strategies and results of atomic force microscopy in the study of cellular adhesion
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Review
Strategies and results of atomic force microscopy in the study
of cellular adhesion
Anne Simon*, Marie-Christine Durrieu
INSERM U577 ‘Biomaterials and tissue repair’, Universite Victor Segalen Bordeaux 2, 33 076 Bordeaux, France
Received 10 February 2005; revised 21 June 2005; accepted 22 June 2005
Abstract
Atomic Force Microscopy (AFM) provides a range of strategies for investigating living cell adhesion to the extracellular matrix, other cells
or biomaterials in their native environment. This review surveys the results obtained from major studies using AFM for mechanical force
evaluation in the cell, morphological visualization of the cell and studies of the cell’s response to chemical or mechanical stress. Recently, the
use of AFM has been broadened to obtain experimental information about cell adhesion molecules. Quantitative measurements of binding
forces between adhesion proteins and their ligands in the cell or on a surface are presented. These analyses provide data on individual
molecules and their resulting collective behaviour at the cell level. They significantly contribute to the characterisation of cellular adhesion
with physical principles relating to biochemistry.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: Atomic force microscopy; Force spectroscopy; Cell adhesion; Cell surface; Cell cytoskeleton; Cell elasticity; Cell–cell interaction
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Principles of an AFM experiment on cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Mechanical properties of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.1. Image processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.2. Force curve analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.3. Force mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4. Cell adhesion to surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.1. Morphologic evaluation of cell adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.2. Adhesion strength measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5. Cell-to-cell adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5.1. Interaction forces between cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5.2. Binding strength between individual cell adhesion molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
0968-4328/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micron.2005.06.006
* Corresponding author. Tel.: C33 5 57571115; fax: C33 5 56900517.
E-mail address: abjsimon@hotmail.com (A. Simon).
1. Introduction
The attachment of cells to other cells or to an extra-
cellular matrix plays a key role in many biological (Basson
et al., 1992; Bourdoulous et al., 1998; Folkman and
Moscona, 1978; Ingber, 1990; Stossel, 1993) and patho-
logical (Klotz, 1992; Pauli et al., 1990) processes such as
embryogenesis, mitosis, leukocyte traffic, motility or
Micron 37 (2006) 1–13
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A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–132
metastasis formation in tumor-bearing patients and throm-
bus formation. In vitro, most cells are anchorage-dependent
and attach firmly to the substrate in order to divide
(Folkman and Moscona, 1978).
The cell’s capacity to make specific contact is mediated
by adhesion proteins (Bongrand, 1998); such as cadherin or
integrin (Kreis and Vale, 1999; Springer and Wang, 2004).
These protein receptors elicit a cellular response upon
ligand binding. Intracellular signalling leads to stabilization
of the contact cell or to cell motility (Hynes, 1992; Ruoslahti
and Pierschbacher, 1987).
Since cellular adhesion involves collective behaviour of
individual proteins, this process is difficult to study.
Experimentally, approaches have been developed to
characterize the process according to cell mechanical
properties as well as cell morphology, for example, and to
study the modulation of the cell properties consistent with
the adhesion receptor attachment to a surface or cytoskeletal
organization.
Quantitative information on the adhesion strength and
mechanical parameters of living cells can be obtain by
applying external forces. With shear flow assays, adherent
cells on a flat surface are submitted to a laminar flow;
weakly attached cells are deformed, and the cell-substrate
contacts are disrupted: cells are carried away and the
remaining cells are counted (Chan et al., 1992). A model
explains the dissipation of the mechanical energy due to the
work of the applied forces. Centrifugal assays may also be
used. They have recently been optimised to better define the
force applied to cells (McClay et al., 1981; Thoumine et al.,
1996). Mechanical properties of cells have also been probed
by other experimental approaches such as micropipettes
(Discher et al., 1994; Evans, 1989), traction forces on cells
(Galbraith and Sheetz, 1997; McClay et al., 1981), new
techniques such as optical tweezers (Ashkin and Dziedzic,
1989; Florin et al., 1997), magnetic tweezers (Bausch et al.,
1998, 1999).
