TECHNICAL NOTE |
Correspondence to: Michel Grandbois, Dept. of Physics and Astronomy, U. of MissouriColumbia, 318 Physics Bldg., Columbia, MO 65211. E-mail: grandboism@missouri.edu
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We used an atomic force microscope (AFM) to produce an image of a mixed layer of group A and O red blood cells with a contrast based only on the measured strength of a specific receptorligand pair. The image was obtained by measuring and plotting for each image pixel the adhesion force between the mixed RBC layer and the AFM tip functionalized with Helix pomatia lectin. The high specificity of that lectin for the N -acetylgalactosamine-terminated glycolipids present in the membrane of group A RBCs enabled us to discriminate between the two cell populations and to produce an image based on affinity contrast. The rupture force of the adhesion events leading to the image formation were quantitatively analyzed and compared to rupture forces measured with the same AFM tip on N-acetylgalactosamine tethered to agarose beads. The mean rupture force was found to be 65 pN when measured on the group A RBCs and 35 pN on the agarose beads. These results show that the adhesion, mediated by only a few receptorligand pairs, produces sufficient contrast for the affinity image formation. (J Histochem Cytochem 48:719724, 2000)
Key Words: atomic force microscope (AFM), affinity, recognition, glycocalix, lectin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The atomic force microscope (AFM) (
The cell glycocalix located at the external membrane side of eucaryotic cells is basically composed of glycosylated molecules. This external sugar envelope contributes to the steric repulsion that prevents undesirable nonspecific cellcell adhesion (
|
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Surface and Tip Preparation
Here we used carboxymethylated amylose for the lectin immobilization on the AFM tip. This protocol was very similar to the one used in the protein immobilization technique developed by
Adhesion and Height Image Acquisition
The adhesion image was obtained by first recording a two-dimensional array (55 x 55) of force vs extension curves over the RBC layer. A typical force vs extension curve is shown in Fig 2 . The contact of the lectin-funtionalized AFM tip with the cell surface can be observed in the first part of the curve (Fig 2a). In Fig 2b, the bridge between the lectin and the AFM tip is stretched, producing a bending of the cantilever which can then be converted into force units (Newton). In Fig 2c one can observe the rupture of the molecular bridge. This bond rupture event could be assigned to the lectinsugar molecular pair. In fact, all the other bonds in the bridge are of covalent nature and their rupture forces have been demonstrated to be at least one order of magnitude higher (
|
|
Force vs Elongation Curves on Agarose Beads
Force vs elongation curves were measured with HPL-functionalized tips and commercially available agarose beads functionalized with galNAc (Sigma). For each experiment, 4000 curves were recorded at a fixed position on one agarose bead. The adhesion force was measured for each force curve as for the RBCs and was plotted in a histogram (Fig 4 ). The indentation force was set to a constant value below 40 pN. The loading rate for each force curve was also 1 nN/sec. A blocking control experiment was performed by exchanging the PBS buffer of the AFM fluid cell with a PBS buffer containing 100 mM free N-acetyl- D-galactosamine.
|
![]() |
Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The epifluorescence image in Fig 1c shows a typical mixed (1:2) group A and O RBC layer used for AFM affinity imaging. On this micrograph, the group A RBCs can be readily identified from their bright fluorescent appearance. The contrast between the two RBC groups was obtained by incubating the cell layer with a fluoresceinlabeled HPL. The high binding specificity of that lectin for group A RBCs enables them to be distinguished in a mixed A and O cell layer. Such a technique is intensively used in histochemistry, where it is very useful for characterization of cell structures. However, this approach may be somewhat destructive because the receptors used for fluorescent contrast imaging are at least partly blocked after the labeling procedure.
In Fig 3 the cell layer was affinity-mapped with an AFM tip functionalized with HPL. The adhesion image in Fig 3a shows several regions on the cell layer in which adhesion events are observed with a very high probability (see bright spots). The arrangement of these regions can be directly correlated to the presence of RBCs as observed on the topographic image ( Fig 3b). Moreover, the ratio of bright cells seen on the affinity image (25%) corresponds very well to the number of group A RBCs present in the cell layer (33%). The small statistical discrepancy between these two values can be explained by the relatively small number of cells scanned in one image (24 cells). The distribution and number of group A RBCs also correspond very well to the one observed on the epifluorescence micrograph shown in Fig 1c . Hence, the affinity image clearly shows that it is possible to observe the receptors present at the surface of a given cell. On the affinity image, it is impossible to extract any information about the distribution of galNAc receptors on the surface of a single group A RBC. Because the lipid membrane receptors of the RBC are well known to be homogeneously distributed and free to diffuse in the experimental time scale, such local distribution was not expected.
