmit the biological signals betweenhe cytoplasm and the extracellular environment, as realized vianteractions with their specific ligands (Antonova et al., 2001;efford and Dubreuil, 2000; Thomas, 1996). Lectins on cell surfaces

ediate cell–cell interactions by recognizing specific and comple-entary carbohydrates on adjacent cells (Brandley and Schnaar,

986). CAMs such as integrin, cadherins and selectins are theransmembrane glycoproteins that mediate cell–cell and cell–ECMdhesions by the recognition of specific receptors on other cells orCM (Edelman, 1983; Edelman and Crossin, 1991). Several impor-ant, fundamental questions remain in our understanding of thesevents: How do receptor proteins interact with their ligands and
nitiate specific transduction pathways? How do lectins bind withheir specific carbohydrates and mediate relevant cell–cell recog-ition and cell–ECM adhesion? How do CAMs interact with ECM
ig. 1. Typical biomolecular interactions that can be measured on the surface ofells.

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and make cells grow on different types of surfaces? What happensif these interactions are blocked by exogenous substances?

All these recognition events on cell surfaces are driven by molec-ular scale interaction forces. The interaction forces of proteins withtheir ligands contain fundamental biophysical data that can enableus to quantitatively elucidate the relevant cellular signal trans-duction processes. Interactions on cell surfaces have fundamentalroles for characterizing various normal processes including cellulargrowth, differentiation, junction formation and polarity (Albeldaand Buck, 1990; Aplin et al., 1998) or pathological processes inliving organisms, such as cellular adhesion, infection and cancercell metastasis (Gorelik et al., 2001; Ohyama et al., 1999; Sharonand Lis, 1989). In addition, these interaction forces provide a meanfor evaluating the selectivity and specificity of various biologicalprobes that can be useful in developing cell-specific bio-analyticaland biomedical devices (Jelinek and Kolusheva, 2004; Turner, 2000;Zheng et al., 2005). Consequently, measuring these forces directlyhas implications in unraveling the molecular basis of relevant bio-logical and pathological processes on cell surfaces, as well as indeveloping new tools for disease diagnosis and detection, or drugscreening at a molecular level (Kim et al., 2007; Yu et al., 2011).

Force spectroscopy as a dynamic analytical technique, allowsthe measurement of interaction forces at the level of individualmolecules, which cannot be obtained from conventional ensem-ble measurements (Hugel and Seitz, 2001). In this context, “forcespectroscopy” is not used in the sense of traditional spectroscopy,which is based on the interaction of radiation with matter (Carvalhoand Santos, 2012). Typically, in this process, the pair-wise inter-action between (bio)molecules is measured by immobilizing one(bio)molecule on a substrate, while the other attached to anothersurface or a probe (for example, a magnetic bead or a stiff can-tilever). The force is then derived from the deviation of the surfaceor the probe from its equilibrium position (Israelachvili et al., 2010;Lin et al., 2005; Molloy and Padgett, 2002; Neuman and Nagy, 2008).The technique of force spectroscopy has been used to study theunfolding of single proteins and nucleic acid structures by mechan-ically stretching the biomolecule across two ends immobilized tosurfaces (Hyeon and Thirumalai, 2007; Zhuang and Rief, 2003). Todate, several different kinds of force spectroscopy techniques havebeen employed for determining these forces of interaction. Theseinclude optical tweezers, magnetic tweezers and atomic forcemicroscopy (AFM) (Gosse and Croquette, 2002; Krasnoslobodtsevet al., 2007; Leckband et al., 1992; Merkel et al., 1999; Moffittet al., 2008), all of which operate within specific limits of sensitivityand range of forces. The principles, applications and limitations ofthese three techniques were recently summarized in an excellentreview (Neuman and Nagy, 2008). Among them, AFM has rapidlyemerged as a versatile tool widely used over the last couple of
decades. The unique advantage of the AFM is the ability not onlyto image single molecules with nanoscale resolution, but also mea-sure inter- and intra-molecular interaction forces with piconewton

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ensitivity (Dufrene and Hinterdorfer, 2008; Florin et al., 1994; Jalilind Laxminarayana, 2004; Li et al., 2002).

The unique abilities of working under near physiological envi-onments combined with excellent force and lateral resolutionake AFM-based force spectroscopy (henceforth referred to asFM-FS in this manuscript) a powerful approach to measure theiomolecular interaction forces not only on fixed non-biologicalubstrates (such as glass or silicon), but importantly, on soft cellurfaces. There have been several reviews on the use of AFM inifferent imaging modalities as well as their use in studying pro-ein structure and function (Engel and Muller, 2000; Fotiadis et al.,002; Santos and Castanho, 2004; Shao et al., 1996). A number ofeviews have also covered the use of AFM-based force spectroscopyo uncover interaction forces as well as unique nanomechanicalignatures obtained by the forced mechanical unfolding of pro-eins and polymers (Borgia et al., 2008; Bujalowski and Oberhauser,013; Hoffmann and Dougan, 2012; Puchner and Gaub, 2009). Inhis review, we specifically focus on the methodology of AFM-FSpplied at static and dynamic cell surfaces. Our focus is primarilyn biomolecules that are either present at cell surfaces or have airect impact on cellular behavior and function. Of special interest ishe in situ application of AFM-FS on cell surfaces, a relatively recentechnique with a great potential to directly probe biomolecules inheir milieu. We then discuss the unique challenges that distinguishhis form of force spectroscopy and summarize recent progressn the study of biomolecules found at the cell surfaces. Probinghe forces that drive biomolecular recognition on cell surfaces willesult in increasing our understanding of the fundamental organi-ation, mechanics, interactions and processes at the cell surface.

. Basic principles of force spectroscopy via AFM

.1. Force spectroscopy

The workings of the AFM have been extensively covered inrior works and will not be reiterated here (Cappella and Dietler,999; Dufrene, 2008; Giessibl, 2003; Lee et al., 2007a; Santos andastanho, 2004). Briefly, an AFM primarily consists of four parts: aoft cantilever with a sharp tip (commonly silicon or silicon nitride),

sample stage on a piezoelectric scanner, a laser diode and a pho-odetector as the optical detection system. In AFM-FS studies, tipsre commonly functionalized with one or a small amount of probeolecules. The cantilever with the modified tip moves vertically

z-axis) toward the surface, contacts the surface, and then retracts.uring this process, the interaction forces between the tip and sam-le cause bending of the cantilever detected by the photodetector.he cantilever deflection (�x) as a function of the vertical displace-ent of the piezoelectric scanner can be recorded. Then the force

an be obtained by applying Hooke’s law (F = −k�x), where F ishe force, k is the spring constant of the cantilever and �x is theeflection of the cantilever. The force of rupture between a pairf molecules can therefore be determined (Hugel and Seitz, 2001;erkel et al., 1999). A typical force curve reflecting the interactions

f the cantilever with a typical sample surface is shown in Fig. 2.The measured rupture forces closely depend on the loading

ates (spring constant of cantilever multiplied by retraction veloc-ty of tip) that are exerted on the molecular complex. With thencrease of loading rates, the forces increase due to their lin-ar relationship with loading rates (Evans and Calderwood, 2007;riddle et al., 2012; Schwesinger et al., 2000). By collecting aange of data over broad loading rates, dynamic force spectroscopy
DFS) as a subset of AFM-FS has emerged as a valuable techniqueor the characterization of dissociation kinetics and energy pro-le of the interacting molecules (Evans, 2001; Hugel and Seitz,001; Hyeon and Thirumalai, 2012). In recent years, the process of
Fig. 2. Probing cell surfaces using AFM based force spectroscopy.

