Chem. Senses 24: 1-6,
1999
© Oxford University Press
The Distribution of Sugar Chains on the Vomeronasal Epithelium Observed with an Atomic Force Microscope
Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-0026 1 Anatomy and Embryology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan
Correspondence to be sent to: Dr Toshiya Osada, Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-0026, Japan. e-mail: tosada{at}bio.titech.ac.jp
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Abstract |
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The distribution of sugar chains on tissue sections of the rat vomeronasal epithelium, and the adhesive force between the sugar and its specific lectin were examined with an atomic force microscope (AFM). AFM tips were modified with a lectin, Vicia villosa agglutinin, which recognizes terminal N-acetyl-D-galactosamine (GalNAc). When a modified tip scanned the luminal surface of the sensory epithelium, adhesive interactions between the tip and the sample surface were observed. The final rupture force was calculated to be ~50 pN based on the spring constant of the AFM cantilever. Distribution patterns of sugar chains obtained from the force mapping image were very similar to those observed using fluorescence-labeled lectin staining. AFM also revealed distribution patterns of sugar chains at a higher resolution than those obtained with fluorescence microscopy. Most of the adhesive interactions disappeared when the scanning solution contained 1 mM GalNAc. The adhesive interactions were restored by removing the sugar from the solution. Findings suggest that the adhesion force observed are related to the binding force between the lectin and the sugars distributed across the vomeronasal epithelium.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The atomic force microscope (AFM) was developed as a scanning probe microscope which gives high resolution images by recording interactions between the scanning probe and sample surface (Binnig et al., 1982
, 1986).
Recently, AFM images using biological materials have been reported. For example, the typical
circular structure of plasmid DNAs and complexes between DNA and proteins have been
resolved with the AFM (Bustamante, et al., 1992; Vesenka et al., 1992; Hansma and Hoh, 1994).
Other biological materials such as proteins (Weisenhorn et al., 1990; Hallett et al., 1995), viruses (Ikai et
al., 1993; Imai et al., 1993) and cell surfaces (Paul et al., 1994; Eppell et al., 1995) have been
extensively studied with the AFM. The AFM can be used for both conductive and insulating
materials both in liquid and in air (Lal and John, 1994), which allows the
AFM to be used in biological systems such as RNA polymerase activity (Kasas et
al., 1997), the clotting process of fibrinogen (Drake et al.,
1989) and cell growth of yeast cells (Gad and Ikai, 1995). As
the AFM tip comes into contact with the sample surface, physical properties of the sample as
well as its topography can be obtained (Ikai, 1996; Mitsui et al., 1996; Brown and Hoh, 1997; Oberleithner et al., 1997; Rief et al., 1997).
AFM can also
be used to measure specific forces between the probe tip and the sample surface. It is possible to
measure the forces between the sample surface and any material selected by modifying the AFM
tip with that material. It has been shown that the binding forces between DNA base pairs (Boland and Ratner, 1995), avidin and biotin (Lee et al., 1994; Ludwig et al., 1997), ligand and
receptor (Florin et al., 1994; Moy et al.,
1994) and antigen and antibody (Dammer et al., 1996; Hinterdorfer et al., 1996; Allen et
al., 1997; You et al., 1997) can be measured
using an AFM.
The vomeronasal organ is located at the base of the nasal septum and originates from the
olfactory placode. The neuroepithelium of the vomeronasal organ (Wysocki, 1979; Halpern, 1987) is similar in structure to the olfactory
epithelium, consisting of supporting cells, receptor neurons and precursor cells. The vomeronasal
organ functions to detect substances such as pheromones associated with social and reproductive
behaviors. These substances may be detected by receptors which are most likely located on the
microvillar surface of vomeronasal sensory neurons.
