1 Department of Chemical Engineering, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139, USA
2 Department of Material Science and Engineering, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, USA
3 Division of Bioengineering and Environmental Health, Massachusetts Institute
of Technology, Cambridge, Massachusetts 02139, USA
4 Biotechnology Process Engineering Center, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, USA
* Author for correspondence (e-mail: griff{at}mit.edu )
Accepted 2 January 2002
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Summary |
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Key words: Cell-substratum adhesion, RGD clustering, Force, Engineered ECM
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Introduction |
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Integrin-mediated cellular behaviors are regulated by a variety of
biochemical and biophysical means. At a basic biochemical level, integrins
differ in their affinities for individual matrix components, allowing cells to
regulate the number of bonds formed with the matrix by the number and type of
integrins expressed. Integrin cytoplasmic domains show a diversity of
functional interactions with intracellular structural and signaling molecules,
and thus the type and number of bonds formed send information about the nature
of the extracellular environment. Integrins are also subject to inside-out
affinity modulation via intracellular signaling pathways set off by growth
factor binding, mechanical stress or other stimuli
(Danen et al., 1998;
Hughes and Pfaff, 1998
;
Humphries, 1996
;
Mould, 1996
).
Biophysical regulation of integrin function appears to occur via diverse
mechanisms, including the physical arrangement of matrix ligands and matrix
rigidity. In forming attachments to the matrix, integrins aggregate in
micron-scale complexes. The cytoplasmic domains of aggregated integrins
nucleate a complex of structural and signaling molecules that link the
integrins to the cytoskeleton, forming a continuous bridge between the
cytoskeleton and the matrix (Critchley,
2000; Giancotti and Ruoslahti,
1999
; Schoenwaelder and
Burridge, 1999
). Several studies have shown that clustering or
aggregation of ligated integrins in this manner is essential to elicit the
full range of integrin-mediated biochemical signaling and subsequent cell
behaviors (Miyamoto et al.,
1995a
; Miyamoto et al.,
1995b
). Integrins restrained from forming clusters via
chain tail mutations show markedly impaired cell adhesion
(Yauch et al., 1997
).
Conversely, cell adhesion is positively regulated by
IIbß3 receptor clustering with a concurrent
increase in tyrosine phosphorylation of pp72 (Syk) and pp 125 (FAK)
(Hato et al., 1998
).
The multimeric structure of ECM molecules such as fibronectin, a dimer with
dual adhesion sites, and tenascin-C, a hexabrachion that presents six
identical cell adhesion domains within 100 nm, suggests that receptor
clustering may be influenced by the physical layout of the extracellular
matrix components. This inference is supported by in vitro studies using
synthetic Arg-Gly-Asp (RGD) adhesion peptides
(Danilov and Juliano, 1989
;
Maheshwari et al., 2000
).
Peptides grafted to albumin at 1-20 peptides per albumin molecule
(Danilov and Juliano, 1989
) or
to synthetic star-configured polyethylene oxide tethers at one to nine
peptides per polymer molecule (Maheshwari
et al., 2000
) are more effective in promoting cell adhesion when
the valency is high. Peptides presented singly (1 peptide per molecule) are
poor substrates for adhesion, whereas peptides presented at cluster sizes of
nine peptides per molecule or higher induce comparable adhesion to matrix
proteins (Danilov and Juliano,
1989
; Maheshwari et al.,
2000
). Further, when RGD adhesion ligands are presented in a
non-clustered fashion, fibroblast migration is significantly impaired, even at
identical average ligand densities
(Maheshwari et al., 2000
).
In addition to sensing ECM spatial organization, cells exert forces on the
matrix and respond to the mechanical properties of their surroundings by
regulating adhesive interactions. Compared with compliant matrices of
identical composition, rigid matrices have been shown to enhance cell-surface
assembly of fibronectin (Halliday and
Tomasek, 1995), provide a preferential substrate for directional
cell migration (Lo et al.,
2000
), regulate the rates of apoptosis and DNA synthesis
(Wang et al., 2000
) and are
associated with increased levels of protein phosphorylation at sites of
cell-matrix contact (Katz et al.,
2000
; Pelham and Wang,
1997
). Compliant matrices, on the other hand, promote cell
motility (Pelham and Wang,
1997
). Cell speeds are also greater on substrates where
fibronectin is adsorbed, and thus compliant, than on substrates where
fibronectin is covalently immobilized and thus inflexible
(Katz et al., 2000
). Since
cell movement requires formation and dissolution of focal adhesions
(Greenwood and Murphy-Ullrich,
1998
), substrate properties that foster focal adhesion turnover
may also favor cell migration.
