T cells use microvilli to search the surfaces of antigen-presenting cells for antigenic ligands. The active motion of scanning microvilli provides a force-generating mechanism that is intriguing in light of single-molecule experiments showing that applied forces on stimulatory receptor-ligand bonds increase their lifetimes (catch-bond behavior). In this work, we introduce a theoretical framework to explore the motion of a microvillus tip above an antigen-presenting surface when receptors on the tip stochastically bind to ligands on the surface and dissociate from them in a force-dependent manner. Forces on receptor-ligand bonds impact the motion of the microvillus, leading to feedback between binding and microvillar motion. We use computer simulations to show that the average microvillus velocity varies in a ligand-dependent manner, that catch bonds generate responses in which some microvilli almost completely stop while others move with a broad distribution of velocities, and that the frequency of stopping depends on the concentration of stimulatory ligands. Typically, a small number of catch bonds initially immobilize the microvillus, after which additional bonds accumulate and increase the cumulative receptor-engagement time. Our results demonstrate that catch bonds can selectively slow and stabilize scanning microvilli, suggesting a physical mechanism that may contribute to antigen discrimination by T cells.

Misregulation of the signaling axis formed by EphA2 and its ligand, ephrinA1, causes aberrant cell-cell contacts and leads to cellular transformation and malignancy in cancer. However, the activation mechanism of EphA2 is poorly understood. Also, solid tumors exhibit the Warburg effect, which results in an acidic extracellular microenvironment. Taking advantage of this property, we have used a novel approach to design TYPE7 (transmembrane tyrosine kinase peptide for Eph), a membrane peptide that targets EphA2. TYPE7 is pH-responsive, a characteristic which provides solubility at neutral pH, but triggers membrane insertion in acidic conditions. Decreasing the pH leads to increased partitioning and insertion of the peptide into the membrane. TYPE7 has a membrane insertion pH50 of 6.2 (the pH point for 50% insertion). However, in the presence of the TM/JM domains of EphA2, the pH50 of TYPE7 increases to 6.9. This effect does not occur in the presence of an unrelated TM domain, indicating specific interaction between TYPE7 and EphA2. Using FRET, we have determined the free energy of dimerization (ΔG) of the TM/JM domains of EphA2; we assess how the presence of TYPE7 alters this value. The activation of EphA2 by TYPE7 shows important differences compared with activation by a cross-linked version of the ephrinA1 (EA1) ligand. Interestingly, TYPE7 activation does not involve phosphorylation of juxtamembrane residues Y588 and Y594, suggesting a novel mechanism to release juxtamembrane inhibition of the EphA2 kinase domain. Furthermore, while TYPE7 and EA1 inhibit cell migration with similar efficiency, EA1 induces formation of larger EphA2 oligomers than TYPE7. This suggests formation of smaller oligomers might be sufficient to fully activate EphA2. These results shed new light on the activation mechanism of EphA2.

Catch bonds are characterized by average lifetimes that initially increase with increasing tensile force. Recently, they have been implicated in T cell activation, where small numbers of antigenic receptor-ligand bonds at a cell-cell interface can stimulate a T cell. Here, we use computational methods to investigate small numbers of bonds at the interface between two membranes. We characterize the time-dependent forces on the bonds in response to changes in the membrane shape and the organization of other surface molecules. We then determine the distributions of bond lifetimes using recent force-dependent lifetime data for T cell receptors bound to various ligands. Strong agonists, which exhibit catch bond behavior, are markedly more likely to remain intact than an antagonist whose average lifetime decreases with increasing force. Thermal fluctuations of the membrane shape enhance the decay of the average force on a bond, but also lead to fluctuations of the force. These fluctuations promote bond rupture, but the effect is buffered by catch bonds. When more than one bond is present, the bonds experience reduced average forces that depend on their relative positions, leading to changes in bond lifetimes. Our results highlight the importance of force-dependent binding kinetics when bonds experience time-dependent and fluctuating forces, as well as potential consequences of collective bond behavior relevant to T cell activation.

Receptors on cell surfaces commonly engage membrane-presented ligands on other cells. An important example is the T cell receptor (TCR), which binds to ligands (pMHC) on antigen-presenting cells to stimulate T cell activation and the adaptive immune system. Recent experiments have shown that stimulatory TCR-pMHC bonds behave like catch bonds, with the average bond lifetime initially increasing with an increasing tensile force. Because T cells are initially stimulated by small numbers of TCR-pMHC complexes that experience a variety of forces, it is important to understand the behavior of small numbers of catch bonds in the presence of other surface molecules (SMs) and thermal fluctuations. To address this problem, we use computational methods to investigate the forces experienced by TCR-pMHC bonds at an intermembrane junction. We describe the energetics of the intermembrane junction with a modified Helfrich Hamiltonian and the SM concentration profile with an advection-diffusion PDE. We use a hybrid deterministic-stochastic algorithm to describe the dynamics, with the membrane locally equilibrating as the SM concentration profile evolves. Changes in membrane shape and SM organization lead to a time-dependent tension on the TCR-pMHC bonds, and fluctuations in the membrane shape lead to fluctuating forces experienced by the bonds. Using an experimentally parameterized model of catch bond kinetics, we construct bond rupture distributions for several different pMHC ligands. We show that the catch bond nature of stimulatory bonds enhances the survival probability in relation to conventional slip bonds in the presence of thermal fluctuations, and hence the catch bonds buffer against force fluctuations. Our computational framework provides a way to systematically study the binding of multiple bonds at generic cell-cell interfaces, and we discuss our results in terms of potential consequences for early T cell signaling.

Positive feedback is a common feature in signal transduction networks and can lead to phenomena such as bistability and signal propagation by domain growth. Physical features of the cellular environment, such as spatial confinement and the mobility of proteins, play important but inadequately understood roles in shaping the behavior of signaling networks. Here, we use stochastic, spatially resolved kinetic Monte Carlo simulations to explore a positive feedback network as a function of system size, system shape, and mobility of molecules. We show that these physical properties can markedly alter characteristics of bistability and stochastic switching when compared with well-mixed simulations. Notably, systems of equal volume but different shapes can exhibit qualitatively different behaviors under otherwise identical conditions. We show that stochastic switching to a state maintained by positive feedback occurs by cluster formation and growth. Additionally, the frequency at which switching occurs depends nontrivially on the diffusion coefficient, which can promote or suppress switching relative to the well-mixed limit. Taken together, the results provide a framework for understanding how confinement and protein mobility influence emergent features of the positive feedback network by modulating molecular concentrations, diffusion-influenced rate parameters, and spatiotemporal correlations between molecules.