A dynamical model for receptor-mediated cell adhesion to surfaces.

We present a dynamical model for receptor-mediated adhesion of cells in a shear field of viscous fluid to surfaces coated with ligand molecules complementary to receptors in the cell membrane. We refer to this model as the "point attachment model" because it considers the contact area between the cell and the surface to be a small, homogeneous region that mediates the initial attachment of the cell to the surface. Using a phase plane analysis of a system of nonlinear ordinary differential equations which govern the changes in free receptor density and bond density within the contact area with time, we can predict the conditions for which adhesion between the cell and the surface will take place. Whether adhesion occurs depends on values of dimensionless quantities that characterize the interaction of the cell and its receptors with the surface and its ligand, such as the bond formation rate, the receptor-ligand affinity, the fluid mechanical force, the receptor mobility, and the contact area. A key result is that there are two regimes in which different chemical and physical forces dominate: a rate-controlled high affinity regime and an affinity-controlled low-affinity regime. Many experimental observations can be explained by understanding which of these regimes is appropriate. We also provide simple approximate analytical solutions, relating adhesiveness to cell and surface properties as well as fluid forces, which allow convenient testing of model predictions by experiment.     

Adhesion energy of receptor-mediated interaction measured by elastic deformation

We investigated the role of receptor binding affinity in surface adhesion. A sensitive technique was developed to measure the surface energy of receptor-mediated adhesion. The experimental system involved a functionalized elastic agarose bead resting on a functionalized glass coverslip. Attractive intersurface forces pulled the two surfaces together, deforming the bead to produce an enlarged contact area. The Johnson-Kendall-Roberts (JKR) model was used to relate the surface energy of the interaction to the elasticity of the bead and the area of contact. The surface energies for different combinations of modified surfaces in solution were obtained from reflection interference contrast microscopy (RICM) measurements of the contact area formed by the bead and the coverslip. Studies with surfaces functionalized with ligand-receptor pairs showed that the relationship between surface energy and the association constant of the ligand binding has two regimes. At low binding affinity, surface energy increased linearly with the association constant, while surface energy increased logarithmically with the association constant in the high affinity regime.     

Focal contact assembly through cytoskeletal polymerization: steady state analysis

For many cell types, initial receptor-mediated attachment to a ligand-coated surface is followed by the formation of focal contacts - strong, specialized, discrete adhesive connections between cell and substrate in which receptors are clustered and simultaneously linked to extracellular ligand and cytoskeletal proteins. Since adhesion affects many aspects of cellular physiology including growth, differentiation, and motility, understanding the biochemical factors which regulate focal contact assembly should enhance our understanding of these phenomena. In this paper, we present a mathematical model to examine how receptor-ligand, receptor-cytoskeleton, and cytoskeleton-cytoskeleton interactions affect the formation of receptor clusters which serve as precursors to mature focal contacts. Receptor clustering is presumed to occur through self-recognition of cytoskeletal elements which induce the polymerization of ligand-receptor-cytoskeleton complexes. Polymerization only occurs when the ligand density is above a critical value and a decrease in the receptor-ligand affinity shifts the critical ligand density to higher values. While cytoskeletal protein expression and receptor-cytoskeleton affinity influence the concentration of monomeric complexes, the formation of polymeric ligand-receptor-cytoskeleton aggregates is most sensitive to changes in the self-association affinity between cytoskeletal proteins. We find that a 100-fold enhancement in the affinity between cytoskeletal elements can produce a substantial increase in the total fraction of adhesion receptors associated with focal contact precursors (from 5% to over 90%). Our results suggest that under physiological conditions, cellular control of focal contact assembly most likely occurs through modulation of specific cytoskeletal proteins to solidify cytoskeleton-cytoskeleton connections within precursor focal contact structures.     

Receptor-mediated binding of IgE-sensitized rat basophilic leukemia cells to antigen-coated substrates under hydrodynamic flow.

