Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, FL 33136, USA
* Author for correspondence (e-mail: vmoy{at}miami.edu)
Accepted 7 March 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cell adhesion, Cell compliance, Integrins, Leukocyte, AFM, Single molecule measurements
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An important attribute of integrins is their ability to modulate the
adhesive states of cells (Dustin and
Springer, 1991; Diamond and
Springer, 1994
). In resting lymphocytes, LFA-1 is expressed in an
inactive, nonadherent state; LFA-1 in this state binds ICAM-1 with low
affinity. Upon engagement by an antigen presenting cell (APC), the T
lymphocyte expresses an activated form of LFA-1 and becomes adherent to the
APC. The interaction between a T lymphocyte and an APC is transient, with the
adherent state lasting long enough for the T lymphocyte to become activated
before it detaches from the APC.
Proposed mechanisms for the regulation of integrin-mediated leukocyte
adhesion include receptor affinity modulation and clustering of integrins
(avidity modulation) (Lollo et al.,
1993; Woska et al.,
1996
; Lupher et al.,
2001
; Stewart and Hogg,
1996
; van Kooyk and Figdor,
2000
). Affinity modulation of LFA-1 is initiated by the engagement
of surface receptors and involves a conformational change in the I domain of
the
chain, leading to an opening of the ICAM-1-binding site
(Shimaoka et al., 2002
). The
mechanism for affinity modulation in activated cells involves an inside-out
signal that acts on the cytoplasmic domain of the ß chain of LFA-1
(Hughes et al., 1996
). The
intramolecular transduction signal crosses the
chain via the ßI
domain/ß-propeller junction, which then activates the I domain
(Lu et al., 2001
). The
requirements for an inside-out signal for the induction of high-affinity LFA-1
can be circumvented by a high concentration of extracellular Mg2+
and by certain antibodies directed against the ß chain
(Ganpule et al., 1997
;
Huth et al., 2000
). Recent
studies suggest that extracellular Mg2+ activates LFA-1 by binding
to the ßI domain, which subsequently induced a conformational change in
the I domain via the ß-propeller (Lu
et al., 2001
).
Although the term avidity is frequently used in the context of cell
adhesion, it remains poorly defined. Generally, avidity modulation refers to
the redistribution or clustering of receptors that results in augmented cell
adhesion. This loose definition of avidity may include several underlying
mechanisms such as polarization of receptors to the zone of cell-cell contact,
clustering of receptors to form focal adhesion sites and dimerization of
receptors. A redistribution of receptors augments adhesion by increasing the
receptor density at the site of cell-cell contact. A clustering of receptors
allows for a more even distribution of the applied force and thus permits the
clustered receptors to support greater forces than when the receptors are
dispersed. In the latter case, there is an increased likelihood that an
applied force will rupture the adhesion complex sequentially. The dimerization
of receptors may result in the formation of a dimeric complex that functions
as a cooperative unit that ruptures simultaneously. Although the relative
contribution of these underlying mechanisms to adhesion still needs to be
resolved, it is well-documented that the initial event in avidity modulation
in leukocyte adhesion is the release of LFA-1 from cytoskeletal constraints
(Stewart et al., 1998). This
is achieved following engagement of the antigen receptor for T lymphocytes and
various surface receptors of migrating leukocytes. Recent studies using
interference-reflection microscopy (IRM) have shown this mobilization of LFA-1
on T lymphocytes in contact with APCs. Once released from the cytoskeleton,
the receptor molecules form ring-like structures termed supramolecular
activation clusters (SMACs). The SMACs consist of antigen receptors localized
at the center of cell-cell contact and LFA-1ICAM-1 complexes in the
periphery (Monks et al., 1998
;
Grakoui et al., 1999
). These
studies suggest that cytoskeletal remodeling plays an important role in T
lymphocyte adhesion.
The mobilization of LFA-1, leading to enhanced adhesion, can be induced by
pharmaceutical agents such as phorbol myristate acetate (PMA)
(Rothlein and Springer, 1986).
PMA is a potent activator of protein kinase C (PKC) and exerts its effects on
intracellular pathways, bypassing the requirement for surface receptor
engagement (Berry and Nishizuka,
1990
). The activation of PKC leads to an increase in intracellular
Ca2+ concentrations, Ins(1,4,5)P3 kinase
activity and phosphorylation of MacMARCKS and L-plastin
(van Kooyk and Figdor, 2000
;
Zhou and Li, 2000
;
Jones et al., 1998
). Elevated
levels of intracellular Ca2+ subsequently activate the
Ca2+-dependent protease calpain, which releases LFA-1 from the
cytoskeleton (Stewart et al.,
1998
). PMA also acts indirectly on the activity of cytohesin-1,
which induces cytoskeletal reorganization and subsequently promotes cell
spreading (Kolanus et al.,
1996
).
