{alpha}5ß1 integrin mediates strong tissue cohesion

Elizabeth E. Robinson, Kathleen M. Zazzali, Siobhan A. Corbett and Ramsey A. Foty*

Department of Surgery, University of Medicine and Dentistry-Robert Wood Johnson Medical School, CAB Room 7070, New Brunswick, NJ 08648, USA

* Author for correspondence (e-mail: fotyra{at}umdnj.edu)

Accepted 16 October 2002


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Integrins and cadherins are considered to have distinct and opposing functions. Integrins are traditionally cited for their role in cell-substratum interactions, whereas cadherins are thought to mediate strong intercellular cohesion. Together, these adhesion systems play crucial roles in a wide variety of cellular and developmental processes including cell migration, morphology, differentiation and proliferation. In this manuscript we present evidence that integrins possess the ability to mediate strong intercellular cohesion when cells are grown as 3D aggregates.

Much of the data elucidating the role of integrins as mediators of cell-extracellular matrix (ECM) interactions have been generated using conventional cell culture techniques in which cells are plated onto ECM-coated 2D surfaces. In vivo, cells are embedded in a 3D meshwork of ECM proteins. We hypothesized that, within this meshwork, integrin-ECM interactions may impart cohesivity to an aggregate of cells by linking adjacent cells together. To test this hypothesis, we transfected Chinese hamster ovary (CHO-B2) cells to express {alpha}5ß1 integrin and found that these cells formed compact, spherical aggregates. We measured aggregate cohesivity using tissue surface tensiometry, a novel technique that quantifies cell-cell cohesivity of spheroids under physiological conditions. We determined that {alpha}5ß1 integrin is capable of conferring strong cohesivity ({sigma}=8.22±0.68 dynes/cm) to aggregates of {alpha}5-integrin-transfected cells. This cohesion was found to be independent of cadherin expression and was significantly greater than the cohesivity conferred onto CHO-B2 cells transfected with N-cadherin ({sigma}=3.14±0.20 dynes/cm, P<=0.0001), a more traditional cell-cell cohesion system.

Fibronectin-null CHO cells that express {alpha}5ß1 integrin but do not secrete endogenous fibronectin do not form aggregates in fibronectin-depleted medium. Addition of increasing amounts of exogenous dimeric fibronectin to these cells resulted in a dose-dependent compaction. However, compaction failed to occur in the presence of fibronectin monomers. These data indicate that fibronectin is required for {alpha}5ß1-mediated compaction and that the dimeric structure of fibronectin is essential for this process. Additionally, aggregate formation of the {alpha}5 integrin transfectants was inhibited by an RGD peptide thus confirming {alpha}5ß1 integrin specificity. Collectively, these data confirm our hypothesis that {alpha}5ß1 integrin acts through fibronectin to link adjacent cells together, thus promoting strong intercellular cohesion in 3D cellular aggregates.

Key words: Integrins, Cadherins, Cohesivity, Tissue surface tensiometry, 3D, Aggregates


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell-cell and cell-extracellular matrix (ECM) interactions are fundamental regulators of both normal and abnormal biological processes including morphogenesis (Fujimori et al., 1990Go), wound healing (Eckes et al., 2000Go) and malignant invasion (Behrens, 1993Go; Foty et al., 1998Go; Foty and Steinberg, 1997Go; Okegawa et al., 2002Go; Shiozaki et al., 1996Go; Steinberg and Foty, 1997Go; Tlsty, 1998Go; Zhou et al., 2000Go). The roles of cadherins and integrins in these processes are well defined but have traditionally been considered as having two distinct and opposing functions. Cadherins regulate strong cell-cell cohesion, whereas integrins are primarily responsible for cell-ECM adhesion.

{alpha}5ß1 integrin binds to fibronectin (FN) and has a well-defined role in cell adhesion, migration and matrix formation. FN is a ubiquitous, multifunctional component of the ECM that exists as a dimer, with the two chains connected by disulfide bonds at the C-terminus (Hynes, 1992Go). Structurally, each FN chain contains a single cell-binding domain including an RGD sequence to which {alpha}5ß1 integrin specifically binds (Akiyama, 1996Go).

Many of the assays measuring the affinity of integrins for their ligands have relied largely upon either measurement of the binding kinetics (Goldmann, 2000Go) or upon assays in which cells, adherent to a 2D ligand-coated substrate, are subjected to either centrifugal force (Koo et al., 2002Go) or to shear stress (Goldstein and DiMilla, 2002Go). Although such assays are useful, they do not take into consideration the 3D nature of tissues and, as such, do not consider the ECM as a potential mechanism by which multicellular tissues may be crosslinked. We reasoned that integrin-mediated adhesion to ECM proteins, when modeled in a 3D system, could cause cells to aggregate and compact into spheroids imparting cohesivity to these aggregates, much as cadherins have been shown to do.

To test this hypothesis, we applied a recently developed method, tissue surface tensiometry (TST), to measure the intensity of intercellular cohesion within tissue-like aggregates under physiological conditions. The biophysical principles underlying TST have been previously described in detail (Foty et al., 1994Go). Briefly, TST measures intercellular binding energy by compressing spherical cellular aggregates between parallel plates in a specially designed device. The shape of the compressed aggregate is recorded, as is the force with which the aggregate resists the compressive force. These parameters are then applied to the Young-Laplace equation (Davies and Rideal, 1963Go), generating measurements of aggregate surface tension, or cohesivity. Using TST, we have previously shown that aggregate cohesivity specifies the spatial positioning of tissues within multicellular aggregates (Foty et al., 1996Go), modulates the rate of tissue spreading on a substratum (Ryan et al., 2001Go) and influences the emigration of cells from a tumor (Foty et al., 1998Go; Foty and Steinberg, 1997Go).

We generated a series of cell lines custom-designed to express {alpha}5ß1 integrin. We also generated cell lines expressing high levels of N-cadherin in order to quantitatively compare the strength of integrin-based cohesivity with that of a more traditional cell-cell cohesion system. We then prepared spherical aggregates of these cells and quantified their cohesivity by TST. We determined that {alpha}5ß1 integrin mediates strong cohesivity in 3D tissue aggregates and that this cohesivity is greater than that conferred by N-cadherin alone. We also determined that this integrin-mediated cohesivity is FN dependent and results from a specific interaction between {alpha}5ß1 integrin and the RGD site on FN. Structurally, we found that dimeric FN is required for aggregate formation and compaction. Therefore, using this 3D culture system and TST, we were able to quantitatively show that {alpha}5ß1 integrin is capable of mediating strong intercellular cohesion. These data are the first to rigorously quantify the cohesivity imparted to tissue-like cellular aggregates by the interaction of {alpha}5ß1 integrin with FN. These data have broad implications in fields influenced by these processes including malignant invasion, embryonic development and wound healing.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell lines
Chinese hamster ovary cells (CHO-B2) express ß1 integrin but do not express the {alpha}5 subunit (Zhang et al., 1993Go). Cells were maintained at 37°C in 95% air/5% CO2 in Dulbecco's Modified Eagle's Medium with 10% fetal calf serum, 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, 2 mM L-glutamine, 100 µg/ml streptomycin sulfate, 100 units/ml penicillin G sodium and 0.25 µg/ml amphotericin B (Gibco-BRL, NY). A CHO cell subclone (CHO-{alpha}5) that expresses high levels of {alpha}5ß1 integrin but does not secrete FN or express cadherin was also used. These cells were maintained as described above.