collecting individual force curves has been further expanded withthe advent of automated scanning modes that allow us to obtainspatial distributions of adhesion forces. These “adhesion force map-ping” methods are based on collecting arrays of force curves on agiven size area, and then displaying the unbinding force values ofall traces (Fig. 3a–c and f). Owing to the collection of spatial andforce information simultaneously, this approach has been partic-ularly useful for mapping recognition sites on the surfaces of livecells (De Pablo et al., 1999; Eaton et al., 2002; Heinz and Hoh, 1999;Kienberger et al., 2006). Another AFM based force mapping methodtermed “dynamic recognition imaging” is also used to identify therecognition sites on cell surfaces. With a ligand functionalized-AFM tip oscillating on the cell surface, cognate receptors can bedetected due to a reduction in the oscillation amplitude whenligand–receptor binding events occur. Although lacking quantita-tive force values, the force maps generated by this approach tend tohave higher spatial and time resolution compared to adhesion forcemaps (Chtcheglova and Hinterdorfer, 2011; Dupres et al., 2007; Hanet al., 1996; Stroh et al., 2004).

2.2. Experimental strategies

Biomolecular interactions can be measured by AFM-FS not onlyon static (often non-biological) substrates, but also directly onthe surfaces of living cells (Dupres et al., 2007). In order to mea-sure the pair-wise interaction forces between biomolecules on thetip and on the sample using AFM, the first step is to immobilizethem on each surface (Neuman and Nagy, 2008). There are severalrequirements that need to be fulfilled during this step in order tocollect correct or meaningful data: (i) firm immobilization to avoiddetachment of the immobilized biomolecules during retraction; (ii)optimal orientation and availability of binding sites of biomoleculesfor recognition; (iii) preservation of their native states, and, (iv)minimization of nonspecific interactions between the functional-ized tip and the substrate (Barattin and Voyer, 2011; Lv et al., 2009;Safenkova et al., 2012). In the case of cells, where the biomoleculebeing probed is in situ at the cell surface, the challenge is to immo-bilize the complementary molecule to the cellular target on thetip only. The basic principles still remain the same as nowadays,it is possible to obtain AFM tips with a wide diversity of materialscoatings including silicon, silicon nitride, aluminum and gold.

Generally, the simplest way to immobilize biomolecules is byphysical absorption. However, the intrinsic lack of control over thisattachment of molecules via this method makes it difficult to fulfillthe requirements discussed above (Barattin and Voyer, 2008; Schon
et al., 2007). In contrast, chemical modification combined withfunctionalized self-assembled monolayers (SAMs) typically showssuperior performance due to good reproducibility and flexibilityin biomolecular immobilization, especially for proteins (Dammer


Fig. 3. Examples of AFM-FS studies on biomolecular recognition on cell surfaces. (a–c) Adhesion force maps in real time showing VEGFRs on cell surface concentratingtoward cell boundaries and clustering rapidly after the addition of the antibody at: (a) 0, (b) 10, and (c) 45 min. Source: Reprinted from (Almqvist et al., 2004) with thepermission of Elsevier. (d) AFM-FS measurements of specific binding forces between HBHA and its receptor HSPG on living host cells (Dupres et al., 2009; Muller et al., 2009).Constant force plateaus at large loading rates on force curves implied that stressed HSPG receptors can potentially detach from the cytoskeleton, leading to extraction of themembrane tethers or nanotube. Source: Reprinted from (Muller et al., 2009) with the permission of Nature Publishing Group. (e, f) Detection of peptidoglycan on surface ofliving Lactococcus lactis cells using adhesion force mapping. (e) Topographic image of two dividing bacterial cells. (f) Adhesion force map (400 nm × 400 nm) recorded with aLysM modified probe in the square area shown in topographic image. The peptidoglycan molecules were detected (bright pixels) to be arranged as lines running parallel tot

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ource: Reprinted from (Andre et al., 2010) with the permission of Nature Publishin

t al., 1996; Miyake et al., 2007; Smith et al., 2004). Common sys-ems include alkylthiolation on gold surfaces or silanization onilicon surfaces. The reaction of alkylthiols with gold and silane withilicon form covalent S H bonds and Si O Si bonds respectively.

H and Si O Si bonds have a higher strength, compared with theinding forces of the biomolecules themselves, which is necessaryor force spectroscopy. After the formation of SAM, the surfaces areften functionalized with the terminated groups of the alkylthiolsnd silanes (Love et al., 2005; Miyake et al., 2007). For example,or protein immobilization, the gold tip and substrate can be func-ionalized with a COOH-terminated SAM (Lv et al., 2010; Wangt al., 2011). Terminated carboxyl groups can then be activatedy 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochlorideEDC), and N-hydroxysulfosuccinimide (NHS). Finally, proteins aremmobilized by the formation of an amide linkage between the acti-ated carboxyl groups and amine groups of lysine on the proteinurface.

The advantage of force spectroscopy on cell surfaces is that theolecules studied are in the milieu of their cell membranes, whichaintains their structural assembly and functional state (Lecuit

nd Lenne, 2007). This enables forces, dynamics and distributionsf specific interactions to be detected in real time (Dufrene andarcia-Parajo, 2012). However, the extremely complex nature ofell membranes, their relative deformability, fragile nature, andack of flatness pose additional challenges in their study by AFM-S. For instance, different targets exposed on cell surfaces mayroduce both specific and nonspecific interactions concurrently,hich makes the interpretation of force curves difficult (Muller

t al., 2009). Similarly, the soft cell membrane may be penetratedy the stiffer cantilevers with high spring constant during the forceollection, which may cause inaccuracies in the force measurement
rocess itself (Kwon et al., 2009).
Beside the tip preparation strategies mentioned above, properell immobilization methods to maintain the natural state ofells are also necessary for accurate force measurements. Typical

up.

substrates for cell immobilization include glass, mica and sili-con. The selection of the substrate depends on the cell type andtarget molecules studied. For instance, for studying membrane pro-teins on animal cells, traditional methods including air-drying andglutaraldehyde immobilization may denature the proteins at thesurface (Kienberger et al., 2006). A simple method by virtue of theirability to spread and stick to solid substrate has been used by mostanimal cell studies. Therefore, AFM-FS can be conducted directlyin cell culture media in a Petri dish (Almqvist et al., 2004). Theadvantage of this method lies in the preservation of normal cellmorphology and viability in a liquid environment. By pre-coatingthe substrate with collagen, fibronectin and polylysine, it is possibleto enhance the immobilization, and separate the cell from the sub-strate to avoid denaturation (Carvalho et al., 2010; Henderson et al.,1992). In contrast, for polysaccharides on plant cell wall surfaces,protein denaturation is not a key issue. Such cells can be immobi-lized directly on glass substrates with epoxy glue and with forcemeasurements performed in a liquid environment (Zhang et al.,2012b). Generally, a fluid cell as a part of the sample stage createsthe needed liquid medium for force measurements. For microbialcells such as bacteria and yeast which cannot adhere to substrateseasily, immobilization is achieved by mechanically trapping them,into porous polycarbonate membranes for example (Andre et al.,2010). The prerequisite of this method is that the pore size of themembrane should be comparable to the dimensions of the cell(Dufrene, 2002; Heinisch et al., 2012; Touhami et al., 2004).