The rupture forces for all the adhesion events observed in the affinity image (Fig 3a) were quantified and plotted in the histogram shown in Fig 4a. This histogram shows a distribution of the rupture forces between 30 and 140 pN, with a maximum centered at 65 pN. One should note here that the majority of the higher rupture force values were observed in the first force curves recorded on a group A RBC (see the bright cell in the left lower corner of Fig 3a). This effect is probably due to detachment of loosely attached lectin. In fact, after the initial measurement of some 50 adhesion events, the rupture forces are found to be close to the maximum at 65 pN. This maximal rupture force on cells compares very well to the rupture forces measured on agarose beads. The histogram in Fig 4b shows the rupture forces calculated from the force vs elongation curves measured on agarose beads functionalized with galNAc. For this experiment, the probability of observing a binding event was approximately 10%. Such a low probability makes it very likely to observe single bindingunbinding events. The maximum at 35 pN can therefore be attributed to the rupture of a single lectinsugar pair. All together, the values measured on group A RBCs and on agarose beads strongly suggest that only a few pairs are required to produce the contrast necessary for affinity imaging.
The locus of failure of the lectinsugar pair remains an open question in our measurements with RBCs. Indeed, the rupture of the lectinglycolipid pair may occur at the receptor ligand binding site, or the membrane receptor may simply be extracted from the cell membrane like a carrot, as proposed in several previous studies (
![]() |
Conclusions |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have demonstrated that it is possible to use specific affinity forces to image a biological relevant surface. It was possible to distinguish between RBC populations having different membrane receptors at their extracellular side. The quantitative analysis of the force curves recorded on RBC and on beads has shown that only a few lectinsugar bonds were necessary to obtain contrast. Wearing out of the tip resulting from pulling the receptor out of the membrane may be an important limiting factor for cell surface imaging. It has been shown recently that a force of a few tens of pN is enough to extract the lipid moiety of a receptor from the membrane. In our case, this problem appeared to be circumvented by the high off-rate of the lectinsugar pair. In that case, the AFM tip seems to be self-cleaned in the time scale of our experiment. Probing of cytoskeleton-anchored transmembrane receptors is believed to be less problematic because their extraction from the membrane is very unlikely. Affinity imaging by AFM has the potential to become a valuable tool for studying membrane receptor expression in cell tissues under different conditions and at different stages of development.
![]() |
Acknowledgments |
---|
Supported by the Deutsche Forschungsgemeinschaft. MG is a recipient of a postdoctoral fellowship from the Canadian NSERC.
Received for publication July 21, 1999; accepted November 24, 1999.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bell GI (1978 ) Models for the specific adhesion of cells to cells. Science 200:618-627 [Medline]
Binnig G , Quate CF , Gerber C (1986 ) Atomic force microscope. Phys Rev Lett 56:930 [Medline]
Bongrand P , Claesson PM , Curtis ASG (1994 ) Studying Cell Adhesion. Berlin , Springer-Verlag
Cleveland JP, Radmacher M, Hansma PK (1994) Atomic scale force mapping with the atomic force microscope: NATO Adv Res Workshop 543549
Dammer U , Hegner M , Anselmetti D , Wagner P , Dreier M , Huber W , Güntherodt H-J (1995 ) Specific antigen/antibody interactions observed by atomic force microscopy. Biophys J 70:2437-2441 [Abstract]
Erlandsson R , Hadziioannou G , Mate CM , McClelland GM , Chiang S (1988 ) Atomic scale friction between the muscovite mica cleavage plane and a tungsten tip. J Chem Phys 89:5190-5193
Evans E , Berk D , Leung A (1991 ) Detachment of agglutinin bonded red blood cells I. Forces to rupture molecular point attachments. Biophys J 59:838-848, a [Abstract]
Evans E , Berk D , Leung A , Mohandas N (1991 ) Detachment of agglutinin-bonded red blood cells II. Mechanical energies to separate large contact areas. Biophys J 59:849-860, b [Abstract]
Feizi T (1998 ) Carbohydrate recognition systems in innate immunity. Adv Exp Med Biol 435:51-54 [Medline]
