Differential segregation in a cell-cell contact interface: the dynamics of the immunological synapse

Receptor-ligand couples in the cell-cell contact interface between a T cell and an antigen-presenting cell form distinct geometric patterns and undergo spatial rearrangement within the contact interface. Spatial segregation of the antigen and adhesion receptors occurs within seconds of contact, central aggregation of the antigen receptor then occurring over 1-5 min. This structure, called the immunological synapse, is becoming a paradigm for localized signaling. However, the mechanisms driving its formation, in particular spatial segregation, are currently not understood. With a reaction diffusion model incorporating thermodynamics, elasticity, and reaction kinetics, we examine the hypothesis that differing bond lengths (extracellular domain size) is the driving force behind molecular segregation. We derive two key conditions necessary for segregation: a thermodynamic criterion on the effective bond elasticity and a requirement for the seeding/nucleation of domains. Domains have a minimum length scale and will only spontaneously coalesce/aggregate if the contact area is small or the membrane relaxation distance large. Otherwise, differential attachment of receptors to the cytoskeleton is required for central aggregation. Our analysis indicates that differential bond lengths have a significant effect on synapse dynamics, i.e., there is a significant contribution to the free energy of the interaction, suggesting that segregation by differential bond length is important in cell-cell contact interfaces and the immunological synapse.     

Selectin-like kinetics and biomechanics promote rapid platelet adhesion in flow: the GPIb(alpha)-vWF tether bond

The ability of platelets to tether to and translocate on injured vascular endothelium relies on the interaction between the platelet glycoprotein receptor Ib alpha (GPIb(alpha)) and the A1 domain of von Willebrand factor (vWF-A1). To date, limited information exists on the kinetics that govern platelet interactions with vWF in hemodynamic flow. We now report that the GPIb(alpha)-vWF-A1 tether bond displays similar kinetic attributes as the selectins including: 1) the requirement for a critical level of hydrodynamic flow to initiate adhesion, 2) short-lived tethering events at sites of vascular injury in vivo, and 3) a fast intrinsic dissociation rate constant, k(0)(off) (3.45 +/- 0.37 s(-1)). Values for k(off), as determined by pause time analysis of transient capture/release events, were also found to vary exponentially (4.2 +/- 0.8 s(-1) to 7.3 +/- 0.4 s(-1)) as a function of the force applied to the bond (from 36 to 217 pN). The biological importance of rapid bond dissociation in platelet adhesion is demonstrated by kinetic characterization of the A1 domain mutation, I546V that is associated with type 2B von Willebrand disease (vWD), a bleeding disorder that is due to the spontaneous binding of plasma vWF to circulating platelets. This mutation resulted in a loss of the shear threshold phenomenon, a approximately sixfold reduction in k(off), but no significant alteration in the ability of the tether bond to resist shear-induced forces. Thus, flow dependent adhesion and rapid and force-dependent kinetic properties are the predominant features of the GPIb(alpha)-vWF-A1 tether bond that in part may explain the preferential binding of platelets to vWF at sites of vascular injury, the lack of spontaneous platelet aggregation in circulating blood, and a mechanism to limit thrombus formation.     

A numerical analysis of forces exerted by laminar flow on spreading cells in a parallel plate flow chamber assay

Exposure of spreading anchorage-dependent cells to laminar flow is a common technique to measure the strength of cell adhesion. Since cells protrude into the flow stream, the force exerted by the fluid on the cells is a function of cell shape. To assess the relationship between cell shape and the hydrodynamic force on adherent cells, we obtained numerical solutions of the velocity and stress fields around bovine aortic endothelial cells during various stages of spreading and calculated the force required to detach the cells. Morphometric parameters were obtained from light and scanning electron microscopy measurements. Cells were assumed to have a constant volume, but the surface area increased during spreading until the membrane was stretched taut. Two-dimensional models of steady flow were generated using the software packages ANSYS (mesh generation) and FIDAP (problem solution). The validity of the numerical results was tested by comparison with published results for a semicircle in contact with the surface. The drag force and torque were greatest for round cells making initial contact with the surface. During spreading, the drag force and torque declined by factors of 2 and 20, respectively. The calculated forces and moments were used in adhesion models to predict the wall shear stress at which the cells detached. Based upon published values for the bond force and receptor number, round cells should detach at shear stresses between 2.5 and 6 dyn/cm(2), whereas substantially higher stresses are needed to detach spreading and fully spread cells. Results from the simulations indicate that (1) the drag force varies little with cell shape whereas the torque is very sensitive to cell shape, and (2) the increase in the strength of adhesion during spreading is due to increased contact area and receptor densities within the contact area. (c) 1993 John Wiley & Sons, Inc.     

