From the Department of Microbiology, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
Binding of integrin receptors to extracellular
ligands is a complex process involving receptor-ligand interactions at
the cell-substrate interface, signals activating the receptors, and assembly of cytoskeletal and adhesion plaque proteins at the
cytoplasmic face. To analyze the contribution of these elements to
overall cell adhesion, we have developed a model system that
characterizes the functional binding characteristic for adhesion
receptors as the force required to separate the integrin-ligand bond. A
spinning disk device was used to apply a range of controlled
hydrodynamic forces to adherent cells. The adhesion of K562
erythroleukemia cells, a cell line expressing a single fibronectin
receptor, integrin
5
1, which was
uniformly activated with the monoclonal antibody TS2/16, to defined
fibronectin surface densities was examined. Cell adhesion strength
increased linearly with receptor and ligand densities. Based on
chemical equilibrium principles, it is shown that adhesion strength is
directly proportional to the number of receptor-ligand bonds. This
analysis provides for the definition of a new physical parameter, the
adhesion constant
, which is related to the bond strength and
binding equilibrium constant and has units of
force-length2. This parameter can be measured by the
experimental system presented and is governed by the activation state
of integrin receptors. This simplified model isolates the integrin
receptor-ligand binding parameters and provides a basis for analysis of
the functions of signaling and cytoskeletal elements in the adhesion
process.
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INTRODUCTION |
Specific receptor-mediated adhesion to surface ligands is a
fundamental property of most cell types. This adhesion is essential to
the formation of multicellular organisms and is tightly regulated during development (1). Many pathological conditions, such as blood
clotting defects and tumor invasion and metastasis, involve abnormal
adhesion processes (2). Understanding of cell adhesion is also critical
to biotechnological applications and to the development of devices for
medical implantation (3-6). Early experiments focused on cell adhesion
as a structural element required for the organization of cells in
multicellular organisms, but recent breakthroughs have shown that
adhesion is directly involved in the transmission of signals essential
for cell proliferation and differentiation (7-9).
The integrin family of heterodimeric receptors provides the dominant
adhesion mechanism for most cells which adhere to extracellular matrix
components, including fibronectin
(Fn)1 and laminin (10, 11).
Many of these receptors have been cloned and sequenced, and their
ligands have been identified (11). Biochemical and functional assays
have indicated that integrin-mediated adhesion is regulated through
several mechanisms, including matrix deposition (12, 13), protease
activity (14), expression of multiple integrins (10), modulation of
receptor-ligand binding affinity (15), and cytoskeletal reorganization
(16, 17). For example, most adherent cells grown in culture use
5
1 integrin as the dominant receptor
binding to Fn synthesized by the cells or exogenously supplied.
5
1 integrin binds to the RGD region in
the 10th type III repeat and to the synergy site in the 9th type III
repeat of Fn (18). This receptor is generally expressed in an inactive
or low binding form on cells that are in suspension or that have been
recently trypsinized (15). Receptor interaction with Fn results in an
energy-dependent binding that may involve a conformational
change in the integrin (15). This binding also causes rapid association
of the receptor-ligand complex with the actin cytoskeleton (17).
Receptor clustering is followed by the association of a complex of
cytoplasmic proteins that include structural proteins, such as vinculin
and talin, and signaling molecules like ras, FAK, and MAPK (19, 20).
Although these changes in conformation and clustering contribute to the
overall adhesion, a mechanistic understanding of the specific
contributions of these factors to receptor-mediated cell adhesion is
still incomplete.
The adhesive properties of adhesion receptors can be defined in terms
of the force required to break the bonds between the receptor and its
extracellular ligand. This is implicit in all adhesion assays. The most
common assay consists of seeding cells onto substrates, washing
"non-adherent" cells off, and counting the remaining cells.
Although these wash assays have contributed to our understanding of
cell adhesion, they are limited in their ability to provide well
defined, sensitive physical measurements. Given the complexity of the
adhesion process, analysis of the biomechanical and biochemical
interactions involved requires the development of both a new
experimental system for measurement and a new approach to the analysis
of these adhesion interactions.