3. AFM-FS of cell surface biomolecules

The cell separates its cytoplasm from the extracellular environ-ment via a cell membrane consisting of a phospholipid bilayer with
embedded proteins and carbohydrates located on the extracellularsurface of the membrane (Fig. 1). The basic function of cell mem-brane is protection of cell cytoplasm from its surroundings. Variouscellular activities are triggered by the biomolecular recognition at

C. Wang, V.K. Yadavalli / Micron 60 (2014) 5–17 9

Table 1Some reported examples of biomolecules on cell surfaces studied via AFM-FS.

Biomolecules on cell surface Functionalized tip

Membrane receptorproteins

Vascular endothelial growth factor receptor (VEGFR)(Almqvist et al., 2004; Lee et al., 2007b)

VEGFR-antibody

Protein tyrosine kinase 7 (PTK7) (O’Donoghue et al., 2012) DNA aptamer sgc8c and PTK7-antibodyCD1d-presented lipid antigens (Bozna et al., 2011) T cell receptor (TCR)Fission yeast pheromone receptor (Sasuga et al., 2012) PheromoneTransforming growth factor ̌ (TGF-ˇ) receptor (Yu et al.,2007)

TGA-ˇ1

Chlorinated ovalbumin (OVA) receptor (Zapotoczny et al.,2012)

Chlorinated OVA

Cell adhesionmolecules

Heparin-binding haemagglutinin adhesin (HBHA) (Dupreset al., 2005)

Heparin

Heparan sulfate proteoglycan receptors (Dupres et al.,2009)

HBHA

Integrin �2�1(Attwood et al., 2013)a Collagen containing specificintegrin-binding motif

Integrin lymphocyte function-associated antigen-1 (Ricoet al., 2010)

Intercellular adhesion molecule-1

�llb�3-related integrin (Carvalho et al., 2010) FibrinogenVascular endothelia cadherin (VE-cadherin) (Chtcheglovaet al., 2010)

VE-cadherin-antibody

Glycans Glycans detected bylectins

Soybean agglutinina (Sletmoen et al., 2009) Porcine submaxillary mucinGlycosylated extracellular domain III of the epidermalgrowth factor receptor (Gunning et al., 2008)

Wheat germ agglutinin

Concanavalin A (Zhang and Yadavalli, 2009)a MannoseMannose and Galactose (Francius et al., 2008) Concanavalin A and Pseudomonas

aeruginosa lectind-galactose (Li et al., 2011b) Ricinus communis agglutinin-120

Glycans on bacterialand plant cell surface

O-antigenic lipopolysaccharides (Handa et al., 2010) Bacteriophage P22 tailspike proteinsNatural crystalline cellulose (Zhang et al., 2012b) Carbohydrate-binding module of

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he cell surface (Andersson et al., 1988; Korn, 1969; Salton, 1967;eagle, 1989). More practicably, the force mapping technique thatombines AFM-FS with the high spatial resolution of the AFM, offershe opportunity to map cell surface recognition sites or biomark-rs that would not be otherwise found by traditional, macroscaleechniques. There have been an increasing number of reports apply-ng AFM-FS to characterizing biomolecular interactions that maye found on the cell surfaces, indicated their potential to pry outnswers to fundamental questions. In the following sections, weiscuss these studies to demonstrate the unique advantages ofFM-based spectroscopy as applied to different kinds of biomolec-lar systems. Table 1 summarizes some of these reports.

.1. Membrane receptor proteins

Membrane proteins play an important role in a variety ofell functions (Dirienzo et al., 1978; Eisenberg, 1984; Tanfordnd Reynolds, 1976). In particular, membrane receptor pro-eins transmit signals between the extracellular and intracellularnvironments. For example, cell adhesion molecules mediateell–cell recognition and cell–ECM adhesion. Their functions areargely determined by physical forces acting on and between theiomolecules. A few examples of membrane proteins studied viaFM-FS are presented below:

.1.1. Vascular endothelial growth factor receptor (VEGFR)VEGFR is a typical transmembrane receptor protein expressed

n vascular endothelial cells (Olsson et al., 2006). The recogni-ions between VEGFR and its ligand are critical to several cellular
ctivities such as focal adhesion turnover, actin cytoskeletal remod-ling, and angiogenesis (Izumi et al., 2003; Rakhmilevich et al.,004). Spatial features such as clustering, distribution of VEGFRn cell surfaces are closely related to endothelial cell growth and
al., 2010) Lysine motif

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migration (Thomas, 1996). Preliminary studies using AFM-FS havetypically involved directly measuring interaction forces betweenthe biomolecular pairs on static substrates prior to their studiesin the more complex cellular environments. Almqvist et al. ini-tially employed AFM-FS to measure the adhesion forces betweenisolated VEGFR adsorbed on the mica substrate and its antibodyimmobilized on an AFM tip (Almqvist et al., 2004). The unbind-ing force of 60 ± 10 pN was consistent with previous studies usingthe antigen–antibody pair (Lee et al., 2007a; Ros et al., 1998). Asdiscussed above, one significant feature of AFM-FS lies in its “adhe-sion force mapping” method allowing mechanical detection of cellsurface recognition sites in real-time and in situ under differentphysiological conditions. Using this approach, the location, den-sity and distribution of VEGFRs on cell surfaces were imaged. Theadhesion force map showed that the VEGFRs tended to concentratetoward the cell boundaries and clustered rapidly after the additionof VEGF or the antibody of VEGFR (Fig. 3a–c). In a later study, a two-step procedure was developed: first locating the VEGFRs followedby force analysis (Lee et al., 2007b). This process used dynamicrecognition imaging method to map VEGFRs on vascular endothe-lial cell surface with antibody-modified AFM tips. Force mapsobtained by this approach revealed the non-uniform distributionof VEGFRs with respect to the underlying cytoskeleton. The forcemaps from dynamic recognition imaging method have a higher spa-tial and time resolution compared to an adhesion force map, as forcecurves are not collected point by point. In order to obtain detailedinformation on the interaction forces, AFM-FS spectroscopy wasthen conducted on the recognition sites to measure the ruptureforce and unbinding times. The distribution of these rupture forces
with two peaks of 33 and 64 pN implied one or two receptorsbinding with a single antibody. This study highlighted the use ofAFM-FS to assess kinetic parameters of binding process on cell sur-faces. From the rupture force and unbinding time acquired, the

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quilibrium dissociation rate koff was determined directly throughhe Bell model (Bell, 1978), koff = 1.05 ± 0.6 × 10−4 s−1. The equilib-ium association rate kon was obtained from a monovalent bindinginetic model (Lauffenburger and Linderman, 1993) by blockingeceptors with soluble antibodies, kon = 5.83 ± 1.48 × 104 s−1 M−1.oth approaches provided spatial visualization of VEGFRs as wells the forces between ligands and their receptors which couldhed light on their functional roles in receptor-mediated cellularehavior.