Beta-1 integrin-mediated adhesion may be initiated by multiple incomplete bonds, thus accounting for the functional importance of receptor clustering

The regulation of cell integrin receptors involves modulation of membrane expression, shift between different affinity states, and topographical redistribution on the cell membrane. Here we attempted to assess quantitatively the functional importance of receptor clustering. We studied beta-1 integrin-mediated attachment of THP-1 cells to fibronectin-coated surfaces under low shear flow. Cells displayed multiple binding events with a half-life of the order of 1 s. The duration of binding events after the first second after arrest was quantitatively accounted for by a model assuming the existence of a short-time intermediate binding state with 3.6 s(-1) dissociation rate and 1.3 s(-1) transition frequency toward a more stable state. Cell binding to surfaces coated with lower fibronectin densities was concluded to be mediated by single molecular interactions, whereas multiple bonds were formed <1 s after contact with higher fibronectin surface densities. Cell treatment with microfilament inhibitors or a neutral antiintegrin antibody decreased bond number without changing aforementioned kinetic parameters whereas a function enhancing antibody increased the rate of bond formation and/or the lifetime of intermediate state. Receptor aggregation was induced by treating cells with neutral antiintegrin antibody and antiimmunoglobulin antibodies. A semiquantitative confocal microscopy study suggested that this treatment increased between 40% and 100% the average number of integrin receptors located in a volume of approximately 0.045 microm(3) surrounding each integrin. This aggregation induced up to 2.7-fold increase of the average number of bonds. Flow cytometric analysis of fluorescent ligand binding showed that THP-1 cells displayed low-affinity beta-1 integrins with a dissociation constant in the micromolar range. It is concluded that the initial step of cell adhesion was mediated by multiple incomplete bonds rather than a single equilibrium-state ligand receptor association. This interpretation accounts for the functional importance of integrin clustering.     

Nano-to-micro scale dynamics of P-selectin detachment from leukocyte interfaces. III. Numerical simulation of tethering under flow

Transient capture of cells or model microspheres from flow over substrates sparsely coated with adhesive ligands has provided significant insight into the unbinding kinetics of leukocyte:endothelium adhesion complexes under external force. Whenever a cell is stopped by a point attachment, the full hydrodynamic load is applied to the adhesion site within an exceptionally short time-less than the reciprocal of the hydrodynamic shear rate (e.g., typically <0.01 s). The decay in numbers of cells or beads that remain attached to a surface has been used as a measure of the kinetics of molecular bond dissociation under constant force, revealing a modest increase in detachment rate at growing applied shear stresses. On the other hand, when detached under steady ramps of force with mechanical probes (e.g., the atomic force microscope and biomembrane force probe), P-selectin:PSGL-1 adhesion bonds break at rates that increase enormously under rising force, yielding 100-fold faster off rates at force levels comparable to high shear. The comparatively weak effect of force on tether survival in flow chamber experiments could be explained by a possible partition of the load amongst several bonds. However, a comprehensive understanding of the difference in kinetic behavior requires us to also inspect other factors affecting the dynamics of attachment-force buildup, such as the interfacial compliance of all linkages supporting the adhesion complex. Here, combining the mechanical properties of the leukocyte interface measured in probe tests with single-bond kinetics and the kinetics of cytoskeletal dissociation, we show that for the leukocyte adhesion complex P-selectin:PSGL-1, a detailed adhesive dynamics simulation accurately reproduces the tethering behavior of cells observed in flow chambers. Surprisingly, a mixture of 10% single bonds and 90% dimeric bonds is sufficient to fully match the data of the P-selectin:PSGL-1 experiments, with the calculated decay in fraction of attached cells still appearing exponential.     

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.     