Quantitative adhesion assays have been developed to apply controlled
detachment forces through centrifugation (21), hydrodynamic fluid flow
(22-30), or micromanipulation (31-33). Although these methods have
provided measurements of adhesion strength, the functional dependence
of adhesion strength on receptor-ligand parameters remains unknown.
Here we present an experimental framework for the analysis of
receptor-ligand interactions involved in cell adhesion. Using a device
that applies a well-defined range of hydrodynamic forces to adherent
cells expressing a single Fn receptor,
5
1
integrin, we obtained specific force measurements of adhesion mediated
by
5
1 integrin-Fn bonds. Furthermore, we
demonstrate that cell adhesion strength increases linearly with the
number of receptor-ligand complexes and is described by a simple
receptor-ligand model. The receptor-ligand interaction is characterized
by a novel experimental parameter, which represents the fundamental
adhesion properties of the specific receptor-ligand pair under
controlled experimental conditions and is governed by the activation
state of the integrin receptor.
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EXPERIMENTAL PROCEDURES |
Cells and Reagents--
K562 cells (ATCC number CCL-243) were
obtained from A. Gerwitz (University of Pennsylvania) and grown in
Dulbecco's modified Eagle's medium supplemented with 10% calf serum
and penicillin-streptomycin. TS2/16 and HFN7.1 hybridomas were obtained
from ATCC (Rockville, MD). AIIB2 and BIIG2 hybridomas were a gift from
C. Damsky. mAbs were affinity purified from supernatants on protein
G-Sepharose columns. Human plasma Fn and cell culture reagents were
purchased from Life Technologies Inc. Ethidium homodimer was obtained
from Molecular Probes (Eugene, OR). Glass coverslips were purchased from Bellco (Vineland, NJ). Sulfo-BSOCOES cross-linker was purchased from Pierce. All other reagents were obtained from Sigma.
Fn Adsorption--
Lyophilized plasma Fn was reconstituted with
sterile distilled H2O to 1 mg/ml. Glass coverslips were
coated with Fn diluted in Dulbecco's phosphate-buffered saline (DPBS;
in mM: 137 NaCl, 2.7 KCl, 4.3 Na2HPO4·7H2O, 1.5 KH2PO4, 0.9 CaCl2·2H2O, 1 MgCl2·6H2O) for 30 min at 22 °C and
blocked in 1% bovine serum albumin (BSA) for 30 min. Adsorbed Fn was
determined for different coating concentrations using Fn iodinated with
the Bolton-Hunter reagent (NEN Life Science Products).
Quantitative Adhesion Assay--
Cell adhesion to adsorbed Fn
was measured using a spinning disk device (30). This apparatus consists
of a disk spinning in large fluid volume
(Fig. 1) and applies a linear range of
forces to adherent cells. K562 cells were washed and resuspended in
DPBS + 2 mM glucose. Cells (400 cells/mm2) were
uniformly seeded onto Fn-coated glass coverslips (25 mm diameter)
mounted on the device and allowed to attach for 15 min in the presence
or absence of mAbs. The chamber of the device was filled with buffer,
and disks were spun for 10 min at constant speed with controlled
acceleration rates. Adherent cells were fixed in 3.7% formaldehyde,
permeabilized with 1% Triton X-100, and stained with ethidium
homodimer. Disks were analyzed by counting the number of nuclei per
microscope field (0.5 mm2) using a motorized stage and
image analysis software (Phase 3 Imaging, Version 3.0, Glen Mills, PA).
Sixty-one fields were analyzed per disk and normalized to the nuclei
count at the disk center for which the applied force is zero. Using a
non-linear computer algorithm, the fraction of adherent cells
(f) was fitted to a sigmoidal curve (f = 1.0/(1.0 + exp [b (
50)]) where
is the surface shear stress, b is the inflection slope, and
50 is the inflection point.

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Fig. 1.
Diagram of spinning disk device:
1, glass coverslip; 2, spinning disk;
3, baffled fluid chamber; 4, shaft connected to
motor.
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For integrin cross-linking experiments, cells were seeded onto
Fn-coated coverslips for 10 min, incubated in 1 mM
sulfo-BSOCOES for 5 min, and then spun and analyzed as before.