.1.2. Protein tyrosine kinase 7(PTK7)PTK7, as a member of the receptor tyrosine kinase family, is

n important surface biomarker in cancer research (Lewis et al.,011). The interactions between PTK7 and its ligand are important

n developing specific bio-analytical and biomedical techniquesZhang et al., 2013a). O’Donoghue et al. focused on the investiga-ion of the binding affinity of a natural antibody and a syntheticNA aptamer with PTK7. AFM-FS provides a high accuracy tool toeasure and compare the rupture forces of PTK7 with its respectiveNA aptamer and antibody (anti-PTK7). The rupture force on liveell membranes between the aptamer and protein was measuredo be 46 ± 26 pN, while the force with the antibody was 68 ± 33 pN.he measured force values with two ligands are very similar indi-ating that DNA aptamer has the potential for targeted delivery ofhemotherapy to tumors. This study also showed that aptamers areseful bio-probes to detect specific biomarkers on various cell sur-aces via functionalization on AFM tips (O’Donoghue et al., 2012).

In addition to specific receptors, AFM-FS was recently appliedor discovery of unknown receptors on cell surface. One exam-le is probing the specific recognition events between chlorinatedvalbumin (OVA) and macrophages (Zapotoczny et al., 2012). Thenique aspect of this research was the data processing, whicheported on the use of adhesion frequency (AF) to detect thenknown receptors on cell surface. The AFM tip modified withhlorinated OVA showed 85% AF on macrophage surface, while theative OVA modified tip as control experiment showed a negli-ible AF value. Blocking of the receptors by the chlorinated OVAignificantly decreased the adhesion between chlorinated OVA andhe cell surface, which confirmed the specificity of the interactions.he results implied this technique could be useful to detection ofndefined recognition sites on cell surfaces.

AFM-FS has been further used in conjunction with other tech-iques to enable efficient data collection and improve experimentalalidity for different systems:

.1.3. Transforming growth factor ˇ1 (TGF-ˇ1) receptorTGF-ˇ1 regulates various cellular biological processes including

ell motility, recognition, proliferation, differentiation, and apo-tosis. The TGF-ˇ1signaling process involves TGF-ˇ1binding to itseceptors including type I TGF- ̌ receptor (TˇRI) and type II TGF-

receptor (TˇRII) (Massague, 1990; Massague and Chen, 2000).sing force spectroscopy, the interactions between TGF-ˇ1and its

eceptors were investigated in vitro and in vivo. Fluorescence imag-ng was used to locate fluorescently-tagged TGF- ̌ receptors onell surface. The AFM results revealed similar unbinding force ofGF-ˇ1with TˇRII on a silicon substrate as well as TˇRII on the cellurface. The co-expression of TˇRI with TˇRII increased the unbind-ng force of TGF-ˇ1 with its receptors, although the expressed TˇRIlone showed no specific binding affinity with TGF-ˇ1 (Yu et al.,007). Here AFM-FS provided a better understanding of the molec-lar mechanism of TGF- ̌ signaling and the fluorescent imagingffered the AFM with an efficient collection of force curves.

.1.4. Invariant natural killer T cell receptor (iNKTCR)Invariant natural killer T (iNKT) cells play a critical role in

he immune system. The functions of iNKT cells are realized by

cron 60 (2014) 5–17

the recognition between iNKTCR on the iNKT membrane withendogenous and exogenous cluster of differentiation 1d (CD1d) –presented lipid antigens (Joyce, 2001). By using iNKTCR function-alized AFM tips, the unbinding forces with CD1d proteins havingdifferent length of the phytosphingosine chain were measuredin vitro and in vivo. The results showed that the CD1d–glycolipidcomplexes immobilized on both mica surfaces and on living cellsurfaces had specific interactions with iNKTCR. Higher unbindingforces were required to dissociate the iNKTCR and CD1d with longerphytosphingosine chains. This study also compared the kinetic rateconstants of the pairwise molecules obtained from AFM-FS withthose obtained using surface plasmon resonance (SPR). The goodconsistency of the two techniques further confirmed the validity ofthe results (Bozna et al., 2011).

3.1.5. Fission yeast pheromone receptorThe fission yeast pheromone receptor is a kind of G protein-

coupled receptor (GPCR), a typical membrane receptor protein oncell surface. These receptors are activated by pheromone bindingand then enable cellular signal transduction (Kohl et al., 2001). Theinteractions between the pheromone and its receptors on yeastcell surfaces were studied by AFM-FS (Sasuga et al., 2012). AFMtips were modified with the pheromone and a pheromone analog(lacking specific binding sites) to measure the interaction forces oncell surfaces. The specific rupture forces between the pheromoneand its receptor were recorded to be ∼120 pN at a pulling velocity of1.74 �m/s. As expected, the pheromone analog modified tips do notshow a force jump in force curves. A report gene assay was carriedout to study their interactions, wherein a green fluorescent proteinlabeled reporter gene was used to monitor the activation level ofsignal transduction following the interactions of pheromone and itsreceptors. Binding of the pheromone to its receptors on cell surfaceinitiated the relevant signaling pathway and increased the fluores-cence intensity, while the nonspecific pheromone analog did notshow positive results. The results of the report gene assay there-fore confirmed the validity of the AFM results, implying that thisapproach has the potential for signaling and screening studies ofreceptors and their ligands.

In addition, cell adhesion molecules (CAMs), as another typeof membrane receptor proteins, are typically transmembrane gly-coproteins that mediate binding to extracellular matrix (ECM)molecules or to counter-receptors on other cells. These moleculesdetermine the specificity of cell–cell or cell–ECM interaction (Aplinet al., 1998):

3.1.6. AdhesinsAdhesins, as a class of CAMs located on bacterial cell surfaces,

facilitate bacterial adhesion to host cell surfaces by the recogni-tion between adhesins and their specific receptors on host cellsurfaces (Jacques and Paradis, 1998). Using AFM-FS, biomolecularinteractions can be measured both in vitro and in vivo, thereby pro-viding complementary information for the system. For instance, thespecific interaction forces between heparin and adhesin (heparin-binding haemagglutinin adhesin, HBHA) were measured and thenthe HBHA distribution were mapped on the surface of Mycobac-terium tuberculosis using adhesion force mapping (Dupres et al.,2005). First, the adhesion forces were measured between HBHAmodified tips and a heparin immobilized surface. The adhesionforce histogram showed a bimodal distribution with forces of 50and 117 pN that could be attributed to one and two binding pairs ofHBHA and heparin. The adhesion force map obtained on living cellsurfaces using heparin-modified tips showed that adhesin tended
to cluster on the cell surfaces, instead of being homogeneouslydistributed. In a later report by the same group, the interactionsbetween HBHA and heparan sulfate proteoglycan (HSPG) recep-tors on living cell surfaces were investigated. Adhesion forces

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easured on the cell surface showed similar force values to theBHA–heparin pairs. The adhesion force map on living cell surface

howed homogeneous distribution of HSPG receptors. Interest-ngly, force curves with constant force plateaus were observed atigher loading rates, which might be interpreted as the extractionf membrane tethers or nanotubes during the pulling process ofFM tip (Dupres et al., 2009; Muller et al., 2009) (Fig. 3d). This par-

icular phenomenon observed from AFM-FS may open new avenuesn pathogenesis research. In addition, the idea of spatially repre-enting force data creates new methods for visualizing biophysicalata on the cellular scale.