The current study employed the atomic force microscope (AFM) to elucidate
how PMA stimulation of leukocytes enhances cell adhesion. AFM measurements of
cell adhesion and cell compliance were carried out using the 3A9 cell line, a
murine T-cell hybridoma that expresses the LFA-1 integrin but no other
receptor for ICAM-1 (Lollo et al.,
1993). LFA-1 of 3A9 cells is constitutively inactive but can be
activated to a high-avidity state by PMA. Our research focused on the initial
interaction between the 3A9 cells and immobilized ICAM-1. The enhanced
adhesion of 3A9 cells following the addition of PMA was immediate and stemmed
from a change in the mechanical properties of the cell rather than a change in
the bond strength of the individual LFA-1ICAM-1 complex.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ICAM-1/Fc chimera consisted of all five extracellular domains of murine
ICAM-1 (Gln 28-Asn 485) and the Fc fragment of human IgG1 and was
purchased from R & D Systems, Inc. (Minneapolis, MN). The ability of this
protein to bind LFA-1 was confirmed using ELISA and adhesion assays. From
these experiments, we were able to conclude that the LFA-1-binding epitope D1
of ICAM-1 is available for binding. Antibodies against LFA-1 (i.e. M17/4.2 and
FD441.8) and against ICAM-1 (i.e. BE29G1) were purified from culture
supernatant by protein G affinity chromatography
(Sanchez-Madrid et al., 1983;
Kuhlman et al., 1991
). Stock
solutions of PMA (10,000x) (Sigma, St. Louis, MO) were prepared at 1 mM
in DMSO.
Protein immobilization
25 µl of ICAM-1/Fc at 50 µg/ml in 0.1 M NaHCO3 (pH 8.6)
was adsorbed overnight at 4°C on the center of a 35 mm tissue culture dish
(Falcon 353001, Becton Dickinson Labware, Franklin Lakes, NJ). Unbound
ICAM-1/Fc was removed and bovine albumin (Sigma) at 100 µg/ml in PBS was
used to block the exposed surface of the dish. A similar protocol was used to
immobilize the anti-LFA-1 antibodies (i.e. FD441.8 and M17/4.2).
AFM measurements of adhesive forces
The AFM force measurements were performed on an apparatus designed to be
operated in the force spectroscopy mode
(Benoit et al., 2000;
Heinz and Hoh, 1999
;
Willemsen et al., 2000
). 3A9
cells were attached to the AFM cantilever by concanavalin A (conA)-mediated
linkages (Zhang et al., 2002
).
To prepare the conA-functionalized cantilever, the cantilevers were soaked in
acetone for 5 minutes, UV irradiated for 30 minutes and incubated in
biotinamidocaproyl-labeled bovine serum albumin (biotin-BSA, 0.5 mg/ml in 100
mM NaHCO3, pH 8.6; Sigma) overnight at 37°C. The cantilevers
were then rinsed three times with phosphate-buffered saline (PBS, 10 mM
PO43-, 150 mM NaCl, pH 7.3) and incubated in
streptavidin (0.5 mg/ml in PBS; Pierce; Rockford, IL) for 10 minutes at room
temperature. Following the removal of unbound streptavidin, the cantilevers
were incubated in biotinylated conA (0.2 mg/ml in PBS; Sigma) and then rinsed
with PBS.
To attach the 3A9 cell to the cantilever, the end of the
conA-functionalized cantilever was positioned above the center of a cell and
carefully lowered onto the cell for approximately 1 second. When attached, the
cell is positioned right behind the AFM tip of the cantilever as illustrated
in Fig. 1. To obtain an
estimate of the strength of the cell-cantilever linkage, we allowed the
attached cell to interact with a substrate coated with conA for 1 minute. Upon
retraction of the cantilever, separation (N>20) always occurred between the
cell and the conA-coated surface. The average force needed to induce
separation was greater than 2 nN. These measurements, thus, revealed that the
linkages supporting cell attachment to the cantilever is greater than 2 nN and
much larger than the detachment force required to separate the bound 3A9 cell
from immobilized ICAM-1 (Zhang et al.,
2002).
|
A piezoelectric translator was used to lower the cantilever/cell onto the
sample. The interaction between the attached 3A9 cell and the sample was given
by the deflection of the cantilever, which was measured by reflecting a laser
beam off the cantilever into a position sensitive 2-segment photodiode
detector. AFM cantilevers were purchased from TM Microscopes (Sunnyvale, CA).