Cell morphology (cell shape and spreading) can be
visualized by microscopy and it is also an initial indication
of adherent cell behaviors. Standard light microscopy is a
straightforward non-invasive method, which can distinguish
distinct morphology classes and state of adhesion: spheroid
cells for a non-adherent cell or spreading cells for an
adherent cell. Massia and Hubbell (1992) also reported SEM
images of fixed cells, and categorized the morphologies of
fibroblast cells as either spheroid cells with zero, one, two,
or more than two filipodia extensions or as flattened cells.
Non-adherent cells are expected to be spheroid with no
filipodia extension. Finally, techniques such as immuno-
fluorescence staining microscopy are capable of identifying
intracellular structures such as cytoskeleton fibers presented
by adherent cells (Charras and Horton, 2002a,b).
Atomic Force Microscopy (AFM) (Binnig et al., 1986) is
emerging as a valuable tool for studying cellular adhesion,
because of its ability to maintain cell viability, so that living
cells may be observed in their culture medium without
inducing irreversible damage (Hansma and Hoh, 1994;
Henderson, 1994; Kasas et al., 1997). AFM results combine
image information about cell morphology with the
mechanical properties of the cell surface such as cellular
elasticity with 2D lateral resolution or measure cell–surface
or cell–cell interaction forces.
The aim of this review is to present the experimental
results of cellular adhesion studies using AFM. On one
hand, AFM allows quantification of mechanical properties.
On the other hand, AFM may be used not only to examine
the cell morphology, but also to mechanically stress the cell
and then, observe the cell’s response to the disturbance and
its influence on cell attachment. Finally, we will review
results from studies on cell-surfaces or cell-types following
the pioneering studies of Sagvolden et al. and Thie et al.
(Sagvolden et al., 1999; Thie and Denker, 1997; Thie et al.,
1998) or the experimental study of ligand–receptor
interaction at the single-molecule level.
2. Principles of an AFM experiment on cells
The principle of AFM will only be briefly described here
as it is presented in depth in many books and review articles
(Binnig et al., 1986; Dufrene, 2003; Henderson, 1994; Hoh
and Hansma, 1992; Jena and Horber, 2002). For further
details, interested readers may refer to them.
The basis of AFM consists of scanning a surface with a
very sharp tip mounted on a cantilever (Fig. 1). The
deformation induced on the tip is recorded with an optical
detection system (Alexander et al., 1989; Meyer and Amer,
1988); and this deformation may be directly translated into a
force with picoNewton accuracy. A piezoelectric scanner
controls motion of the surface with sub-Amstrong precision.
Topographic information about the surface can be
obtained in two modes: intermittent contact and contact
(Fig. 2). In the intermittent contact mode (Nagao and
Dvorak, 1999; Tamayo et al., 2001; Vie et al., 2000), the
cantilever is vibrated on the sample. Because the tip is in
intermittent contact with the sample surface, the frictional
force and the contact time can be reduced and it can be used
to image fragile or loosely attached samples (Hansma et al.,
1994; Putman et al., 1994). Some studies have examined the
topography of unfixed cells using this approach (Butt et al.,
1990; Horber et al., 1992; Le Grimellec et al., 1994, 1998).
In the contact mode, the tip is brought into continuous
contact as it scans the surface. The tip–sample interaction is
kept constant throughout the imaging process. Since the
biological sample observed is not flat and since the tip can
interact differently with different areas on the sample, the
vertical position of the sample varies in order to maintain
interaction while the tip is scanning the specimen. To
prevent the tip from moving the cells, cells can be trapped in
pores of filters (Kasas and Ikai, 1995). They may
spontaneously adhere to the surface of a glass coverslip. It
is also possible to coat a glass or a silica wafer with
Fig. 1. AFM tip geometries. (A) Pyramidal cantilever (bar is 10 mm) and small tip (inset, bar is 1 mm). (Kumar and Hoh, 2001, Probing the machinery
trafficking with the Atomic Force Microscope, Traffic, q 2001 Blackwell Publishing) (B) Microbead glued to the cantilever. (C) Coated with human
trophoblast-type cells. Scale bar is 5 mm. (Thie et al., 1998).