.1.7. IntegrinsIntegrins form another class of CAMs that mediate cell–cell

dhesion and cell–matrix interactions. The integrins as adhesioneceptors on cell surfaces are involved in a number of biolog-cal processes, including cellular signal-transduction, embryonicevelopment, inflammation, cancer metastasis and wound healingAlbelda and Buck, 1990). For example, the recognition betweenlatelet transmembrane receptor, integrin �2�1, and collagen isrucial for hemostasis (Knight et al., 2000). Using AFM-FS, thenteraction forces between integrin �2�1 and a collagen con-aining specific integrin-binding motif Gly-Phe-Hyp-Gly-Glu-ArgGFOGER) were measured. Control experiments were conductedy using a collagen containing Gly-Pro-Pro (GPP) which lacks theFOGER motif. The force curves showed multiple unbinding events

or both GFOGER and GPP indicating that the binding sites between2�1 and collagen are not only at the GFOGER motif, but alsot other parts of the collagen molecule (Attwood et al., 2013). Innother study, the temperature effect on integrin-mediated celldhesion was investigated using AFM-FS (Rico et al., 2010). TheFM tip was functionalized with intercellular adhesion molecule-1

ICAM-1) and was used to probe the receptor integrin lympho-yte function-associated antigen-1 (LFA-1) on the cell surface. Theesults indicated that cell adhesion was suppressed by a decreasef temperature. This implies that a reduction of temperature con-ributes to controlling swelling and inflammation.

.1.8. Vascular endothelial cadherin (VE-cadherin)VE-cadherin, a single-pass transmembrane glycoprotein, is the

ajor adhesion molecule of endothelial adherent junctions andnown to be primarily responsible for mechanical linkage betweenndothelial cells. By using a VE-cadherin-Fc-functionalized AFMip and dynamic force mapping, a recognition map of the dis-ribution of VE-cadherin on microvascular endothelial cells wasbtained. The VE-cadherins were observed to not be uniformly dis-ributed, but located mostly along F-actin filaments (Chtcheglovat al., 2010). This study showed that the clustered VE-cadherinolecules are linked through their cytoplasmic domain to the

ctin-based cytoskeleton, which is important for the understand-ng of mechanism of VE–VE adhesion from the view of their cellurface architecture.

.1.9. Fibrinogen adhesion receptorsThe interactions of fibrinogen and its adhesion receptors on

rythrocyte membrane are the primary reason for erythrocyteggregation, which increases the incidence of cardiovascular anderebrovascular diseases. The interaction forces between fibrino-en and its receptors on erythrocyte membrane were initiallyetected using AFM (Maeda et al., 1987). The unbinding force of7 pN was close to the force between fibrinogen and a platelet.he interactions were inhibited by eptifibatide (an �llb�3 specific
nhibitor), which implied the possibility of �llb�3-related integ-in as the adhesion receptors on the cell surfaces. Subsequently,orce spectroscopy was conducted on cells from a patient withlanzmann thrombasthenia, a disease caused by �llb�3 deficiency.
cron 60 (2014) 5–17 11

The decreased unbinding forces further confirmed the presence of�llb�3-related integrin on the erythrocyte surfaces (Carvalho et al.,2010). These examples showed the suitability and versatility ofAFM-FS for studies on cell–cell adhesion and cell–matrix inter-actions from a mechanical perspective. Simultaneous imaging ofspecific binding sites down to the level of a few nanometers wasalso enabled.

Besides the molecular scale studies discussed above, AFM-basedsingle-cell force spectroscopy (SCFS) has been developed as anexpanded tool for studying CAMs and the dynamics of regulatedadhesion processes in living cells. By attaching a single cell ontoan AFM cantilever, a living cell can be converted to a “cell-probe”to detect the adhesion force between whole cells or to CAMsimmobilized on substrates (Helenius et al., 2008). The depiction ofcell–cell adhesion measurements and typical force–distance curvesare shown in Fig. 4a and b. For example, a lectin functionalized sur-face was used to immobilize Dictyostelium discoideum cells onto anAFM cantilever and substrate so that cell–cell adhesion could bestudied (Benoit et al., 2000). Developmental regulation and EDTAresistance indicated that the measured force of 23 pN may be dueto unbinding of contact site A (csA) molecules on the cell surfaces.Further genetic manipulation demonstrated that csA is the princi-pal source of the intercellular adhesion. Similarly, the interactionof LFA-1, expressed on Jurkat T cells, with intercellular adhesionmolecules (ICAM) were also studied using SCFS (Wojcikiewicz et al.,2006; Zhang et al., 2002). In the latter report, the interaction ofLFA-1 with ICAM-1 and ICAM-2 were conducted at varying load-ing rates (Wojcikiewicz et al., 2006). An AFM cantilever attachedwith a Jurkat T cell was allowed to interact with the protein-coated substrates. The results indicated that the dissociation ofboth complexes involved overcoming a steep inner, and a wideouter activation barrier. ICAM-1 exhibited stronger binding to thehigh-affinity form of LFA-1. Differences in the kinetic profiles andintermolecular potentials of ICAM-1 and ICAM-2 could therefore beattributed to structural differences in their binding site. For integ-rin and collagen interactions, early steps of �2�1 integrin-mediatedcell adhesion to a collagen type I matrix were studied using SCFS.Increasing the contact time to a certain extent showed elevatedoverall cell adhesion, suggesting a change from single to coopera-tive receptor binding (Taubenberger et al., 2007). This SCFS studyprovided new insights into temporal and mechanistic aspects ofearly integrin binding events.

3.2. Cell surface glycans

3.2.1. Glycans detected using lectin-probesCell-surface glycans have been studied as significant factors

in various cellular physiology and diseases, including immunerecognition, cell adhesion, cell migration, bacterial infections,inflammation and cancer (Inatani et al., 2003; Ohyama et al., 1999;Pilobello and Mahal, 2007). Lectins are proteins having a specificaffinity to glycans. By virtue of their interactions with specific gly-cans, lectins have evolved as efficient molecular probes to detectspecific glycans on cell surfaces, which in turn, are essential toa variety of cell functions and pathological processes (Grandboiset al., 2000; Kim and Varki, 1997). Several works have focused onthe interactions between lectins–glycans both in vitro and in vivo byusing AFM-FS. For instance, soybean agglutinin (SBA) as a molecularprobe showed its unique ability to analyze the structural prop-erty of glycoproteins by force curve analysis. The unbinding forcesbetween SBA on a mica surface and mucin on AFM tip were mea-sured (Sletmoen et al., 2009). Force curves with multiple force
jumps implied several unbinding events, consistent with the struc-ture of the carbohydrate chains of mucin. Moreover, the unbindingdistance shown on force curves was equal to the length of the mucinchain. These results combined with calculated kinetic parameters