The largest triangular cantilever (320 µm long and 22 µm wide) from a
set of five on the cantilever chip was used in our measurements. These
cantilevers were calibrated by analysis of their thermally induced fluctuation
to determine their spring constant (Hutter
and Bechhoefer, 1993). The experimentally determined spring
constants were consistent with the nominal value of 10 mN/m given by the
manufacturer.
AFM force measurements of individual LFA-1ICAM-1
complexes
Measurements of unitary LFA-1-ICAM-1-unbinding forces were obtained under
conditions that minimized contact between the 3A9 cell and the sample. An
adhesion frequency of <30% in the force measurements ensured that there is
a >85% probability that the adhesion event is mediated by a single
LFA-1ICAM-1 bond (Zhang et al.,
2002).
Data were corrected for hydrodynamic drag. Our determination of the
hydrodynamic force was based on the method used by Tees et al. and Evans et
al. (Tees et al., 2001;
Evans et al., 2001
). We allowed
the cantilever to undergo free movement at different speeds, and the
hydrodynamic force for each speed was measured. These data suggest that the
hydrodynamic force acts in the opposite direction of cantilever movement, and
its magnitude is proportional to the cantilever movement speeds. In this
report the data obtained with cantilever retraction speeds higher than 1
µm/s were corrected by adding the hydrodynamic force.
To determine the damping coefficient, we plotted the hydrodynamic force
versus speed of cantilever movement. The damping coefficient is the slope of
the linear fit and is about 2 pNs/µm
(Zhang et al., 2002).
AFM measurements of cell elasticity
In this report, the AFM served a dual purpose: first as a pulling device to
detach adherent cells (described above), and secondly, as a microindenter that
probes the mechanical properties of the cell. In the cell elasticity
measurements, the bare AFM tip is lowered onto the cell surface at a set rate,
typically 2 µm/second. After contact, the AFM tip exerts a force against
the cell that is proportional to the deflection of the cantilever. The
deflection of the cantilever was recorded as a function of the piezoelectric
translator position during the approach and withdrawal of the AFM tip. The
force-indentation curves of the cells were derived from these records using
the surface of the tissue culture dish to calibrate the deflection of the
cantilever. Estimates of Young's modulus were made on the assumptions that the
cell is an isotropic elastic solid and the AFM tip is a rigid cone
(Wu et al., 1998;
Hoh and Schoenenberger, 1994
;
Radmacher et al., 1996
).
According to this model, initially proposed by Hertz, the force
(F)-indentation (
) relation is a function of Young's modulus
of the cell, K, and the angle formed by the indenter and the plane of
the surface,
, as follows:
![]() | (1) |
Young's modulus was obtained by least square analysis of the
force-indentation curve using routines in the Igor Pro (WaveMetrics, Inc.,
Lake Oswego, OR) software package. The indenter angle, , and Poisson
ratio,
, were assumed to be 55° and 0.5, respectively.
Measurements of cell adhesion and elasticity were carried out at 25°C
in fresh tissue culture medium supplemented with 10 mM HEPES buffer. Cells
were stimulated by 5 mM MgCl2 plus 1 mM EGTA or 100 nM PMA. The
activation of 3A9 by Mg2+ was immediate. 3A9 cells were exposed to
PMA for 5 minutes at 37°C prior to the start of the experiments. All
experiments involved making contact with the same cell up to 50 times. There
was no dependence on previous contacts observed in either the elasticity or
adhesion AFM studies.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
AFM measurements of the detachment of the cell from the ICAM-1-coated
dishes revealed a complex process. The measurements showed that the cells
detached through a series of jumps (as indicated by arrows in
Fig. 2) in force before final
separation. Each of the force jumps (breakage force) in the retraction trace
were interpreted as the breakage of one or more LFA-1ICAM-1 bonds of
>50 pN (Zhang et al.,
2002). The number of LFA-1ICAM-1 complexes depended on
various factors, including the compression force that pressed the cell against
the substrate and the duration of cell-substrate contact. During the
retraction of the cantilever, the cell is stretched with elongations of
several microns, possibly stemming from tether formations
(Benoit, 2002
).