Cytoplasm dynamics and cell motion: two-phase flow models

The motion of amoeboid cells is characterized by cytoplasmic streaming and by membrane protrusions and retractions which occur even in the absence of interactions with a substratum. Cell translocation requires, in addition, a transmission mechanism wherein the power produced by the cytoplasmic engine is applied to the substratum in a highly controlled fashion through specific adhesion proteins. Here we present a simple mechano-chemical model that tries to capture the physical essence of these complex biomolecular processes. Our model is based on the continuum equations for a viscous and reactive two-phase fluid model with moving boundaries, and on force balance equations that average the stochastic interactions between actin polymers and membrane proteins. In this paper we present a new derivation and analysis of these equations based on minimization of a power functional. This derivation also leads to a clear formulation and classification of the kinds of boundary conditions that should be specified at free surfaces and at the sites of interaction of the cell and the substratum. Numerical simulations of a one-dimensional lamella reveal that even this extremely simplified model is capable of producing several typical features of cell motility. These include periodic 'ruffle' formation, protrusion-retraction cycles, centripetal flow and cell-substratum traction forces.     

A dynamical model for receptor-mediated cell adhesion to surfaces in viscous shear flow

We present a dynamical model for receptor-mediated cell adhesion to surfaces in viscous shear flow when the surfaces are coated with ligand molecules complementary to receptors in the cell membrane. This model 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 for a system of nonlinear ordinary differential equations that 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, including the effects of temperature and receptor mobility on adhesiveness, 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.     

Synthesis and activity of a cyclo-heptapeptide containing Lys-Gly-Asp-sequence as a novel anti-platelet agent

Linear hepta-peptide Cys-Lys-Gly-Asp-Trp-Asp-Cys was synthesized first and then disulfide bond was formed between the Cys1 and Cys7 to develop cyclo-heptapeptide containing Lys-Gly-Asp-sequence. Structural simulation showed that Lys-Gly-Asp-motif (KDG) displayed functional conformation. The cyclo-heptapeptide exhibited potent anti-platelet aggregation activity based on specific recognition and interaction with the GPIIb-IIIa receptor on platelet cell membrane. The specific and potent anti-platelet activity makes the KGD-containing cyclo-heptapeptide a possible therapeutic agent.     

Cell-cell conjugation. Transient analysis and experimental implications.

In the present study we investigate the transient conjugation of cell pairs by using a mathematical model. Macromolecules responsible for adhesion (bonds) are assumed to exist in two reversible states, attached and unattached, and exert a force elastic in nature only when they cross-link the two cell surfaces (attached state). Bonds form a link between the two cell surfaces only in the attached form. The unattached bridges are assumed laterally mobile in the plane of the cell membrane. Lateral mobility of attached bonds may be limited by structures on the undersurface of the cell membrane. Using this model we show that the bond density distribution between a cytotoxic T-cell (F-1) and a cancer cell (JY:HLA-A2-B7-DR4, W6) approaches equilibrium within 10 min, the incubation period used in experiments by Sung, K.L.P., L.A. Sung, M. Crimmins, S.J. Burakoff, and S. Chien (1986. Science [Wash. DC]. 234:1405-1408). If the diffusion coefficient of attached bonds is set equal to zero in the computations the model predictions indicate accumulation of bonds at the edge of conjugation. This prediction is consistent with present experimental data on lectin-induced red blood cell aggregation (Vayo, M., R. Skalak, P. Brunn, S. Usami, and S. Chien. 1987. Fed. Proc. 46:1043). It is concluded that significant features of micromanipulation data on specific adhesion can be explained by the diffusivity properties of bonds responsible for adhesion.     

Exact solutions to a spatially extended model of kinase-receptor interaction

B and Mast cells are activated by the aggregation of the immune receptors. Motivated by this phenomena we consider a simple spatially extended model of mutual interaction of kinases and membrane receptors. It is assumed that kinase activates membrane receptors and in turn the kinase molecules bound to the active receptors are activated by transphosphorylation. Such a type of interaction implies positive feedback and may lead to bistability. In this study we apply the Steklov eigenproblem theory to analyze the linearized model and find exact solutions in the case of non-uniformly distributed membrane receptors. This approach allows us to determine the critical value of receptor dephosphorylation rate at which cell activation (by arbitrary small perturbation of the inactive state) is possible. We found that cell sensitivity grows with decreasing kinase diffusion and increasing anisotropy of the receptor distribution. Moreover, these two effects are cooperating. We showed that the cell activity can be abruptly triggered by the formation of the receptor aggregate. Since the considered activation mechanism is not based on receptor crosslinking by polyvalent antigens, the proposed model can also explain B cell activation due to receptor aggregation following binding of monovalent antigens presented on the antigen presenting cell.     