The integrated molecular mechanisms underlying these observed
integrin-mediated responses to ECM properties are being illuminated using
approaches that probe mechanical as well as chemical stimuli. A direct role
for a coupling of mechanical and signaling factors in the regulation of focal
adhesion dynamics has been shown using a permeabilized cell system to control
the local molecular environment at individual focal adhesions
(Crowley and Horwitz, 1995).
Control of the phosphorylation levels of cytoskeleton-associated proteins
using ATP and/or phosphatases and control of cell contraction using a peptide
inhibitor of the actin-myosin interaction suggest that both tyrosine
phosphorylations and tension mediate the release of adhesions
(Crowley and Horwitz, 1995
).
In addition to tension-regulated adhesion turnover, cells respond to external
mechanical stimuli by modulating the strength of adhesion sites.
Adhesion-ligand-coated magnetic beads, twisted in a magnetic field after cell
binding to apply defined stresses to integrin-cytoskeletal linkages, induce
increased cytoskeletal stiffness proportional to increases in applied strain
(Wang and Ingber, 1995
).
Localized stress on integrin-ligand bonds, applied by an optical trap pulling
on fibronectin-coated beads bound to lamellipodia, leads to reinforcement in
adhesion to ECM (Choquet et al.,
1997
). Subsequent optical trap studies have revealed that
reinforcement mediated specifically through the vitronectin receptor is
regulated by the tyrosine kinase Src
(Felsenfeld et al., 1999
).
Mechanotransduction via integrin-cytoskeleton linkages has also been cited in
the process of microtubule assembly in smooth muscle cells, thereby directly
affecting cellular structure and phenotype
(Putnam et al., 2001
).
We have previously shown that cell adhesion and migration on substrates
presenting a minimal RGD sequence, YGRGD, require ligand clustering on the
50 nm scale (Maheshwari et al.,
2000
). In these previous studies, we used a branched from of
polyethylene oxide (PEO) to present the ligand in local clusters of one, five
or nine peptides per PEO molecule against a background otherwise inert towards
cell and protein adhesion. Here, we demonstrate that cell adhesion to the
GRGDSPK peptide is reinforced upon application of a distraction force to cells
when the ligand is presented in a clustered arrangement. In this work, we use
a comb-shaped copolymer comprising a poly(methyl methacrylate) (PMMA) backbone
with short PEO side chains (six to nine ethylene oxide units) to present the
ligand (Irvine et al., 2001
).
The hydrophobic PMMA portion establishes a stable surface film in an aqueous
environment, whereas the hydrophilic PEO side chains are extended at the
water-polymer interface to present adhesion peptides as well as to prevent
non-specific protein adsorption (Fig.
1A). The average ligand density and ligand cluster size can be
independently tuned by controlling the number of ligands per comb polymer
molecule and mixing modified and unmodified combs in defined proportions.
|
We used this system to examine cell adhesion responses to three RGD peptide
cluster sizes (n=5.4, 3.6 and 1.7 RGD/comb) with overall RGD surface
densities ranging from 5270 to 190 RGD/µm2. In this RGD density
regime, we found that ligand clustering increased cell adhesion strength for a
given average ligand density, consistent with previous studies at higher
ligand densities (Maheshwari et al.,
2000). Furthermore, we found that cells reinforced their integrin
linkages to withstand stronger detachment force in a manner that depended on
ligand clustering. Our findings implicate nanoscale ligand distribution as an
important additional mechanism in controlling the mechanically induced cell
adhesion response.
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Materials and Methods |
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Preparation of clustered RGD adhesion substrates
Polymers were synthesized and modified with RGD-containing adhesion
peptides as previously described (Irvine
et al., 2001). Briefly, comb terpolymer was synthesized with free
radical polymerization using methyl methacrylate, polyethylene glycol
methacrylate (HPOEM, Mn
360 g/mol), polyethylene glycol methyl
ether methacrylate (POEM, Mn
475 g/mol);
Azo(bis)isobutyronitrile (AIBN) was used as an initiator. The molecular weight
and polydispersity of the resulting polymer were determined by gel permeation
chromatography to be Mn=93,900 g/mol and PDI=2.0, respectively, on
the basis of the polystyrene standards
(Irvine et al., 2001
). The
weight ratios of the three monomers in the resulting polymer were determined
by NMR to be 66:16:18 (MMA:HPOEM:POEM). Comb polymer was carboxylated via
hydroxyl groups of HPOEM using succinic anhydride and was activated using
dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS). NHS-activated
polymer was stored at -20°C until used. GRGDSPK peptide (American Peptide
Company) was coupled to the NHS-activated HPOEM side chains of the polymer in
solution via the N-terminus. An average number of peptides per comb molecule
(n) was determined on the basis of the elemental analysis performed
by Quantitative Technologies, Inc. Variations in peptide cluster size were
achieved by varying the peptide to polymer concentration ratio during the
solution coupling reaction.
