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Institute of Food Research, Norwich Research Park, Colney, Norwich, UK
2Correspondence: Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK. E-mail: patrick.gunning{at}bbsrc.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: AFM • Caco-2 • force mapping • lectin-carbohydrate interactions
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. However, it is also instrumental in determining the uptake of nutrients and other bioactive molecules within the body and, through uptake of particulate material, signals to the underlying immune system the presence of potentially harmful pathogens or beneficial commensal microflora (3
4
5
6)
. Specific receptors within the gut provide sites for targeted delivery of drugs or vaccines (7)
. Within this scenario, the ability to measure and map specific interactions between molecules of biological and biomedical relevance, with specific receptors expressed on the surface of the intestinal epithelial cells, would represent an important achievement.
Atomic force microscopy (AFM) is a powerful tool (8)
to measure both intramolecular and intermolecular forces associated with biological systems (9
10
11)
. Furthermore, it has the unique ability to measure adhesion events that result from individual molecular interactions in intact cells under physiological conditions, which provides both a high spatial and force resolution (12
13
14)
. The advantage of the use of AFM measurements over simple labeling approaches is that, in addition to locating the specific receptors, the AFM data give added information on the magnitude of specific and nonspecific binding forces involved in the interactions. This has the prospect to yield important biological information, which would be hard or impossible to obtain by other methods.
In this study we aim to demonstrate the potential of this approach by measuring and mapping a specific interaction between a lectin and its carbohydrate receptor expressed on the surface of living human intestinal epithelial cell monolayers formed by Caco-2 cells, a cell line that represents the most commonly used system to study intestinal epithelial cell biology and transport (15)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
The Caco-2 human colon carcinoma cell line was purchased from the European Collection of Cell Cultures (Salisbury, Wiltshire, UK) and maintained between passage 33 and 37. Cells were grown in DMEM containing 10% fetal calf serum, 1 mM sodium pyruvate, 1x nonessential amino acids, 100 U penicillin, and 100 µg ml–1 streptomycin with 0.25% trypsin-EDTA used for subculturing. Caco-2 monolayers were grown on 40 mm diameter plastic Petri dishes (Nalge Nunc International, Rochester, NY, USA) by seeding Caco-2 cells at a level of 2 x 104 cells cm–2.
Functionalization of silica particles
Silica beads with a mean diameter of 6.8 µm were purchased from Bangs Laboratories (Bangs Laboratories, Fishers, IN, USA). The beads were cleaned prior to functionalization by washing in the following series of solvents: ultrapure water (3x1 ml, 18.2 M
, Elga Ltd, Marlow, Bucks, UK), very low residue ethanol (3x1 ml), and toluene (3x1 ml, dried over a molecular sieve 4A). After each washing stage, the samples were spun down in an Eppendorf centrifuge (Eppendorf UK Ltd, Cambridge, UK) and the supernatant was discarded. Functionalization was performed following the protocol described by Bhatia and co-workers (16)
. The beads were incubated in a 2% solution of 3-mercaptopropyltrimethoxy silane (MTS) in dry toluene for 2 h at room temperature (20°C). Next, the beads were washed with dry toluene (3x1 ml), followed by very low-residue ethanol (3x1 ml). At each stage, the beads were centrifuged and the supernatant was discarded. The beads were then incubated in a 2 mM solution of n-
-maleimidobutyryloxy succinimide ester (GMBS, Pierce, Rockford, IL, USA) in ethanol for 2 h (20°C). The reagent was removed by washing with very low residue ethanol, and then a 1 µl aliquot of the dispersion was dropped onto a clean Petri dish. The sample was allowed to dry in air. The functionalized beads were transferred onto the ends of silicon nitride AFM cantilevers by the following method: the very end of the cantilever was dipped carefully (using the fine approach motor of the AFM) into a freshly prepared 2-part epoxy resin (40 min cure time, Permatex Inc, Hartford, CT, USA), which had been streaked over a clean microscope slide. The onboard camera allowed the moment of jump-to-contact of the lever to the surface to be seen. Once this had occurred, the cantilever motion was immediately reversed, which allowed the amount of adhesive transferred to be kept to a minimum. Capture of silica spheres onto the apex of the cantilevers was essentially a repeat of the procedure, but this time the cantilever containing the glue was gently placed onto a deposited sphere (jump-to-contact point, but no further pressing) and retracted. Once the epoxy resin had cured, the beads on the end of the cantilever were incubated with 0.1 mg ml–1 solutions of the relevant protein (Alexa-WGA or BSA) in phosphate buffered saline (Dulbecco’s PBS) for 1 h (20°C), followed by washing in PBS. The fully functionalized "tips" were then inserted into the liquid cell of the AFM and maintained under PBS solution. After completion of the binding experiments on cell monolayers, the quality of each tip was examined by scanning electron microscopy (SEM).