In a previous paper (Osada et al., 1998) we described a new
method for the preparation of vomeronasal tissue sections which allowed us to obtain AFM
images comparable to images obtained using the transmission electron microscope (TEM). Most
subcellular structures of the vomeronasal epithelium observed with the TEM were also observed
with the AFM. In this report we describe another potential AFM application for the study of the
vomeronasal organ. As the AFM tip makes contact with the sample surface, it is possible to
measure the binding force between pheromone substances and their receptors. Since rat
pheromones are not yet available, we used Vicia villosa agglutinin (VVA), which is
reported to label the luminal surface of vomeronasal sensory epithelium (Takami et al., 1994).
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Tissue sections
Sprague–Dawley rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused with physiological saline followed by fixation with 4% paraformaldehyde in 0.1 M phosphate buffer. The vomeronasal organs were removed and kept in the fixative solution overnight. The organs were cut into 20-µm-thick sections using a freezing microtome. Sections were mounted onto gelatin-coated slides and stored at –80°C until used.
Lectin staining
The sample sections were rinsed with phosphate-buffered saline (PBS) and then incubated with fluorescein isothiocyanate (FITC)-labeled VVA (Sigma, St Louis, MO) for 1 h followed by washing in three changes of PBS for at least 10 min each. Sections were coverslipped with water-based mountant (PermaFlour Lipshaw) and observed with a confocal laser scanning microscope (MRC-600, Bio-Rad, Tokyo, Japan).
Preparation of AFM tips
Free sulfhydryl groups were introduced onto VVA by derivatization with long-chain succinimidyl 6-[3'-(2-pyridyldithio)-propionamido] (Pierce, IL, USA) followed by the reduction with dithiothreitol (DTT). The sample solution was applied to Sephadex G-25 (Pharmacia, Uppsala, Sweden) to remove DTT. About four thiol groups were introduced on VVA as estimated from the determination of generated pyridine-2-thione with a spectrophotometer. Gold-coated AFM tips (OMCL-TR400PB; OLYMPUS, Tokyo, Japan) with a nominal spring constant of 0.025 N/m were modified with the derived VVA through Au–S bonds.
Atomic force microscopy
AFM was performed in force volume mode using an NVB100 AFM (OLYMPUS) in combination with an inverted optical microscope. All experiments were done in Petri dishes filled with PBS. The scan area was divided into 16 x 16 sub-areas, where force curves were obtained in each sub-area. Both the horizontal and vertical scan rates were 1 Hz. The maximum pressure between the tip and the sample surface was controlled using the trigger mode, so that the maximum bending of the cantilever was limited to 10 nm.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In previous studies FITC-labeled VVA was reported to stain only the microvillar surface of the vomeronasal sensory epithelium (Takami et al., 1994
). Figure 1a confirms these results and illustrates the
intense fluorescence observed at the microvillar surface. Figure 1b shows
an AFM cantilever over the section of the vomeronasal epithelium with a tip located at its end,
although this cannot be seen in the figure. The position of the tip was controlled using an optical
microscope.
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Figure 2
a shows a schematic drawing of the AFM force curve. The
ordinate is the
deflection of the cantilever and the abscissa is the distance moved by the piezoelectronics that
drives the cantilever up or down relative to the sample surface. When the cantilever approaches,
the force curve is initially flat until it reaches the sample surface, where it starts to be deflected
linearly. After the cantilever starts to retract at the left end of the diagram, the curve closely
follows the previous approach curve until the deflection returns to the initial level. When the
cantilever is withdrawn, the adhesion force between the tip and the surface causes the cantilever
to bend towards the sample for some distance. This distance can be used to estimate the rupture
force required to break the adhesion (force = II x spring constant of the cantilever).
The length of the sugar chain pulled by cantilever can be obtained by subtracting II from I. Force
curves were obtained from 256 sub-areas to map the distribution of the specific sugar. Force
curves are classified into three categories. The first group includes force curves with rupture
events (Figure 2b), which indicates adhesion events between the lectin
molecules and sugar chains. The second group includes force curves without rupture events
(Figure 2), which shows no interaction between the tip and the sample.
The last group contains force curves that appear when the tip could not reach the sample surface
(data not shown). This indicates that the sample has a groove deeper than the vertical scan range
(1 µm) of the force curve. These force curves were observed in this experiment when the
tip
scanned over the lumen of the vomeronasal epithelium.