Fig. 4. (a) Schematic of cell based force spectroscopy experiments: During the approach (green arrows), the cell probe is pressed onto the cell on substrate until a set forceis reached and held for a specific contact time. The cell is then retracted from the substrate (blue arrows) and the detachment forces between the probe and sample can bemeasured from the bending of the cantilever. (b) Before detachment, two types of molecular unbinding events can occur. In the first, the receptor remains anchored in thecell cortex and unbinds as the force increases (jumps). The second unbinding event occurs when receptor anchoring is lost and membrane tethers are pulled out of the cell(

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ere compatible with a binding model in which lectin moleculesend to “bind and jump” from a glycan residue to another along theolypeptide chain of mucin before dissociating. In another report,heat germ agglutinin (WGA) was used as the probe to detect

pecific receptors on a cell surface. Gunning et al. measured thenteraction forces between a WGA coated tip and a glass surfaceunctionalized with N,N′,N′′-triacetylchitotriose (a known receptoror WGA) (Gunning et al., 2008). The rupture force between WGAnd its receptor (the glycosylated extracellular domain III of the epi-ermal growth factor receptor) was measured on a Caco-2 humanolon carcinoma cell. Adhesive events were subsequently used toap the location of recognition sites on the cell surface revea-

ing heterogeneity in their distribution. Similarly, Ricinus communisgglutinin-120 (RCA120) was used as a probe to directly measurehe interaction forces between RCA120 and galactosyl residues oniving HeLa cell surfaces (Li et al., 2011b). The unbinding force wasbserved to be 43 pN at loading rate of 0.4 nN s−1. The blockingxperiment performed in free d-galactose solution confirmed thepecificity of the lectin–carbohydrate interactions.

Concanavalin A (Con A) is another widely used lectin thatinds specifically to �-d-mannosyl and �-d-glucosyl residues.hang et al. used an efficient one-step amination reaction strategyo fabricate carbohydrate arrays based on mixed self-assembled

onolayers and then probe the specific Con A-mannose inter-ctions. The results showed the value of the rupture force for aingle Con A-mannose bond to be 95 ± 10 pN (Zhang and Yadavalli,009). The aggregation state of Con A was regulated by pH, result-

ng in different dominant rupture forces. Con A was also used as probe to detect specific carbohydrates on cell surface. Franciust al. used a Con A modified tip to probe mannose immobilized ongarose beads. The corresponding adhesion force histogram dis-layed a rupture force peak at 57 ± 19 pN (Francius et al., 2008).his and another functionalized tip (Pseudomonas aeruginosa lectin,A-1 that interacts with galactose) were used to map the sur-ace polysaccharides of the Lactobacillus rhamnosus strain GG (LGG)ild-type and mutant strains using adhesion force mapping. Theaps showed the mannose and galactose properties (distribution,

dhesion, extension) of the LGG wild-type are markedly differ-
nt from those of the mutant strains. Similarly, the presence ofacterial glycogen on the surface of Pseudomonas fluorescens wasetermined by combining infrared spectroscopy, AFM-FS and flu-rescence microscopy (Quiles et al., 2012). Con A was used as
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the bio-probe in both AFM-FS and fluorescence experiments. Theresults showed the increase of glycogen production with time. Theconformational change of the glycogen with time, revealed throughits infrared spectral signature, was further confirmed by analysis ofthe force curves using a Freely Jointed Chain polymer model, whichindicated an increase in glycogen contour length. The combina-tion of these techniques provided the bulk and surface informationof chemical composition at both the macroscale and molecularscale. These results demonstrate AFM-FS as a powerful techniqueto explore the recognition and force of lectin–carbohydrate inter-action on both static substrates and cell surfaces in their nativeenvironments.

3.2.2. Other glycans on bacterial and plant cell surfacesAFM-FS is also suitable for the study of the specific recognition

between proteins and polysaccharides on bacterial cell walls suchas lipopolysaccharides (LPS) and peptidoglycans. Generally, the LPScovers the outer membrane of Gram-negative bacteria, while inGram-positive bacteria, a thick layer of peptidoglycan constitutesthe main body of the cell wall. These bacterial polysaccharidesplay crucial roles in various processes such as adhesion, infec-tion and inflammation via biomolecular recognition (Beveridge andGraham, 1991; Costerto et al., 1974). Handa et al. measured theinteractions between bacteriophage P22 tailspike proteins (TSPs)and O-antigenic LPS of the Gram-negative bacteria Salmonellatyphimurium. AFM tips were modified with TSPs and an LPS bilayerdeposited on a solid substrate by vesicle fusion (Handa et al.,2010). The results showed strong and multivalent binding of immo-bilized TSPs to supported LPS. A stable unbinding force undervarying environmental conditions also demonstrated that phagesare less sensitive to pH and temperature fluctuations. This makesthem attractive candidates as immunosensors. In another reporton Gram-positive bacteria, a Lysine Motif (LysM), a protein mod-ule that specifically binds peptidoglycan, was used to modify theAFM tip (Andre et al., 2010). Adhesion forces were then collectedon living Lactococcus lactis cells. The force distribution showed asingle peak at 71 ± 16pN, which reflected the rupture force of thespecific LysM–peptidoglycan complex. The recorded high adhesion
areas on adhesion force maps (Fig. 3f) correlated well with the cor-responding features on topographic images (Fig. 3e), implying thatthe peptidoglycans were arranged as lines running parallel to theshort cell axis. This study showed the AFM-FS based adhesion force

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In recent cellulose research, both AFM imaging and force spec-roscopy were performed to study the recognition between thearbohydrate-binding module (CBM3a) of a cellulolytic enzymend the natural crystalline cellulose on the cell wall. First, using AFMon-contact imaging, CBM3a-modified gold nanoparticles werebserved to bind with the cellulose on cell surface. In order to con-rm the affinity of CBM3a and cellulose, a CBM3a-functionalized tipas used to map the plant cell wall surface using dynamic recogni-

ion imaging. CBM3a was observed to bind to the cellulose surface,losely aligning along the cellulose extension. AFM-FS was thensed to measure the interaction forces between the cellulose andBM3a. The unbinding force was measured to be 44.96 ± 18.80 pNt loading rate of 67.2 nN/s. This research provided a better under-tanding of biomass–enzyme interactions which is valuable for theesign of high-efficiency cellulolytic enzymes (Zhang et al., 2012b).

. Applications of cell-surface AFM-FS

As shown above, AFM-FS has been widely applied to charac-erize diverse biomolecular recognition processes on cell surfaceswing to advantages of high force sensitivity and the ability to oper-te under different physiological conditions. Knowledge of theseorces contribute to a better understanding of the molecular basisf molecular recognition events. Specifically, such data has directmplications in areas such as cancer research and drug screening.

e discuss some examples of AFM-FS applications in these areas:

.1. Probing cancer related biomarkers

A significant application of interest is the prospect of probing cellurface biomarkers that relate to cancer etiology and progression.

.1.1. Prostate specific membrane antigen (PSMA)PSMA is a possible therapeutic target expressed on the surface of

rostate cancer cells (Chang et al., 1999; Israeli et al., 1994). Laidlert al. used AFM tips with anti-PSMA antibodies to detect PSMA onifferent types of prostate cancer cell surfaces. The specific recog-ition events showed the effect of estradiol and basic fibroblastrowth factor in the expression of PSMA. The identical unbindingorce values for different cell lines implied the antigenic similarityf the membrane form of PSMA (Laidler et al., 2005).

