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Department of Biomedical Engineering, Boston University, Boston, Massachusetts
Correspondence: Address reprint requests to Joyce Y. Wong, Dept. of Biomedical Engineering, Boston University, 44 Cummington Street, Boston, MA 02215. Tel.: 617-353-2374; Fax: 617-353-6766; Email: jywong{at}bu.edu.
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ABSTRACT |
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) with a maximum at 
200 molecules/µm2, whereas the total traction force increases and reaches a plateau as a function of . In addition to these qualitative similarities, there are significant quantitative differences between fibroblasts adhered to FN and RGD. For example, fibroblasts on FN have a spread area that is on average greater by 200 µm2 over a 40-fold change in . In addition, fibroblasts on FN exert 3–5 times more total force, which reaches a maximum at a value of 5 times less than for cells adhered to RGD. The data also indicate that the differences in traction are not simply a function of the degree of spreading. In fact, fibroblasts on FN ( 2000 µm–2) and RGD ( 200 µm–2) have both similar spread area (600 µm2) and migration speed (11 µm/h), yet the total force production is five times higher on FN than RGD (0.05 dyn compared to 0.01 dyn). Thus, the specific interactions between fibroblasts and FN molecules must inherently allow for higher traction force generation in comparison to the interactions between fibroblasts and RGD. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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; Zamir et al., 1999; Geiger et al., 2001). These interactions collectively give rise to even more complex physiological phenomena such as tumor metastasis, wound healing, and tissue morphogenesis (Trinkaus, 1969; Harris, 1986; Lauffenburger and Horwitz, 1996). Cells exert tractional forces through adhesive contacts (e.g., focal adhesions), and tractional forces in turn are required for cell migration. However, cell migration is not required for traction force generation. In fact, cells can exhibit low migration rates and yet exert substantial traction forces through contractile proteins. Moreover, both positive and negative correlations between traction and migration have been observed, depending on the cell type (Shreiber et al., 2003).
The relationship between traction and migration also depends on the nature of the ECM, and numerous studies have investigated how cell-ECM interactions control adhesion and migration (Brandley and Schnaar, 1988; Massia and Hubbell, 1990; DiMilla et al., 1993; Garcia et al., 1999; Maheshwari et al., 2000; Hersel et al., 2003). In contrast, there have been relatively few studies that have directly probed the effect of specific cell-ECM interactions on traction force generation (Gaudet et al., 2003; ReinhartKing et al., 2003) or directly compared adhesion, traction, and migration (Gaudet et al., 2003). These types of studies are critical for engineering the cell-biomaterial interface to recapitulate phenomena such as wound healing and tissue morphogenesis. Here, we directly compare cellular response on the well-studied protein fibronectin (FN) and its cell adhesion peptide mimic Arg-Gly-Asp (RGD). Although there have been numerous qualitative studies comparing effects of RGD and FN, to our knowledge, the relationships between cell spreading, migration, and traction force generation on RGD- versus FN-modified substrata have not yet been thoroughly explored.
The cellular response to FN depends not only on the surface density of the molecule, but also its conformation. For example, recent in vitro studies have shown that the conformation of adsorbed FN is not only dependent on the properties of the substrate, but that these changes in FN conformation also lead to differences in cellular response (Garcia et al., 1999). Furthermore, the precise control of the surface conformation of ECM proteins such as FN requires site-specific immobilization and is rather challenging. In contrast, it is much easier to manipulate and preserve the conformation of short synthetic peptide sequences. Therefore, peptide sequences are frequently used in place of the native protein. The best example is RGD, which is found in the cell-binding domain of FN (Pierschbacher and Ruoslahti, 1984; Ruoslahti, 1996) and has been incorporated into a wide variety of otherwise nonadhesive substrata to promote cell adhesion, spreading, and focal contact formation (Massia and Hubbell, 1990, 1991). However, several studies have also shown that RGD-modified substrata do not completely recapitulate the same phenotype and behavior observed in cells adhered to FN-modified substrata. For instance, cells adhered to RGD-modified substrata exhibit significant differences in migration speed (Maheshwari et al., 2000), spreading and focal contact formation (Streeter and Rees, 1987) compared to cells adhered to FN-modified substrata.