In the current study, we investigated the mechanisms of enhanced cell adhesion to immobilized ICAM-1 following cell activation. Enhanced cell adhesion to immobilized ICAM-1 was induced by either 5 mM MgCl2/1 mM EGTA or 100 nM PMA. Fig. 2 presents a series of measurements carried out under identical conditions (i.e. 200 pN compression force, 5 seconds contact and 2 µm/second retraction speed) with resting (first trace), Mg2+-treated (second trace) and PMA-stimulated (third trace) cells. Both Mg2+-treated and PMA-stimulated cells adhered more to immobilized ICAM-1 than resting cells, as was evident by the larger maximum force required to dislodge the cell (detachment force, fde). Moreover, the number of rupture events is significantly greater for PMA-stimulated cells than is the case of resting cells.
An alternative measure of adhesion is the work done by the cantilever to detach the cell from immobilized ICAM-1. The work of de-adhesion includes work done to break the LFA-1ICAM-1 complexes and to stretch the cell during this process. Work was derived by integrating the adhesive force over the distance traveled by the cantilever. Fig. 3 shows that the enhancement in cell adhesion is more pronounced following cell activation when adhesion is expressed in terms of work of de-adhesion rather than in terms of detachment force. To demonstrate that the adhesion of both resting and activated cells is mediated by interactions of the LFA-1ICAM-1 complex, we showed that cell adhesion is inhibited by antibodies against either LFA-1 or ICAM-1 and by 5 mM EDTA. Furthermore, 3A9 cells did not adhere to immobilized BSA. It should be emphasized that both the work of de-adhesion and the detachment force are functions of multiple parameters, including compression force, the contact duration and the retraction rate of the measurements. Hence, the comparison of the work of de-adhesion and the detachment force for different cases is valid for measurements carried out under identical conditions and should only be used to determine relative adhesion.
|
To determine if enhanced adhesion of PMA-stimulated cells is associated
with an increase in receptor cooperativity (i.e. a simultaneous unbinding of
two or more complexes), the magnitudes of the force transitions detected in
the force-displacement traces were measured and plotted in the histograms
presented in Fig. 4. Both
resting and PMA-stimulated cells revealed a force distribution centered at
45 pN. These force transitions are consistent with forces attributed to
the unbinding of individual LFA-1ICAM-1 complexes
(Zhang et al., 2002
). An
observation that is more relevant to the current discussion is that the force
distributions for resting and PMA-stimulated cells are nearly identical. On
the basis of these measurements, there is no evidence for the simultaneous
unbinding of multiple LFA-1ICAM-1 complexes following PMA stimulation
of the cells. If PMA did increase receptor cooperativity, then we would have
detected a shift in the force distribution of the stimulated cells toward
higher forces (Chen and Moy,
2000
).
|
In parallel experiments, we investigated the interactions between 3A9 cells
and immobilized antibodies against LFA-1
(Fig. 5). The anti-LFA-1
monoclonal antibodies used in this study, M17/4.2 and FD441.8, recognized
eptiopes on the chain and the complexed
ß chains,
respectively. Our force measurements revealed enhanced adhesion to immobilized
M17/4.2 and immobilized FD441.8 for cells stimulated by PMA but not by
Mg2+. These results, along with the experiments described in the
previous paragraphs, are consistent with observations made using more
conventional cell adhesion assays (Stewart
et al., 1996
). Enhanced cell adhesion to immobilized ICAM-1 in
response to Mg2+ can be attributed to an induced conformational
change in LFA-1 that results in a high-affinity conformer of LFA-1. In
contrast, the absence of enhanced adhesion to immobilized anti-LFA-1 suggested
that the monoclonal antibodies bind to eptiopes that do not change following
affinity modulation of LFA-1. However, enhanced cell adhesion to both ICAM-1
and anti-LFA-1 was detected in cells stimulated by PMA. These results revealed
that enhanced adhesion to the antibodies following PMA is not the result of a
conformational change in the ICAM-1-binding site of LFA-1.
|
Measurements of the unbinding forces of individual LFA-1ICAM-1
complexes
To assess the bond strength of the individual LFA-1ICAM-1 complexes,
contact between the 3A9 cell and immobilized ICAM-1 was minimized by reducing
both contact duration (50 milliseconds) and compression force (
60
pN). Examples of measurements acquired under these conditions are given in
Fig. 6A. In contrast to the
measurements presented in Fig.
2, these measurements frequently revealed no adhesion. When
adhesion did take place, the AFM force-displacement trace revealed a linear
increase in force, followed by a single sharp transition that signaled the
breakage of a single LFA-1ICAM-1 complex. The unbinding force of the
individual LFA-1ICAM-1 complex was derived from the magnitude of the
force transition with corrections for hydrodynamic drag. In order to determine
the loading rate, the force spectra were plotted as force versus piezo
displacement. We measured loading rates by multiplying the slope of the force
versus displacement curve with the cantilever retraction speed.