To vary overall RGD density independently of cluster size, ligand-modified
combs were mixed with unmodified combs in defined proportions to achieve a
total combined final comb polymer concentration of 2 mg/ml in 50:50
water:ethanol. Polymer films were prepared in 96-well plates by solvent
casting from water/ethanol and drying at room temperature for 5 hours followed
by drying in vacuo for 24 hours. Films cast at this concentration had a
thickness 150 nm. The diameter of an expanded quasi-2D comb `island' at
the interface (Fig. 1B) was
approximated to be
32 nm (Irvine et
al., 2001
), which can accommodate roughly 10 closely packed
integrin receptors. For all cluster sizes examined in our study, the estimated
center-to-center distance between two RGD peptides within an RGD-bearing comb
disk ranged from 14 to 25 nm (Table
1); therefore most tethered RGD peptides should be available for
binding to integrin receptors (
10 nm head diameter) without steric
hindrance. The average densities of RGD peptides on the substrates used in
adhesion studies were determined using quantitative reaction with a
fluorescent reporter and are summarized in
Table 1.
|
Fibronectin surface preparation
Fibronectin diluted in phosphate buffered saline (PBS) (0.1, 0.3 µg/ml)
was allowed to adsorb to Nunc tissue-culture-treated polystyrene 96-well
plates for 18 hours at 4°C. Wells were washed twice with cold PBS and
blocked for 1 hour at 37°C with 1 mg/ml heat-inactivated bovine serum
albumin (BSA) in PBS. Wells were washed twice with warm PBS prior to cell
plating.
Adhesion assay
Cell adhesion strength was determined using a centrifugation assay
(Chu et al., 1994). Cells were
incubated in serum-free medium containing 1 mg/ml BSA instead of FBS for 12
hours post-seeding (5,000 cells per well on RGD-comb surfaces and 2,000 cells
per well on fibronectin-coated surfaces). Serum-free medium was then changed
to HEPES assay medium containing 1% dialyzed serum (dserum) and cells were
incubated for 8 hours. This incubation protocol with its particular time
points was designed to correspond to other assays we plan to perform in the
future to study cell migration and growth factor signaling on the RGD-comb
substrate (Maheshwari et al.,
2000
). At the end of the incubation, the wells were filled with 1%
d-serum medium and covered with sealing tape to avoid medium loss and air
bubbles during spinning. Inverted plates were spun in a Sorvall centrifuge for
10 minutes at 25°C. For each adhesion experiment performed on RGD-comb
films, control wells containing surfaces with 5.4 RGD/comb and 1050
RGD/µm2 were subjected to gravity (1 g) by
simple inversion to account for inter-experiment variations. After
centrifugation, the number of cells left adherent in the wells was quantified
using the CyQuant nucleic assay according to the manufacturer's instructions.
A cell adhesion index (CAI) was used to measure surface adhesiveness. To
obtain a CAI, the post-assay number of adherent cells left in sample wells was
divided by the number of adherent cells left in the control wells. Each
surface condition was examined at least in triplicate in every experiment, and
each error bar indicates the standard deviation of the mean. The assay was
repeated for selected points (labeled with an asterisk `*' in data
plots). Post-centrifugation, the RGD-substrates usually retained 25% or less
of the original cell seeding number. The normal detachment force was
calculated using the equation f=RCF x V x (
c -
m), where f is the force exerted on a cell, RCF is the
relative centrifugal force, V is the cell volume (
500 µm3),
c is the density of the cell (
1.07 g/ml) and
m is the density of the medium (
1.00 g/ml).
For adhesion studies performed on fibronectin-coated surfaces, each experiment was repeated independently three times, and each surface condition was examined in triplicate in every experiment. Control surfaces coated with 1 µg/ml of fibronectin in PBS were subjected to gravity (1 g) by simple inversion in each experiment to account for inter-experiment variations. The number of cells left adherent post-assay was determined as described previously. The number of cells in sample wells was normalized by the number of cells in control wells. The resultant percentage of cells adherent was used to measure surface adhesiveness.
To ascertain the statistical significance of adhesion reinforcement and strengthening owing to mechanical stimuli and ligand clustering, a student t-test was performed with a 95% confidence level on data points adjacent to either side of the reinforcement peak for a given substrate and on adhesion data derived from different substrates at a given detachment force.