Construction of an {alpha}5 integrin cDNA expression vector
A 1.8 kb BamHI-XhoI fragment encoding the N-terminal portion of the human {alpha}5 cDNA in pLJ (kindly provided by Jean Schwarzbauer, Princeton University) was cloned into pcDNA 3.1(+) (pcDNA 3.1 {alpha}5-N, Invitrogen, CA). A 2.5 kb fragment containing the C-terminus of the human {alpha}5 cDNA was then ligated into an XhoI digest of pcDNA 3.1 {alpha}5-N to yield a complete human {alpha}5 cDNA in pcDNA 3.1.

Transfection
Cells were transfected by electroporation in 400 µl of transfection medium (RPMI, 0.1 mM DTT, 10 mM dextrose) at 200 volts and 960 µF in a 0.4 cm electroporation cuvette using a BioRad Gene Pulser II apparatus. For expression of human {alpha}5 integrin, CHO-B2 cells were transfected with 20 µg of pcDNA3 plasmid containing the coding region for human {alpha}5 integrin and resistance to G418. For expression of chicken N-cadherin, CHO-B2 cells were transfected with 20 µg of the N-cadherin expression plasmid pMiwcN (Fujimori et al., 1990Go) (kindly provided by M. Takeichi, Kyoto University) and 5 µg of the zeocin resistance plasmid pZeoSV (Invitrogen, CA). Cells were grown to confluence in medium supplemented with either 800 µg/ml G418 or 500 µg/ml zeocin. Empty vector control cells (CHO-P3; pcDNA3 only) were generated by the same transfection and selection processes. {alpha}5-integrin-transfected (CHO-A5) and N-cadherin-transfected (CHO-Ncad) cells were bulk sorted by FACS.

FACS and flow cytometry
CHO-A5 cells were detached with trypsin-EDTA (Gibco-BRL, NY), washed three times with ice-cold Hanks' balanced salt solution (HBSS) and incubated with an anti-human {alpha}5 integrin antibody (CD49e, PharMingen, CA) at 5 µg/ml on ice for 45 minutes. Cells were again washed with cold HBSS and incubated on ice for an additional 45 minutes with a FITC-conjugated goat-anti-mouse secondary antibody (Zymed, CA). Cells expressing {alpha}5 integrin were FACS sorted (EPICS ALTRA, Beckman Coulter, FL) and expanded. CHO-Ncad cells were detached from tissue culture plates with trypsin-calcium (0.1% trypsin/5 mM Ca2+) to preserve cadherin receptor integrity (Hyafil et al., 1981Go) and sorted to express levels of N-cadherin similar to {alpha}5 integrin expression by the CHO-A5 cells. CHO-Ncad cells were incubated with 10 µg/ml anti N-cadherin primary antibody (NCD2, Zymed, CA), followed by a FITC-conjugated goat-anti-rat secondary antibody (Zymed, CA). Cells were sorted three times to generate pure populations. Receptor expression was confirmed monthly by flow cytometry.

Western blot analysis
Expression of both {alpha}5 integrin and N-cadherin was confirmed by western blot analysis using standard protocols. Cell lysates were prepared from near-confluent 10 cm tissue culture plates. Cell monolayers were washed twice with ice-cold HBSS then lysed by the addition of 500 µl RIPA lysis buffer (150 mM NaCl, 50 mM TRIS pH 7.5, 1% NP40, 0.25% DOC) containing a protease inhibitor cocktail (Calbiochem, CA), EDTA and sodium vanadate. The lysates were transferred to microcentrifuge tubes, rotated at 4°C for 1 hour, then passed through a Qia-shredder (Qiagen, CA) and centrifuged at 14,000 g for 15 minutes at 4°C. Cell lysates from 3D aggregates were prepared in the same manner except that aggregates were disrupted by sonication in RIPA lysis buffer. Protein concentrations were determined using the BCA Protein Assay Kit (Pierce, IL). 20 µg of protein was separated on a 7% SDS-PAGE gel and electroblotted to nitrocellulose using standard protocols. Blots were blocked overnight at 4°C in either Membrane Blocking Solution (CHO-Ncad, Zymed, CA) or 5% nonfat dry milk in TBS-0.2% TWEEN 20 (CHO-A5). Blots were incubated at room temperature for 1 hour in either an N-cadherin primary antibody (3B9 at 0.5 µg/ml, Zymed, CA) or in an {alpha}5 integrin primary antibody (AB1928 at 1 µl/ml, Chemicon, CA), washed three times with TBS-0.2% TWEEN 20, followed by an additional 1 hour incubation in either horseradish-peroxidase-conjugated goat anti-mouse secondary antibody (CHO-Ncad) or goat-anti-rabbit secondary antibody (CHO-A5). Blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce, IL) and exposed to X-ray film. All blots were then stripped in 62.5 mM Tris HCl pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol for 30 minutes at 50°C and re-probed with an anti-actin antibody (Sigma, MO) to confirm equal lane loading.

Fast aggregation assay
Aggregation assays, performed in the presence and absence of calcium, measure rapid, calcium-dependent aggregation (Takeichi, 1977Go). This technique was used to assess cadherin function of the CHO-B2, CHO-P3, CHO-A5 and CHO-Ncad cell lines. Cells were detached from near-confluent 10 cm plates by trypsin-calcium, washed in PBS, and stained with PKH26 (Sigma, MO) red fluorescent membrane intercalating dye according to the manufacturer's instructions. Stained cells were washed three times in HBSS containing either 2 mM CaCl2 (HBSS + Ca2+) or in Ca2+/Mg2+-free HBSS (HBSS-Ca2+). Cells were counted and resuspended at 1.0x106 cells/ml of HBSS±Ca2+. 3 ml of each cell suspension was transferred to individual 10 ml shaker flasks (Belco Glass, NJ) and incubated on an orbital shaker at 120 rpm for one hour at 37°C. The degree of aggregation was assessed by fluorescence microscopy.