Receptor Binding to Soluble Fn--
Measurements of receptor
binding to soluble Fn were performed (15). Briefly, K562 cells were
washed and resuspended in DPBS + 2 mM glucose + 1 mg/ml
BSA. Binding experiments were conducted in 200-µl volumes, consisting
of 100 µl of cell suspension (1.3 × 106 cells), 50 µl of 125I-Fn, and 50 µl of TS2/16 mAb, buffer, or
inhibitor (EDTA + NaN3). After incubating for 30 min at
22 °C under gentle agitation, 50-µl aliquots were layered in
triplicate onto 300 µl of 20% sucrose. Bound Fn was separated from
unbound by centrifugation at 12,000 × g for 3 min and
quantified. For nonspecific binding, cells were incubated in 5 mM EDTA + 0.01% NaN3. Binding data was
analyzed for absolute number of receptors per cell and equilibrium
binding constant using a non-linear computer algorithm for monovalent receptor-ligand binding.
Overexpression of
5
1
Integrin--
K562 cells were transfected with pRSVneo
5 (34) by electroporation at 300 V/500 microfarad
(GenePulser II, Bio-Rad, Hercules, CA). Colonies were selected for G418
antibiotic resistance and analyzed by flow cytometry using the
anti-
5 rat mAb BIIG2 or anti-
1 rat mAb
AIIB2. Briefly, cells were resuspended in DPBS + 0.1% BSA + 0.01%
NaN3. Cells were incubated in primary mAb, washed,
incubated in fluorescein isothiocyanate-conjugated anti-rat IgG, and
analyzed in a FACScan (Becton Dickinson, San Jose, CA). Receptor values
for the transfected cells were converted to absolute numbers by
combining cytometry data with soluble Fn binding measurements for
untransfected cells.
 |
RESULTS |
Analytic Approach to Measure the Force Required to Detach
Cells--
The objective of this analysis was to develop an
experimental framework to reduce the number of variables associated
with adhesion measurements and provide measurements of the force
required to disrupt specific receptor-ligand interactions. We used a
spinning disk device to apply a range of hydrodynamic forces to
adherent cells (30). For this configuration, the applied force varies linearly along the surface of the disk allowing the application of a
wide range of forces in a single experiment under uniform chemical
conditions. The flow patterns in this device were validated using an
electrochemical method over the full range of speeds used in the
adhesion experiments. As shown in Equation 1, the applied shear stress
(force/area) at any point on the surface of the disk varies
linearly with radial position and is given,
|
(Eq. 1)
|
where r is radial position from the disk center,
and µ are fluid density and viscosity, respectively, and
is
angular speed.
Cells were seeded on a coverslip mounted on the device and spun at a
constant speed. Fluid flow over the cells on the disk produces a
detachment force that is proportional to the hydrodynamic shear force
in Equation 1. Cells at the center experience negligible force, and
cell numbers decrease toward the outside of the disk as the applied
force increases. Thus, for a single disk, a linear range of forces is
applied to a large cell population producing a cell detachment profile
that allows the calculation of a mean detachment force. We define this
mean detachment force as the adhesion strength.
The applied hydrodynamic force is dependent on cell shape; for
spherical cells, exact solutions have been derived (35, 36). K562 cells
were chosen because they remain spherical even when plated on Fn-coated
surfaces. From a biochemical perspective, this cell line is an ideal
model because it allows the examination of a single receptor-ligand
interaction. These cells express a single Fn receptor, integrin
5
1, and no other
chains that associate with
1 (37). The
5
1 receptor on K562 cells is expressed in
a low binding state based on its reduced ability to bind Fn in solution
and weak cell adhesion to Fn. To increase Fn binding,
5
1 integrin was activated by addition of
mAb TS2/16 (38). TS2/16 was added at saturating levels to provide for
uniform activation of all
5
1.