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Results |
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Here, we quantified cell attachment to these highly specific RGD-modified substrates using a centrifugal detachment assay over a wide range of forces. Cell adhesion was quantified by enumerating the cells left adherent after a given detaching force had been applied for 10 minutes, and the results were plotted as a cell adhesion index (as described in Materials and Methods) versus normal detachment force to generate an adhesion profile for each set of conditions. The expected profile was a monotonically decreasing curve of adhesion index with increasing force. Surprisingly, a definitive peak in the WT NR6 adhesion strength profile was found on most of the RGD-comb substrates investigated (Fig. 2A,B). Peaks occurred within a detachment force range of 70 to 150 pN/cell, depending on the average cluster size and overall RGD density.
|
The presence of a peak in adhesion strength implies a reinforcement of cell connections to the substrate, namely the RGD-integrin and/or integrin-cytoskeletal linkages, in response to externally applied mechanical forces. Reinforcement was most prominent on surfaces with the highest peptide density and the highest cluster size (Fig. 2A; n=5.4 RGD/comb, 5270 RGD/µm2). However, mechanically induced adhesion reinforcement occurred at all densities varying from 260 to 5270 RGD/µm2 when five RGD peptides were clustered in 32 nm islands. Following reinforcement, cell adhesion decreased monotonically and eventually leveled off with increasing detachment force. A similar reinforcement phenomenon was found on 3.6 RGD/comb surfaces, with densities ranging from 190 to 2100 RGD/µm2. The position of the reinforcement peak remained relatively insensitive to RGD density and cluster size. However, the amplitude of the peak increased with increasing ligand density. For each clustered surface (n=5.4, 3.6 RGD/comb), the peak presence was verified with a student t-test at a 95% confidence level as described in the Materials and Methods.
Adhesion reinforcement was not detectable on surfaces with the lowest RGD cluster size (n=1.7), even when overall RGD densities were comparable to those of the clustered substrates (Fig. 2C). Thus adhesion reinforcement appears to be a phenomenon promoted by ligand clustering.
Cell adhesion depends on ligand density and ligand clustering
For higher cluster sizes, cell adhesion to RGD-comb surfaces increased with
greater values of overall RGD density (Fig.
2A,B). The baseline at which the cell adhesion profile leveled off
at high detachment forces also exhibited a density dependence. Usually, a
baseline in the cell adhesion profile is expected to occur when all cells are
removed from the substrate. However, cell detachment on comb surfaces reached
a plateau at a non-zero value. Furthermore, this baseline value increased with
increasing peptide density. This observation further suggested that cells were
actively responding to the applied forces in addition to the simple process of
physical detachment in a substrate-dependent manner.
To discriminate the effect of ligand clustering from that of ligand density, we examined adhesion strength measured on RGD substrates with comparable average ligand density but different average cluster size (Fig. 3). To most effectively demonstrate clustering-promoted cell adhesion, we compared surfaces with highest (n=5.4) and lowest (n=1.7) cluster size. At all three densities examined (260-1660 RGD/µm2), nanoscale ligand clustering enhanced cell attachment to the substrate when the ligand density remained the same or was slightly lower.
|
Cell adhesion reinforcement is not observed on fibronectin
substrates
Clustering-dependent cell response to a mechanical stimulus suggested a
modulatory function of ligand distribution in the integrin-ligand interaction
and the subsequent signaling processes. To test this phenomenon in a
physiological context, we measured cell adhesion on substrates adsorbed with
fibronectin, a natural ECM adhesion ligand, using a protocol similar to that
described for measurement of adhesion on RGD-polymer substrates. Cell adhesion
was quantified after a 12 hour serum starvation period followed by an 8 hour
incubation in the presence of 1% d-serum. The adhesion profiles for the
various surface conditions were nearly indistinguishable from one another, and
the number of cells attached remained essentially unchanged within the range
of detachment forces applied (Fig.
4). Cell adhesion dependence on fibronectin density was greatly
diminished. Furthermore, while 70% to 80% of the cells remained adherent on
fibronectin after centrifugation, heat-inactivated BSA, a protein that does
not support cell adhesion, retained 60% of the cells. Thus under the given
conditions, we were not able to determine whether fibronectin could promote
reinforcement in the presence of serum. Unlike the synthetic RGD polymer
substrates, proteins could adsorb to the substrates used in these experiments.
Therefore we conclude that adhesion proteins present in the 1% d-serum have
contributed to the increased adhesion on these substrates.
|
Since this adhesion assay entailed two incubation periods, a 12-hour serum-starvation phase to quiesce cells and an additional 8 hour incubation in 1% d-serum, we examined adhesion to fibronectin at the end of the first phase, when protein adsorption from serum would not be present to influence the results. In the absence of serum, the adhesion strength profile for WT NR6 cells exhibited a monotonic decrease without reaching a plateau as the detachment force was increased (Fig. 5). As expected, cell adhesion also depended on surface fibronectin concentration. When the applied force was under 300 pN/cell, substrates adsorbed with 0.3 µg/ml fibronectin solution retained four to five times more cells than those coated with 0.1 µg/ml solution. Thus, in the absence of serum, a physiological ECM protein such as fibronectin failed to induce adhesion-strength reinforcement. Additionally, this result confirmed that the peak observed in adhesion to RGD was not an artefact of the centrifugation assay or due to changes in the viscoelastic properties of the cells.