Fig. 6B summarizes the force
distribution for the separation of the LFA-1ICAM-1 complex at loading
rates of 100 pN/second, 1000 pN/second and 30,000 pN/second. At a loading rate
of 100 pN/second, the average force distribution was 30±2 pN (s.e.m.)
for resting cells and became shifted to 64±1 when LFA-1 was activated
by Mg2+. Interestingly, the average force distribution did not
change when the cells were stimulated by PMA (30±2 pN). At a loading
rate of 1000 pN/s, the average force distribution was 54±2 pN (s.e.m.)
for resting cells and was also shifted towards higher values averaging
100±3 pN for Mg2+-activated LFA-1. Again, no change in the
force distribution was detected when the cells were stimulated by PMA
(51±2 pN). At 30,000 pN/s, histogram peaks had similar values in all
three cases (240±6 pN for resting cells, 230±5 pN for
PMA-stimulated cells and 240±7 pN for Mg2+-activated
cells).
|
As reported previously (Zhang et al.,
2002), the average unbinding force of the LFA-1ICAM-1
complex increases over three orders of magnitude change in loading rate
(Fig. 7). Two loading regimes
in the LFA-1ICAM-1 interactions were evident in the force spectrum
(plot of unbinding force versus loading rate). There was a gradual increase in
unbinding force with an increasing loading rate up to about 10,000 pN/second.
Beyond this point, there was a second loading regime that exhibited a faster
increase in unbinding force. Induction of high-affinity LFA-1 by
Mg2+/EGTA resulted in higher LFA-1ICAM-1 unbinding forces,
which were pronounced in the slow loading regime. There was no significant
difference in the dynamic response of the low- and high-affinity complexes in
the fast loading regime (i.e. loading rates >10,000 pN/seconds). Cells that
were activated with PMA, however, did not express a form of LFA-1 that
exhibits a higher affinity for immobilized ICAM-1. As shown in
Fig. 7, the force spectrum of
the LFA-1ICAM-1 complex acquired from PMA-stimulated 3A9 cells was
superimposable on the measurements obtained from resting cells. Thus,
measurements of the unbinding force of individual LFA-1ICAM-1 complexes
revealed that the observed enhanced cell adhesion following PMA stimulation
(Figs 2 and
3) did not stem from a change
in the intrinsic properties of the LFA-1ICAM-1 interaction.
|
Elasticity of stimulated cells
Changes in elasticity of the cell may allow the cell to spread, leading to
the formation of more adhesion complexes and, hence, greater adhesion. The
mechanical properties of 3A9 cells were measured by AFM indentation
measurements of cell elasticity. Fig.
8A presents typical force versus indentation curves obtained from
resting and stimulated cells. The measurements were made with indentations of
<1 µm and an indentation force of <1 nN in order to probe the cell
without damaging it. (Forces >2 nN can damage the cell.) The fitted curves
using the Hertz model were overlaid on the measurements. The average value for
Young's modulus (N>300; 20-25 different cells) of the resting,
Mg2+/EGTA-treated and PMA-stimulated cells are 1.4±0.04 kPa,
3.0±0.09 kPa, and 0.30±0.01 kPa, respectively
(Fig. 8C). Cells exposed to the
equivalent amount of DMSO, used as a carrier in the PMA stimulation
measurements, are unaffected. Thus, our measurements of cell elasticity
revealed that the PMA-stimulated cells are softer than both resting and
Mg2+-treated cells. Fig.
8B includes histograms for resting, PMA-stimulated and
Mg2+-treated cells in order to show the data distribution for these
experiments. The histogram for the resting cells revealed a peak around 0.6
kPa and another at around 1.5 kPa. The PMA-stimulated cell data histogram had
a peak around 0.1 kPa and another around 0.35 kPa. This could indicate that
the PMA caused a shift in the two populations of cells or that only some of
the cells responded to PMA. The Mg2+/EGTA-activated cells exhibited
a broad data distribution.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
LFA-1 polarization has been suggested as a mechanism for PMA-induced
enhanced cell adhesion (Kupfer et al.,
1990). However, it is unlikely that LFA-1 polarization is
responsible for the enhanced adhesion observed in the time scale (
5
seconds) of AFM measurements following PMA-stimulation. A lateral
redistribution of receptors is expected to change the local density of
receptors, resulting in a higher receptor density in some areas and lower in
others. If this were the case, we would have great irregularities in the AFM
force measurements as areas of high and low receptor densities would
contribute to high and low adhesion. We never detected a decrease in cell
adhesion following PMA stimulation. The absence of a systematic decrease in
cell adhesion suggested that the lateral redistribution of LFA-1 remained
random and not directed toward or away from the area of cell contact. Since
the average work of de-adhesion from many measurements was significantly
higher for PMA-stimulated cells than for resting cells, we conclude that
enhanced adhesion did not stem from a simple lateral redistribution of
LFA-1.