Although these studies clearly show differences in cell behavior on FN- versus RGD-modified substrata, they do not give any information regarding the traction stresses exerted by these cells. The lack of quantitative comparisons of cell traction on FN- and RGD-modified substrata can be attributed to the fact that methods to quantify cell traction have only recently been developed (Dembo and Wang, 1999; Balaban et al., 2001; Tan et al., 2003). Because focal contact formation has been linked to traction force generation (Balaban et al., 2001; Tan et al., 2003) and also because cells exert traction forces during migration (Lee et al., 1994; Oliver et al., 1994; Munevar et al., 2001b), we hypothesize that cellular traction force generation will differ on FN- and RGD-modified substrata.
To quantify the differences and similarities between RGD and FN on cellular response, we directly compare the spread area, contractility, and migration of balb/c fibroblasts that are adhered to polyacrylamide substrata modified with varying surface densities of RGD and FN. Polyacrylamide substrata are utilized because they are nontoxic, elastic, and require the covalent attachment of a specific ligand to promote cell adhesion. Thus, we are able to follow the effects of both ligand type and surface density on three different aspects of cellular behavior. Spread area and contractility are measured using traction force microscopy (Dembo and Wang, 1999; Lo et al., 2000; Beningo et al., 2001; Munevar et al., 2001a,b; Wang et al., 2001; Gaudet et al., 2003; Marganski et al., 2003a; Doyle et al., 2004). Cell migration is quantified using a random walk model (Dunn, 1983; DiMilla et al., 1992b). This systematic approach provides a quantitative functional comparison of cellular behavior on RGD- and FN-modified substrata.
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MATERIALS AND METHODS |
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; Brandley et al., 1990). Specifically, the polymerization mixture contains 8% acrylamide, 0.04% bis-acrylamide, 10 µmol/ml acrylic acid NHS ester, 2 ml 0.25 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Sigma-Aldrich), 200 µl 0.75 µm-diameter fluorescent marker beads (Fluoresbrite carboxylate microspheres; Polysciences Inc., Warrington, PA), and 15 µL TEMED (N,N,N',N'- tetramethylethylenediamine; BioRad). The pH of the solution is adjusted to 6.0 by careful addition of 1 M HCl: it is critical to use a pH of 6.0, since a lower pH can inhibit polymerization whereas a higher pH can hydrolyze the NHS ester. Polymerization is initiated by adding 10% aqueous ammonium persulfate and the solution is cast onto glass cover slips that are activated with 3-aminopropyltrimethoxysilane and glutaraldehyde (Wang and Pelham, 1998). FN or the hexapeptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) is covalently linked to the substratum through the NHS ester. Various plating concentrations (Table 1) of human plasma FN (Invitrogen, Carlsbad, CA) and the hexapeptide GRGDSP (Invitrogen) are prepared in cold, sterile PBS (phosphate-buffered saline; Invitrogen) and HEPES (pH = 8). The PAAM substrata are incubated in the peptide or protein solution at 4°C for 24 h. After the incubation period, ethanolamine (1 µl/ml in 50 mM HEPES and 10% ethanol) is added to react (15 min) with the remaining hydrolyzed ester groups to prevent nonspecific cell adhesion. Lastly, before cell culture, the substrata are rinsed in cold deionized water for 1 h and sterilized under UV radiation for 30 min.
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Measurement of the mechanical properties of PAAM substrata
The mechanical properties of PAAM substrata are determined by a bulk tensile measurement (Pelham and Wang, 1997). Briefly, PAAM samples are polymerized to form cylindrical structures with a diameter of 1 cm and length 10 cm. The Young's modulus is determined by the stress-strain curve obtained by adding known weights to the bottom of a suspended PAAM sample and then measuring the changes in the length of the sample.
Cell culture and microscopy for cell area and traction
Balb/c 3T3 fibroblasts (American Type Culture Collection, Manassas, VA) are maintained at 37°C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) supplemented with penicillin-streptomycin-L glutamine (Invitrogen) and 10% calf serum (Hyclone, Logan, UT). For cell area and traction measurements, cells are plated on the RGD- and FN-PAAM substrata at a low cell density (1000 cells/cm2) in the presence of 10% calf serum. After 15 h of incubation, the substratum is mounted on a Zeiss Axiovert S 100 microscope equipped with a 40x, 0.75 N.A. phase objective, a motorized stage (Ludl, Model No. 99D008-Z1, Hawthorne, NY), and a stage incubator that maintains the environment at 37°C and 5% CO2. Phase-contrast images of the cell and fluorescent images of the marker beads within the substratum are collected using a cooled CCD camera (Princeton Instruments, Trenton, NJ) and the Metamorph imaging software (Universal Imaging, Downingtown, PA).