The physiological function of many cells is dependent on their ability to adhere via receptors to ligand-coated surfaces under fluid flow. We have developed a model experimental system to measure cell adhesion as a function of cell and surface chemistry and fluid flow. Using a parallel-plate flow chamber, we measured the binding of rat basophilic leukemia cells preincubated with anti-dinitrophenol IgE antibody to polyacrylamide gels covalently derivatized with 2,4-dinitrophenol. The rat basophilic leukemia cells' binding behavior is binary: cells are either adherent or continue to travel at their hydrodynamic velocity, and the transition between these two states is abrupt. The spatial location of adherent cells shows cells can adhere many cell diameters down the length of the gel, suggesting that adhesion is a probabilistic process. The majority of experiments were performed in the excess ligand limit in which adhesion depends strongly on the number of receptors but weakly on ligand density. Only 5-fold changes in IgE surface density or in shear rate were necessary to change adhesion from complete to indistinguishable from negative control. Adhesion showed a hyperbolic dependence on shear rate. By performing experiments with two IgE-antigen configurations in which the kinetic rates of receptor-ligand binding are different, we demonstrate that the forward rate of reaction of the receptor-ligand pair is more important than its thermodynamic affinity in the regulation of binding under hydrodynamic flow. In fact, adhesion increases with increasing receptor-ligand reaction rate or decreasing shear rate, and scales with a single dimensionless parameter which compares the relative rates of reaction to fluid shear.     

Effect of receptor-ligand affinity on the strength of endothelial cell adhesion.

The objective of this study was to determine the effect of receptor-ligand affinity on the strength of endothelial cell adhesion. Linear and cyclic forms of the fibronectin (Fn) cell-binding domain peptide Arg-Gly-Asp (RGD) were covalently immobilized to glass, and Fn was adsorbed onto glass slides. Bovine aortic endothelial cells attached to the surfaces for 15 min. The critical wall shear stress at which 50% of the cells detached increased nonlinearly with ligand density and was greater with immobilized cyclic RGD than with immobilized linear RGD or adsorbed Fn. To directly compare results for the different ligand densities, the receptor-ligand dissociation constant and force per bond were estimated from data for the critical shear stress and contact area. Total internal reflection fluorescence microscopy was used to measure the contact area as a function of separation distance. Contact area increased with increasing ligand density. Contact areas were similar for the immobilized peptides but were greater on surfaces with adsorbed Fn. The dissociation constant was determined by nonlinear regression of the net force on the cells to models that assumed that bonds were either uniformly stressed or that only bonds on the periphery of the contact region were stressed (peeling model). Both models provided equally good fits for cells attached to immobilized peptides whereas the peeling model produced a better fit of data for cells attached to adsorbed Fn. Cyclic RGD and linear RGD both bind to the integrin alpha v beta 3, but immobilized cyclic RGD exhibited a greater affinity than did linear RGD. Receptor affinities of Fn adsorbed to glycophase glass and Fn adsorbed to glass were similar. The number of bonds was calculated assuming binding equilibrium. The peeling model produced good linear fits between bond force and number of bonds. Results of this study indicate that 1) bovine aortic endothelial cells are more adherent on immobilized cyclic RGD peptide than linear RGD or adsorbed Fn, 2) increased adhesion is due to a greater affinity between cyclic RGD and its receptor, and 3) the affinity of RGD peptides and adsorbed Fn for their receptors is increased after immobilization.     

Simulation of cell rolling and adhesion on surfaces in shear flow. Microvilli-coated hard spheres with adhesive springs.

The adhesion of cells to ligand-coated surfaces in viscous shear flow is an important step in many physiological processes, such as the neutrophil-mediated inflammatory response, lymphocyte homing, and tumor cell metastasis. This article describes a calculational method that allows simulation of the interaction of a single cell with a ligand-coated surface. The cell is idealized as a microvilli-coated hard sphere covered with adhesive springs. The distribution of microvilli on the cell surface, the distribution of receptors on microvilli tips, and the forward and reverse reaction between receptor and ligand are all simulated using random number sampling of appropriate probability functions. The velocity of the cell at each time step in the simulation results from a balance of hydrodynamic, colloidal, and bonding forces; the bonding force is derived by summing the individual contributions of each receptor-ligand tether. The model can simulate the effect of many parameters on adhesion, such as the number of receptors on microvilli tips, the density of ligand, the rates of reaction between receptor and ligand, the stiffness of the springs, the response of springs to extension, and the magnitude of hydrodynamic stresses. By varying these parameters, the model can successfully recreate the entire range of expected and observed adhesive phenomena, from completely unencumbered motion, to rolling, to transient attachment, to firm adhesion. Also, the model can provide meaningful statistical measures of adhesion, including the mean and variance in velocity, rate constants for cell attachment and detachment, and the frequency of adhesion. We find a critical modulating parameter of adhesion is the fractional spring slippage, which relates the extension of a bond to its rate of breakage; the higher the slippage, the faster the breakage for the same extension. Changes in the fractional spring slippage can radically change the adhesive behavior of a cell. We show that stiffer springs will only serve to increase adhesion if the fractional slippage remains small. In addition, our simulations emphasize the importance of reaction rates between receptor and ligand, rather than affinity, as being the key determinant of adhesion under flow. These results suggest reaction rates and response to stress of adhesion molecules must be independently measured to understand how adhesion is controlled at the molecular level.     