|
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Discussion |
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The phenomenon we observe here appears to be similar to the adhesion
reinforcement observed in response to forces exerted on integrins by pulling
on ligand-coated beads using optical tweezers after bead attachment to
lamellipodia (Choquet et al.,
1997; Felsenfeld et al.,
1999
). In these studies, Sheetz and colleagues simulated
extracellular attachment sites with different rigidities by using an optical
trap to restrain the movement of beads that presented covalently linked
fibronectin type III repeat 7-10 fragments and had formed linkages to the
retrograde-moving cytoskeleton through integrins
(Choquet et al., 1997
). In
response to increased restraining force, integrin-cytoskeleton linkages
stiffened and strengthened proportionally. This adhesion reinforcement is an
acute, sustained and localized phenomenon that requires both receptor
occupancy and aggregation. Only a 10-second induction time is required after a
bead initially contacts the cell surface in order to elicit such a
strengthening response to restraining force. The reinforcement phenomenon is
inhibited by phenylarsine oxide (PAO), an inhibitor of tyrosine phosphatases,
and is inhibited by the tyrosine kinase Src when adhesion is mediated by the
vitronectin receptor (Felsenfeld et al.,
1999
).
From the perspective of this work, an interesting facet of the results
presented by Sheetz and colleagues arises from the method used to
systematically vary ligand density on the beads in optical trap force
experiments (Choquet et al.,
1997). For one set of the ligand-modified beads, fibronectin
fragments were linked onto albumin molecules to achieve several peptides per
albumin molecule. Then, multivalent peptide-modified albumin was mixed with
unmodified albumin in defined proportions and coupled onto the bead surface to
vary overall average ligand density. This is similar in principle to the
methodology employed in making the clustered RGD-polymer surface for this
study, where peptide-modified synthetic polymers were mixed with unmodified
polymers to create clusters on a flat adhesion substrate. It also bears
resemblance to the use of RGD-peptide modified albumin in the study by Danilov
and Juliano, where the number of peptides per albumin was systematically
varied over a wide range and coated onto substrates at defined average
densities (Danilov and Juliano,
1989
). Thus, ligand clustering could conceivably be a key
parameter in the reinforcement phenomenon observed by Sheetz and colleagues,
although they do not explicitly discuss this possibility. Results with beads
prepared in this fashion were pooled with results obtained using a more
homogeneous method of ligand attachment, and thus it is difficult to tease out
clustering effects from the data presented
(Choquet et al., 1997
;
Felsenfeld et al., 1999
).
To our knowledge, the phenomenon of adhesion strength reinforcement under detachment stress has not been reported previously in published shear flow or radial flow detachment assays using surfaces adsorbed with natural ECM adhesion proteins. The phenomenon we observe depends on both ligand density and nanoscale ligand clustering. Reinforcement occurs at very low centrifugal forces; hence, this phenomenon could easily be missed in such an assay unless a comprehensive range of forces is used.
Despite the differences in methodology used in the optical trap experiments
conducted by Sheetz and colleagues and the detachment studies reported here,
we can compare the forces associated with adhesion reinforcement by estimating
the approximate force per bond in each case. In the bead assay, restraining
forces ranging from 5 pN to 60 pN were applied to 1 µm diameter beads
coated with peptides at densities of up to 5000 ligands/bead. Assuming uniform
ligand distribution, hemispherical contact with the cell membrane and full
engagement of available ligands with integrins, a simple calculation of force
per integrin-ligand bond for the `high density' ligand regime (1000-5000
ligands/bead) where adhesion reinforcement was reliably observed in the
optical trap assays, leads to an estimate of 0.002 pN to 0.12 pN per bond. In
a centrifugation assay, detachment force encountered by a single cell is
calculated using the equation f=RCF x V x
(c-
m), where f is the force exerted on a cell,
RCF is the relative centrifugal force, V is the cell volume,
c
is the density of the cell, and
m is the density of the
medium. Typical integrin expression is of the order of 100,000 receptors per
cell (Akiyama and Yamada,
1985a
). If we presume that
50,000 integrins/cell (
50
bonds/µm2 for a spreading cell) are engaged, forces that induce
adhesion reinforcement during centrifugation range from 0.0028 pN to 0.0042 pN
per bond. Here we assume that most available integrins are engaged, hence the
force range calculated is likely to be a substantial underestimate.
Nevertheless, these forces are within the estimated range of those observed in
the optical trap assays.