Calculation of cellular traction forces
Traction forces exerted by individual balb/c 3T3 fibroblasts are measured using traction force microscopy (Dembo and Wang, 1999; Lo et al., 2000; Beningo et al., 2001; Munevar et al., 2001a,b; Wang et al., 2001; Gaudet et al., 2003; Marganski et al., 2003a; Doyle et al., 2004). Briefly, an individual cell is identified on the substratum that is isolated from other cells. A phase image of the cell is taken to capture its morphology. In addition, fluorescent images of the marker beads within the substratum are recorded while the cell is adherent (the so-called "strained" image) and after it has been removed using trypsin (the so-called "unstrained" image). The deformation of the substratum due to the activity of the cell is measured using an optical flow-based algorithm that matches patterns of beads between the unstrained and strained fluorescent images (Marganski et al., 2003b). The cellular traction field is then calculated by solving an ill-posed inverse problem based on linear elasticity theory when given the set of substratum deformations, the Young's modulus and Poisson's ratio of the substratum, and the boundary of the cell (Dembo et al., 1996; Dembo and Wang, 1999).
The overall force production of a cell can be determined by computing the magnitude of the total traction force 
In this expression, T(x,y) = [Tx(x,y), Ty(x,y)] represents the continuous field of traction vectors acting at spatial positions (x,y) within the cell boundary. The cell area, A, is determined by integrating the region that is enclosed by the boundary of the cell. Dividing |F| by A yields the average traction magnitude, |T|, a measure of the average absolute force per unit area exerted within the cell.
Quantifying cell migration
Time-lapse microscopy equipped with a motorized stage is used to capture phase-contrast images of single cells at 15 min intervals up to 12 h. Fields are chosen at random and cells that come into contact with each other, undergo mitosis, or leave the field of view are not analyzed. The centroid of each cell is tracked using Metamorph imaging software, and mean-squared displacements (
d2
) of the centroid of each cell are calculated. The speed (S) and persistence time (P) of each cell are obtained by fitting d2 as a function of time (t) to a random walk model equation 
(Dunn, 1983; DiMilla et al., 1992b). The Levenberg-Marquandt method for nonlinear least squares fitting is used to obtain S and P. The parameter P represents the mean period of time over which a cell continues to move in a particular direction before changing direction by 60°. Here, only values for S are reported.
Cytoskeletal staining
Immunofluorescence co-staining is performed to simultaneously observe actin filaments and focal adhesions containing paxillin. Cells are first fixed for 15 min in a 0.75% glutaraldehyde (Polysciences) solution in PBS. Sodium borohydride is then added to quench autofluorescence and, finally, 0.5% Triton X-100 is added to permeabilize the cell membranes. All solutions used thereafter in the staining protocol are prepared in a 1% BSA in PBS. To prevent nonspecific binding, goat serum (Sigma-Aldrich) is added at room temperature for 1 h. The cells are then incubated at 37°C with a mouse anti-paxillin monoclonal antibody (Chemicon, Temecula, CA) for a total period of 4 h. Rhodamine-phalloidin (Molecular Probes, Eugene, OR) is added 2 h subsequent to the addition of the primary paxillin antibody. Finally, a FITC-conjugated anti-mouse IgG secondary antibody (Sigma-Aldrich) is added to detect the presence of paxillin. The fluorescently-labeled samples are stored in a solution containing 1:3 (v/v) glycerol in PBS. Images are obtained with a Zeiss Axiovert S100 inverted microscope equipped with a cooled CCD digital camera and Metamorph imaging software.
Statistics
All quantitative data are expressed as a mean ± SE. The statistical significance between sets of data was determined using the Student's t-test. A value of p<0.05 was taken as statistically significant.
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RESULTS |
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30 kdyn-cm–2 for all substrata used in this study. We have also measured the Young's modulus using a Hertzian-based microindentation technique (Landau and Lifshitz, 1986) and find that the values for the Young's modulus obtained from these two different techniques are in good agreement (data not shown). The thickness of the polyacrylamide substrate is estimated to be 100 µm and a Poisson ratio of 0.3 is assumed (Li et al., 1993).
Quantification of the surface density of FN and RGD
We observe a strong linear correlation between input and immobilized mass for both FN and RGD (Fig. 1). This linearity persists over an 10-fold change in input FN and over an 80-fold change in input RGD. Fitting each data set with a linear curve illustrates that the binding efficiency is 6.8% for FN and 3.5% for RGD. One reason for the low coupling efficiency is that the NHS-ester can be hydrolyzed in water (Lomants and Fairbanks, 1976; Brinkley, 1992).
