Timo Bihr

Switching from Ultraweak to Strong Adhesion

Nature switches from weak to strong adhesion at the cellular level to spectacular effect – for example, for incredibly sensitive recognition and decisive action during immune response. [ 1 ] If realized in an artifi cial system, such a switching could one day be harnessed as a powerful tool to manipulate weakly interacting objects. The fi rst step towards realizing such a system involves understanding how to create and detect ultraweak adhesion and how to then switch-on a strong interaction. So far, in the context of model membranes, weak adhesion has been achieved only with a ligand-receptor of intrinsically low binding affi nity. [ 2 ] Whatever, the intrinsic strength of the bonds, so far they were usually found to be arranged in compact stable domains. [ 3 ] Here, we present experiments and simulations that indicate how to create and detect ultraweak adhesion in the context of fl uid two dimensional membranes interacting via specifi c ligand/receptor bonds. Thus, specifi c adhesion is mediated by transient domains consisting of sparsely distributed bonds. Amazingly, we demonstrate that the avidin/biotin pair – famous for forming the strongest receptor/ligand bond known in nature, mediates ultraweak adhesion under suitable circumstances. This choice of binders allows us to switch on strong binding once sensitive detection is achieved – without resorting to a second binding pair – something not possible with intrinsically weak binders. However, this goal necessitates an appropriate design strategy elaborated below. The in vitro system consists of two membranes: a solid supported lipid bilayer (SLB) and the freely fl uctuating membrane of a giant unillamelar vesicle (GUV). A GUV is a two

Association rates of membrane-coupled cell adhesion molecules.

Thus far, understanding how the confined cellular environment affects the lifetime of bonds, as well as the extraction of complexation rates, has been a major challenge in studies of cell adhesion. Based on a theoretical description of the growth curves of adhesion domains, we present a new (to our knowledge) method to measure the association rate k(on) of ligand-receptor pairs incorporated into lipid membranes. As a proof of principle, we apply this method to several systems. We find that the k(on) for the interaction of biotin with neutravidin is larger than that for integrin binding to RGD or sialyl Lewis(x) to E-selectin. Furthermore, we find k(on) to be enhanced by membrane fluctuations that increase the probability for encounters between the binders. The opposite effect on k(on) could be attributed to the presence of repulsive polymers that mimic the glycocalyx, which points to two potential mechanisms for controlling the speed of protein complexation during the cell recognition process.

Two intertwined facets of adherent membranes: membrane roughness and correlations between ligand–receptors bonds

We study equilibrium fluctuations of adherent membranes by means of Langevin simulations in the case when the interaction of the membrane with the substrate is twofold: a non-specific homogeneous harmonic potential is placed at large distances, whereas discrete ligand–receptor interactions occur at short distances from the flat substrate. We analyze the correlations between neighboring ligand–receptor bonds in a regime of relatively strong membrane fluctuations. By comparison with the random distribution of bonds, we find that the correlations between the bonds are always positive, suggesting spontaneous formation of domains. The equilibrium roughness of the membrane is then determined by fluctuations in the number density of bonds within the domains. Furthermore, we show that the excess number of bonds arising due to correlations and the instantaneous roughness of the membrane both follow master curves that depend only on the instantaneous bond density and not on the intrinsic binding strength of the ligand–receptor pair. The master curves show identical trends, further corroborating the link between membrane roughness and bond correlations.

Coexistence of dilute and densely packed domains of ligand-receptor bonds in membrane adhesion

We analyze the stability of micro-domains of ligand-receptor bonds that mediate the adhesion of biological model membranes. After evaluating the effects of membrane fluctuations on the binding affinity of a single bond, we characterize the organization of bonds within the domains by theoretical means. In a large range of parameters, we find the commonly suggested dense packing to be separated by a free energy barrier from a regime in which bonds are sparsely distributed. If bonds are mobile, a coexistence of the two regimes should emerge, which agrees with recent experimental observations.

Signature of a Nonharmonic Potential as Revealed from a Consistent Shape and Fluctuation Analysis of an Adherent Membrane

The interaction of fluid membranes with a scaffold, which can be a planar surface or a more complex structure, is intrinsic to a number of systems - from artificial supported bilayers and vesicles to cellular membranes. In principle, these interactions can be either discrete and protein mediated, or continuous. In the latter case, they emerge from ubiquitous intrinsic surface interaction potentials as well as nature-designed steric contributions of the fluctuating membrane or from the polymers of the glycocalyx. Despite the fact that these nonspecific potentials are omnipresent, their description has been a major challenge from experimental and theoretical points of view. Here we show that a full understanding of the implications of the continuous interactions can be achieved only by expanding the standard superposition models commonly used to treat these types of systems, beyond the usual harmonic level of description. Supported by this expanded theoretical framework, we present three independent, yet mutually consistent, experimental approaches to measure the interaction potential strength and the membrane tension. Upon explicitly taking into account the nature of shot noise as well as of finite experimental resolution, excellent agreement with the augmented theory is obtained, which finally provides a coherent view of the behavior of the membrane in a vicinity of a scaffold.

Multiscale approaches to protein-mediated interactions between membranes—relating microscopic and macroscopic dynamics in radially growing adhesions

Macromolecular complexation leading to coupling of two or more cellular membranes is a crucial step in a number of biological functions of the cell. While other mechanisms may also play a role, adhesion always involves the fluctuations of deformable membranes, the diffusion of proteins and the molecular binding and unbinding. Because these stochastic processes couple over a multitude of time and length scales, theoretical modeling of membrane adhesion has been a major challenge. Here we present an effective Monte Carlo scheme within which the effects of the membrane are integrated into local rates for molecular recognition. The latter step in the Monte Carlo approach enables us to simulate the nucleation and growth of adhesion domains within a system of the size of a cell for tens of seconds without loss of accuracy, as shown by comparison to

Crowding of receptors induces ring-like adhesions in model membranes

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Nucleation of ligand-receptor domains in membrane adhesion.

times more expensive Langevin simulations. To perform this validation, the Langevin approach was augmented to simulate diffusion of proteins explicitly, together with reaction kinetics and membrane dynamics. We use the Monte Carlo scheme to gain deeper insight to the experimentally observed radial growth of micron sized adhesion domains, and connect the effective rate with which the domain is growing to the underlying microscopic events. We thus demonstrate that our technique yields detailed information about protein transport and complexation in membranes, which is a fundamental step toward understanding even more complex membrane interactions in the cellular context.

Facile colloidal coating of polystyrene nanospheres with tunable gold dendritic patches

The dynamics of formation of macromolecular structures in adherent membranes is a key to a number of cellular processes. However, the interplay between protein reaction kinetics, diffusion and the morphology of the growing domains, governed by membrane mediated interactions, is still poorly understood. Here we show, experimentally and in simulations, that a rich phase diagram emerges from the competition between binding, cooperativity, molecular crowding and membrane spreading. In the cellular context, the spontaneously-occurring organization of adhesion domains in ring-like morphologies is particularly interesting. These are stabilized by the crowding of bulky proteins, and the membrane-transmitted correlations between bonds. Depending on the density of the receptors, this phase may be circumvented, and instead, the adhesions may grow homogeneously in the contact zone between two membranes. If the development of adhesion occurs simultaneously with membrane spreading, much higher accumulation of binders can be achieved depending on the velocity of spreading. The mechanisms identified here, in the context of our mimetic model, may shed light on the structuring of adhesions in the contact zones between two living cells. This article is part of a Special Issue entitled: Mechanobiology.