It is conceivable that microclustering of LFA-1 can promote enhanced adhesion by distributing the applied force more evenly among the LFA-1ICAM-1 complexes within the microclusters. The formation of these clusters may lead to cooperativity among the LFA-1ICAM-1 complexes during forced unbinding. However, our force measurements provided no evidence for cooperative unbinding of multiple LFA-1ICAM-1 complexes. During the detachment of PMA-stimulated cells from immobilized ICAM-1, the majority of the rupture events (as indicated by a sharp transition in force, Fig. 6A) corresponded to the breakage of a single LFA-1ICAM-1 complex (based on analysis of the magnitude of the force transitions). These results suggest that enhanced cell adhesion following PMA stimulation stemmed from an increase in the number of adhesion complexes formed between the cells and immobilized ICAM-1 rather than from the cooperative rupture of multiple adhesion complexes. It should be pointed out that a clustering of LFA-1 may still augment adhesion by distributing the applied force among the clustered LFA-1ICAM-1 complexes even in the absence of cooperative unbinding of the LFA-1ICAM-1 complexes.
In light of these observations, we explored other mechanisms by which cells
can promote adhesion. Since there is good evidence that PMA promotes cell
spreading, we postulated that PMA changes the elasticity of the cell, which,
in turn, would allow the cell to deform and form greater contact with the
apposing surface for a given applied force. Using the AFM, we measured the
elasticity of resting, PMA-stimulated and Mg2+-treated 3A9 cells.
Our data did show that the PMA-stimulated cells were softer than both resting
and Mg2+-activated cells (Fig.
8C). The histograms for this elasticity data shown in
Fig. 8B also revealed that
there were two populations of resting and PMA-stimulated cells. We attribute
this to two possible factors. One factor being the variability in membrane
elasticity and the other changes in elasticity as a result of cell cycle
changes. Our observations are consistent with a recent study by Matzke et al.,
who found using AFM in force mapping mode that fibroblast cells exhibit varied
elasticities depending on their cell cycle stage
(Matzke et al., 2001). Cells
in interphase exhibited an elasticity of 1.0±0.4 kPa, cells in
metaphase 1.7±0.9 kPa and cells at the onset of anaphase 3.0±2.5
kPa. The histogram for the Mg2+-treated cells showed a broad data
distribution with no major peak.
These AFM measurements of 3A9 cell elasticity revealed that the Young's
modulus of the resting cell is 1.4 kPa
(Fig. 8C). An estimate of
contact area Ac for a given compression force
F, cell radius R and Young's modulus K is given by
the Hertz model, that is,
![]() |
Assuming that the work of de-adhesion is proportional to the area of
cell-substrate contact, changes in the elasticity of PMA-stimulated cells may
give rise to a 280% increase in cell adhesion. It should be noted that another
important component of the work of de-adhesion stems from the elastic
deformation of the cell. An increase in cell compliance will increase the work
of de-adhesion because softer cells are stretched more than stiffer cells
during the detachment force of the LFA-1ICAM-1 complexes. If the cell
behaves like a linear system, the total work, W, done to deform the
cell just before detachment is inversely proportional to
kc (i.e.
W=F2/2kc). Hence, the
measured fivefold increase in cell compliance following PMA activation is
expected to result in a
fivefold increase in the work of de-adhesion.
However, this may be an overestimate (>20%) because a softer system will
reduce the loading rate of the forced dissociation of the LFA-1ICAM-1
complexes and hence lower the rupture force of the complexes.
In conclusion, the current study highlights the importance of cell compliance in leukocyte adhesion. Changes in the mechanical properties of the cell may result in an increase in the contact area, leading to greater adhesion. Moreover, increases in the compliance of the cell will increase the work required to induce separation of the LFA-1ICAM-1 complexes. These changes in the mechanical properties of the cell can contribute to a >10-fold increase in the work of de-adhesion.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Benoit, M. (2002). Cell adhesion measured by force spectroscopy on living cells. Methods Cell Biol. 68, 91-114.[Medline]
Benoit, M., Gabriel, D., Gerisch, G. and Gaub, H. E. (2000). Discrete interactions in cell adhesion measured by single-molecule force spectroscopy. Nat. Cell Biol. 2, 313-317.[CrossRef][Medline]
Berry, N. and Nishizuka, Y. (1990). Protein kinase C and T cell activation. Eur. J. Biochem. 189,205 -214.[Abstract]
Chen, A. and Moy, V. T. (2000). Cross-linking
of cell surface receptors enhances cooperativity of molecular adhesion.