Aggregate formation and hanging drop cultures
Cells were removed from near-confluent 10 cm plates with trypsin-EDTA, washed, counted and resuspended at a concentration of 2.5x106 cells/ml in complete medium supplemented with 2 mM CaCl2. 15 and 20 µl aliquots of this suspension were deposited on the underside of a 10 cm tissue culture dish lid. The lid was then inverted over 10 ml of 1x phosphate-buffered saline creating hanging drops on the upper lid. Drops were incubated under tissue culture conditions for 2-3 days, allowing the cells to coalesce at the base of the droplets and form sheets. The sheets from hanging drop culture were then transferred to 10 ml shaker flasks (Belco Glass, NJ) in 3 ml complete medium and placed on an orbital shaker at 110 rpm for 2-3 days. This encouraged cell rearrangement and 3D spheroid formation. Spheroids ranged in size from 540-800 µm in diameter.

In order to determine the molecular mechanisms of {alpha}5ß1-mediated aggregate compaction, we performed hanging drop assays in FN-depleted medium. Serum was depleted of FN by incubation with collagen sepharose beads as previously described (Corbett et al., 1997Go). Depletion was confirmed by western blot analysis. Hanging drops of CHO-A5 cells were cultured in the presence of a 100 µM cyclic RGD-blocking peptide (FR-1, Calbiochem, CA). Hanging drops of the CHO-{alpha}5 cells were cultured in FN-depleted medium, with 3-300 µg/ml exogenous rat plasma FN, or in the presence of a recombinant monomeric FN fragment.

Synthesis of recombinant fibronectin monomer
Construction of recombinant FN monomers was performed in the baculovirus vector pVL1393 as previously described. Briefly, a terminal codon was generated by adding an XbaI linker to a StuI site located at position 6860 in the FN{Delta}III1-7 cDNA, yielding a FN molecule structurally identical to FN{Delta}III1-7 but lacking the dimerization site (Corbett and Schwarzbauer, 1999Go; Sechler et al., 1996Go). This fragment was inserted between the BamHI and XbaI sites in pVL1393. The construct was confirmed by restriction enzyme digest analysis. Recombinant protein production was performed as previously described (Sechler et al., 1997Go).

Tissue surface tensiometry
Aggregate cohesivity was measured by TST as previously described (Foty et al., 1994Go; Foty et al., 1996Go). Aggregates ranging in size from 540-800 µm were transferred to the inner chamber of the tensiometer and positioned on the lower compression plate (LCP, Fig. 1A). The inner chamber contained pre-warmed, de-gassed CO2-independent medium at 37°C. The upper compression plate (UCP), attached to a nickel-chromium wire, was then positioned above the aggregate and connected to a Cahn electrobalance. The weight of the UCP was zeroed to establish a pre-compression UCP weight baseline. In order to minimize adhesion of cell aggregates to the compression plates, both the lower and upper plates were pre-coated with poly 2-hydroxyethylmethacrylate (poly-HEMA), a polymeric material to which cells do not adhere (Folkman and Moscona, 1978Go). Compression was initiated by raising the LCP until the aggregate became compressed against the UCP. Adjusting the height of the LCP controlled different degrees of compression. The force with which the aggregates resisted compression was monitored by the Cahn recording electrobalance. Aggregate geometry was monitored through a 25x Nikon dissecting microscope equipped with a CCD video camera and connected to a Macintosh Power PC computer. Images of aggregates were captured, digitized and their geometries were analyzed using NIH Image software. Each aggregate was subjected to two different degrees of compression, the second greater than the first Measurements of aggregate geometry (Fig. 1B) and the force of resistance to the compressive force were then applied to the Young-Laplace equation (Davies and Rideal, 1963Go), producing numerical values of apparent tissue surface tension ({sigma}) (Eqn. 1):

1



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Fig. 1. (A) Parallel plate compression device. The apparatus (not drawn to scale) contains inner and outer rectangular Plexiglas chambers. The outer chamber (OC) is connected to a thermostatted circulating water pump and serves to regulate the temperature of the inner chamber (IC). The lower assembly (LA) screws into the base of the inner chamber. The position of its central core (CC), whose tip is the lower compression plate (LCP), can be adjusted vertically by a screw thread to set the distance between the two plates. The upper compression plate (UCP) is a cylinder about 15 mm long suspended from the arm of a Cahn recording electrobalance, labeled as B, by a 0.15 mm diameter nickel-chromium wire (NCW). Its position can be adjusted horizontally to place the UCP directly above the LCP. Both plates are coated with poly-HEMA before each use. During an experiment, a spheroidal cell aggregate, labeled as A, is positioned on the lower plate and raised until it contacts the upper plate. Compression of the aggregate reduces the load measured by the balance by an amount equal to the force acting upon the cell aggregate. (B) Diagram of a liquid droplet compressed between two parallel plates at shape equilibrium. R1 and R2 are the two primary radii of curvature, at the droplet's equator and in a plane through its axis of symmetry, respectively. R3 is the radius of the droplet's circular area of contact with either compression plate. H is the distance between the upper and lower compression plates. Because R1, R2 and H can all be directly measured with greater accuracy than R3, the latter parameter was calculated using Eqn. 2.

 

Because R1, R2 and H can all be directly measured with greater accuracy than R3, the latter parameter was calculated using Eqn. 2:

2

A true surface tension is one in which the measured {sigma} is invariant of the applied force, as would be expected of a true liquid surface tension. Only those measurements of surface tension exhibiting this behavior were used to calculate aggregate cohesivity.

The two likely material states to be considered as they apply to tissue aggregates are liquidity and elasticity. The calculated surface tension of a liquid aggregate, when subjected to two compressions, the second greater than the first, will remain constant. By contrast, the calculated surface tension of an elastic aggregate will obey Hooke's law and increase in proportion to the applied force. For example, we have previously shown that when an elastic poly-acrylamide sphere is subjected to two successive compressions, the calculated surface tension increased in proportion to the applied force (Foty et al., 1996Go). By contrast, several liquid systems have been described in which surface tension remains constant irrespective of the applied force. Such examples include compression of aggregates of embryonic tissues (Davis et al., 1997Go; Foty et al., 1994Go; Foty et al., 1996Go), HT1080 human fibrosarcoma (Foty et al., 1998Go), Lewis lung carcinoma (Foty and Steinberg, 1997Go) and genetically engineered cells (Duguay et al., 2002Go).

In order to confirm the validity of our TST measurements, we calibrated our tissue surface tensiometer by compressing an air bubble in culture medium and comparing the calculated surface tension with that obtained by the de Noüy ring method (Davies and Rideal, 1963Go). Surface tension measured by TST and by the de Noüy technique were essentially identical, 39 dynes/cm and 42 dynes/cm, respectively (Foty et al., 1994Go). On the basis of this result, we are confident that the measured surface tensions of our transfected aggregates represent absolute values of aggregate cohesivity.