Weak effect of membrane diffusion on the rate of receptor accumulation at adhesive contacts

To assess if membrane diffusion could affect the kinetics of receptor recruitment at adhesive contacts, we transfected neurons with green fluorescent protein-tagged immunoglobin cell adhesion molecules of varying length (25-180 kD), and measured the lateral mobility of single quantum dots bound to those receptors at the cell surface. The diffusion coefficient varied within a physiological range (0.1-0.5 microm(2)/s), and was inversely proportional to the size of the receptor. We then triggered adhesive contact formation by placing anti-green fluorescent protein-coated microspheres on growth cones using optical tweezers, and measured surface receptor recruitment around microspheres by time-lapse fluorescence imaging. The accumulation rate was rather insensitive to the type of receptor, suggesting that the long-range membrane diffusion of immunoglobin cell adhesion molecules is not a limiting step in the initiation of neuronal contacts.     

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.     

Adhesion of leukocytes under oscillating stagnation point conditions: a numerical study

Leukocyte recruitment from blood to the endothelium plays an important role in atherosclerotic plaque formation. Cells show a primary and secondary adhesive process with primary bonds responsible for capture and rolling and secondary bonds for arrest. Our objective was to investigate the role played by this process on the adhesion of leukocytes in complex flow. Cells were modelled as rigid spheres with spring like adhesion molecules which formed bonds with endothelial receptors. Models of bond kinetics and Newton's laws of motion were solved numerically to determine cell motion. Fluid force was obtained from the local shear rate obtained from a CFD simulation of the flow over a backward facing step.In stagnation point flow the shear rate near the stagnation point has a large gradient such that adherent cells in this region roll to a high shear region preventing permanent adhesion. This is enhanced if a small time dependent perturbation is imposed upon the stagnation point. For lower shear rates the cell rolling velocity may be such that secondary bonds have time to form. These bonds resist the lower fluid forces and consequently there is a relatively large permanent adhesion region.     

Creation of intercellular bonds by anchoring protein ligands to membranes using the diphtheria toxin T domain

We describe the creation of cell adhesion mediated by cell surface engineering. The Flt3-ligand was fused to a membrane anchor made of the diphtheria toxin translocation domain. The fusion protein was attached to the surface of a cell by an acid pulse. Contact with another cell expressing the receptor Flt3 lead to its activation. This activity involved direct cell-cell contact. A mean force of 20 nN was needed to separate functionalized cells after 5 min of contact. Overall, we showed that it is possible to promote specific cell-cell adhesion by attaching protein ligands at the surface of cells.     

Minimum energy analysis of membrane deformation applied to pipet aspiration and surface adhesion of red blood cells.

An experimental procedure is demonstrated which can be used to determine the interfacial free energy density for red cell membrane adhesion and membrane elastic properties. The experiment involves micropipet aspiration of a flaccid red blood cell and manipulation of the cell proximal to a surface where adhesion occurs. A minimum free energy method is developed to model the equilibrium contour of unsupported membrane regions and to evaluate the partial derivatives of the total free energy, which correspond to the micropipet suction force and the interfacial free energy density of adhesion. It is shown that the bending elasticity of the red cell membrane does not contribute significantly to the pressure required to aspirate a flaccid red cell. Based on experimental evidence, the upper bound for the bending or curvature elastic modulus of the red cell membranes is 10-12 ergs (dyn-cm). Analysis of the adhesion experiment shows that interfacial free energy densities for red cell adhesion can be measured from a lower limit of 10-4 ergs/cm2 to an upper limit established by the membrane tension for lysis of 5-10 ergs/cm2.     

The activation of cell aggregation by phosphorylation in Dictyostelium discoideum

Single amoebae of D. discoideum are phosphorylated in the presence of external ATP. Phosphorylation is catalyzed by a cAMP independent cell membrane bound protein kinase. As a result of phosphorylation cell aggregation is induced and the chemotactic sensitivity of the amoebae to a cAMP gradient decreased. Cell membrane phosphorylation may be involved in triggering cell aggregation in vivo. The fact that the number of free phosphorylable sites per cell decreases at the onset of aggregation gives support to this hypothesis. The existence of a plasma membrane bound phosphoprotein phosphatase suggests a possible regulator role for this enzyme on the phosphorylation of the amoebae. Finally, ATP inhibits intercellular contact sites outside the aggregation center. Despite this inhibiting effect on cell adhesiveness, amoebal movement toward an aggregation center maintains its normal periodicity.