Florin E-L , Moy VT , Gaub HE (1994 ) Adhesive forces between individual ligand-receptor pairs. Science 264:415-417 [Medline]
Florin EL , Rief M , Lehmann H , Ludwig M , Dornmair C , Moy VT , Gaub HE (1995 ) Sensing specific molecular interactions with the atomic force microscope. Biosensors Bioelectron 10:895-901
Frisbie CD , Rozsnyai LF , Noy A , Wrighton MS , Lieber C (1994 ) Functional group imaging by chemical force microscopy. Science 265:2071-2074, a
Frisbie CD , Rozsnyai LF , Noy A , Wrighton MS , Lieber CM (1994 ) Functional group imaging by chemical force microscopy. Science 265:2071-2074, b
Gabius HJ , Gabius S (1997 ) Glycosciences. Weinheim , Chapman & Hall
Grandbois M , Beyer M , Rief M , ClausenSchaumann H , Gaub H (1999 ) How strong is a covalent bond? Science 283:1727-1730
Hinterdorfer P , Baumgartner W , Gruber HJ , Schilcher K , Schindler H (1996 ) Detection and localization of individual antibody-antigen recognition events by atomic force microscopy. Proc Natl Acad Sci USA 93:3477-3481
Hoh J , Engel A (1993 ) Friction effects on force measurements with an atomic force microscope. Langmuir 9:3310-3312
Jarvis SP , Yamamoto SI , Yamada H , Tokumoto H , Pethica JB (1997 ) Tip-surface interactions studied using a force controlled atomic force microscope in ultrahigh vacuum. Appl Phys Lett 70:2238-2240
Johnsson B , Löfas S , Lindquist G (1991 ) Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal Biochem 198:268-277 [Medline]
Leckband DE , Mueller W , Schmitt F-J , Ringsdorf H (1995 ) Molecular mechanisms determining the strength of receptor-mediated intermembrane adhesion. Biophys J 69:1162-1169 [Abstract]
Lee GU , Kidwell DA , Colton RJ (1994 ) Sensing discrete streptavidin biotin interactions with the atomic force microscope. Langmuir 10:354-357
Leng Y , Williams CC (1993 ) Molecular charge mapping with the electrostatic force microscope . SPIE Scann Probe Microsc II 185:35-39
Ludwig M , Dettmann W , Gaub HE (1997 ) AFM imaging contrast based on molecular recognition. Biophys J 72:445-448 [Abstract]
Manne S , Gaub HE (1995 ) Molcular organization of surfactants at solid-liquid interfaces . Science 270:1480-1482 [Abstract]
Marti O , Colchero J , Mlynek J (1990 ) Friction and Forces on an Atomic Scale. Nanotechnology 1:141-144
Moiseev YN , Panov VI , Savonov SV (1991 ) The effect of local friction on an atomic force microscope image of surface structure. Sov Tech Phys Lett 17:360-362
Moy VT , Florin EL , Gaub HG (1994 ) Intermolecular forces and energies between ligands and receptors . Science 266:257-259 [Medline]
Radmacher M , Fritz M , Cleveland JP , Walters DA , Hansma PK (1994 ) Imaging adhesion forces and elasticity of lysozyme adsorbed on mica with the atomic force microscope. Langmuir 10:3809-3814
Radmacher M , Fritz M , Kacher CM , Cleveland JP , Hansma PK (1996 ) Measuring the visco-elastic properties of human platelets with the atomic force microscope. Biophys J 70:556-567 [Abstract]
Ros R , Schwesinger F , Anselmetti D , Kubon M , Schäfer R , Plueckthun A , Tiefenauer L (1998 ) Antigen binding forces of individually adressed single-chain Fv antibody molecules. Biophysics 95:7402-7405
Rotsch C , Radmacher M (1997 ) Mapping local electrostatic forces with the atomic force microscope . Langmuir 13:2825-2832
Torres BV , McCrumb DK , Smith DF (1988 ) Glycolipid-lectin interactions: reactivity of lectins from Helix pomatia, Wisteria floribunda and Dolichos biflorus with glycolipids containing N-acetylgalactosamine. Arch Biochem Biophys 262:1-11 [Medline]
Torres BV , Smith DF (1988 ) Purification of Forssman and human blood group A glycolipids by affinity chromatography on immobilized Helix pomatia lectin. Anal Biochem 170:209-219 [Medline]
Weis W , Drickamer K (1996 ) Structural basis of lectin-carbohydrate recognition. Annu Rev Biochem 65:441-473 [Medline]
Weis RM, McConnell HM (1982) Molecular basis of cell-cell recognition. Cell Funct Differ [A] 331336
Weisenhorn AL , Hansma PK , Albrecht TR , Quate CF (1989 ) Forces in atomic force microscopy in air and water. Appl Phys Lett 54:2651-2653
Weisenhorn AL , Maivald P , Butt H-J , Hansma PK (1992 ) Measuring adhesion, attraction, and repulsion between surfaces in liquids with an atomic force microscope. Phys Rev 45:11226-11232. [B]
Yip CM , Yip CC , Ward MD (1998 ) Direct force measurements of insulin monomer-monomer interactions . Biochemistry 37:5439-5449[Medline]