    Results
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 Materials and Methods
 Results
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 References
 
{alpha}5ß1 integrin and N-cadherin expression by transfectants
Stable populations of CHO-A5, CHO-Ncad and CHO-P3 (empty vector control) cells were created by bulk sorting and antibiotic selection. Receptor expression was confirmed by flow cytometry (Fig. 2A). A significant and comparable uniform shift in fluorescence intensity represents {alpha}5 and N-cadherin expression in the CHO-A5 and CHO-Ncad cells, respectively. Receptor expression was further confirmed by western blot analysis. Strong 130 kDa and 120 kDa bands were detected on the CHO-A5 and CHO-Ncad blots, respectively, representing the published weights of {alpha}5 integrin and N-cadherin (Fig. 2B).



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Fig. 2. (A) Flow cytometric analysis of CHO-Ncad and CHO-A5 transfectants. Note the significant shift in mean channel fluorescence of positive cells, representing approximately a 100-fold increase in protein expression. (B) Western blot analysis of CHO-Ncad and CHO-A5 transfectants. 25 µg of protein from cell lysates of CHO-Ncad and CHO-A5 cells were separated by SDS-PAGE, blotted to PVDF and subjected to immunoblot analysis using appropriate antibodies. Enhanced chemiluminescence detected a weak 130 kDa band corresponding approximately to the known average molecular weight for N-cadherin in the CHO-B2 parent line. A much stronger 130 kDa band is evident in the CHO-Ncad transfectant. {alpha}5 integrin was undetectable in CHO-B2 cells but was strongly expressed by the CHO-A5 cell line as represented by a 120 kDa band.

 

CHO-A5 cells formed compact spheroids
All cell lines were cultured in hanging drops and transferred to shaker flasks as previously described. CHO-B2 and CHO-P3 cells did not form spherical aggregates but instead remained as thick, flat sheets (Fig. 3A). CHO-A5 cells, however, formed compact spheroids (Fig. 3B). CHO-Ncad cells also formed spherical aggregates (Fig. 3C). CHO-Ncad aggregates were used as a comparison for the CHO-A5 aggregates for two reasons. First, they represent a traditional cell-cell cohesion system, and second, they formed spheroids and therefore their cohesivity could be quantified by TST.



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Fig. 3. Aggregate formation of CHO-B2, CHO-A5 and CHO-Ncad cell lines. Hanging drop cultures containing 2.5x106 cells/ml were incubated for 2-3 days then transferred to shaker flasks and incubated for another 2-3 days. Note that CHO-B2 (A) aggregates only formed thick, flat sheets, whereas aggregates of CHO-A5 (B) and CHO-Ncad (C) formed spheres. Bar, 1.67 mm.

 

{alpha}5ß1 integrin confers stronger aggregate cohesivity than N-cadherin
When subjected to TST, CHO-A5 aggregates were found to have a surface tension of 8.22±0.68 dynes/cm. These data were generated from 18 aggregates each subjected to two successive compressions, the second greater than the first (Table 1). By contrast, CHO-Ncad cells were found to have a surface tension of 3.14±0.20 dynes/cm (n=20), a value significantly lower than that measured for CHO-A5 cell aggregates (P<=0.0001, Student's unpaired t-test, Table 1). These data suggest that {alpha}5ß1 integrin confers stronger cohesivity to 3D tissue aggregates than does the expression of comparable levels of N-cadherin.


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Table 1. Aggregate surface tension values for aggregates of CHO-Ncad and CHO-A5

 

Assessment of liquid behavior of CHO-A5 and CHO-Ncad aggregates
To confirm that our TST measurements represented true surface tension, we demonstrated that {sigma} was size and force independent, as would be expected of a true liquid system (Davies and Rideal, 1963Go). As surface tension is an inherent physical property of a liquid, size independence is an absolute requirement. Air bubbles should therefore have the same surface tension irrespective of whether they are large or small if measured under similar experimental conditions. Surface tension of CHO-Ncad aggregates remained constant over a threefold range of volumes. A linear regression analysis of {sigma} versus volume generated a correlation coefficient (r2) of 0.099 for CHO-Ncad aggregates, demonstrating no statistical correlation between the two parameters. This was confirmed by performing a t-test to compare the confidence intervals. We found that for an n of 20, the calculated r value (0.315) for CHO-Ncad is below the critical value of r for testing P=0, (0.378, {alpha}=0.05), proving the null hypothesis that the slope of the linear regression is zero. This indicates that, for CHO-Ncad aggregates, surface tension is size independent.

A similar analysis was generated for CHO-A5 aggregates yielding an r2 value of 0.402, suggesting a possible weak relationship between {sigma} and volume (Fig. 4). A t-test comparing the confidence intervals in this data set generated an r value (0.634) greater than the critical value of r (0.400, n=18, {alpha}=0.05), thus confirming a correlation between surface tension and volume. We have previously shown that aggregate surface tension can become variable as a function of time in culture (Foty et al., 1996Go). To determine if this increased variability was a possible explanation for the correlation described above, we analyzed both CHO-Ncad and CHO-A5 TST data as a function of time in culture. Whereas {sigma} of CHO-Ncad remained relatively constant from 4-6 days in culture and exhibited relatively little scatter in the data, {sigma} of CHO-A5 nearly doubled over the same time period (Fig. 5). Linear regression analysis of {sigma} versus volume for CHO-A5 aggregates at 5 days in culture generated an r2 value of 0.164 (Fig. 6). The calculated r value (0.405, n=9) is below the critical value for r (0.582, {alpha}=0.05), indicating that, for 5 days in culture, surface tension is size independent. The 6 day data, however, were much more scattered and yielded an r value of 0.741 (r2=0.550, n=6), which is greater than the critical value for r (0.729), indicating size dependence. We conclude from this analysis that, although the CHO-A5 aggregates behave as liquids, the properties of these aggregates can, over time, become more variable, possibly owing to matrix deposition or a transition to elasticity.



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Fig. 4. Linear regression analysis of surface tension versus volume for CHO-Ncad and CHO-A5 aggregates. Aggregates of CHO-Ncad ({Delta}) ranging in volume from 0.1 to 0.3mm3 were subjected to TST. Linear regression analysis of the data generated a correlation coefficient (r2) of 0.099, indicating that no statistically significant correlation exists between {sigma} and volume. A similar analysis of CHO-A5 (O) aggregates produced an r2 value of 0.402, suggesting a possible weak relationship between {sigma} and volume.