Mathematical model for the effects of adhesion and mechanics on cell migration speed.

Migration of mammalian blood and tissue cells over adhesive surfaces is apparently mediated by specific reversible reactions between cell membrane adhesion receptors and complementary ligands attached to the substratum. Although in a number of systems these receptors and ligand molecules have been isolated and identified, a theory capable of predicting the effects of their properties on cell migration behavior currently does not exist. We present a simple mathematical model for elucidating the dependence of cell speed on adhesion-receptor/ligand binding and cell mechanical properties. Our model can be applied to propose answers to questions such as: does an optimal adhesiveness exist for cell movement? How might changes in receptor and ligand density and/or affinity affect the rate of migration? Can cell rheological properties influence movement speed? This model incorporates cytoskeletal force generation, cell polarization, and dynamic adhesion as requirements for persistent cell movement. A critical feature is the proposed existence of an asymmetry in some cell adhesion-receptor property, correlated with cell polarity. We consider two major alternative mechanisms underlying this asymmetry: (a) a spatial distribution of adhesion-receptor number due to polarized endocytic trafficking and (b) a spatial variation in adhesion-receptor/ligand bond strength. Applying a viscoelastic-solid model for cell mechanics allows us to represent one-dimensional locomotion with a system of differential equations describing cell deformation and displacement along with adhesion-receptor dynamics. In this paper, we solve these equations under the simplifying assumption that receptor dynamics are at a quasi-steady state relative to cell locomotion. Thus, our results are strictly valid for sufficiently slow cell movement, as typically observed for tissue cells such as fibroblasts. Numerical examples relevant to experimental systems are provided. Our results predict how cell speed might vary with intracellular contractile force, cell rheology, receptor/ligand kinetics, and receptor/ligand number densities. A biphasic dependence is shown to be possible with respect to some of the system parameters, with position of the maxima essentially governed by a balance between transmitted contractile force and adhesiveness. We demonstrate that predictions for the two alternative asymmetry mechanisms can be distinguished and could be experimentally tested using cell populations possessing different adhesion-receptor numbers.     

Cell membrane alignment along adhesive surfaces: contribution of active and passive cell processes

Cell adhesion requires nanometer scale membrane alignment to allow contact between adhesion receptors. Little quantitative information is presently available on this important biological process. Here we present an interference reflection microscopic study of the initial interaction between monocytic THP-1 cells and adhesive surfaces, with concomitant determination of cell deformability, using micropipette aspiration, and adhesiveness, using a laminar flow assay. We report that 1), during the first few minutes after contact, cells form irregular-shaped interaction zones reaching approximately 100 micro m(2) with a margin extension velocity of 0.01-0.02 micro m/s. This happens before the overall cell deformations usually defined as spreading. 2), These interference reflection microscopic-detected zones represent bona fide adhesion inasmuch as cells are not released by hydrodynamic forces. 3), Alignment is markedly decreased but not abolished by microfilament blockade with cytochalasin or even cell fixation with paraformaldehyde. 4), In contrast, exposing cells to hypotonic medium increased the rate of contact extension. 5), Contacts formed in presence of cytochalasin, after paraformaldehyde fixation or in hypotonic medium, were much more regular-shaped than controls and their extension matched cell deformability. 6), None of the aforementioned treatments altered adhesiveness to the surface. It is concluded that adhesive forces and passive membrane deformations are sufficient to generate initial cell alignment to adhesive surfaces, and this process is accelerated by spontaneous cytoskeletally-driven membrane motion.     