Several factors may also contribute to the more modest force range observed
in our study. (1) The centrifugation assay measures the average response from
a cell population, whereas the optical trap method determines a single-cell
response. For example, focal adhesion development and dynamics are drastically
different for mobile and stationary cells
(Balaban et al., 2001;
Smilenov et al., 1999
), and
such a distinction is not feasible to identify when averaged over a population
of cells. (2) Trapped beads only measure a localized cell response from the
upper surface of the lamella, but the strength of
fibronectin-integrin-cytoskeleton linkages are regionally specific, especially
in motile cells where preferential binding at the leading edge and release at
the tail are usually observed (Galbraith
and Sheetz, 1997
; Nishizaka et
al., 2000
). (3) The optical trap exerts a combination of normal
and shear forces as opposed to the pure normal force applied in the
centrifugation method. (4) Short linear RGD peptides such as the one used in
this work are known to have lower affinity for integrin receptors than their
parental proteins or larger fragments of the parental proteins such as the
fibronectin 7-10 fragment used in the optical trap experiment. Further, the
isolated adhesion domain contained in an RGD peptide is likely to elicit
incomplete signaling responses compared to the fibronectin 7-10 fragment,
which contains both RGD and adhesion synergy motifs
(Leahy et al., 1996
). Finally,
we note that the time scale of adhesion strengthening in the optical trap
studies is
10 seconds. This is consistent with the time scale observed in
our centrifugation study, where reinforcement must develop before equilibrium
conditions for detachment are achieved (possibly on the order of seconds to
minutes) and must be stable over the course of force application (10
minutes).
Reinforcement is most prominent on the substrate with the largest cluster
size and the highest surface peptide density, and it diminishes when either
parameter is reduced. One mechanism for mechanotransduction in adhesion
modulation is the ability of the cell to dynamically form, mature, sustain and
disassemble adhesion structures in response to chemical and mechanical
signals. Visualization of GFP-tagged vinculin or paxillin dynamics has
provided direct evidence of focal adhesion growth in response to local
centripetal force (Riveline et al.,
2001). To further understand whether focal adhesion size
correlates to force transmission, Geiger and colleagues have measured forces
exerted by stationery cells on a deformable substrate
(Balaban et al., 2001
). A
striking finding is that local forces are related linearly to focal adhesion
area, and a constant force of the order of 1 pN is estimated for each integrin
bond. Also consistent with the time scales observed in adhesion reinforcement
studies, local adhesion assembly was extremely rapid, on a time scale below
seconds (Balaban et al.,
2001
).
In light of the striking effect that clustered RGD peptides had on cell
adhesion in response to mechanical stimulus, we wanted to investigate possible
adhesion reinforcement induced by multidomain adhesion proteins such as
fibronectin. Fibronectins are dimers of two similar polypeptide chains, each
containing an RGD recognition moiety with a nearby synergy site. Thus a
cluster of adhesion motifs is effectively present in each fibronectin molecule
over a molecular dimension of hundreds of nanometers. In addition to the
molecule's multimeric structure, Monte Carlo simulation has predicted that
random adsorption at low fibronectin densities may provide stochastic
clustering of fibronectin molecules when they are randomly deposited in close
proximity (Irvine et al.,
2002a). Using fibronectin coating concentrations of 0.1 and 0.3
µg/ml under the coating conditions described in this paper, approximately
50 and 100 fibronectin molecules/µm2 were adsorbed onto the
substrate (Asthagiri et al.,
1999
). These densities are lower than the average RGD densities
used (500-5000 molecules/µm2) in order to compensate for the
much higher integrin-binding affinity of fibronectin compared with its
peptidyl counterpart. According to competitive inhibition studies, there is a
10- to 100-fold difference in affinity between fibronectin and linear RGD
peptides such as GRGDSP (Akiyama and
Yamada, 1985b
; Hautanen et
al., 1989
; Pierschbacher and
Ruoslahti, 1984
), suggesting that significantly fewer fibronectin
molecules are required on the surface in order to form the same number of
bonds with integrin receptors. In light of these findings, we selected a
fibronectin density range that would generate comparable surface adhesive
properties to the RGD-comb substrates by using commensurably lower fibronectin
densities. Furthermore, the fibronectin densities chosen provided a wide range
of cell adhesion responses to the range of forces we applied;
50% of the
cells seeded adhered to the 0.3 µg/ml substrates at low distraction forces,
whereas only
15% adhered at 0.1 µg/ml. Higher fibronectin densities
would not provide as large a dynamic range in the response and were not
investigated. Although in our results we observed an absence of adhesion
reinforcement on fibronectin after serum-free treatment, we were not able to
determine the effect of serum, as serum-derived adhesion proteins apparently
confounded substrate chemistry through non-specific adsorption. Consistent
with our finding, detachment of erythroleukemia cells from
fibronectin-adsorbed surfaces by shear stress under serum-free conditions
exhibits sigmoidal detachment profiles without reinforcement
(Garcia et al., 1998a
;
Garcia et al., 1998b
).