 


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Fig. 5. Surface tension versus days in culture for CHO-Ncad and CHO-A5 aggregates. Surface tension of CHO-Ncad aggregates (white bars) remained relatively constant between 4 and 6 days in culture. The surface tension of CHO-A5 aggregates (shaded bars), however, increased between 4 and 6 days in culture. On each day, the surface tension of the CHO-A5 aggregates was greater than that of the CHO-Ncad aggregates.

 


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Fig. 6. Linear regression analysis of surface tension versus volume of CHO-A5 aggregates after 5 ({circ}) and 6 ({Delta}) days in culture. The surface tension of CHO-A5 aggregates cultured for 5 days remained relatively constant over a threefold range in volume. Linear regression analysis generated a correlation coefficient (r2) value of 0.164, indicating that surface tension at 5 days is size independent. At 6 days in culture, however, a greater degree of scatter in the data gave rise to an r2 value of 0.550, indicating size dependence.

 

In true liquid systems, {sigma} must also be independent of the applied force. An air bubble, compressed in liquid, should have the same surface tension irrespective of the degree of compression. Therefore, the ratio of the applied forces (F2/F1) will be greater than 1, whereas the ratio of the measured surface tensions ({sigma}2/{sigma}1) should be equal to 1. As Table 1 demonstrates, {sigma} of CHO-Ncad and CHO-A5 aggregates is indeed independent of the applied force as surface tension measured after two different compressive forces was not significantly different based on a student's t-test (CHO-A5, P=0.47; CHO-Ncad, P=0.74; Table 1). Moreover, the ratio of F2/F1 relative to {sigma}2/{sigma}1 was approximately 1.3:1, indicating that increasing the force at compression 2 had no effect on surface tension (Table 1). This force independence further establishes our {sigma} values as representative of true aggregate cohesivity.

CHO-A5 aggregate cohesion is cadherin independent
Transfection of {alpha}5 integrin into chick myoblasts has previously been shown to upregulate expression of N-cadherin (Huttenlocher et al., 1998Go). One possible explanation for the observed cohesivity of the CHO-A5 aggregates is upregulation of cadherin expression or function. CHO-B2 cells naturally express low levels of N-cadherin. No upregulation of N-cadherin was detected in the CHO-A5 cells by western blot analysis, irrespective of whether cells were grown as 2D tissue cultures or as 3D aggregates (Fig. 7A). Moreover, no upregulation of N-cadherin function was noted as CHO-B2, CHO-P3 and CHO-A5 cells failed to undergo calcium-dependent aggregation (Fig. 7B). As expected, CHO-Ncad cells aggregated in a calcium-dependent manner. Thus, upregulation of N-cadherin expression or function was not responsible for CHO-A5 aggregate formation.



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Fig. 7. (Top panel) Western blot analysis of N-cadherin expression of CHO-B2 and {alpha}5 integrin transfected cell lines grown as 2D or 3D cultures. Cell lysates were prepared from cells grown on tissue culture plastic and cells grown as 3D spheroids. N-cadherin was detected by immunoblot analysis. Note the presence of a 130 kDa band, corresponding to the published molecular weight of N-cadherin. Note also that transfection of CHO cells with {alpha}5 integrin did not result in increased N-cadherin expression irrespective of whether cells were grown in conventional tissue culture or as spheroids. (Bottom panel) Assessment of cadherin function by fast aggregation assay. Cells from near-confluent plates of CHO-B2 (A,B), CHO-P3 (C,D), CHO-A5 (E,F) and CHO-Ncad (G,H) were detached by trypsin/calcium (0.05% trypsin/2 mM CaCl2) treatment. Cells were stained with the membrane intercalating dye PKH-2 and resuspended at a concentration of 1x106 cells/ml in 3 ml of either calcium/magnesium-free HBSS (A,C,E,G) or HBSS with 2 mM Ca2+ (B,D,F,H), transferred to shaking flasks and placed on a gyratory shaker at 37°C and 120 rpm. Aggregation was monitored 1 hour later and imaged by fluorescence microscopy. Note that only the CHO-Ncad cell line aggregated in a calcium-dependent manner (G,H).

 

Cohesivity of CHO-A5 aggregates is fibronectin dependent
FN is the primary ligand of {alpha}5ß1 integrin. As {alpha}5ß1 integrin is not known to form strong homophilic interactions, we hypothesized that the strong cohesivity observed in the CHO-A5 tissue aggregates was due to {alpha}5ß1 integrin's interaction with FN. To test this hypothesis, we generated hanging drop cultures of CHO-A5 cells in FN-depleted medium. Such cultures produced small irregularly shaped, loosely associated clusters of cells (Fig. 8A). The normal physiological range of plasma FN is 100-1000 µg/ml (Ouaissi et al., 1986Go; Swisher and Rannels, 1997Go). When 300 µg/ml of plasma FN was added to the FN-depleted medium, the ability of CHO-A5 cells to form compact spheroids was restored (Fig. 8B). These data suggest that FN plays a role in {alpha}5ß1-integrin-mediated aggregate formation and compaction. As CHO cells are known to secrete small amounts of FN (Rajaraman et al., 1980Go), we postulated that this endogenous FN production could contribute to the basal level of aggregation of CHO-A5 cells observed in FN-depleted medium.



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Fig. 8. FN-dependent aggregation of CHO-A5 cells. CHO-A5 cells secrete low levels of endogenous FN. When cultured in hanging drops in FN-depleted tissue culture medium, cells formed loose sheets (A). Addition of 300 µg/ml of exogenous FN resulted in compact spheroid formation (B). Bar, 1.0 mm.

 

We further confirmed the role of FN in {alpha}5ß1-integrin-mediated cohesivity by utilizing the CHO-{alpha}5 cell line that expresses {alpha}5ß1 integrin but does not produce endogenous FN (Sechler et al., 1996Go). Hanging drop cultures of these cells did not aggregate in the absence of exogenous FN (Fig. 9A) but progressively formed more compact sheets as FN concentration was increased from 3-100 µg/ml (Fig. 9B-D). The addition of 300 µg/ml of FN resulted in the formation of spheroids (Fig. 9E). Spheroid formation was dependent purely on integrin expression since this subclone does not express cadherin and does not aggregate in a calcium-dependent manner (E.E.R., S.A.C. and R.A.F., unpublished). Moreover, de novo N-cadherin expression was not detected in 3D spheroids (data not shown). These data demonstrate that {alpha}5ß1-integrin-mediated cohesivity of the CHO-A5 aggregates is dependent upon FN.



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Fig. 9. Dose-dependent aggregation of FN-null CHO-{alpha}5 cells. CHO-{alpha}5 cells express high levels of {alpha}5 integrin but do not express N-cadherin or secrete FN. Cells were cultured in hanging drops either in the absence of FN (A) or in 3 (B), 30 (C), 100 (D) or 300 (E) µg/ml rat plasma FN. Note the dose-dependent aggregation and compaction of CHO-{alpha}5 cells in response to the addition of exogenous FN. Bar, 0.5 mm.