Fig. 2. AFM imaging modes. (A) Intermittent contact mode. (B) Contact
mode (Kumar and Hoh, 2001, Probing the machinery trafficking with the
Atomic Force Microscope, Traffic, q 2001 Blackwell Publishing).
A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–13 3
positively charged material such as poly-lysine, or adhesive
proteins in order to improve the adhesion of the cells to a flat
surface (Altankov and Groth, 1994; Liu et al., 2003; Puleo
and Nanci, 1999; Ruardy et al., 1995). Prior to cell imaging,
the scanning force is adjusted with force curves. The vertical
force applied is adjusted to a minimum force. Typically, the
force applied is situated between 20 pN and 1 nN (Le
Grimellec et al., 1998). It is set in the force calibration mode
and collected by measuring the cantilever deflection as the
sample is moved toward the tip. Note that AFM is
minimally disruptive to cells using a standard AFM tip
(Parpura et al., 1995) or when used for short periods of time,
around a few hours (You et al., 2000). Many cell types such
as living neurons and glia (Parpura et al., 1993), endothelial
cells (Mathur et al., 2000, 2001), epithelial cells (Hassan et
al., 1998; Henderson and Oberleithner, 2000; Hoh and
Schoenenberger, 1994), and fibroblasts (Rotsch and
Radmacher, 2000) have been imaged using AFM. Nuclei,
mitochondria and cytoskeletal filaments have been
identified.
A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–134
AFM can be carried out either in air or in near-
physiological liquid conditions (Drake et al., 1989; Gould
et al., 1990). Better resolution is obtained in air, on fixed
cells (Hoh and Schoenenberger, 1994), but dynamic cell
behavior and the mechanical features of the cells are not
accessible.
The results obtained by AFM in studies of living cells let
expected, in future work, an improvement in time
resolution. In order to follow processes in situ, with AFM,
the data acquisition time is limited as it takes a few minutes
to capture an image, whereas many biological and
biochemical processes occur in less than a minute. Faster
scanning rates should be developed in the future.
3. Mechanical properties of cells
Cell mechanical properties are controlled mainly by the
cytoskeleton and by membrane elasticity (Sackmann, 1994).
As intracellular cytoskeleton organization is connected to
the surface by adhesion receptors, it is associated with
cellular adhesion. The atomic force microscope has
emerged as a powerful apparatus for obtaining mechanical
information about the sample under observation. The
mechanical properties of the sample are obtained by
image processing, by force curve analysis or by force
modulation methods. We shall now review these tree
methodologies.
3.1. Image processing
One method consists in recording height images at a
constant load. These combine topographical and mechanical
Fig. 3. Cytoskeletal fiber height by plotting a cell profile and AFM images. I
properties of the imaged cell. From the height profile
(Fig. 3) of the scanned cell, one can extract the tip
indentation depth at different locations on the cell from
which the elastic modulus G is deduced:
F Z kd Z PGf=ð1Kn2Þ (1)
The variation of the elastic modulus G as a function of
the indentation d is due in part to the cell heterogeneity
and to the finite size of the tip. It is a suitable way to
establish a correlation between the cell’s compliance and
its local structure. In an attempt to model of cell local
mechanical properties deduced from the analysis of
height images (Simon et al., 2004), a weak adherent
cell appeared to exhibit a more homogeneous elastic
response than a cell adhering firmly to the surface, as
expected.
3.2. Force curve analysis
The force curves give the variation of the cantilever
deflection, dc, as a function of the imposed vertical
displacement of the sample, D. They can be fitted with the
relation dcZSD, where dc is the cantilever deflection, D is
the ceramic displacement, and S is the slope of the force
curve containing the mechanical properties of the cell
studied.