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FN) and RGD (RGD) were calculated by first converting the immobilized mass amounts into the number of immobilized molecules. The molecular weights used were 440 kDa for FN and 588 Da for RGD. The number of immobilized molecules of FN and RGD were then normalized by the surface area over which they are distributed. FN is a large molecule and therefore cannot penetrate the surface of the substratum. Evidence supporting low penetration of FN is that we do not observe significant diffusion of FITC-dextran (20 kDa; Stokes radius 3.3 nm, Kodama et al., 2000) when it is entrapped in the gel (data not shown). The dimensions of FN have been observed to be 1), an elongated structure (150 x 3 nm, H. Erickson, personal communication) on hydrophilic surfaces (Bergkvist et al., 2003; Erickson and Carrell, 1983; Johnson et al., 1999); or 2), cylindrical with 60 nm length and 6 nm base diameter (DiMilla et al., 1992a). Therefore, it is unlikely that FN will penetrate the gel. In contrast, RGD is a small peptide that can penetrate the surface and is therefore distributed throughout the substratum. However, it has been shown that an adherent cell can only access the top 10 nm of a polyacrylamide substratum (Brandley and Schnaar, 1988). We therefore calculated the RGD concentration only in the top 10 nm of the gel. Table 1 provides the immobilized surface densities of FN and RGD for the various input concentrations of ligand used in this study. These values were obtained using Fig. 1 and the calculations discussed in the above paragraph. The units are in molecules-µm–2, and from here on are abbreviated as µm–2.
Traction force microscopy
An illustration of the traction force microscopy technique is given in Fig. 2, A and B, for a balb/c 3T3 fibroblast adhered to a FN-modified substratum with a surface density of 200 µm–2. The cell shown was selected because it is closest to the population mean (n = 15) in terms of A, |F|, and |T| (denoted as A, |F|, and |T|, respectively). Fig. 2 A displays a vector plot of the substratum displacements overlaid on top of the outline of the cell and its nucleus. Each displacement vector indicates the direction and amount of movement that a particular location within the substratum underwent due to the mechanical activity of the cell. The most-likely traction field that fits the set of substratum displacements is shown in Fig. 2 B. Each vector of the traction field indicates the direction and amount of force exerted at a specific location within the cell. Most of the significant tractions are exerted at the lamellae on the top, bottom, and right side of the cell with very small tractions within the interior in regions near the nucleus. Furthermore, the traction vectors along the edge of the cell tend to be directed toward the cell interior and appear to be contracting the cell inward. Overall, this cell is characterized by A 900 µm2, |T| 8.7 kdynes/cm2, and |F| 0.08 dyn.
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200 µm–2. Note that the length scale, displacement vector scale, and traction vector scale are all the same as those illustrated in Fig. 2, A and B, and furthermore, that the cell is closest to the population mean (n = 10) in terms of A, |T|, and |F|. Many aspects of the most typical cell on RGD 200 µm–2 are similar to the most typical cell on FN 200 µm–2. For instance, the cell on RGD also has three or four lamellae with significant tractions oriented toward the interior. Moreover, tractions near the nucleus are minimal. Although both the morphology and the spatial distribution of the tractions of these two most typical cells are similar, there are quantitative differences. These differences are apparent if one considers that the most typical cell on RGD with RGD 200 µm–2 is characterized by A 430 µm2, |T| 3.8 kdyn/cm2, and |F| 0.02 dyn. Therefore, even though the ligand density is the same, there are systematic differences in the overall cell properties due to the type of ligand.
Cell-to-cell variability in projected cell area and contractility
To completely characterize the effects of both surface density and ligand type on cellular behavior, experiments such as those depicted in Fig. 2 were carried out on a large sample size of cells at various surface densities of FN and RGD. See Table 1 for the surface densities studied and the sample size at each density. The cell-to-cell variability in the data at a low (50 µm–2), medium (200 µm–2), and high (2000 µm–2) surface density of FN and RGD is illustrated in Table 2. In general, the variance in |F|, A, and |T| is quite large at all three surface densities and both ligand types. For example, A varies 3.5-fold, |F| varies 25-fold, and |T| varies by 10-fold. Despite the large variance in the data, two specific trends are apparent. First, at any particular surface density, the population means |A|, |F|, and |T| are significantly greater for fibroblasts adhered to FN- compared to RGD-modified substrata (p < 0.05 for six out of nine comparisons). Second, for each ligand type, both |F| and |T| increase as increases: |F| increases 2.6-fold for FN and 4.7-fold for RGD, and |T| increases 2.2-fold for FN and 3.7-fold for RGD.