Functionalization of glass slides
The functionalization of glass slides with N,N',N''-triacetylchitotriose followed the same procedure as for the silica beads, but the hetero-bifunctional crosslinker 4-(4-N-maleimidophenyl)butyric acid hydrazide hydrochloride (MPBH, Pierce) was used in place of GMBS. MPBH contains a hydrazide group, which reacts with carbonyl groups such as the aldehydes to form stable hydrazone bonds (17)
. In the absence of an oxidation step during functionalization this limits the reaction to the reducing end of the oligosaccharide, leaving the majority of the molecule unbound and free to interact with the functionalized silica bead on the AFM tip.
Fluorescence microscopy of Caco-2
To visualize the carbohydrate receptors, cell cultures were sampled at different stages of cell growth and differentiation, incubated with 10 µg ml–1 fluorescent WGA-Alexa488 conjugate in PBS for 30 min at 37°C, and then washed in PBS (3x3 ml). Alternatively differentiated cells were incubated with WGA-Alexa Fluor 488 coated beads and then washed in PBS (3x3 ml). This system was used to assess the effect of N,N',N''-triacetylchitotriose on bead binding to the monolayer surface. Lectin or bead localization was visualized using an Olympus IX-70 microscope (Olympus UK Ltd, Watford, Herts, UK), and fluorescence images were recorded through a dichroic filter set (optimized for FITC) using an ART285 camera (Artemis CCD, Norwich, Norfolk, UK).
AFM and force spectroscopy
The force spectroscopy studies were made using a combined AFM-inverted optical microscope (TM Lumina, Veeco, Santa Barbara, CA, USA). A small CCD camera attached to the AFM head was used to position the AFM tip on the surface of selected cells within the monolayers maintained under PBS in a Petri dish. AFM images were captured at scan rates of 0.5–1 Hz using either standard NanoProbeTM V-shaped cantilever tips (200 µm long thin, Veeco) or the same cantilevers with silica beads attached adjacent to the tip. The set-point of the instrument was kept at the minimum tracking force necessary for stable imaging (typically 0.2–0.5 nN). All binding measurements on functionalized glass surfaces or Caco-2 monolayers were performed under PBS or 0.5 mM N,N',N''-triacetylchitotriose in PBS. The instrument was fitted with a bespoke heating stage (Babraham Technix, Cambridge, UK), which maintained the sample temperature at 37°C. Data were captured in the layered imaging mode (at a rate of 10 µm s–1 in the z direction and a scan rate of 1 Hz). In this mode the instrument ramps the Z piezo element of the scanner by a predetermined amount at each "imaging" point over a selected scan area and records the subsequent deflection of the cantilever as it is pushed into and then retracted away from the sample surface. This produces a series of images in which the gray level of each pixel represents the value of cantilever deflection at a given Z piezo distance in the cycle. It also yields a matrix of curves of cantilever deflection vs. distance that relate to the image coordinates. This allows features in the deflection-distance data to be matched to sample topography. The layered images presented here are either 50 x 50 or 25 x 25 pixels laterally, and the associated matrix of deflection-distance curves contains either 164 or 220 points, respectively. Raw data of cantilever deflection-piezo displacement captured with the AFM were converted into force-distance by a bespoke Excel macro using experimentally determined values for INVOLS (18)
and spring constant, k (19
, 20)
.