 

CHO-A5 aggregate formation and compaction requires a specific interaction between {alpha}5ß1 integrin and fibronectin
FN is a complex, dimeric ECM protein that binds both cells and other ECM proteins including heparin, fibrin and collagen. {alpha}5ß1 integrin is known to interact specifically with an RGD site located at type III repeat 10 in the cell-binding domain. We earlier demonstrated that the {alpha}5ß1-integrin-mediated cohesivity was dependent upon FN. This could be due to two possible mechanisms: {alpha}5ß1 integrin specifically binding to FN at the cell-binding domain or, alternatively, an interaction between FN and other cell surface receptors, such as syndecans. To define the mechanism by which FN promotes aggregate formation, we incubated the CHO-A5 cells in hanging drop culture with an RGD-blocking peptide. This peptide competes for the ligand-binding pocket of {alpha}5ß1 integrin and specifically interferes with its ability to bind FN (Akiyama, 1996Go). Hanging drop cultures formed in the presence of RGD peptide produced only dispersed cell clusters (Fig. 10). These data support the role of a specific interaction between {alpha}5ß1 integrin and FN in mediating cohesivity. No change was observed with the addition of RGD peptide to hanging drop cultures of the CHO-P3 cells in FN depleted medium (data not shown).



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Fig. 10. Aggregate formation and compaction of CHO-A5 cells in the presence of RGD peptide. CHO-A5 cells secrete low levels of endogenous FN. Culturing these cells in FN-depleted medium resulted in formation of cellular sheets (A). With the addition of 100 µM RGD peptide CHO-A5 cells failed to form aggregates (B). Bar, 1.0 mm.

 

Dimeric fibronectin is required for aggregate formation and compaction
FN exists as a dimer, each arm containing a single cell-binding site. The dimeric structure of FN is essential for its assembly into a fibrillar matrix. We hypothesized that this dimeric structure was a necessary component in the linkage of adjacent cells and that monomeric FN would be unable to provide such a linkage. To test this hypothesis, we used a recombinant FN monomer that lacks the dimerization site but that contains all the structural elements necessary for FN matrix assembly. CHO-{alpha}5 cell hanging drops were cultured in FN-depleted medium with 100 µg/ml of either monomeric recombinant FN or dimeric rat plasma FN. Aggregates failed to form in the presence of the recombinant monomer (Fig. 11). These data demonstrate that the dimeric structure of FN is essential for {alpha}5ß1-integrin-mediated aggregate formation and is required to link adjacent cells together.



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Fig. 11. Aggregate formation and compaction of CHO-{alpha}5 cells in the presence of recombinant monomeric FN. CHO-{alpha}5 cells were cultured in FN-depleted medium with 100 µg/ml of either monomeric (A) or dimeric (B) rat plasma FN. Note that aggregates failed to form in the presence of FN monomers (A). Bar, 1.0 mm.

 


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell-cell and cell-substratum adhesions involving cadherins and integrins have been widely studied and have been demonstrated to be vital contributors to normal and abnormal biological processes such as embryonic development, wound healing and malignant invasion. Cadherins have long been considered the primary mediators of tissue cohesivity, whereas integrins have traditionally been considered as mediators of cell-substratum interactions. In this manuscript we show that {alpha}5ß1 integrin is also capable of conferring strong cohesivity to 3D cellular aggregates. We demonstrate that interaction of {alpha}5ß1 integrin with its ligand, FN, confers greater cohesivity to an aggregate of cells than does N-cadherin, a typical cell-cell adhesion system.

This observation was made possible due, in large part, to application of TST, a method that measures the strength of intercellular cohesion of multicellular aggregates under physiological conditions. The main advantage of TST is that this method measures intercellular adhesive intensity within 3D aggregates and therefore more accurately mimics in vivo cellular interactions than do conventional 2D assays. Cells in tissues establish contacts and assume shapes more reminiscent of 3D foams than of the typical `fried egg' configurations adopted when cells are placed on 2D substrate-coated surfaces. Consequently, TST is able to accurately measure the effect of both direct intercellular cohesion (as mediated by cadherins), as well as indirect intercellular contacts mediated by the ECM in which cells are embedded.

It is known that some integrins, including {alpha}1ß2, {alpha}4ß7 and {alpha}Mß2, recognize integral membrane proteins of the IgG superfamily such as ICAM-1, ICAM-2 and VCAM-1 and are thus able to mediate direct cell-cell adhesion (Hynes, 1992Go). Others, such as integrin {alpha}IIbß3, are almost exclusively responsible for platelet-platelet interaction through GPIb/V/IX (Schoenwaelder et al., 2000Go). Typically, however, such interactions are not known to contribute to tissue cohesivity. The more classically defined ECM binding heterodimers, such as {alpha}5ß1, function principally to promote the adhesive events required for cell motility and tension-generated matrix remodeling. However their contribution to the overall cohesivity of a tissue has never been rigorously quantified. This work is the first to rigorously quantify the cohesivity imparted by the interaction of {alpha}5ß1 integrin with FN in 3D tissue-like aggregates.

{alpha}5ß1 integrin is unique amongst the FN-binding integrins in that it is the only integrin that naturally assembles FN into a matrix. Studies have defined many of the structural requirements for FN matrix assembly. These include FN's dimer structure, the N-terminal assembly domain and FN-binding sites in the first two type III repeats (Schwarzbauer, 1991Go; Sechler et al., 2001Go). Integrin binding to the RGD sequence in the cell-binding domain is also an essential requirement for matrix assembly (Sechler et al., 1996Go). FN-integrin interactions promote intermolecular association between the FN dimers, leading to the formation of fibrils. The fact that FN monomers failed to support aggregate formation supports the concept that FN matrix assembly may contribute to aggregate cohesivity by creating a scaffold, or an organized 3D matrix, which functionally links the cells to each other and promotes force generation. In the absence of solid structural support, the net effect of FN-mediated force generation is tissue compaction and remodeling, processes that are essential for a variety of biological functions.

The demonstration that the {alpha}5ß1-integrin—FN interaction confers strong intercellular cohesivity in 3D cellular aggregates significantly impacts several areas of interest. The study of malignant invasion, for example, represents a model system in which the forces of adhesion and cohesion influence cellular behavior (Behrens, 1993Go; Foty et al., 1998Go; Foty et al., 1994Go; Foty and Steinberg, 1997Go; Shiozaki et al., 1996Go; Steinberg and Foty, 1997Go; Tlsty, 1998Go; Zhou et al., 2000Go). Normal E-cadherin expression and function is considered to be important in maintaining tumor integrity whereas overexpression of various integrins has been associated with increased potential for invasion and migration. {alpha}5ß1 integrin is often found to be downregulated in metastatic cancer, and overexpression has been shown to rescue a transformed phenotype (Giancotti and Ruoslahti, 1990Go). These observations diverge from the commonly held view of integrins as metastasis-promoting molecules and suggest a possible role for {alpha}5ß1 integrin as a potential invasion suppressor molecule, much as has been reported for E-cadherin. Being able to quantify the contribution of integrins in regulating tumor cohesivity will provide information necessary for the development of strategies aimed at promoting intercellular cohesivity, thus discouraging dissemination of cancer cells.