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Figure 3
shows the distribution of these three kinds of force curves.
As shown in Figure 3a,b the adhesive points were distributed at the
boundary of a groove and rarely existed on internal area as one can see in Figure 3c. The distributions shown in Figure 3a,b were
reproduced in sequential scanning of the same area. Based on microscopic observation of the tip
position during force mapping, it was determined that the zone of adhesion was on the
microvillar surface of the sensory epithelium. The distribution of the adhesion observed with
AFM corresponded to the fluorescence image in Figure 1
a, except that
the AFM adhesion zone seemed to make two lines where the fluorescence did not. To confirm
this result we observed the VVA FITC-stained tissue section using a confocal microscope. When
we focused on a thin layer at the top surface of the sample, we observed two lines of fluorescence
(Figure 3d).
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In order to confirm that the observed adhesion was due to he specific binding of the lectin to N-acetyl-D-galactosamine (GalNAc), a competitive experiment was carried out. Figure 4
a shows the force mapping within a 500 x
500 nm scanning area in the adhesive zone. Then GalNAc (1 mM) in PBS was added to the Petri
dish and incubated for 30 min at room temperature. The frequency of the adhesion was
dramatically decreased, as shown in Figure 4b. The adhesion was
recovered after the solution in the Petri dish was washed with PBS three times. Given these
observations, together with the results illustrated in Figure 3
showing the position specificity of the adhesion, we concluded that the measured adhesion force
was due to specific binding between VVA and GalNAc.
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The advantage of using AFM to detect ligand distribution lies not only in its high resolution but also in its ability to detect the strength of the binding and the length of the interaction. We analyzed the force curves that show rupture events from this point of view. The binding force and length were determined as shown in Figure 2
a. The
histogram of the binding force (Figure 5a) showed a peak around 50 pN.
This value represents the force needed to rupture the binding between the lectin and the sugar.
Some very strong rupture forces may represent a multiple binding effect. The histogram of the
length (Figure 5b) showed a more dispersed distribution, suggesting that
the bound sugar chains were pulled for some length (~800 nm).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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AFM tips modified with VVA have been employed to map the distribution of terminal GalNAc on vomeronasal epithelial sections. The results shown in Figure 3
a,b illustrate the high resolution of specific sugar mapping with an AFM. The
distribution of the sugar forming two bands was not seen with fluorescence microscopy. This
illustrates the unique potential of the AFM method in determining the fine distribution of ligands
or receptors. Findings suggest that GalNAc is enriched in the apical and basal thirds of microvilli
but not in middle third. This distribution pattern of sugars has not been reported before.
However, we cannot exclude the possibility that these are artificial structures due to sample
preparation.
When the epithelial sections were mapped with only the gold-coated AFM tips without
any modification, many adhesion forces were observed randomly (data not shown). This is due to
non-specific interaction between gold and the ample surface as reported previously (
Allen et al., 1997). When the gold-coated AFM tips were
modified with bovine serum albumin or other lectins which cannot stain vomeronasal epithelial
sections, the non-specific interactions disappeared (data not shown). The modified tip with VVA
can be used not only to detect specific binding between VVA and the sugar but also to reduce
non-specific binding between gold and the sample surface. We confirmed that the adhesive
forces observed here was specific binding between VVA and the sugar using competitive
experiments, as shown in Figure 4.
Figure 5 suggests that this method has the ability to examine
properties of the detected
binding. As we succeeded in measuring binding forces, it is possible to distinguish and compare
the sites with different binding forces. This is important when several similar receptors with
different binding forces to the same ligand are distributed in a tissue. Our method will open a
new way to map individual receptors. The length of interaction was distributed divergently.
This may come from the wide distribution of the length of the detected sugar chains. The other
possibility is that the membrane where the sugar chains were anchored as deformed differently
when pulled. Further investigation is needed to determine the cause of the observed distribution.