Cell densities for individual fields exhibited a Poisson distribution
prior to spinning, and it is expected that the ability of individual
cells to withstand a specific detachment force will be a normally
distributed property over the population. The combination of these
distributions predicts a sigmoidal adhesion curve with a mean adhesion
strength given by the shear stress for 50% detachment (
50). As expected, the fraction of adherent cells
(f) decreased non-linearly with shear stress (
), and the
data was fitted to a sigmoidal curve
(Fig. 2). The sigmoidal model accurately
described the experimental data (mean R2 = 0.90 ± 0.10). Since all scored fields were used to calculate
50, this value represents the measurement of the effect
of the force field on >12,000 cells. The values determined by this
assay have been reproducible within 10% over a 10-month period.

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Fig. 2.
Characteristic detachment profile. Shown
is fraction of adherent cells (f) as a function of surface
shear stress ( ) for 10 µg/ml Fn in the presence of TS2/16;
experiment ( ), sigmoidal fit ( ). Curve-fit parameters:
50 = 64.6 dyne/cm2,
R2 = 0.94 (10 dyne/cm2 = 1 N/m2).
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The Detachment Model Is Specific for the
5
1 Integrin-Fn Bond--
Experiments at
different rotational speeds for the same Fn concentration demonstrated
that the observed sigmoidal decrease in cell numbers was a function of
applied force. By varying the rotational speed, the detachment profile
shifted along the disk surface; however, the shear stress for 50%
detachment remained the same for all speeds (data not shown).
Experiments using different cell seeding densities revealed that, for
the cell densities examined (200-800 cells/mm2), the
detachment profile and attachment strength were independent of cell
density (data not shown).
Experiments were conducted for different seeding times to determine
whether TS2/16-activated K562 cells exhibit adhesion strengthening upon
receptor binding. There were no differences in adhesion strength between 5 and 15 min or at 15 min in the presence of NaN3
to inhibit energy-dependent processes (data not shown),
demonstrating no evident strengthening response at these initial times
and validating this cell model for analyzing the initial integrin
receptor-Fn interaction.
The mechanism of detachment was examined using the
membrane-impermeable, homobifunctional NHS-ester sulfo-BSOCOES to
cross-link integrins bound to Fn. Cross-linking bound integrins in
TS2/16-activated cells resulted in >2-fold increase in adhesion
strength compared with uncross-linked controls, shown as a right shift
in the detachment profile (Fig. 3).
Cross-linking cells without activated receptors yielded background
levels of adhesion (data not shown), indicating that the adhesive force
is specifically provided by the bound receptors. The significant
increase in adhesion strength as a result of cross-linking bound
receptors demonstrates that detachment occurs at the integrin-Fn
junction, and the assay, therefore, measures the strength of this
interaction.

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Fig. 3.
Detachment mechanism. Shown is fraction
of adherent cells (f) as a function of shear stress ( )
for 5 µg/ml Fn in the presence of TS2/16: control ( ), cross-linked
( ), CD-treated ( ), cross-linked and CD-treated ( ).
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Disruption of the actin cytoskeleton with cytochalasin D (CD, 1 µM) altered the failure mechanism by compromising
cellular integrity. CD treatment of cells cross-linked to the substrate reduced adhesion to the same levels as uncross-linked cells (Fig. 3),
suggesting that failure occurs somewhere other than at the integrin-Fn
junction. Moreover, CD-treated cells ruptured and left behind
considerable cellular debris after detachment; this was not observed in
untreated cells. These findings suggest that treatment with CD is not
appropriate for examining the role of actin cytoskeleton in cell
adhesion because it is not specific to the actin-adhesion complex
interaction and introduces a mechanical artifact.
To examine the relationship between adhesion strength and ligand
density, disks coated with different levels of Fn were analyzed. Fig. 4 shows a family of detachment
profiles for different Fn densities. As expected, increasing the Fn
coating concentration generates a family of sigmoids that shift to the
right with increasing concentration, indicating a direct relationship
between ligand density and adhesion strength. To further analyze the
specificity of the interaction, the mAbs HFN7.1 that interacts with the
cell binding domain of Fn (39) and BIIG2 which reacts with the
5 chain and inhibits its binding to Fn (40) were
examined. In the presence of TS2/16, each of these mAbs reduced K562
adhesion to levels similar to those in the absence of TS2/16 or surface Fn (Fig. 5). The low level of adhesion in
the presence of these inhibitory mAbs or in the absence of TS2/16
represents nonspecific adhesion due primarily to electrostatic
interactions.