Biophys. J. 78,2814
-2820.
Diamond, M. S. and Springer, T. A. (1994). The dynamic regulation of integrin adhessiveness. Curr. Biol. 4,506 -517.[Medline]
Dustin, M. L. and Springer, T. A. (1991). Role of lymphocyte adhesion receptors in transient interactions and cell locomotion. Annu. Rev. Immunol. 9, 27-66.[CrossRef][Medline]
Evans, E., Leung, A., Hammer, D. and Simon, S.
(2001). Chemically distinct transition states govern rapid
dissociation of single L-selectin bonds under force. Proc. Natl.
Acad. Sci. USA 98,3784
-3789.
Ganpule, G., Knorr, R., Miller, J. M., Carron, C. P. and Dustin, M. L. (1997). Low affinity of cell surface lymphocyte function-associated antigen-1 (LFA-1) generates selectivity for cell-cell interactions. J. Immunol. 159,2685 -2692.[Abstract]
Grakoui, A., Bromley, S. K., Sumen, C., Davis, M. M., Shaw, A.
S., Allen, P. M. and Dustin, M. L. (1999). The immunological
synapse: a molecular machine controlling T cell activation.
Science 285,221
-227.
Heinz, W. F. and Hoh, J. H. (1999). Spatially resolved force spectroscopy of biological surfaces using the atomic force microscope. Trends Biotechnol. 17,143 -150.[CrossRef][Medline]
Hoh, J. H. and Schoenenberger, C. A. (1994).
Surface morphology and mechanical properties of MDCK monolayers by atomic
force microscopy. J. Cell Sci.
107,1105
-1114.
Hughes, P. E., Diaz-Gonzalez, F., Leong, L., Wu, C., McDonald,
J. A., Shattil, S. J. and Ginsberg, M. H. (1996). Breaking
the integrin hinge. A defined structural constraint regulates integrin
signaling. J. Biol. Chem.
271,6571
-6574.
Huth, J. R., Olejniczak, E. T., Mendoza, R., Liang, H., Harris,
E. A., Lupher, M. L., Jr, Wilson, A. E., Fesik, S. W. and Staunton, D. E.
(2000). NMR and mutagenesis evidence for an I domain allosteric
site that regulates lymphocyte function-associated antigen 1 ligand binding.
Proc. Natl. Acad. Sci. USA
97,5231
-5236.
Hutter, J. L. and Bechhoefer, J. (1993). Calibration of atomic-force microscope tips. Rev. Sci. Instrum. 64,1868 -1873.[CrossRef]
Hynes, R. O. (1992). Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25.[Medline]
Israelachvili, J. N. (1992). Intermolecular and surface forces. London: Academic Press.
Jones, S. L., Wang, J., Turck, C. W. and Brown, E. J.
(1998). A role for the actin-bundling protein L-plastin in the
regulation of leukocyte integrin function. Proc. Natl. Acad. Sci.
USA 95,9331
-9336.
Kolanus, W., Nagel, W., Schiller, B., Zeitlmann, L., Godar, S., Stockinger, H. and Seed, B. (1996). Alpha L beta 2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule. Cell 86,233 -242.[Medline]
Kuhlman, P., Moy, V. T., Lollo, B. A. and Brian, A. A.
(1991). The accessory function of murine intercellular adhesion
molecule-1 in T lymphocyte activation. Contributions of adhesion and
co-activation. J. Immunol.
146,1773
-1782.
Kupfer, A., Burn, P. and Singer, S. J. (1990). The PMA-induced specific association of LFA-1 and talin in intact cloned T helper cells. J. Mol. Cell Immunol. 4, 317-325.[Medline]
Lollo, B. A., Chan, K. W., Hanson, E. M., Moy, V. T. and Brian,
A. A. (1993). Direct evidence for two affinity states for
lymphocyte function-associated antigen 1 on activated T cells. J.
Biol. Chem. 268,21693
-21700.
Lu, C., Shimaoka, M., Zang, Q., Takagi, J. and Springer, T.