A theoretical analysis for the effect of focal contact formation on cell-substrate attachment strength.

For many cell types, growth, differentiation, and motility are dependent on receptor-mediated adhesion to ligand-coated surfaces. Focal contacts are strong, specialized, adhesive connections between cell and substrate in which receptors aggregate and connect extracellular ligand to intracellular cytoskeletal molecules. In this paper, we present a mathematical model to examine how focal contact formation affects cellular adhesive strength. To calculate adhesive strength with and without focal contacts, we use a one-dimensional tape peeling analysis to determine the critical tension necessary to peel the membrane. Receptor-ligand bonds are modeled as adhesive springs. In the absence of focal contacts, we derive analytic expressions for the critical tension at low and high ligand densities and show how membrane morphology affects adhesion. Then, focal contacts are modeled as cytoplasmic nucleation centers which bind adhesion receptors. The extent of adhesive strengthening upon focal contact formation depends on the elastic rigidity of the cytoskeletal connections, which determines the structural integrity of the focal contact itself. We consider two limits to this elasticity, very weak and rigid. Rigid cytoskeletal connections give much greater attachment strengths. The dependence of attachment strength on measurable model parameters is quite different in these two limits, which suggests focal contact structure might be deduced from properly performed adhesion experiments. Finally, we compare our model to the adhesive strengthening response reported for glioma cell adhesion to fibronectin (Lotz et al., 1989. J. Cell Biol. 109:1795-1805). Our model successfully predicts the observed detachment forces at 4 degrees C and yields values for the number of fibronectin receptors per glioma cell and the density of cytoskeletal connection molecules (talin) involved in receptor clusters which are consistent with measurements for other cell types. Comparison of the model with data at 37 degrees C suggests that while cytoskeletal cross-linking and clustering of fibronectin receptors significantly increases adhesion strength, specific glioma cell-substratum attachment sites possess little mechanical rigidity and detach through a peeling mechanism, consistent with the view that these sites of < or = 15 nm cell-substrate separation are precursors to fully formed, elastically rigid focal contacts.     

A response calculus for immobilized T cell receptor ligands

To address the molecular mechanism of T cell receptor (TCR) signaling, we have formulated a model for T cell activation, termed the 2D-affinity model, in which the density of TCR on the T cell surface, the density of ligand on the presenting surface, and their corresponding two-dimensional affinity determine the level of T cell activation. When fitted to T cell responses against purified ligands immobilized on plastic surfaces, the 2D-affinity model adequately simulated changes in cellular activation as a result of varying ligand affinity and ligand density. These observations further demonstrated the importance of receptor cross-linking density in determining TCR signaling. Moreover, it was found that the functional two-dimensional affinity of TCR ligands was affected by the chemical composition of the ligand-presenting surface. This makes it possible that cell-bound TCR ligands, despite their low affinity in solution, are of optimal two-dimensional affinity thereby allowing effective TCR binding under physiological conditions, i.e. at low ligand densities in cellular interfaces.     

Morphology of cell-substratum adhesion

Many cell types modulate growth, differentiation, and motility through changes in cell substrate adhesion, including regulation of focal contact formation. Clustering of cell surface adhesion receptors is an essential early step in the development of focal contacts, and thus may influence cell physiology. In this paper, we present a theoretical framework to examine how cell surface chemistry affects receptor clustering. Our one-dimensional tape-peeling model couples the equations of mechanical equilibrium for a cell membrane with kinetic receptor-ligand binding relations. We considered two distinct model scenarios: Adhesion mediated by multiple receptor-ligand interactions of different length and specific binding of a single receptor type occurs in the presence of van der Waals attraction and nonspecific repulsion. In each case, nonuniform (wave-like) membrane morphologies are observed in certain parameter ranges that support the clustering of adhesion receptors. The formation of these morphologies is described in terms of a balance of membrane stresses; when cell-surface potential as a function of separation distance is symmetric between two potential energy minima, nonuniform morphologies are obtained. Increases in the chemical binding energy between receptor and ligand (e.g., increases in ligand density) or decreases in the membrane rigidity result in smaller wavelengths for nonuniform interfaces. Additionally, we show wave-like geometries appear only when the mechanical compliance of receptor-ligand bonds is within an intermediate range, and examine how the mobility of “repellers”—glycocalyx molecules that exert a nonspecific repulsive force—influences membrane morphology. We find fully mobile repellers always redistribute to prevent nonuniform morphologies.     