Serum-derived non-specific adhesion also greatly increases substrate
adhesiveness such that no significant changes are observed in cell detachment
within the force range employed in this study. Thus, it remains inconclusive
whether or not fibronectin promotes adhesion reinforcement, although in the
absence of serum such a phenomenon was not established. It is also possible
that spatial arrangement of the ligand clustering is indeed required, as in
the case of RGD-comb substrates.
Using a star polymer system, we have previously demonstrated that adhesion
enhancement induced by ligand clustering was concurrently accompanied by a
higher degree of focal adhesion and stress fiber formation
(Maheshwari et al., 2000).
Numerous studies have shown that such cytoskeletal changes are mediated by Rho
family GTPases (Hall and Nobes,
2000
; Tapon and Hall,
1997
). In particular, integrin-promoted Rho activity drives actin
stress fiber assembly and focal adhesion maturation
(Clark et al., 1998
;
Hotchin and Hall, 1995
). Rho
may also play a key role in adhesion phenomena induced by ligand clustering.
Reinforcement observed in the optical trap studies also exhibits dependence on
ligand concentration and can be inhibited by low doses of PAO, suggesting the
involvement of a ligand-mediated enzymatic signaling cascade
(Choquet et al., 1997
).
Biochemical studies to gain mechanistic understanding of clustering-mediated
cell response in our system are in progress. In our study, the monotonically
decreasing adhesion profile of long-term cell adhesion to fibronectin under
serum-free conditions supports the notion that reinforcement on RGD-comb is
not an artefact of the system or the assay, but rather is a biochemically
based phenomenon.
We propose a mechanism by which a mechanical stimulus might trigger intracellular signaling leading to adhesion reinforcement (Fig. 6). The adhesion profile for an attached object undergoing physical detachment by an external force is expected to decrease monotonically as a function of detachment force until all the objects are removed (blue solid line). However, for living cells there may exist signaling cascades that lead to the strengthening of cell attachment to a substratum, for example, through strengthening of ligand-receptor and/or receptor-cytoskeleton linkages. This signaling is induced by mechanical stimuli such as an external force or substrate rigidity. In our model, signaling reinforces cell adhesion in response to force in a sigmoidal fashion (pink solid line), similar to an `on/off' switch. When this signaling effect is compounded to the purely physical process of detachment (blue solid line), an adhesion profile characterized by an initial drop followed by a distinct reinforcement peak is developed (red solid line, region A). Further, if signaling for reinforcement itself can be reinforced at a higher mechanical threshold, then we will observe a non-zero plateau in the resultant adhesion profile (red solid line, region B). We are planning future studies to test these ideas.
|
Using a multi-arm YGRGD-modified star PEO polymer with ligand tethers of
60 nm (fully extended length), we have previously demonstrated enhanced
cell adhesion to nanoscale clustered adhesion ligands compared with singly
presented ligands, but adhesion reinforcement was not studied in this system
(Maheshwari et al., 2000
).
Although both star and comb systems focus on the regime of small clusters
(<10 ligand/polymer molecule) that are approximately 30-50 nm in diameter,
there are marked distinctions in their structures, properties and display of
ligand that may influence cell responses. A quasi-2D comb polymer at the
interface with short
2 nm ligand tethers provides a more rigid substrate
than a 3D star polymer with flexible long tethers linked to a 300 nm thick
hydrogel base, and substrate flexibility modulates cell behaviors such as
focal adhesion formation and motility (Lo
et al., 2000
; Pelham and Wang,
1997
). In addition to substrate mechanical properties, tether
length also affects ligand mobility and spacing. For systems with long PEO
tethers such as the star polymer, the ligand is in constant, rapid motion over
a volume defined by the
60 nm extended length of the highly flexible
tether, leading to a high probability that multiple ligands on the same star
can `adjust' their positioning in response to integrin clustering. In
comparison, the short (
2 nm) PEO tether of the combs constrains ligand
mobility; therefore integrin spacing in integrin-ligand clusters formed on the
comb systems closely matches that of the side-chain spacing in the comb
itself. We speculate that there may be specific minimum peptide densities
within a cluster to achieve appropriately close ligand spacing, which in turn
allows proper integrin clustering. The average spacing between adjacent
ligands within a cluster ranged from approximately 14 nm (n=5.4) to
17 nm (n=3.6) to 25 nm (n=1.7)
(Table 1). For comparison, the
head dimension of an integrin receptor is of the order of 10 nm
(Erb et al., 1997
). We are
currently using the comb system to vary ligand spacing and the number of
peptides per cluster independently.