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A) on for balb/c 3T3 fibroblasts adhered to FN- and RGD-modified substrata (Fig. 3). In both cases, A has a biphasic dependence on , reaches a maximum at 200 µm–2, and changes at most by a factor of 1.8 as is varied 40-fold. Despite these similarities there is one significant difference in the average spread area for cells on FN- and RGD-modified substrata. Specifically A is significantly higher by 200 µm2 for cells on FN compared to cells on RGD over almost the entire range of surface densities studied (p < 0.05 after exceeds 50 µm–2). Thus, even if the number of available FN and RGD molecules presented to a cell are equal, there is something inherent in the FN molecule that allows a cell to spread more effectively on FN than on RGD.
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|F|) increases and reaches a plateau as a function of for cells on both FN and RGD (Fig. 4). However, there are significant differences between the two data sets. For the case of RGD, |F| is 0.004 dyn at 50 µm–2, increases to 0.025 dyn at 500 µm–2, and maintains this value as increases to 2000 µm–2. In contrast, |F| is significantly higher (3–5-fold) for cells on FN compared to cells on RGD when exceeds 50 µm–2. Furthermore, |F| reaches a plateau at 100 µm–2 for cells on FN, which is 5 times less than the surface density needed for |F| to reach a plateau for cells on RGD.
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|F| and A data, there are only statistically significant differences in the population average traction magnitude (|T|) for cells adhered to FN- and RGD-modified substrata at low surface densities (Fig. 5). For instance when < 500 µm–2, |T| is 2–5-fold larger (p < 0.05) for cells adhered to FN-modified substrata. As reaches and exceeds 500 µm–2, the differences in |T| between cells adhered to FN- and RGD-modified substrata are no longer statistically significant and, therefore, are effectively eliminated. Specifically, as reaches 2000 µm–2, |T| asymptotes to 6–8 kdyn/cm2 for cells on FN and RGD.
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S) with are similar for balb/c 3T3 fibroblasts adhered to FN- and RGD-modified substrata (Fig. 6). In both cases, cells migrate fairly slowly at low surface densities (S 6–8 µm/h at 50 µm–2). However, as is increased to 1000 µm–2, S rises steadily eventually approaching 13–14 µm/h. In the case of cells adhered to FN-modified substrata, S decreases slightly after exceeds 1000 µm–2.
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DISCUSSION |
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A has a biphasic dependence on for both FN and RGD. We reported this biphasic dependence for the same cells on collagen and proposed a receptor-saturation model to explain our results (Gaudet et al., 2003). Our data also indicate that fibroblasts on FN are 2–5 times more contractile and have a spread area 200 µm2 greater than on RGD over a 40-fold change in ligand density. One may expect that the differences in contractility are simply due to the fact that cells on FN are spread to a greater degree. However, there is not a simple correlation between spread area and the amount of force production. As shown in Table 2, |F| is an increasing function of , whereas A is a biphasic function of for both FN and RGD. Moreover, to remove the influence of spread area on force generation, one could compare the overall force production of fibroblasts at particular surface densities of FN and RGD at which A is equal. This situation occurs at FN 2000 and RGD 200 µm–2 where A is 600 µm2 for both ligand types (see Fig. 3). We can compare the cells closest to the population mean at these two surface densities (Fig. 7). The phase-contrast images (Fig. 7, A and B) indicate that both cells are spread to the same extent and have polarized morphology with a few lamellae. However, the traction fields (Fig. 7, C and D) indicate that there are significant differences in the overall contractile output. Specifically, the cell on the FN-modified substratum generates twice as much total force (0.04 dyn) than the cell on the RGD-modified substratum (0.02 dyn). Thus, these data show that ligand type directly controls contractile activity and, furthermore, this control is independent of the amount of spreading.