Adhesion maps
Adhesion maps were generated from the adhesion data derived from the analysis of the force vs. distance curves contained within the "layered images" using the contour surface plot function in Excel 2003TM (Microsoft, Redmond, WA, USA). This takes the 50 x 50 or 25 x 25 matrix data array and produces a surface chart viewed from above with colors representing discrete ranges of data values.
Scanning electron microscopy
Silica bead cantilever samples were coated with a 5 nm layer of gold using an Emitech 550 plasma coater (Quorum Technologies, Newhaven, East Sussex, UK) and imaged on a Stereoscan 360 scanning electron microscope (Leica Microsystems, Milton Keynes, UK), operating at 10 kV and a working distance of 11 mm.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To monitor these changes in WGA binding during monolayer formation and subsequent polarization, cells were incubated with fluorescently labeled WGA and imaged using epifluorescence microscopy (Fig. 1
). The images were taken through a FITC dichroic filter block so that only those regions of the cells with bound fluorescent lectin would appear bright. Figure 1a
, taken 24 h after seeding the cells onto the Petri dish, shows largely individual isolated cells roughly spherical in shape. The degree of labeling is relatively uniform, with the brighter regions probably due to out-of-plane contributions at the cell peripheries. In Fig. 1b
, taken at 5 days postseeding, a confluent monolayer of flattened cells appears. The images reveal considerable cell-to-cell heterogeneity in the level of binding of the labeled WGA. For example, the center of the image in Fig. 1b
shows a group of cells that are noticeably darker than their neighbors. Careful examination of the image in Fig. 1b
also reveals a heterogeneity in the lectin binding on individual cells. In Fig. 1c
, taken at 12 days postseeding, the appearance of the fluorescently stained cells has altered in a more subtle fashion. At day 12, the intracell heterogeneity appears to have become more pronounced than the intercell variation. In addition, the samples contain regions (domes) where the cells bulge upwards out of the Petri dish. It has been reported that typically 50 domes/cm2 are observed in cultures over 9 days postseeding (15)
. Such domed regions were avoided in AFM studies, and areas containing just adherent cells were chosen for imaging. All the subsequent experiments were performed on 12 day postseeding cell cultures.
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Figure 2
presents data obtained from binding experiments between silica spheres functionalized with fluorescent lectin or fluorescent BSA and Caco-2 cell monolayers. Figure 2a
shows that the lectin-functionalized beads have a strong affinity for the cell monolayer. The marked heterogeneity of binding is consistent with that observed for the free lectin (Fig. 1c
). The observed heterogeneity of the binding and the retention of the bound spheres after washing suggest that the binding is specific. However, in order to rule out the possibility of nonspecific interactions due to other effects, such as electrostatic and hydrophobic attraction, a control experiment was carried out. Silica beads coated with BSA, a protein with no specific affinity for Caco-2 cell receptors, were incubated with a Caco-2 cell monolayer. The results of this experiment (Fig. 2b
) show only minimal binding to the monolayer surface. This finding suggests that the binding of the lectin-coated beads to Caco-2 monolayers is unlikely to arise from nonspecific interactions. A further control experiment was performed in order to demonstrate the biological specificity of the lectin-functionalized beads toward the Caco-2 monolayer surface: incubation was performed with Alexa 488 WGA-tagged beads in the presence of free N,N',N''-triacetylchitotriose, a known receptor for wheat germ agglutinin (22)
. Under these conditions, Fig. 2c
demonstrates that binding of the tagged spheres is significantly reduced, although not eliminated entirely.
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Having demonstrated the functionality and specificity of the lectin-coated silica beads toward Caco-2 monolayers, it is necessary to optimize the procedure for attaching the functionalized beads to the AFM-cantilever assembly, in order to ensure that the functionality is preserved on attachment. Figure 3
a shows an SEM image of an AFM cantilever following the attachment of a silica bead. SEM imaging of the bead-bearing cantilevers was found to be an important step in verifying optimal positioning and gluing of the silica spheres to the AFM tip–cantilever assembly. Figure 3a
shows optimal binding with the sphere nestling against the pyramidal tip. The image also demonstrates that the glue is localized between the sphere and cantilever at the point of attachment and has not spread over the remaining surface of the sphere.