The comb system used here and the star system used in the previous study also differed in their biochemical constitutions. In this study, we used a higher affinity peptide (GRGDSPK) and examined a different ligand-density regime (200 to 5,000 RGD/µm2) than in our previous study (YGRGD; 1,000-200,000 RGD/µm2). Thus, ligand-clustering-promoted cell adhesion was consistently observed in two markedly different substrate systems. Our present study additionally demonstrated adhesion strength reinforcement under applied force - another unexpected cell adhesion response that is dependent on nanoscale ligand organization.
It is conceivable that ligand clustering enhances local ligand avidity,
which in turn promotes integrin receptor aggregation to form focal adhesions.
Focal adhesions are specialized cell attachment sites where ligated integrin
receptors aggregate to link the ECM to a dynamic cytoskeletal architecture,
transmitting both force and signals
(Critchley, 2000;
Sastry and Burridge, 2000
;
Yamada and Geiger, 1997
). To
account for cell-substrate adhesion enhancement owing to focal adhesions where
receptor clustering is promoted, Ward and Hammer developed a biophysical model
incorporating the effects of ligand density, receptor density and
intracellular talin polymerization at focal adhesion sites
(Ward and Hammer, 1993
). It
predicts that receptor aggregation within focal adhesions would be greatest at
high ligand densities and becomes negligible below a certain minimum density.
In agreement with their model, Massia and Hubbell have shown that human skin
fibroblasts failed to form focal adhesions and stress fibers when the density
of RGD peptide on the substrate fell below 60 peptide/µm2, which
corresponds to an RGD spacing of 140 nm
(Massia and Hubbell, 1991
).
Although in our study average ligand densities ranged from 200 to 5,000
peptides/µm2, a more meaningful parameter characterizing overall
ligand distribution is the RGD cluster spacing
(Table 1). Presenting adhesion
ligands in discrete nanoscale islands (5 RGD/comb) enhances cell adhesion even
when RGD cluster spacing is increased to 290 nm
(Fig. 3C;
Table 1). The substrates
prepared for this study have inappropriate optical characteristics for
high-resolution fluorescence microscopy, and thus we did not examine focal
adhesion formation. We are currently modifying the preparation protocols to
facilitate such studies.
Cell adhesion studies are often carried out using protein-coated surfaces,
and adhesion strength is measured after short-term cell attachment (within
several hours of cell plating). Serum-free conditions and protein synthesis
inhibitors are usually used to ensure negligible non-specific adhesion
mediated through deposition of serum-derived or cell-secreted matrix
molecules. These requirements place grave restrictions on long-term studies of
cell adhesion, multi-molecular synergy and other adhesion-based cell behaviors
such as motility. Adhesion profiles presented in
Fig. 4 and
Fig. 5 demonstrate the
`masking' of a bio-specific surface by nonspecific molecules. Applying a cell
detachment assay after a 12 hour incubation in serum-free media shows that
cell adhesion exhibits unambiguous dependence on fibronectin density and
detachment force. However, an 8 hour incubation in 1% dialyzed serum following
serum deprivation nearly abolishes fibronectin density dependence. Even
substrates coated with heat-denatured BSA, which does not promote cell
adhesion specifically, became non-specifically adhesive after an 8 hour
incubation in 1% d-serum. Thus protein-coated substrates fail to provide a
robust system for long-term studies in which ligand density and specificity
are of interest. The PEO/PMMA comb polymer surface is rationally designed to
provide a cell inert background on which covalently tethered biological
signals remain specific over time (Banerjee
et al., 2000; Irvine et al.,
2001
; Irvine et al.,
2002b
). Therefore, over the course of our experiment (>20
hours), surface biochemistry has remained constant and specific in the
presence of serum and cell-secreted proteins.
Finally, we noted that the RGD-comb substrates used in the present work were of relatively moderate adhesiveness since the number of cells attached to them was low. However, the development of an adhesion profile is inherently due to a distribution of cell adhesive properties within a cell population. Therefore, even when the percentages of retained cells are moderate, the observed phenomena are still reflective of the behavior of a heterogeneous cell population.
Results from this study emphasize the integrated governance of cell behavior through biochemical and biophysical cues that are present in the ECM. Delineation of the individual biochemical and biophysical contributions remains challenging, and we present here a new tool to allow independent control over ligand clustering and ligand density. Our findings suggest that these two substrate properties control cell adhesion synergistically. Nanoscale ligand clustering enhances cell adhesion even when the ligand density remains constant. In addition, cell adhesion is modulated by mechanical forces transduced through cell-ECM contacts in a manner that requires both ligand distribution and intracellular signaling. These results have implications for rational design of biomaterials that modulate cell adhesion, allowing the efficiency of peptide use to be improved. In this paper, we presented the initial work of an integrated cell behavioral investigation using the comb substrates. We are currently investigating the role of ligand clustering in cell motility and and in the crosstalk between integrin and growth-factor-mediated signaling pathways.
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