Simulation of cell rolling and adhesion on surfaces in shear flow

The adhesion of cells to ligand-coated surfaces in viscous shear flow is an important step in many physiological processes, such as the neutrophil-mediated inflammatory response, lymphocyte homing, and tumor cell metastasis. This article describes a calculational method that allows simulation of the interaction of a single cell with a ligandcoated surface. The cell is idealized as a microvilli-coated hard sphere covered with adhesive springs. The distribution of microvilli on the cell surface, the distribution of receptors on microvilli tips, and the forward and reverse reaction between receptor and ligand are all simulated using random number sampling of appropriate probability functions. The velocity of the cell at each time step in the simulation results from a balance of hydrodynamic, colloidal, and bonding forces; the bonding force is derived by summing the individual contributions of each receptor-ligand tether. The model can simulate the effect of many parameters on adhesion, such as the number of receptors on microvilli tips, the density of ligand, the rates of reaction between receptor and ligand, the stiffness of the springs, the response of springs to extension, and the magnitude of hydrodynamic stresses. By varying these parameters, the model can successfully recreate the entire range of expected and observed adhesive phenomena, from completely unencumbered motion, to rolling, to transient attachment, to firm adhesion. Also, the model can provide meaningful statistical measures of adhesion, including the mean and variance in velocity, rate constants for ceil attachment and detachment, and the frequency of adhesion. We find a critical modulating parameter of adhesion is the fractional spring slippage, which relates the extension of a bond to its rate of breakage; the higher the slippage, the faster the breakage for the same extension. Changes in the fractional spring slippage can radically change the adhesive behavior of a cell. We show that stiffer springs will only serve to increase adhesion if the fractional slippage remains small. In addition, our simulations emphasize the importance of reaction rates between receptor and ligand, rather than affinity, as being the key determinant of adhesion under flow. These results suggest reaction rates and response to stress of adhesion molecules must be independently measured to understand how adhesion is controlled at the molecular level.     

Morphology of cell-substratum adhesion. Influence of receptor heterogeneity and nonspecific forces.

Many cell types modulate growth, differentiation, and motility through changes in cell substrate adhesion, including regulation of focal contact formation. Clustering of cell surface adhesion receptors is an essential early step in the development of focal contacts, and thus may influence cell physiology. In this paper, we present a theoretical framework to examine how cell surface chemistry affects receptor clustering. Our one-dimensional tape-peeling model couples the equations of mechanical equilibrium for a cell membrane with kinetic receptor-ligand binding relations. We considered two distinct model scenarios: Adhesion mediated by multiple receptor-ligand interactions of different length and specific binding of a single receptor type occurs in the presence of van der Waals attraction and nonspecific repulsion. In each case, nonuniform (wave-like) membrane morphologies are observed in certain parameter ranges that support the clustering of adhesion receptors. The formation of these morphologies is described in terms of a balance of membrane stresses; when cell-surface potential as a function of separation distance is symmetric between two potential energy minima, nonuniform morphologies are obtained. Increases in the chemical binding energy between receptor and ligand (e.g., increases in ligand density) or decreases in the membrane rigidity result in smaller wavelengths for nonuniform interfaces. Additionally, we show wave-like geometries appear only when the mechanical compliance of receptor-ligand bonds is within an intermediate range, and examine how the mobility of "repellers"--glycocalyx molecules that exert a nonspecific repulsive force--influences membrane morphology. We find fully mobile repellers always redistribute to prevent nonuniform morphologies.     

Simulation of cell rolling and adhesion on surfaces in shear flow: general results and analysis of selectin-mediated neutrophil adhesion.