This method may be useful for examining the interaction between pheromone and its receptor
and the distribution of the receptors on the luminal surface of the sensory epithelium.
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Acknowledgments |
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The authors thank Dr R. Costanzo for critically reviewing the manuscript and for helpful discussion. We also thank N. Iwasaki for her technical assistance. This research was supported by CREST (J.S.T.).
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References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Allen, S., Chen, X., Davies, J., Davies, M.C., Dawkes, A.C., Edwards, J.C., Roberts, C.J., Sefton, J., Tendler, S.J.B. and Williams, P.M. (1997) Detection of antigen–antibody binding events with the atomic force microscope. Biochemistry, 36,7457 –7463.[Medline]
Binnig, G., Quate, C.F., Gerber, C.H. and Weibel, E. (1982) Surface studies by scanning tunneling microscopy. Phys. Rev. Lett., 49, 57–61.
Binnig, G., Rohrer, H. and Gerber, C.H. (1986) Atomic force microscopy. Phys. Rev. Lett., 56,930 –933.[Web of Science][Medline]
Boland, T. and Ratner, B.D. (1995) Direct
measurement of hydrogen binding in DNA nucleotide bases by atomic force microscopy. Proc. Natl Acad. Sci. USA, 92, 5297–5301.
Brown, H.G. and Hoh, J.H. (1997) Entropic exclusion by neurofilament sidearms: a mechanism for maintaining interfilament spacing. Biochemistry, 36, 15035–15040.[Medline]
Bustamante, C.J., Vesenka, J., Tang, C.L., Rees, W., Guthold, M. and Keller, R. (1992) Circular DNA molecules imaged in air by scanning force microscopy. Biochemistry, 31, 22–26.[Medline]
Dammer, U., Hegner, M., Anselmetti, D., Wagner, P., Dreier, M., Huber, W. and Guntherodt, H.J. (1996) Specific antigen/antibody interaction measured by force microscopy. Biophys. J., 70, 2437–2441.[Web of Science][Medline]
Drake, B., Orater, C.B., Weisenhorn, A.L., Gould, S.A.C., Albrecht, T.R.,
Quate, C.F., Cannell, D.S., Hansma, H.G. and Hansma, P.K. (1989) Imaging
crystals, polymers and processes in water with the atomic force microscope. Science, 243, 1586–1589.
Eppell, S.J., Simmons, S.R., Albrecht, R.M. and Marchant, R.E. (1995) Cell surface receptors and proteins of platelet membranes imaged by scanning force microscopy using immunogold contrast enhancement. Biophys. J., 68, 671–680.[Web of Science][Medline]
Florin, E.L., Moy, V.T. and Gaub, H.E. (1994) Adhesion forces
between individual ligand pairs. Science, 264, 415–417.
Gad, M. and Ikai, A. (1995) Method for immobilizing microbial cells on gel surface for dynamic AFM studies. Biophys. J., 69, 2226–2233.[Web of Science][Medline]
Hallett, P., Offer, G. and Miles, M.T. (1995) Atomic force microscopy of the myosin molecule. Biophys. J., 68, 1604–1606.[Web of Science][Medline]
Halpern, M. (1987) The organization and function of the vomeronasal system. Annu. Rev. Neurosci., 10,325 –362.[Web of Science][Medline]
Hansma, H.G. and Hoh, J. (1994) Biomolecular imaging with the atomic force microscope. Annu. Rev. Biophys. Biomol. Struct., 23, 115–139.[Web of Science][Medline]
Hinterdorfer, P., Baumgartner, W., Gruber, H.J., Schilcher, K. and Schindler, H. (1996) Detection and localization of individual
antibody– antigen
recognition events by atomic force microscopy. Proc. Natl Acad. Sci. USA, 93, 3477–3481.
Ikai, A., Yoshimura, K., Arisaka, F., Ritani, A. and Imai, K. (1993) Atomic force microscopy of bacteriophage T4 and its tube–baseplate complex. FEBS Lett., 326, 39–41.[Web of Science][Medline]
Ikai, A. (1996) STM and AFM of bio/organic molecules and structure. Surface Sci. Rep., 26, 261–332.