To extract an elastic modulus from the curve, a model, as
specified in several references (Domke et al., 2000;
Radmacher, 1997; Simon et al., 2003; Vinckier and
Semenza, 1998), and based on the theory of Hertz and of
Sneddon is used to describe the elastic indentation of the cell
surface by the tip (Hertz, 1882; Sneddon, 1965).The local
stiffness ks of the cell surface is given by: ksZGf, where G
mage scan size: 130!130 mm. Z-scale: 0–15 mm (Simon et al., 2003).
A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–13 5
and f are the elastic modulus and the diameter of the contact
area, respectively. The key point is determining the
geometric factor that shows the relation between the
indentation depths, d, and the diameter of the contact
area, f. The geometry of the tip is widely considered to
resemble a cone shape (Hertz, 1882; Sneddon, 1965).
This approach has been widely used in similar situations
(Boschung et al., 1994; Domke and Radmacher, 1998;
Domke et al., 2000; Haga et al., 2000; Mathur et al., 2000;
Rotsch and Radmacher, 2000).
With the help of two equations, one corresponding to the
equality of the force between the elastic medium and the
cantilever, kc dcZks ds and the other giving the vertical
displacement as the sum of the cantilever deflection and
indentation depth, DZdcCds one can immediately calculate
the expression of the slope, S, of the force curve (the plot of
dc versus D) that contains information on the elastic
properties (Fig. 4).
Many experimental reports evaluate the elastic properties
of different cells. Mathur et al. (2001) determined the
mechanical properties of three different cell types. They
Fig. 4. (A) Forces curves obtained on the hard surface and on a soft material, su
surface (symbols) and theoretical calculations (line) (Simon et al., 2003). (C) T
Reprinted from Colloids and Surfaces B: Biointerfaces, 19, Domke et al., Substra
investigated by atomic froce microscopy, 367–379. q 2000 with permission from
showed that cardiac cells were the stiffest (100 kPa),
skeletal muscle cells were intermediate (25 kPa) and
endothelial cells were the softest (1.4–7 kPa) depending
on the location of the cell surface tested, consistent with the
function of these cells. As reviewed in this article (Fig. 5), a
wide range of values of elastic modulus (0.6 kPa–0.67 MPa)
can be obtained, depending on the cell types and on the
location of the cell surface.
By combining AFM and Total Internal Reflection
Fluorescence Microscopy (TIRFM), the mechanical force
transmission measurements were simultaneously examined
with focal contact dynamics in cultured cells monolayer
(Mathur et al., 2000). In this way, the study provided
evidence of force transmission from the apical cell
membrane to the basal cell membrane globally, resulting
in rearrangement of contacts on the basal surface.
3.3. Force mapping
The force mapping mode has been used to access local
structure information. The sample is scanned laterally,
rface cell. (B) Forces curves obtained on different zones of the cell-bound
wo-dimensional map of the elasticity of the sample (Domke et al., 2000.
te dependent differences in morphology and elasticity of living ostecblasts
Elsevier).
Fig. 5. Cell mechanical properties measured by AFM. Literature (Mathur et al., 2001. Reprinted from Journal of Biomechanics, Endothelial, cardiac muscle
and skeletal muscle exhibit different viscous and elastic properties as determined by atomic force microscopy, 1545–1553. q 2001, with permission from
Elsevier).
A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–136
and force curves are taken at each point. The force curves
are analysed to calculate a quantitative value for the local
elastic modulus at each location on the cell. Due to
hydrodynamic interaction and the travel scale, the scan
rate is limited and fast cellular processes cannot be tracked
in this way; and it is useful in this case, to process images.
Nevertheless, the result is a two-dimensional map of the
elasticity of the sample, as shown in Fig. 4C (Domke et al.,
2000). The substrate appears stiffer than the soft part of the
cell. The cytoskeletal fibers present increase elastic modulus
as compared with the body of the cell. Rotsch and
Radmacher (2000) affected the integrity of different
components of the cytoskeleton using drugs and measured
the elastic modulus of the cell. They showed the importance
of the actin network compared to the microtubule network
on the cell’s elastic properties and on the mechanical
stability of living cells.