A. (2001). Locking in alternate conformations of the integrin
alpha Lbeta 2 I domain with disulfide bonds reveals functional relationships
among integrin domains. Proc. Natl. Acad. Sci. USA
98,2393
-2398.
Lupher, M. L., Jr, Harris, E. A., Beals, C. R., Sui, L. M.,
Liddington, R. C. and Staunton, D. E. (2001). Cellular
activation of leukocyte function-associated antigen-1 and its affinity are
regulated at the I domain allosteric site. J. Immunol.
167,1431
-1439.
Marlin, S. D. and Springer, T. A. (1987). Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1). Cell 51,813 -819.[Medline]
Matzke, R., Jacobson, K., Radmacher, M. (2001). Direct, high-resolution measurement of furrow stiffening during division of adherent cells. Nat. Cell Biol. 3, 607-610.[CrossRef][Medline]
Monks, C. R., Freiberg, B. A., Kupfer, H., Sciaky, N. and Kupfer, A. (1998). Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82-86.[CrossRef][Medline]
Radmacher, M., Fritz, M., Kacher, C. M., Cleveland, J. P. and Hansma, P. K. (1996). Measuring the viscoelastic properties of human platelets with the atomic force microscope. Biophys. J. 70,556 -567.[Abstract]
Rothlein, R. and Springer, T. A. (1986). The requirement for lymphocyte function-associated antigen 1 in homotypic leukocyte adhesion stimulated by phorbol ester. J. Exp. Med. 163,1132 -1149.[Abstract]
Sanchez-Madrid, F., Simon, P., Thompson, S. and Springer, T. A. (1983). Mapping of antigenic and functional epitopes on the alpha- and beta-subunits of two related mouse glycoproteins involved in cell interactions, LFA-1 and Mac-1. J. Exp. Med. 158,586 -602.[Abstract]
Shimaoka, M., Takagi, J. and Springer, T. A. (2002). Conformational regulation of integrin structure and function. Annu. Rev. Biophys. Biomol. Struct. 31,485 -516.[CrossRef][Medline]
Springer, T. A. (1990). Adhesion receptors of the immune system. Nature 346,425 -434.[CrossRef][Medline]
Stewart, M. and Hogg, N. (1996). Regulation of leukocyte integrin function: affinity vs. avidity. J. Cell Biochem. 61,554 -561.[CrossRef][Medline]
Stewart, M. P., Cabanas, C. and Hogg, N. (1996). T cell adhesion to intercellular adhesion molecule-1 (ICAM-1) is controlled by cell spreading and the activation of integrin LFA-1. J. Immunol. 156,1810 -1817.[Abstract]
Stewart, M. P., McDowall, A. and Hogg, N.
(1998). LFA-1-mediated adhesion is regulated by cytoskeletal
restraint and by a Ca2+-dependent protease, calpain. J.
Cell Biol. 140,699
-707.
Tees, D. F., Waugh, R. E. and Hammer, D. A.
(2001). A microcantilever device to assess the effect of force on
the lifetime of selectin-carbohydrate bonds. Biophys.
J. 80,668
-682.
van Kooyk, Y. and Figdor, C. G. (2000). Avidity regulation of integrins: the driving force in leukocyte adhesion. Curr. Opin. Cell Biol. 12,542 -547.[CrossRef][Medline]
Willemsen, O. H., Snel, M. M., Cambi, A., Greve, J., de Grooth,
B. G. and Figdor, C. G. (2000). Biomolecular interactions
measured by atomic force microscopy. Biophys. J.
79,3267
-3281.
Woska, J. R., Jr, Morelock, M. M., Jeanfavre, D. D. and Bormann,
B. J. (1996). Characterization of molecular interactions
between intercellular adhesion molecule-1 and leukocyte function-associated
antigen-1. J. Immunol.
156,4680
-4685.
Wu, H. W., Kuhn, T. and Moy, V. T. (1998). Mechanical properties of L929 cells measured by atomic force microscopy: effects of anticytoskeletal drugs and membrane crosslinking. Scanning 20,389 -397.[Medline]
Zhang, X., Wojcikiewicz, E. and Moy, V. T.
(2002). Force spectroscopy of the leukocyte function-associated
antigen-1/intercellular adhesion molecule-1 interaction. Biophys.
J. 83,2270
-2279.
Zhou, X. and Li, J. (2000). Macrophage-enriched
myristoylated alanine-rich C kinase substrate and its phosphorylation is
required for the phorbol ester-stimulated diffusion of beta 2 integrin
molecules. J. Biol. Chem.
275,20217
-20222.