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Fig. 4.
Dependence of adhesion strength on Fn coating
concentration (µg/ml): 0 ( ), 1 ( ), 2 ( ), 5 ( ), 10 ( ).
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Fig. 5.
Blocking experiments demonstrating
specificity of 5 1 integrin-Fn
interaction: 5 µg/ml Fn + TS2/16 ( ); 0 Fn + TS2/16 ( ); 5 µg/ml Fn ( ); 5 µg/ml Fn + HFN7.1 + TS2/16 ( ); 5 µg/ml Fn + TS2/16 + BIIG2 ( ).
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Receptor-Ligand Adhesion Model--
Our analysis is based on the
theoretical work of Bell (41) as later refined by Hammer and
Lauffenburger (36). The adhesion model considers a spherical cell with
a single class of receptors attaching to a surface through uniformly
distributed receptor-ligand complexes. The force per unit area or shear
stress for detachment,
d, is given by the
following.
|
(Eq. 2)
|
This equation consists of five elements: (i) a geometric parameter
(G) related to the total force exerted by the bonds in the
contact area to resist the hydrodynamic force applied to the cell. (ii)
The adhesion constant
is a novel experimental parameter specific to
the receptor-ligand interaction.
is related to the bond strength
and receptor-ligand affinity and has units of
force-length2. This parameter is analogous to the
equilibrium binding constant used for describing receptor-ligand
interactions in solution. (iii) NR represents
the receptor density. (iv) NL is adsorbed ligand
density. (v)
represents the nonspecific adhesion between the cell
and the surface, arising largely from electrostatic interactions. This
model predicts that adhesion strength is determined directly by the
number of receptor-ligand complexes in the contact area and that the
constant
is a measure of bond strength. For this model,
is
independent of geometry and ligand and receptor densities but is
dependent on integrin activation state. If valid, this experimental
approach could be used to provide a direct measurement of the
activation state of integrin receptors.
The ability to perform this analysis on whole cells ensures proper
presentation of the receptors on the surface and provides a system that
is amenable to genetic manipulation and potentially applicable to a
wide range of adhesion receptors. The relationships predicted by the
model shown in Equation 2 were examined individually.
Dependence on Ligand Density
NL--
125I-Fn was adsorbed to glass surfaces
at different coating concentrations, and adsorbed ligand was quantified
(Fig. 6). Fn surface density is
relatively linear up to about 10 µg/ml coating concentration, after
which it saturates. These values are in general agreement with previous
measurements (42-44). The saturation density represents the
approximate amount of Fn required for a monolayer coating based
on estimates of the dimensions of the Fn molecule (45).
Combining measurements for adsorbed Fn density with adhesion profiles
for different ligand densities, mean adhesion strength (
50) was plotted as a function of Fn surface density
(NL) (Fig. 7).
Mean adhesion strength increased linearly with Fn surface density in
the presence of activating mAb TS2/16. In the absence of TS2/16,
adhesion strength was independent of Fn surface density and was
indistinguishable from nonspecific binding measured in EDTA + NaN3. This linear relationship is consistent with the model and the difference in the two conditions results from differences in
bond strength and affinity for the integrin-Fn bond arising from
binding of activating mAb. Differences in slope demonstrate differences
in the activation state of integrin
5
1
that are reflected by differences in
since all other parameters are
held constant.

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Fig. 7.
Cell adhesion strength ( 50,
mean ± S.D.) as a function of Fn surface density (NL)
in the presence ( ) and the absence ( ) of mAb TS2/16. Linear
regression: TS2/16: 50 = 0.41 NL + 1.4, R2 = 0.95; control: 50 = 0.02 NL + 2.9, R2 = 0.38.