The receptor-mediated adhesion of cells to ligand-coated surfaces in viscous shear flow is an important step in many physiological processes, such as the neutrophil-mediated inflammatory response, lymphocyte homing, and tumor cell metastasis. This paper describes a calculational method which simulates the interaction of a single cell with a ligand-coated surface under flow. The cell is idealized as a microvilli-coated hard sphere covered with adhesive springs. The distribution of microvilli on the cell surface, the distribution of receptors on microvilli tips, and the forward and reverse reaction between receptor and ligand are all simulated using random number sampling of appropriate probability functions. The velocity of the cell at each time step in the simulation results from a balance of hydrodynamic, colloidal and bonding forces; the bonding force is derived by summing the individual contributions of each receptor-ligand tether. The model can simulate the effect of many parameters on adhesion, such as the number of receptors on microvilli tips, the density of ligand, the rates of reaction between receptor and ligand, the stiffness of the resulting receptor-ligand springs, the response of springs to strain, and the magnitude of the bulk hydrodynamic stresses. The model can successfully recreate the entire range of expected and observed adhesive phenomena, from completely unencumbered motion, to rolling, to transient attachment, to firm adhesion. Also, the method can generate meaningful statistical measures of adhesion, including the mean and variance in velocity, rate constants for cell attachment and detachment, and the frequency of adhesion. We find a critical modulating parameter of adhesion is the fractional spring slippage, which relates the strain of a bond to its rate of breakage; the higher the slippage, the faster the breakage for the same strain. Our analysis of neutrophil adhesive behavior on selectin-coated (CD62-coated) surfaces in viscous shear flow reported by Lawrence and Springer (Lawrence, M.B., and T.A. Springer 1991. Cell. 65:859-874) shows the fractional spring slippage of the CD62-LECAM-1 bond is likely below 0.01. We conclude the unique ability of this selectin bond to cause neutrophil rolling under flow is a result of its unique response to strain. Furthermore, our model can successfully recreate data on neutrophil rolling as function of CD62 surface density.     

Visualization of CD2 interaction with LFA-3 and determination of the two-dimensional dissociation constant for adhesion receptors in a contact area

Many adhesion receptors have high three-dimensional dissociation constants (Kd) for counter-receptors compared to the KdS of receptors for soluble extracellular ligands such as cytokines and hormones. Interaction of the T lymphocyte adhesion receptor CD2 with its counter- receptor, LFA-3, has a high solution-phase Kd (16 microM at 37 degrees C), yet the CD2/LFA-3 interaction serves as an effective adhesion mechanism. We have studied the interaction of CD2 with LFA-3 in the contact area between Jurkat T lymphoblasts and planar phospholipid bilayers containing purified, fluorescently labeled LFA-3. Redistribution and lateral mobility of LFA-3 were measured in contact areas as functions of the initial LFA-3 surface density and of time after contact of the cells with the bilayers. LFA-3 accumulated at sites of contact with a half-time of approximately 15 min, consistent with the previously determined kinetics of adhesion strengthening. The two-dimensional Kd for the CD2/LFA-3 interaction was 21 molecules/microns 2, which is lower than the surface densities of CD2 on T cells and LFA-3 on most target or stimulator cells. Thus, formation of CD2/LFA-3 complexes should be highly favored in physiological interactions. Comparison of the two-dimensional (membrane- bound) and three-dimensional (solution-phase) KdS suggest that cell- cell contact favors CD2/LFA-3 interaction to a greater extent than that predicted by the three-dimensional Kd and the intermembrane distance at the site of contact. LFA-3 molecules in the contact site were capable of lateral diffusion in the plane of the phospholipid bilayer and did not appear to be irreversibly trapped in the contact area, consistent with a rapid off-rate. These data provide insights into the function of low affinity interactions in adhesion.     

The relative importance of topography and RGD ligand density for endothelial cell adhesion

The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×10(2)-6×10(11) RGD/mm(2). We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×10(5) RGD/mm(2) on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×10(8) RGD/mm(2) irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry.     

Functional incorporation of integrins into solid supported membranes on ultrathin films of cellulose: impact on adhesion

Biomimetic models of cell surfaces were designed to study the physical basis of cell adhesion. Vesicles bearing reconstituted blood platelet integrin receptors alpha(IIb)beta(3) were spread on ultrathin films of cellulose, forming continuous supported membranes. One fraction of the integrin receptors, which were facing their extracellular domain toward the aqueous phase, were mobile, exhibiting a diffusion constant of 0.6 micro m(2) s(-1). The functionality of receptors on bare glass and on cellulose cushions was compared by measuring adhesion strength to giant vesicles. The vesicles contained lipid-coupled cyclic hexapeptides that are specifically recognized by integrin alpha(IIb)beta(3). To mimic the steric repulsion forces of the cell glycocalix, lipids with polyethylene glycol headgroups were incorporated into the vesicles. The free adhesion energy per unit area deltag(ad) was determined by micro-interferometric analysis of the vesicle's contour near the membrane surface in terms of the equilibrium of the elastic forces. By accounting for the reduction of the adhesion strength by the repellers and from measuring the density of receptors one could estimate the specific receptor ligand binding energy. We estimate the receptor-ligand binding energy to be 10 k(B)T under bioanalogue conditions.     