4. Cell adhesion to surface
4.1. Morphologic evaluation of cell adhesion
AFM is a powerful experimental approach for the
morphological characterization of the cell adhesion. The
morphologic evaluation of bone cells by AFM will be taken
as an illustration, since understanding the interaction of
bone cells with implants is of obvious interest for
manufacturing bone materials (Puleo and Nanci, 1999).
The biocompatibility of materials is also related to the way
cells behave in contact with them.
The study of the adhesion of bone cells with different
materials has been performed using AFM by several teams
(Charras and Horton, 2002a,b; Domke et al., 2000;
Lehenkari and Horton, 1999; Simon et al., 2003). These
studies have provided an initial characterization of cell
adhesion by AFM images which show different cell shapes
and flattening: cell spreading could be distinguished
(Fig. 6A). The shape of the cells can also be defined by
parameters such as cell area, cell height or the flatness shape
factor, as shown by Domke et al. (2000). As in other studies,
it is assumed that cell shape gives a strong indication of the
quality of adhesion (van Wachem et al., 1989). Moreover, it
is accepted that flatness is directly correlated with good
adhesion, while this is not the case for more compact and
higher cells. Finally, the presence of an extensive network
of fibers inside the cell which constitutes part of the
cytoskeletal structure indicated, in addition to the shape and
the spreading of the cells, direct establishment of cell
adhesion (Simon et al., 2003).
These characterizations are consistent with those
obtained by SEM (Simon et al., 2003) and by immuno-
fluorescence staining microscopy which confirm the nature
of the fibers observed on AFM images since F-actin was
stained in this experiment (Fig. 6B; Charras and Horton,
2002a,b).
Mechanical load as well as drugs (Rotsch and
Radmacher, 2000) are also known to influence the
organization of the cell cytoskeleton bundles. Therefore,
to evaluate effective cellular adhesion, a large external force
can be applied with the AFM tip in situ (Fig. 7). When a cell
was stable and really adherent to a surface, exerting a force
on the cell did not disturb it (Simon et al., 2003). The
difference in cellular adhesion could explain the effect of the
different loads exerted by the tip on the cell.
Morphological characterization of cellular adhesion may
be revealed by AFM at different levels, first distinguished by
the presence of developed and structured stress fibers and by
the cell’s stability when the cell undergoes an applied load
or the effect of drugs.
Fig. 6. (A) Morphological aspect of osteoblast spreading observed by contact-mode AFM. Image scan size: 130!130 mm. Z-scale: 0–15 mm (Simon et al.,
2003). (B) Characterization of cytoskeleton actin fibers and focal contact protein paxillin, observed by immunofluorescence staining microscopy. Scale bar is
10 mm (Reprinted from Biophysical Journal, Charras and Horton, 2002a, with permission from the Biophysical society).
A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–13 7
4.2. Adhesion strength measurements
Sagvolden et al. (1999) reported a very interesting AFM
study of the quantification of cell adhesion forces. They
adapted AFM and introduced a manipulation force
microscope. The principle is shown on Fig. 8. An inclined
Fig. 7. Detachment of osteoblasts observed by contact-mode AFM. Imag
AFM cantilever is used like a piston fitted with a spring, to
laterally dislodge a cell. Before displacement, the inclined
cantilever was aligned with the cell using an inverted optical
microscope. No force was recorded after all cell-substrate
bonds had been removed. This apparatus measures the force
necessary to displace an object adhering to a substrate.
e scan size: 130!130 mm. Z-scale: 0–15 mm. (Simon et al., 2003).
Fig. 8. AFM adapted and cell displacement (cartoon of the cell displacement, cell as viewed in the optical microscope and the applied force versus
displacement) (Sagvolden et al., 1999; q 1999 National Academy of Sciences, U.S.A.).
A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–138
The force measurement, when displacing a cell adhering
to a substrate, is obtained by recording the laser beam
deflection versus the lateral cantilever displacement.