.1.2. Tenascin-CThe high expression of tenascin-C can be used as a tumor

iomarker and target in glioblastoma diagnosis and therapyReardon et al., 2007; Yokoyama et al., 2000; Zagzag et al., 1995).sing a tenascin-C DNA aptamer modified AFM tip, the interaction

orces between the aptamer and tenascin-C on living cells wereeasured at different loading rates. Several dynamic parameters

cquired including the association rate constant, dissociation rateonstant and dissociation constant provide a better understand-ng of the binding mechanisms of the aptamer to its ligand as wells potential diagnostic and therapeutic targets. The results alsohowed that the interactions depend on the presence of magne-ium ions: The unbinding force did not change apparently whilehe binding probability increased in the presence of Mg2+ (Li et al.,013b).

.1.3. P-selectinP-selectin, which belongs to the family of selectin adhesion

olecules, has been studied as a common therapeutic targetor cardiovascular disorders, inflammation and tumor metasta-is (Ludwig et al., 2007). Platelets protect tumor cells from themmune cells by their adhesion to the tumor cell (Borsig et al.,

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2001). P-selectin expressed on the activate platelets surface medi-ate tumor cells metastasis by their interactions with the ligands(CD44 variant isoforms, CD44v) on tumor cell surface (Hanleyet al., 2006). P-selectin functionalized AFM tips were used toprobe the interactions between P-selectin and CD44v incorpo-rated into lipid vesicles. By comparing the interactions betweenfibrin and CD44v, P-selectin and CD44v showed higher tensilestrength, which explains their better binding ability at elevatedshear stresses. These studies provided biophysical insight intothe shear-dependent receptor–ligand binding involved in cancermetastasis (Raman et al., 2011).

4.1.4. GlycansAs discussed above, glycans on cell surfaces play important roles

in various biological processes. Alterations in glycans have beendemonstrated to be related to many diseases such as bacterial infec-tions, inflammation and tumor metastasis (Dwek, 1996; Sharon andLis, 2004). For instance, Lekka et al. studied the glycans expres-sion on two human bladder cells (cancer and nonmalignant ones)with AFM-FS by using lectins as probes (Lekka et al., 2006). Theresults showed differences in glycan expression between cancercells and the nonmalignant cells. Both the number of given glycantypes (characterized by binding probability) and their structure (ascharacterized by unbinding force) were observed to be differentin the two cell types. In particular, for the lectin from Phaseolusvulgaris, a much larger unbinding force indicated a distinct struc-ture of the binding site in cancer cells. These results highlightthe applicability of AFM for investigating alteration of the gly-cans during the cancer transformation. In another report, AFM-FScombined with fluorescence microscopy was utilized to charac-terize glycans on pathogenic and non-pathogenic yeast cell walls(El-Kirat-Chatel et al., 2013). Specific fluorescent-labeled lectinsand antibodies were used to detect glycans on cell surfaces usingfluorescence microscopy. Both AFM-FS and fluorescence resultsshowed consistent differences in cell glycan properties includingdensity, distribution, and extensions between pathogenic and non-pathogenic yeasts.

4.2. Drug design and detection of action mechanisms at themolecular scale

4.2.1. AntibioticsVancomycin is a member of glycopeptide antibiotics in the treat-

ment of methicillin resistant Staphylococcus aureus. It binds to theterminal d-alanyl-d-alanine moieties of peptidoglycan precursorswith a high affinity and specificity, thus preventing the synthe-sis of bacterial cell wall (Nieto and Perkins, 1971; Walsh, 2000;Williams, 1996). The interaction forces between d-alanyl-d-alanineimmobilized on mixed self-assembled monolayers and vancomycinmodified on AFM tips were measured (Gilbert et al., 2007). Theadhesion force histogram displayed a single maximum of 98 pN,which reflected a single rupture pair. A complementary adhesionforce map obtained on a cell surface showed that the specific bind-ing sites were mainly detected in the septum region, implying thatthe newly formed peptidoglycan was inserted in these regions.

AFM-FS also provided a new opportunity to study antibi-otic resistance by investigating the nanoscale effects of drugs onbacteria. Two kinds of P. aeruginosa, ATCC 27853 as the non-drug resistant strain, and PaR3 as a multi-drug resistant strain,were submitted to the antibiotics ticarcillin, tobramycin and para-guanidinoethylcalix[4]arene (CX1). Lectin-functionalized tips wereused to detect specific carbohydrates on cell wall surface. PaR3
showed flat force curves after treatment with ticarcillin andtobramycin, while ATCC 27853 showed specific interactions, whichimplied the cell wall of PaR3was not disorganized by the twoantibiotics and no molecules could be pulled out from the surface.

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nterestingly, both the PaR3 and ATCC 27853 after treatment withX1 showed long distance pull-off forces, which could imply thatX1 disorganized the bacterial cell such that long glycans (lectinecognition molecules) were pulled off from the cell wall (Formosat al., 2012).

.2.2. RituximabRituximab is a monoclonal antibody against cluster of differ-

ntiation 20 (CD20) on most B cell lymphomas. By recognitionith CD20 on a cell surface, rituximab is able to trigger lysis of

he dysfunctional B cell, and thus treat the lymphomas (Cartront al., 2004). Winiarksa et al. first measured the interactions ofituximab attached on AFM tip and CD20 on Raji cell and lovas-atin pre-treated Raji cell surfaces (Winiarska et al., 2008). Theecreased unbinding force on the lovastatin pre-treated cell sur-ace implied potential conformational changes of CD-20 induced bytatins. Combined with other biochemical methods, this researchhowed statins could interfere with both the detection of CD20, asell as the anti-lymphoma activity of rituximab, which had sig-ificant clinical implications. Subsequently, Li et al. used rituximabodified AFM tips to measure the rupture force between rituximab

nd CD-20 on mica, obtaining a single pair force value of 121 pN (Lit al., 2011a). Then the forces were measured on Raji cells and lym-homa patient B cells, which showed ruptures of 89 pN and 126 pNespectively. This research provided essential biophysical data onhe molecular interactions for the development of a monoclonalntibody drug. In recently reported research from the same group,uorescence microscopy was used to differentiate cancer cells fromormal cells by virtue of ROR1 fluorescence labeling on cancer cells,ince ROR1 is a specific biomarker expressed on the cancer cells, butot on normal cells. Interaction forces of rituximab and CD20 on theurfaces of B cell lymphomas could then be measured with the guid-nce of fluorescence labeling. Here, the application of fluorescenceicroscopy as a complementary tool improved the efficiency and

cope of data collection (Li et al., 2013a).