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; Aota et al., 1991; Redick et al., 2000). The cell binding domain of FN consists of the type III domains 7–10 (denoted as FN III 7–10). In addition to the RGD sequence in the cell binding domain, the peptide sequence Pro-His-Ser-Arg-Asn (PHSRN) on FN III domain 9 (Aota et al., 1994) has also been shown to have a synergistic effect on cell adhesion. More recently, a putative binding site Lys-Asn-Glu-Glu-Asp (KNEED) on FN III domain 8 has been shown to modulate cell spreading (Wong et al., 2002). Finally, recent reports in the literature (Ohashi et al., 1999; Baneyx et al., 2001, 2002; Gao et al., 2003) have shown that FN is a highly extensible molecule and that traction forces exerted by cells can expose additional cryptic binding sites. For instance, NIH 3T3 fibroblasts cultured on FN-coated glass surfaces have been shown to extend and remodel the FN within 1–4 h (Baneyx et al., 2001). In our studies, data on contractility were measured 15 h post-cell plating, allowing sufficient time for the fibroblasts to expose additional cryptic binding sites in FN by exerting tractional forces.
Another mechanism that may be responsible for the differences in contractility is the spatial distribution of the ligands on the substratum. For instance, FN molecules can aggregate and assemble at the cell-substratum interface (Magnusson and Mosher, 1998), leading to the presentation of multiple cell-binding sites. In the case of RGD, there is no driving force for ligand clustering on the substratum, and one would expect a random distribution of RGD ligands on the surface. However, Maheshwari et al. (2000) were able to precisely control the spatial distribution of RGD ligands at the nanoscale level, and by increasing the degree of clustering, they were able to enhance cytoskeletal organization and formation of actin stress fibers on clustered RGD to levels close to that found on FN. We point out that they still found significant differences in cell migration, even with clustered RGD: they did not observe a biphasic dependence of cell migration speed on ligand density for clustered RGD. Thus, it is clear that the synergistic sites have a significant role in governing cellular response.
The constrained conformation of the RGD site within the FN molecule has been proposed to account for some differences observed between linear RGD and FN. There are differences in the affinities of ligands to cyclic and linear RGD, and recent studies have also observed differential cellular response (Xiao and Truskey, 1996; Kantlehner et al., 1999). A recent study (Kato and Mrksich, 2004) of 3T3 Swiss Albino cells found twice as many focal adhesions on surfaces modified by cyclic RGD compared to linear RGD. The focal adhesions were smaller in size on cyclic RGD and a higher distribution of focal adhesions was found in the interior of the cell. One would therefore expect to find differences in contractility and migration for cells on cyclic RGD-modified substrata. Such studies that quantify contractility and migration on substrata modified by cyclic RGD would provide valuable insights into the mechanisms of integrin activation and signal transduction.
Focal adhesions have also recently been directly related to traction force generation. Traction forces have been shown to be directly correlated to focal adhesion size (Balaban et al., 2001; Tan et al., 2003) as long as the adhesions are greater than 1 µm (Tan et al., 2003). There is clearly a dynamic aspect to this dependency because nascent focal adhesions have been shown to have decreased traction forces when they mature (Beningo et al., 2001). Presumably this change in focal adhesion structure would be reflected in cytoskeletal reorganization events. Thus, co-immunostaining of the cytoskeleton and focal adhesion proteins would give further insight into differences between FN- and RGD-modified substrata. For instance, we can compare the cytoskeletal organization of fibroblasts at particular surface densities of FN and RGD at which A is equal but |T| (or |F|) is different. As discussed earlier, this situation occurs at FN 2000 and RGD 200 µm–2, and cells co-stained for actin and paxillin at these two surface densities are shown in Fig. 8. We find that fibroblasts on FN exhibit well-defined actin stress fibers aligned through the entire interior of the cell (Fig. 8 A). In contrast, fibroblasts adhered to RGD exhibit actin fibers that are extremely fine and located primarily along the periphery of the cell (Fig. 8 C). Punctate regions enriched in paxillin (1–3 µm2) are observed on the cell periphery on FN (Fig. 7 B). Such features are not observed for cells on RGD (Fig. 7 D). These findings suggest that cells exert lower tractional forces on RGD because of the difficulty in forming focal adhesions and stress fibers.
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ACKNOWLEDGEMENTS |
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This project was supported by the Whitaker Foundation (RG-98-0506; TF-02-0026) and a National Science Foundation CAREER Award (BES-9985338) to J. Y. Wong, and the Computational Science Graduate Fellowship Program of the Office of Scientific Computing and Office of Defense Programs in the Department of Energy under contract DE-FG02-97ER25308 to W. A. Marganski.
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FOOTNOTES |
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Padmavathy Rajagopalan's present address is Center for Engineering in Medicine, Harvard Medical School, Massachusetts General Hospital, Boston, MA.
Submitted on November 11, 2003; accepted for publication June 18, 2004.
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REFERENCES |
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