A force–distance curve is obtained. The baseline to peak
force DF, the peak width Dx and the work DW, were
calculated from the data. As emphasized by the authors,
the measured force depends on the direction and the speed at
which the cell is displaced. This approach also estimates the
time before adhesion forces are observed and the time over
which adhesion saturates (Fig. 9). Cervical carcinoma cells
attached faster and stronger at 37 8C than at 23 8C and better
on a hydrophilic than on a hydrophobic surface. The force
was of the order of 100–200 nN, depending on the coating
substrate (Fig. 9). As studied by Yamamoto et al. (1998), the
force between 300 and 400 nN was necessary to dislodge
murine fibroblast cells. In this case, the technique used was
similar to the manipulation force microscope. Interestingly,
these observed forces are similar to those obtained by other
techniques. By high-speed centrifugation, the force required
to dissociate epithelial cells from the surface was 100 nN
(Thoumine et al., 1996). The measurements with an adapted
AFM give detailed information on the adhesion forces as
a function of time, temperature and the hydrophobicity
of the substrates.
5. Cell-to-cell adhesion
5.1. Interaction forces between cells
Force spectroscopy investigates cell-to-cell interaction
down to the molecular level. This approach has emerged
Fig. 9. Cell-substrate adhesion forces on cell-culture surfaces. Median forces as a function of time for cells seeded on hydrophilic (Phil) polystyrene coated with
bovine serum albumin (BSA), fibronectin, or laminin. Table of cell adhesion measurements from Sagvolden et al. (1999) work q 1999 National Academy of
Sciences, U.S.A.
A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–13 9
recently to measure the behaviour of a molecule or a cell
under stretching mechanical force. As reviewed earlier
(Fotiadis et al., 2002), single biomolecules can be imaged,
manipulated and dissected in a controlled manner, and
inter- and intra molecular forces can be measured (Benoit
and Gaub, 2002; Hinterdorfer et al., 1996; Lehenkari and
Horton, 1999). Although much work has been done to
quantify the binding forces of interactions between protein
receptors and their ligand plated on a surface or on cell using
AFM, it is not easy to provide quantitative data on specific
interaction forces between cell layers. An ideal experiment
has done by Thie et al. (1998) and consisted of (1) bringing
cells into contact with monolayers of cells at know
indentation threshold force, (2) waiting for some time, and
then, (3) separating them. Repulsive forces exerted between
cells could be determined in step 1. In the next step, after the
initial contact between cells adhesive interactions develop
slowly, and are measured in step 3. This procedure of n
‘approach and separation’ cycles could be repeated a
sufficient number of times to obtain reproducible results
because of the complexity of living cells. These series of
experiments could also be repeated for different values of
contact times.
In this study, Thie et al. used this approach to study cell–
cell adhesion (Thie and Denker, 1997; Thie et al., 1998). As
a force-sensor, a microbead is mounted on cantilevers
(Johnsson et al., 1991) (Fig. 1b). This AFM sensor was
functionalized with human trophoblast-type cells (JAR)
(Fig. 1c). JAR-coated force sensors were brought into
contact with confluent monolayers of human uterine
epithelial cells (HEC-1-A, RL95-2), for different periods
of time (Fig. 10).
The approach force curves displayed repulsive inter-
actions for both HEC-1-A and RL95-2 cells. But RL95-2
cells showed lower values for the repulsive regime than
HEC-1-A cells. This repulsion is attributed by the authors to
the peripheral structure of the cell (e.g. glycocalyx). They
have previously shown that HEC-1-A cells posses a thicker
glycocalix than RL95-2 cells (Thie and Denker, 1997; Thie
et al., 1998). After having overcome repulsive interactions,
the initial contact was followed by adhesive interactions
(Fig. 10). The adhesion increased with increasing contact
time. For prolonged contact times (20 and 40 min), both cell
types showed a marked difference in the cell separation
force curves; particularly after the maximum adhesion
force. The force between JAR and HEC decreases
continuously in small steps of !200 pN upon separation,
while RL showed specific JAR binding with repeated
rupture events in the range of 1–3 nN, attributed to the
formation of strong cell-to-cell bonds.