The {alpha}4, {alpha}5 and ß1 integrin subunits have been shown to be essential for embryonic development. The knockout of these genes in mouse embryos always leads to embryonic lethality (Beauvais-Jouneau and Thiery, 1997Go). Davis et al. have shown that the cohesivity of the germ layers in amphibian gastrulae correlate perfectly with their spatial position (Davis et al., 1997Go). Injection of RGD peptide into amphibian blastulae has been shown to block gastrulation by disrupting cell interactions with FN and preventing formation of the meshwork of FN fibrils involved in migration (Boucaut et al., 1984Go). If {alpha}5ß1-integrin-FN interaction is indeed capable of conferring cohesivity onto tissues, particularly very early in development when embryos are essentially aggregate-like, then it is possible that {alpha}5ß1-integrin-FN interactions also have the capacity to specify the relative cohesivity of cells within the gastrula. FN-mediated compaction has also been shown to be important later in development. For instance, Downie and Newman demonstrated that a correlation exists between FN secretion and pre-cartilage mesenchymal condensation during wing and leg bud development in chick embryos (Downie and Newman, 1995Go), with high FN secretion correlating with compact and spheroidal condensation. Modulation of tissue cohesivity through differential cadherin expression during tissue development provides one possible mechanism of tissue assembly (Gumbiner, 2000Go; Steinberg and McNutt, 1999Go). Modulation of {alpha}5ß1 integrin and FN provides yet another potential cell-cell cohesion mechanism available to the embryo during the self-assembly process. Accordingly, molecular pathways regulating either the expression or function of {alpha}5ß1 integrin or FN have the capacity to function as potential morphogens, modulating tissue behavior, perhaps independently of cadherins.

During wound healing, alterations in integrin expression coincide with the increase in cell migration required for the repair process. Cells in the wound milieu alter their integrin expression to interact with ligands of the newly formed provisional ECM, whose primary structural components include fibrinogen and FN. The {alpha}5ß1-integrin-FN interaction has also been shown to be vital for retraction of 3D FN-fibrin clot matrices (Corbett and Schwarzbauer, 1999Go), a process crucial to early wound healing and tissue remodeling. It is thought that engagement of {alpha}5ß1 integrin with FN generates the tractional force required for clot retraction. We propose that {alpha}5ß1-FN-mediated intercellular cohesivity also contributes to clot retraction, in much the same way as the apparent `retraction' or compaction observed for CHO-A5 aggregates in response to increased concentrations of FN (Fig. 9).

The information presented in this manuscript is, to our knowledge, the first quantitative demonstration that the interaction between {alpha}5ß1 integrin and FN can give rise to strong intercellular cohesivity of 3D aggregates. Our data bring into question the undisputed role of cadherins as the prime mediators of tissue cohesivity and provide an alternative or additional mechanism by which the balance of cohesive and adhesive forces can be used to alter tissue behavior. Further understanding of these forces is important, for example, in such recent developments as the use of ligand-coated microparticles to compact complex cellular aggregates for tissue engineering and biomedical applications (Dai et al., 1994Go; Saltzman and Olbricht, 2002Go). The balance between cell-cell cohesion and cell-substratum adhesion, the regulatory mechanisms involved in maintaining this balance, and our ability to quantify them are important for developing unifying principles governing such processes as embryonic development, wound healing, malignant invasion, tissue regeneration and tissue engineering.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Akiyama, S. K. (1996). Integrins in cell adhesion and signaling. Hum. Cell 9, 181-186.[Medline]

Beauvais-Jouneau, A. and Thiery, J. P. (1997). Multiple roles for integrins during development. Biol. Cell 89,5 -11.[CrossRef][Medline]

Behrens, J. (1993). The role of cell adhesion molecules in cancer invasion and metastasis. Breast Cancer Res. Treat. 24,175 -184.[Medline]

Boucaut, J. C., Darribere, T., Poole, T. J., Aoyama, H., Yamada, K. M. and Thiery, J. P. (1984). Biologically active synthetic peptides as probes of embryonic development: a competitive peptide inhibitor of fibronectin function inhibits gastrulation in amphibian embryos and neural crest cell migration in avian embryos. J. Cell Biol. 99,1822 -1830.[Abstract]

Corbett, S. A. and Schwarzbauer, J. E. (1999). Requirements for alpha(5)beta(1) integrin-mediated retraction of fibronectin-fibrin matrices. J. Biol. Chem. 274,20943 -20948.[Abstract/Free Full Text]

Corbett, S. A., Lee, L., Wilson, C. L. and Schwarzbauer, J. E. (1997). Covalent cross-linking of fibronectin to fibrin is required for maximal cell adhesion to a fibronectin-fibrin matrix. J. Biol. Chem. 272,24999 -25005.[Abstract/Free Full Text]

Dai, W., Belt, J. and Saltzman, W. M. (1994). Cell-binding peptides conjugated to poly(ethylene glycol) promote neural cell aggregation. Biotechnology (NY) 12,797 -801.[Medline]

Davies, J. T. and Rideal, E. K. (1963). Interfacial Phenomena. New York: Academic Press.

Davis, G. S., Phillips, H. M. and Steinberg, M. S. (1997). Germ-layer surface tensions and "tissue affinities" in Rana pipiens gastrulae: quantitative measurements. Dev. Biol. 192,630 -644.[CrossRef][Medline]

Downie, S. A. and Newman, S. A. (1995). Different roles for fibronectin in the generation of fore and hind limb precartilage condensations. Dev. Biol. 172,519 -530.[CrossRef][Medline]

Duguay, D., Foty, R. A. and Steiberg, M. S. (2002). Cadherin mediated cell adhesion and tissue segregation: qualitative and quantitative determinants. Dev. Biol. (in press).