Imai, K., Yoshimura, K., Tomitori, M., Nishikawa, O., Kokawa, R., Yamamoto, M., Kobayashi, M. and Ikai. A. (1993) Scanning tunneling and atomic force microscopy of T4 bacteriophage and tobacco mosaic viruses. Jpn. J. Appl. Phys., 32, 2962–2964.[Web of Science]
Kasas, S., Thomson, N.H., Smith, B.L., Hansma, H.G., Zhu, X., Guthold, M., Bustamante, C., Kool, E.T., Kashlev, M. and Hansma, P.K. (1997) Escherichia coli RNA polymerase activity observed using atomic force microscopy. Biochemistry, 36,461 –468.[Medline]
Lal, R. and John, S.A. (1994) Biological
applications of atomic force microscopy. Am. J. Physiol., 266, C1–C21.
Lee, G.U., Kidwell, D.A. and Colton R.C. (1994) Sensing discrete streptavidin–biotin interactions with atomic force microscopy. Langmuir, 10, 354–357.[Web of Science]
Ludwig, M., Dettmann, W. and Gaub, H.E. (1997) Atomic force microscope imaging contrast based on molecular recognition. Biophys. J., 72,445 –448.[Web of Science][Medline]
Mitsui, K., Hara, M. and Ikai, A. (1996) Mechanical unfolding of alpha-2-macroglobulin molecules with atomic force microscope. FEBS Lett., 385,29 –33.[Web of Science][Medline]
Moy, V.T., Florin, E.L. and Gaub, H.E. (1994) Intermolecular forces and energies between ligands and receptors. Science, 266,257
–259.
Oberleithner, H., Schneider, S.W. and Henderson, R.M. (1997) Structual activity of a cloned potassium channel (ROMK1) monitored
with the atomic force microscope: the `molecular-sandwich' technique. Proc. Natl Acad. Sci. USA, 94, 14144–14149.
Osada, T., Arakawa, H., Ichikawa, M. and Ikai, A. (1998) Atomic force microscopy of histological sections using a new electron beam etching method. J. Microsc., 189, 43–49.
Paul, J.K., Nettikadan, S.R., Ganjeizadeh, Yamaguchi, M. and Takeyasu, K. (1994) Molecular imaging of Na+, K+-ATPase in purified kidney membranes. FEBS Lett.,346 , 289–294.[Web of Science][Medline]
Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J.M. and Gaub,
H.E. (1997) Reversible unfolding of individual titin immunoglobulin
domains by AFM. Science, 276, 1109–1112.
Takami, S., Getchell, M.L. and Getchell, T.V. (1993) Lectin histochemical localization of galactose, N-acetylgalactosamine and N-acetylglucosamine in glycoconjugates of the rat vomeronasal organ, with comparison to the olfactory and septal mucosae. Cell Tissue Res., 277, 211–230.[Web of Science]
Vesenka, J., Guthold, M., Tang, C.L., Keller, D., Delaine, E. and Bustamante, C. (1992) A substrate preparation for reliable imaging of DNA molecules with the scanning force microscope. Ultramicroscopy, 42/44, 1243–1249.
Weisenhorn, A.L., Drake, B., Prater, C.B., Gould, S.A.C., Hansma, P.K., Ohnesorge, F., Egger, M., Heyn, S.P. and Gaub, H.E. (1990) Immobilized proteins in buffer imaged at molecular resolution by atomic force microscopy. Biophys. J., 58,1251 –1258.[Web of Science][Medline]
Wysocki, C.J. (1979) Neurobehavioral evidence for the involvement of the vomeronasal system in mammalian reproduction. Neurosci. Biobehav. Rev., 3, 301–341.[Web of Science][Medline]
You, H. and Yu, L. (1997) Investigation of the image contrast of tapping-mode atomic force microscopy using protein-modified cantilever tips. Biophys. J., 73, 3299–3308.[Web of Science][Medline]
Accepted September 7, 1998
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