This force spectroscopy on live cells was applied to a
variety of different cell adhesion systems such as bacterial
adhesion (Razatos et al., 1998), and leukocyte–endothelial
interaction (Zhang et al., 2004).
5.2. Binding strength between individual cell adhesion
molecules
A pioneering study was performed by Dammer et al.
measuring the binding strength of proteoglycan to
Fig. 10. (A). Force–distance plot and schematic illustration of the experiment (reprinted from Methods in Cell Biology, 68, Jena and Horbes, AFM in cell
biology, 91–114, q 2002, with permission from Elsevier). (B) Typical adhesive forces curves for HEC and RLG cells resulting when a bovine serum albumin-
coated microbead or a JAR-coated microbead (Thie et al., 1998). (Reprinted from Methods in Cell Biology, 68, Jena and Horbes, AFM in cell biology 91–114,
q 2002, with permission from Elsevier.)
A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–1310
proteoglycan interactions (Dammer et al., 1995; Popescu
and Misevic, 1997). Glyconectin 1 proteoglycans purified
from marine sponge species were covalently cross-linked to
the cantilever and to a flat mica surface. Like cell-to-cell
interaction force spectroscopy, the approach–retract cycles
were repeated and an intermolecular force of about of
400 pN resulting from about 10 individual interactions of
40 pN each was found. Moreover, glyconectin 1–glyconec-
tin 1 interactions were inhibited with block 2 anti-
glyconectin 1 monoclonal antibodies (which were shown
to completely block glyconectin promoted cell and bead
adhesion). It clearly established that these proteoglycan–
proteoglycan interactions mediate cell adhesion and
produce fundamental cell cohesion-forces in the sponge,
like protein-carbohydrate binding immunoglobulin, cad-
herin, lectin and extracellular matrix families of cell
adhesion molecules. In another study, Oberhauser et al.
(1998) studied the mechanical properties of the extracellular
A. Simon, M.-C. Durrieu / Micron 37 (2006) 1–13 11
matrix protein tenascin, which mediates the attachment and
the rolling of cells in shear flow. They showed the dynamic
extensibility of tenascin: single molecules of tenascin could
be stretched to several times its resting length. This property
could allow the tenascin-receptor bond to persist over long
extensions. Similar results were obtained in studies of
the giant muscle protein titin (Rief et al., 1997). Authors
showed that the elasticity of titin is determined by
the composition of elastic Ig domains within the protein
structure which in turn determine the macroscopic
mechanical behavior of this protein.
In a very interesting study, Marshall et al. (2003)
compared lifetime measurements from AFM and flow
chamber assays. They reported that at low forces
(!50 pN), the selectin-PSGL1 (P-selectin glycoprotein
ligand 1) bond lifetime increases with force, whereas at
high forces (O50 pN), it decreases with force. They show
that the mechanical response of the selectin-PSGL1 bond
depends on the force history.
These quantitative measurement examples on the single
molecule level investigate fundamental principles of
adhesion proteins structure and function.
6. Conclusion
This review has dealt with cellular adhesion studies using
AFM. With this technique living cells can be investigated in
their physiological environment through the examination of
the mechanical properties as well as the morphology of the
cells. AFM allows the exploration and the measurement of
mechanical properties of cells related to the local cell
structure. Elastic modulus can be collected. It is possible to
image the adhesion state of cells under native condition and
also to follow cellular processes such as deadhesion by
applying a mechanical load with the AFM tip or by injecting
drugs. An investigation of different factors (temperature,
substrate, etc.) involved in cellular adhesion can be
evaluated by a direct measurement of the force necessary
to detach a cell from a surface. AFM must be adapted for
these measurements, by inclining the cantilever. Finally,
AFM provides direct measurements of cell-to-cell inter-
action down to the molecular level. In the future, these
experimental developments will find applications in
investigations of mutated cell adhesion proteins or the
coupling of cellular adhesion molecule with the cytoskele-
ton on the affinity of cell for materials or other cells.
Acknowledgements
The authors would like to thank Dr A. Fuchs for helpful
discussions and for critical reading of the manuscript.
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