.2.3. Design of a drug delivery systemOne strategy to achieve cancer cell targeting is by integrating

ligand moiety in a drug delivery system, capable of recognizingpecific binding sites on the surfaces of cancer cells (Dharap et al.,005). It had been found that the receptors for luteinizing hormone-eleasing hormone (LHRH-R) are over-expressed in breast, ovarian,nd prostate cancer cells (Dharap and Minko, 2003). LHRH canherefore be used as a target for drug delivery systems to release therug to malignant cancer cells and reduce the concentration of toxicrug for normal cells. As a promising anticancer target drug, theHRH-P. aeruginosa exotoxin 40 (LHRH-PE40) was constructed byhe fusion of both LHRH and PE40 genes using genetic engineeringn vitro. Via AFM-FS, the recognition force of LHRH-PE40/LHRH-R

as compared with that of LHRH/LHRH-R. The results showed thathe recombinant protein LHRH-PE40 preserves the LHRH moiety’sbility to bind to LHRH-Rs on a living cell surface, thereby validatingts capability as a promising target drug (Zhang et al., 2012a).

.3. Evaluating the effects of therapeutic agents at cell surfaces

As discussed above, the ability of AFM-FS to probe biomolecu-ar interactions in various liquid and cellular media, including theresence of drugs, provides a unique tool to evaluate the effectf therapeutic agents or exogenous substances on biomolecularnteractions. A few interesting examples are discussed below:

.3.1. AtorvastatinInflammatory responses from vascular endothelial cells which

nvolve monocyte adhesion to the vascular endothelial cells, play

cron 60 (2014) 5–17

an important role in the pathogenesis of atherosclerosis. Adhe-sion of monocytes to the endothelial cells occurs by means of theinteractions between intercellular cell adhesion molecule-1 (ICAM-1) on endothelial cells and integrin CD1b on monocytes (Hentzenet al., 2000; Libby, 2000). Atorvastatin, as a commonly used statindrug for atherosclerosis therapy, has been recently reported for itsanti-inflammatory potential (Weitz-Schmidt, 2002). Using CD1bmodified tips, the interaction forces of ICAM-1 and CD1b weremeasured on endothelial cells in the absence and presence of ator-vastatin. The results showed atorvastatin did not inhibit bindingof ICAM-1 to CD1b. Flow cytometry results indicated that atorvas-tatin decreased the expression of ICAM-1, which may contributeto its anti-inflammatory effects. The combination of AFM and flowcytometry here provided a new approach to study the mechanismsof drug action (Li et al., 2010).

4.3.2. Effects of cigarette smoke extract (CSE)As a vital anticoagulation cofactor, thrombomodulin (TM)

located on the endothelial cell surface regulates intra-vascularcoagulation by binding to thrombin. The effects of CSE on TM-thrombin recognition were measured using AFM-FS (Wei et al.,2012). First, the unbinding forces of TM and thrombin were mea-sured in vitro in the absence and presence of CSE by AFM-FS. Theresults showed that the unbinding force values were similar withand without CSE treatment, but CSE tended to reduce the bindingprobability. Unbinding forces measured on the living-cell surfaceshowed that the CSE could reduce the binding probability of throm-bin to its specific receptor TM. These results provided new insightinto the understanding of thrombosis induced by smoking, andthe potential of AFM-FS as a tool to study the effects of clinicallyrelevant drugs and to screen candidates based on biomolecularrecognition at the cell surface.

4.3.3. Trastuzumab and PertuzumabHER2, as a member of the epidermal growth factor receptor

family, is often over-expressed on several human tumor cells,especially breast cancer cells (Slamon et al., 1987). Trastuzumaband Pertuzumab are two monoclonal antibodies targeting differentextracellular domains of HER2 for cancer therapy (Ross et al., 2009).By using AFM-FS on living cell surfaces, the effects of these twoantibodies on HER2-modulated epidermal growth factor (EGF)-epidermal growth factor receptor (EGFR) interactions were studied.The results demonstrated that the binding of EGF and EGFR wasmore stable on the cells co-expressing EGFR and HER2, and thatthis binding enhancement in the presence of HER2 was inhibitedby either Trastuzumab or Pertuzumab. The research showed AFM-FS offers a new approach to study the molecular mechanism ofanti-cancer drugs at the cellular level (Zhang et al., 2013b).

5. Challenges and future directions

The wide-ranging examples reviewed above show how AFM-FS is a label-free, highly sensitive tool for the study of biomolecularrecognitions on cell surfaces. We have confined our discussion hereto the vitally important and emerging field of investigations onthe surface of living cells. The unique capabilities of this modalityinclude not only measuring interaction forces between cellularlyrelevant biomolecules, but also being able to directly map recogni-tion sites on cell surfaces. Importantly, these measurements can betaken at physiological conditions and on live cells, which are sig-nificant for unraveling key biological and pathological questions
from the nanoscale perspective of molecular mechanics. Changesin the extracellular environment can be directly probed for theireffect on specific cell surface targets. However, there are still severalchallenges that need to be addressed for the broader application

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f AFM-FS for cellular systems. The first is the complicated liq-id environments of cell surface. It is often difficult to differentiatehe nonspecific binding events from specific interactions owing tohe multicomponent and soft cell surface. Several sets of controlxperiments therefore need to be conducted to confirm the specificnteractions and improve the signal to noise ratio. The typical con-rol experiments for instance, involve using different kinds of tipsncluding bare tip and tips modified with non-specific molecules,r by directly adding free blocking agents in the liquid media dur-ng force measurement. A higher quality of sample preparation andquilibration of the system is also critical to improve the accuracy ofata collection. Data interpretation is a crucial factor that will deter-ine the results of the experiments (Muller and Dufrene, 2011;uller et al., 2009). The current trend to automate collection and

nalysis of force-spectroscopy data has the ability to streamline thisrocess.

The second challenge lies in balancing requirements of time ofata collection vs. spatial resolution, especially for adhesion forceapping. Higher spatial resolutions of force maps imply the col-

ection of more force curves and therefore more time requiredo conduct the experiments. Given the highly dynamic nature ofells, this is poses an added technical challenge. Many biologi-al processes may occur faster than the time required by AFM toollect force curves. During the long period of time of data col-ection, particularly in liquid environments, instruments undergohermal drift, which result in the inaccuracies in the force mapsbtained. This implies that both the spatial and time resolutionhould be taken into account for high quality of force map. The usef softer (lower spring constant) and sharper (lower radius of tips)ips will facilitate to detect smaller interaction forces and smallerecognition sites on cell surface, which cannot probed by conven-ional AFM tips (Viani et al., 1999). Finally, there is a good potentialor combining AFM-FS with other analysis methods, such as flu-rescence microscopy, surface plasmon resonance, quartz crystalicrobalance and Raman spectroscopy (Wei and Latour, 2010; Yeo

t al., 2009; Zhang and Yadavalli, 2011). These multifunctional tech-iques will provide more biophysical and biochemical informationf biomolecular recognitions on cell surfaces. In the future, AFM-FSs expected to be increasingly applied in biomolecular research onell surfaces, especially for molecular recognition related patholog-cal process and specific biomarker on cancer cells. The quantitativeharacterization of biophysical information will contributes to theesign and screening of drugs-modulating biomolecular recogni-ion processes on cell surface. Overall, this tool is only beginning tocratch the surface of vast potential at and inside complex cellularnvironments.

cknowledgement

The authors would like to thank Dr. Y.R. Sarma for valuable editsnd critiques of the manuscript.

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Documents

Investigating biomolecular recognition at the cell surface using atomic force microscopy