Eckes, B., Zigrino, P., Kessler, D., Holtkotter, O., Shephard, P., Mauch, C. and Krieg, T. (2000). Fibroblast-matrix interactions in wound healing and fibrosis. Matrix Biol. 19,325 -332.[CrossRef][Medline]

Folkman, J. and Moscona, A. (1978). Role of cell shape in growth control. Nature 273,345 -349.[Medline]

Foty, R. A. and Steinberg, M. S. (1997). Measurement of tumor cell cohesion and suppression of invasion by E- or P-cadherin. Cancer Res. 57,5033 -5036.[Abstract]

Foty, R. A., Forgacs, G., Pfleger, C. M. and Steinberg, M. S. (1994). Liquid properties of embryonic tissues: Measurement of interfacial tensions. Phys. Rev. Lett. 72,2298 -2301.[CrossRef][Medline]

Foty, R. A., Pfleger, C. M., Forgacs, G. and Steinberg, M. S. (1996). Surface tensions of embryonic tissues predict their mutual envelopment behavior. Development 122,1611 -1620.[Abstract/Free Full Text]

Foty, R. A., Corbett, S. A., Schwarzbauer, J. E. and Steinberg, M. S. (1998). Dexamethasone up-regulates cadherin expression and cohesion of HT-1080 human fibrosarcoma cells. Cancer Res. 58,3586 -3589.[Abstract]

Fujimori, T., Miyatani, S. and Takeichi, M. (1990). Ectopic expression of N-cadherin perturbs histogenesis in Xenopus embryos. Development 110,97 -104.[Abstract]

Giancotti, F. G. and Ruoslahti, E. (1990). Elevated levels of the alpha 5 beta 1 fibronectin receptor suppress the transformed phenotype of Chinese hamster ovary cells. Cell 60,849 -859.[Medline]

Goldmann, W. H. (2000). Kinetic determination of focal adhesion protein formation. Biochem. Biophys. Res. Commun. 271,553 -557.[CrossRef][Medline]

Goldstein, A. S. and DiMilla, P. A. (2002). Effect of adsorbed fibronectin concentration on cell adhesion and deformation under shear on hydrophobic surfaces. J. Biomed. Mater. Res. 59,665 -675.[Medline]

Gumbiner, B. M. (2000). Regulation of cadherin adhesive activity. J. Cell Biol. 148,399 -404.[Abstract/Free Full Text]

Huttenlocher, A., Lakonishok, M., Kinder, M., Wu, S., Truong, T., Knudsen, K. A. and Horwitz, A. F. (1998). Integrin and cadherin synergy regulates contact inhibition of migration and motile activity. J. Cell Biol. 141,515 -526.[Abstract/Free Full Text]

Hyafil, F., Babinet, C. and Jacob, F. (1981). Cell-cell interactions in early embryogenesis: a molecular approach to the role of calcium. Cell 26,447 -454.[Medline]

Hynes, R. O. (1992). Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25.[Medline]

Koo, L. Y., Irvine, D. J., Mayes, A. M., Lauffenburger, D. A. and Griffith, L. G. (2002). Co-regulation of cell adhesion by nanoscale RGD organization and mechanical stimulus. J. Cell Sci. 115,1423 -1433.[Abstract/Free Full Text]

Okegawa, T., Li, Y., Pong, R. C. and Hsieh, J. T. (2002). Cell adhesion proteins as tumor suppressors. J. Urol. 167,1836 -1843.[Medline]

Ouaissi, M. A., Neyrinck, J. L. and Capron, A. (1986). Development of a competitive radioimmunoassay for human plasma fibronectin. Int. Arch. Allergy Appl. Immunol. 81, 75-80.[Medline]

Rajaraman, R., Sunkara, S. P. and Rao, P. N. (1980). Morphological reverse transformation of Chinese hamster ovary (CHO) cells and surface fibronectin. Cell Biol. Int. Rep. 4,897 -906.[Medline]

Ryan, P. L., Foty, R. A., Kohn, J. and Steinberg, M. S. (2001). Tissue spreading on implantable substrates is a competitive outcome of cell-cell vs. cell-substratum adhesivity. Proc. Natl. Acad. Sci. USA 98,4323 -4327.[Abstract/Free Full Text]

Saltzman, W. M. and Olbricht, W. L. (2002). Building drug delivery into tissue engineering. Nat. Rev. Drug Discov. 1,177 -186.[Medline]

Schoenwaelder, S. M., Yuan, Y. and Jackson, S. P. (2000). Calpain regulation of integrin alpha IIb beta 3 signaling in human platelets. Platelets 11,189 -198.[CrossRef][Medline]

Schwarzbauer, J. E. (1991). Identification of the fibronectin sequences required for assembly of a fibrillar matrix. J. Cell Biol. 113,1463 -1473.[Abstract]

Sechler, J. L., Takada, Y. and Schwarzbauer, J. E. (1996). Altered rate of fibronectin matrix assembly by deletion of the first type III repeats. J. Cell Biol. 134,573 -583.[Abstract]

Sechler, J. L., Corbett, S. A. and Schwarzbauer, J. E. (1997). Modulatory roles for integrin activation and the synergy site of fibronectin during matrix assembly. Mol. Biol. Cell 8,2563 -2573.[Abstract/Free Full Text]

Sechler, J. L., Rao, H., Cumiskey, A. M., Vega-Colon, I., Smith, M. S., Murata, T. and Schwarzbauer, J. E. (2001). A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly. J. Cell Biol. 154,1081 -1088.[Abstract/Free Full Text]

Shiozaki, H., Oka, H., Inoue, M., Tamura, S. and Monden, M. (1996). Ecadherin mediated adhesion system in cancer cells. Cancer 77,1605 -1613.[CrossRef][Medline]

Steinberg, M. S. and Foty, R. A. (1997). Intercellular adhesions as determinants of tissue assembly and malignant invasion. J. Cell Physiol. 173,135 -139.[Medline]

Steinberg, M. S. and McNutt, P. M. (1999). Cadherins and their connections: adhesion junctions have broader functions. Curr. Opin. Cell Biol. 11,554 -560.[CrossRef][Medline]

Swisher, J. W. and Rannels, D. E. (1997). Assembly of exogenous fibronectin into type II cell extracellular matrix. Am. J. Physiol. 272,L908 -L915.[Abstract/Free Full Text]

Takeichi, M. (1977). Functional correlation between cell adhesive properties and some cell surface proteins. J. Cell Biol. 75,464 -474.[Abstract]

Tlsty, T. D. (1998). Cell-adhesion-dependent influences on genomic instability and carcinogenesis. Curr. Opin. Cell Biol. 10,647 -653.[CrossRef][Medline]

Zhang, Z., Morla, A. O., Vuori, K., Bauer, J. S., Juliano, R. L. and Ruoslahti, E. (1993). The alpha v beta 1 integrin functions as a fibronectin receptor but does not support fibronectin matrix assembly and cell migration on fibronectin. J. Cell Biol. 122,235 -242.[Abstract]

Zhou, J., Sargiannidou, I. and Tuszynski, G. P. (2000). The role of adhesive proteins in the hematogenous spread of cancer. In Vivo 14,199 -208.[Medline]