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Covalent attachment of the lectin to the sphere on the AFM tips was verified by fluorescence microscopy (Fig. 3b
). The presence of the fluorescently labeled WGA on the bead is clear, since it appears as a bright spot on the end of the cantilever. To allow the cantilever to be seen in the fluorescence image, a low-level white light illumination from the AFM head was also used. When this was turned off, all that was visible was the labeled lectin-coated bead.
To demonstrate that it was possible to measure the interaction between the lectin and the carbohydrate receptor, initial studies were made of the interaction between a lever-bound lectin-coated bead and a glass surface functionalized with covalently bound N,N',N''-triacetylchitotriose in PBS. Figure 4
a shows a typical force-distance curve. The force required to detach the functionalized bead from the functionalized glass surface is determined by quantifying the difference in the approach and retract curves at the pull-off point. Figure 4b
shows a histogram of the adhesive interactions seen in the experiment. The data reveal a distribution of adhesion forces in the range 0–4 nN with a modal value of 1770 pN. Figure 4c
shows data collected following titration of free carbohydrate into the liquid cell sufficient to saturate the binding sites on the lectin. After addition of the free carbohydrate (N,N',N''-triacetylchitotriose) the measurable adhesion was significantly reduced, with the majority of the data occupying the lowest available bin in the histogram.
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Figure 5
shows the results of adhesion measurements obtained on confluent monolayers of the Caco-2 cells in PBS probed with an Alexa 488 WGA-functionalized silica sphere cantilever. Adhesive events were detected only on certain regions of the cell surfaces. The histogram was obtained from an analysis of 2500 force-distance runs and is shown with the 0 bin omitted for clarity (1474 of the curves exhibited no measurable adhesion). Adhesive events in the range of 0–1650 pN were detected, with a modal value of 125 pN.
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To understand the topography of the Caco2 cell monolayers, a typical AFM deflection mode image obtained with a silica bead tip is presented in Fig. 6
a. The image illustrates the roughness of the cell surfaces with variations in height up to 5.5 µm. The image presented in Fig. 6a
only really shows the prominent boundary regions between adjacent cells, and relatively little surface detail is resolved. This is mainly due to the bluntness of the tip–the silica bead has a much lower radius of curvature (3.4 µm) than a standard AFM tip (10–30 nm). An additional factor is the deformability of the cells, which leads to distortion and blurring of the surface detail. Even with a standard tip (Fig. 6b
) it is difficult to resolve surface detail such as surface receptors due to the deformation of the cells. A combination of adhesion maps with the AFM deflection images should provide information on the location of receptors on the cell surface.
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Figure 7
a, b shows a series of adhesion maps obtained on Caco-2 cell monolayers alongside the associated AFM deflection mode images. These are true deflection images obtained with the feedback loop switched off during force mapping using the functionalized beads. The color scale of the adhesion maps has been set in 100 pN ranges, using the same colors for each of the examples. The effect of adding free N,N',N''-triacetylchitotriose to the liquid cell of the AFM is shown in the adhesion map presented in Fig. 7c
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Most of the WGA bound to Caco-2 membranes (85%) is associated with the glycosylated extracellular domain III of the epidermal growth factor receptor (23)
. In the present studies the appropriate model for the role of specific interactions in the gut is the binding to confluent monolayers of cells.
Between becoming confluent and 12 days postseeding, Caco-2 cells undergo polarization and acquire features of epithelial cells, including microvilli and brush border enzyme activities on the apical surface (24)
. The data presented in Fig. 1c
using fluorescently labeled WGA indicate that the carbohydrate receptor is still expressed on the polarized Caco-2 cells and retains the lectin binding capacity but that the localization appears much more heterogeneous.