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Dependence on Receptor Density NR--
The absolute
number of receptors per cell was determined from binding curves of
125I-Fn to K562 cells in suspension in presence of TS2/16
(Fig. 8). This analysis revealed 1.0 ± 0.08 × 105 receptors per cell and
KD equal to 98 ± 16 nM
(R2 = 0.94). In the absence of activating mAb,
binding of soluble Fn was indistinguishable from cells treated with
EDTA + NaN3 (nonspecific binding). Thus, as for cell
detachment, solution binding of Fn to the untreated
5
1 receptor expressed on K562 cells was
below the detection limits of this method. The receptor number and
binding constant values obtained for these cells are in good agreement with previous measurements (15, 46).

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Fig. 8.
Cell binding to soluble Fn (mean ± S.D.) in the presence ( ) or the absence ( ) of TS2/16 or in the
presence of EDTA+NaN3 for nonspecific binding
( ).
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To vary receptor density, K562 cells were transfected with the pRSVneo
5 vector and screened for increased levels of surface expressed
5
1. The adhesion strength for
stable transfectants expressing
5
1
integrin at 1.9, 2.3, and 5.6 times the levels on parental cells was
measured for a fixed Fn surface density (110 ng/cm2).
Adhesion strength increased linearly with receptor density (Fig. 9), as predicted by the model. The
linear increases in adhesion strength with both ligand and receptor
densities suggest that adhesion strength is a function of the number of
bonds formed in the contact area. For a monovalent receptor-ligand
interaction, the number of bonds formed per unit area
(NB) is as follows,
|
(Eq. 3)
|
where K is an equilibrium affinity constant and
NR and NL are the
receptor and ligand densities. Fig. 10
shows that cell adhesion strength increased linearly with the product
of receptor and ligand densities as predicted by Equation 2. Therefore,
adhesion strength varies linearly with the number of receptor-ligand
complexes.

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Fig. 9.
Cell adhesion strength ( 50,
mean ± S.D.) as a function of 5 1
integrin receptor density (NR),
50 = 0.094 NL + 1.4, R2 = 0.91.
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Fig. 10.
Cell adhesion strength ( 50,
mean ± S.D.) as a function of ligand density-receptor density
product (NL * NR),
50 = 6.2 × 10 5
NL * NR + 6.4, R2 = 0.91.
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DISCUSSION |
In this research, we have integrated engineering and cell biology
principles in order to analyze cell adhesion. From the engineering perspective, we have applied the concept that, under well-defined mechanical conditions, the force required to detach a cell could be
used to evaluate the strength of the receptor-ligand bond. This
principle was implemented using a spinning disk device to apply a range
of forces to adherent cells in order to determine the mean adhesion
strength. From the cell biology perspective, it was necessary to
isolate the parameters so that adhesion due to a specific
receptor-ligand pair would dominate the analysis. The majority of the
published adhesion data does not provide ligand or receptor dependence
relationships that can be interpreted in terms of a single
receptor-ligand pair, suggesting that additional factors related to the
physical configuration of the assay (e.g. a poorly defined
mechanical environment) and/or parameters related to cell handling or
expression of multiple elements contribute to the adhesion. The K562
cell system represents a cell with a single Fn receptor,
5
1 integrin, which is expressed in an
inactive state but which can be activated by specific mAbs (15). The ability to use this passive activation system provides cell surface receptors that are uniformly activated. Adhesion strength increased linearly with both ligand and receptor densities over the full range of
adsorbed Fn densities, consistent with a theoretical model based on a
monovalent receptor-ligand interaction. Blocking experiments with mAbs
directed against both ligand and receptor demonstrated that the assay
measured the
5
1 integrin-Fn interaction. Furthermore, adhesion strength increased linearly with the product of
receptor and ligand densities, indicating that adhesion strength is
directly proportional to the number of
5
1-Fn bonds, as predicted by chemical
equilibrium principles. This result is in agreement with Palecek
et al. who observed increases in adhesion strength with the
product of receptor-ligand densities (47).
A critical assumption of the adhesion model is uniformly stressed bonds
within the contact area (36). In reality, the applied force results in
bond loading that is position-dependent with maximum
stressing at the upstream edge of the contact area and decreasing
toward the downstream edge. Several complex models incorporating
nonuniform bond loading predict a non-linear dependence of adhesion
strength on the product of receptor-ligand densities (48-50). Our
experimental findings do not support these predictions. A simple
explanation for this difference is that the portion of the contact area
in which the bonds are maximally stressed constitutes an effective
contact area for this experimental system. Once the bonds in this
effective contact area are broken, the remaining bonds are insufficient
to restrain the cell from detaching. At this point, we have no
independent means of determining the proportion of the theoretical
contact area that contributes to the effective contact area.