Hepatocyte adhesion to carbohydrate-derivatized surfaces. II. Regulation of cytoskeletal organization and cell morphology

Rat hepatic lectins mediate adhesion of isolated rat hepatocytes to synthetic surfaces derivatized with galactosides. Initial weak adhesion is followed by rapid adhesion strengthening. After hepatocytes contact galactose-derivatized gels, the hepatic lectins move rapidly into an inaccessible patch at the adhesive surface (Weisz, O. A., and R. L. Schnaar. 1991. J. Cell Biol. 115:485-493). Hepatic lectin patching, which occurs both at 37 degrees C and 4 degrees C, is not responsible for adhesion strengthening, which does not occur at 4 degrees C. Of various cytoskeletal and metabolic perturbants tested, only a combination of hyperosmotic medium, colchicine, and cytochalasin caused a marked (72%) reduction of adhesion strengthening (without reducing weak cell adhesion). Clathrin and actin were readily detected in the adhesive patch by immunofluorescence microscopy. Rat hepatocytes also adhered avidly to surfaces derivatized with asialofetuin, a high-affinity ligand for the rat hepatic lectins. However, hepatic lectin molecules did not migrate into a patch on the asialofetuin-derivatized surface, suggesting that hepatic lectin-asialofetuin binding may have resulted in the rapid formation of a ring of essentially irreversibly adherent receptors that prevented diffusion of additional lectin molecules into the contact site. The cells were unable to increase their adhesive contact area by flattening onto the derivatized surface. Treatment of cells with cytochalasin, however, did result in an increase in the size of the contact area. Cells adhering to surfaces derivatized with an adhesion-promoting peptide (containing an arg-gly-asp sequence) had larger contact areas than those adhering to galactoside-derivatized surfaces. A model is proposed in which carbohydrate-mediated adhesion causes specific reorganization of cytoskeletal components, leading to strengthened adhesion and a characteristic spherical cell morphology.     

Influence of type I collagen surface density on fibroblast spreading, motility, and contractility

We examine the relationships of three variables (projected area, migration speed, and traction force) at various type I collagen surface densities in a population of fibroblasts. We observe that cell area is initially an increasing function of ligand density, but that above a certain transition level, increases in surface collagen cause cell area to decline. The threshold collagen density that separates these two qualitatively different regimes, approximately 160 molecules/ microm(2), is approximately equal to the cell surface density of integrin molecules. These results suggest a model in which collagen density induces a qualitative transition in the fundamental way that fibroblasts interact with the substrate. At low density, the availability of collagen binding sites is limiting and the cells simply try to flatten as much as possible by pulling on the few available sites as hard as they can. The force per bond under these conditions approaches 100 pN, approximately equal to the force required for rupture of integrin-peptide bonds. In contrast, at high collagen density adhesion, traction force and motility are limited by the availability of free integrins on the cell surface since so many of these receptors are bound to the surface ligand and the force per bond is very low.     

Neutrophil adhesive contact dependence on impingement force

Neutrophil capture and recruitment from the circulation requires the formation of specific receptor/ligand bonds under hydrodynamic forces. In the present study we examine bond formation between beta2-integrins on neutrophils and immobilized ICAM-1 while using micropipettes to control the force of contact between the cell and substrate. Magnesium was used to induce the high affinity conformation of the integrins, and bond formation was assessed by measuring the probability of adhesion during repeated contacts. Increasing the impingement force caused an increase in the contact area and led to a proportional increase in adhesion probability (from approximately 20 to 50%) over the range of forces tested (50-350 pN). In addition, different-sized beads were used to change the force per unit area in the contact zone (contact stress). We conclude that for a given contact stress, the rate of bond formation increases linearly with contact area, but that increasing contact stress results in higher intrinsic rates of bond formation.