For the adhesion studies the cell surface was probed using functionalized silica spheres glued to the tip-cantilever assembly. The use of silica spheres and fluorescently labeled ligands enabled the success of the coupling chemistry to be monitored and allowed precise control over the positioning of the active species, limiting it to the apex of the AFM cantilever. This is not the case when the tip-cantilever is functionalized directly, since the entire assembly will possess active sites. This is a particular issue on rough samples such as cell monolayers, where the large sample topography increases the possibility that the cantilever may make contact with the sample in addition to the tip in certain regions. Furthermore, spherical probes are less likely to damage the cell surfaces than sharp AFM tips because they distribute the loading over a larger surface area (25)
. Despite the expected potential loss in lateral resolution it has been found that spherical probes have been used successfully to detect nonspecific interactions between colloidal spheres, used as models for drug delivery systems and living cell surfaces (26
, 27)
. In the present studies, functionalized spheres were used to probe specific intermolecular interactions at the cell surface. We believe that micrometer-sized spheres provide a realistic model for the interaction between particulate materials and the gut epithelium. This is relevant to bacterial–gut interactions, uptake mechanisms for food particles in the gut, and targeted delivery systems.
Before performing the force spectroscopy experiments on Caco-2 cell monolayers with the AFM, a number of important steps were performed to ensure the validity of the final data. The first of these was a demonstration that attachment of the proteins to the silica spheres did not interfere with the protein–carbohydrate binding. To this end the attachment of labeled spheres to Caco-2 monolayers (12 days postseeding) was visualized using Alexa 488 WGA-functionalized silica beads in place of free lectin. The data in Fig. 2a
show that the beads have a strong affinity for the cell surfaces. The significant reduction in the number of bound beads after addition of free N,N',N''-triacetylchitotriose (Fig. 2c
) demonstrates that the binding of the lectin-coated silica beads was specific, and due to the lectin-carbohydrate interaction, since the free carbohydrate would be expected to compete for, and at sufficiently high concentration, saturate the lectin sites on the beads, which rendered the beads essentially inactive toward the cell surface. However, since ligand-receptor binding is essentially a stochastic process, in which bound and unbound states exist in some form of equilibrium, the total elimination of binding events would not be expected.
Verification that AFM force spectroscopy with functionalized silica beads was capable of detecting lectin-carbohydrate interactions is shown in Fig. 4
. The adhesion values seen in the data are relatively large, but this is to be expected when one considers the relatively large size of the silica sphere and the high density of covalently bound receptor on the glass surface: The detachment forces measured in the experiment are likely to arise from multiple lectin-carbohydrate interactions. The large modal value demonstrates that the lectin-functionalized silica spheres produced using Bhatia’s protocol were highly active. Determination of the single bond strength in such circumstances, once thought to be describable by simple Poisson statistics (28
, 29)
, has more recently been shown to be highly complex (30)
and to require an independent means of determining the number of bonds (31)
. Touhami et al. (32) report values of 96 ± 55 pN for single lectin (Concanavalin A) -carbohydrate (glucan) rupture events. These authors also reported rupture forces of 121 ± 53 pN (33)
for lectin-carbohydrate interactions on live yeast cells. The reduction in adhesion on addition of free carbohydrate to the system, seen in Fig. 4c
, is consistent with the results shown in Fig. 2c
and demonstrates that the adhesion measured by force spectroscopy is specific and attributable to lectin-carbohydrate rupture events. It also suggests that any nonspecific interactions are significantly smaller than the observed specific interaction. The concentration of N,N',N''-triacetylchitotriose required to reduce WGA binding to Caco-2 cells is in good agreement with published values (22)
.