Validation of the proposed conceptual framework for cell adhesion
through parametric analysis of the effects of ligand and receptor
densities on adhesion strength demonstrates that the strength of the
specific receptor-ligand interaction can be described by a new
parameter, the adhesion constant
. In the Hammer-Lauffenburger model, the geometric parameter G is equal to
0.03(a/R)3, where R is the
cell radius and a is the radius of the contact area. In the
present analysis, for a contact radius of 1 µm,
for
TS2/16-activated
5
1 integrin has a value
of 2.8 × 10
18 dyne-cm2 (2.8 × 10
27 N-m2). This experimental
parameter is the fundamental descriptor for the specific
receptor-ligand interaction and is independent of receptor and ligand
densities. The adhesion constant
is analogous to the chemical
dissociation constant KD except that in the later
case the dissociation is measured under conditions of free diffusion
and in the former under mechanical loading. While it is likely that
there is some relationship between these parameters, the exact
relationship is not yet clear. One report suggests a logarithmic
relationship based on the analysis of the binding of protein A to Fc
regions of IgG from different species (50). This analysis is limited to
a single interaction type and may not, for example, apply when
comparing IgGs and integrins.
These data address a fundamental property of integrin adhesion
receptors, the relationship between receptor-ligand bonds and adhesion
strength. Following the initial encounter of a cell expressing integrin
receptors with a substrate containing an appropriate ligand, there is
an activation of the receptor which is thought to involve cell
signaling and a change in the conformation of the receptor resulting in
initial binding (15, 51). Over time, there is the accumulation of
structural proteins, including vinculin, talin and
-actinin, and
signaling molecules, including FAK, src, paxillin, to the sites of
adhesion (19, 20, 52). Actin stress fibers connect, and the complex
assembles into an adhesion plaque. This is thought to involve a
redistribution of integrin receptors to concentrate at these sites
(53). It has been proposed that this assembly contributes to the total
adhesion strength (54). Since these complexes are fully within the
cytoplasmic domain, they can do so only indirectly through the
transmembrane receptors either by increasing their affinity for ligand
or inducing some form of cooperative binding through receptor
aggregation. In the former case, changes in receptor binding would be
reflected in increases in
or the slope of the cell strength-Fn
density plot. In the latter case, cooperative binding would change the
shape of the binding plot from linear or first order to higher order. In fact, mathematical models considering receptor clustering and adhesion plaque development predict this non-linear behavior (55, 56).
In the model system used here, addition of TS2/16 provides a uniform
passive activation of
5
1 integrin
resulting in a change in the slope of the cell strength-Fn density plot
due to a switch to a new
value for the receptor-ligand interaction
itself. The absence of cooperative binding effects in this simple model
is expected since K562 cells do not assemble focal contacts and do not
spread. It is possible that cooperative binding will be observed in
more complex systems; however, the simple model is necessary to
establish a baseline from which deviations can be measured.
The experimental analysis presented provides a direct means for
measuring the adhesion constant
characteristic for the interaction of a specific receptor conformation with a ligand of specific conformation. This is important because integrin receptors can exist in
different conformations (10) that represent different activation states
and different ligand binding properties (57). This approach provides a
direct method of accessing these interactions in intact cells under
conditions of adhesion. In other experiments (42, 44, 58-60), it has
been shown that the conformation of adsorbed Fn is dependent on the
physicochemical characteristics of the surface. These differences in Fn
conformation lead to differences in the strength of the Fn-integrin
bond (58). The analytic approach presented here provides the basis for
analysis of biochemical factors and signaling events that contribute to
the receptor-mediated adhesion of cells.
We thank C. Damsky for generously providing
integrin antibodies, A. F. Horwitz for
5 vectors,
P. Ducheyne for the use of the spinning disk device, and J. Huang for
technical assistance.