Having demonstrated that AFM can be used to measure specific interactions of the lectin-coated beads with immobilized receptor, and shown specific binding of functionalized beads to Caco-2 cells as well as inhibition of this binding with the free N,N',N''-triacetylchitotriose, it was possible to use the functionalized beads to probe binding sites on the Caco-2 cell surfaces with a high degree of confidence. The range of adhesive interactions observed in Fig. 5
demonstrates that the system worked and could quantify the adhesion over different regions of the cell monolayer. The modal value for adhesion seen in the measurements, 125 pN, is notable, as it is similar to the 121 pN value reported for the lectin-carbohydrate interactions in flocculating yeast cells (33)
and the 100 pN value reported elsewhere for single molecule antibody–antigen recognition events on the surface of G6D3 cells (34)
. In fact, it is close to typical values reported for individual receptor–ligand interactions in general, such as 160 pN for avidin–biotin interactions (10)
and 112 pN for antibody–antigen interactions (35)
. This finding suggests that the majority of the forces measured in the present experiment may well represent single lectin-carbohydrate rupture events. The fact that, in the main, single-molecule interactions were observed may be due to restricted access to the carbohydrate at the cell surface, due to the curvature of the cell surface or the mode of presentation of the carbohydrate on the surface. Both may effectively reduce the contact area and allow single molecule events to be detected, despite the large size of the bead. Furthermore the time that the sphere and cell were in contact was less than 0.2 s, conditions that have been shown previously to favor the observation of single rupture events in measurements of the unbinding forces on separation of Dictyostelium discoideum cells, where multiple binding sites exist (12)
.
A significant advantage of capturing adhesion data in a layered imaging mode is that each force-distance curve is assigned to a physical location in the area sampled. By combining this information with the adhesion calculated from each curve, it is possible to generate an adhesion map relating to the sample topography. In effect the dry statistical information from the adhesion histograms is converted to a visual impression of the stickiness of the sample surface to a particular probe. This approach has many advantages, not least of which is the ability of the observer to match shapes intuitively. This provides excellent confirmation of the validity of the data, since the adhesion, if genuine, should correlate to some extent with sample topography. In particular the adhesion maps should show the heterogeneity of the distribution of the receptors observed previously for the binding of the fluorescently labeled WGA. The adhesion maps presented in Fig. 7a, b
show that in each case some correlation exists between the cell surface topography and its corresponding adhesion map. Furthermore, they also show that the distribution of the receptor sites on the cells appears to be heterogeneous. The data obtained following the addition of free carbohydrate, shown in Fig. 7c
, provide further proof of the efficacy of this approach. The relatively narrow range of colors and the relatively small area that they occupy when compared with the data obtained for the "active" examples in Fig. 7a, b
are striking. These data reveal that the addition of free carbohydrate reduced both the number and magnitude of binding events significantly. This finding isconsistent with the data observed in Fig. 2c
, where only a small number of bound beads are observed on the Caco-2 cell monolayer after addition of N,N',N''-triacetylchitotriose. It is also consistent with the force spectroscopy validation experiment shown in Fig. 4c
, where a reduction in lectin–carbohydrate interactions is seen following addition of free carbohydrate.
In this article, AFM-based methodology has been developed to measure the specific interaction of ligands with receptor sites on the surface of confluent monolayers of living Caco-2 cells in PBS. To our knowledge, this is the first example of the use of AFM to measure specific molecular interactions on a realistic in vitro model of the human gut epithelium. A series of control steps have been developed to validate the methodology. Measurement of the interaction of the ligand with the receptor and mapping of the receptors on the cell surfaces has been achieved through the use of functionalized silica beads attached to an AFM tip cantilever assembly. The beads were functionalized using covalent coupling of the ligand to the silica sphere. The methodology was demonstrated by measuring the binding of the lectin WGA to the surface of Caco-2 cells. Adhesion events were used to map the location of the binding sites on the cell surface. The binding of WGA to Caco-2 cell surfaces is attributed predominantly to binding to the glycosylated extracellular domain III of the epidermal growth factor receptor (23)
.
Cultured monolayers of Caco-2 cells are the accepted in vitro model for gut epithelium cells. In culture, Caco-2 cells can be induced to differentiate and mimic the structure and properties of M cells. Cocultures of human Caco-2 cells with human Raji B cells are now being widely used as in vitro models to investigate lympho-epithelial interaction in the gut (36)
. The methodology has the potential for the investigation of the specificity of molecular interactions with receptors on cell surfaces that are believed to be of importance in explaining the bioactivity of food components or drugs. The methods can be used to investigate the binding of commensal bacteria or the attachment of pathogenic bacteria within the gut. Optimizing molecular attachment of particulate material is of importance in the design of targeted delivery and release systems for drugs or vaccines in the gut (7)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication November 5, 2007. Accepted for publication January 10, 2008.
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