Evan Evans

Forces and Bond Dynamics in Cell Adhesion

Adhesion of a biological cell to another cell or the extracellular matrix involves complex couplings between cell biochemistry, structural mechanics, and surface bonding. The interactions are dynamic and act through association and dissociation of bonds between very large molecules at rates that change considerably under stress. Combining molecular cell biology with single-molecule force spectroscopy provides a powerful tool for exploring the complexity of cell adhesion, that is, how cell signaling processes strengthen adhesion bonds and how forces applied to cell-surface bonds act on intracellular sites to catalyze chemical processes or switch molecular interactions on and off. Probing adhesion receptors on strategically engineered cells with force during functional stimulation can reveal key nodes of communication between the mechanical and chemical circuitry of a cell.

Dynamic tension spectroscopy and strength of biomembranes.

Rupturing fluid membrane vesicles with a steady ramp of micropipette suction produces a distribution of breakage tensions governed by the kinetic process of membrane failure. When plotted as a function of log(tension loading rate), the locations of distribution peaks define a dynamic tension spectrum with distinct regimes that reflect passage of prominent energy barriers along the kinetic pathway. Using tests on five types of giant phosphatidylcholine lipid vesicles over loading rates(tension/time) from 0.01-100 mN/m/s, we show that the kinetic process of membrane breakage can be modeled by a causal sequence of two thermally-activated transitions. At fast loading rates, a steep linear regime appears in each spectrum which implies that membrane failure starts with nucleation of a rare precursor defect. The slope and projected intercept of this regime are set by defect size and frequency of spontaneous formation, respectively. But at slow loading rates, each spectrum crosses over to a shallow-curved regime where rupture tension changes weakly with rate. This regime is predicted by the classical cavitation theory for opening an unstable hole in a two-dimensional film within the lifetime of the defect state. Under slow loading, membrane edge energy and the frequency scale for thermal fluctuations in hole size are the principal factors that govern the level of tension at failure. To critically test the model and obtain the parameters governing the rates of transition under stress, distributions of rupture tension were computed and matched to the measured histograms through solution of the kinetic master (Markov) equations for defect formation and annihilation or evolution to an unstable hole under a ramp of tension. As key predictors of membrane strength, the results for spontaneous frequencies of defect formation and hole edge energies were found to correlate with membrane thicknesses and elastic bending moduli, respectively.

Observing force-regulated conformational changes and ligand dissociation from a single integrin on cells

A biomembrane force probe visualizes force-regulated reversible switches between bent and extended conformations of αLβ2 integrin on the surface of a living cell.

Dynamic strength of fluid membranes

Abstract Rupturing fluid membrane vesicles with a steady ramp of micropipette suction yields a tension distribution that images the kinetic process of membrane failure. When plotted on a log scale of tension loading rate, the distribution peaks (membrane strengths) define a dynamic tension spectrum with distinct regimes that reflect passage of prominent energy barriers along the pathway to rupture. Demonstrated here by tests on giant PC lipid vesicles over loading rates from 0.06–60 mN/m/s, the stochastic process of rupture can be modelled as a causal sequence of two thermally-activated transitions where each transition governs membrane strength on separate scales of loading rate. Under fast ramps of tension, a steep linear regime appears in each spectrum at high strengths which implies that failure requires nucleation of a rare nanoscale defect. The slope and projected intercept yield defect size and spontaneous production rate respectively. However, under slow ramps of loading, the spectrum crosses over to a shallow-curved regime at lower strength, which is consistent with the kinetic impedance to opening an unstable hole in a fluid film. The dependence of rupture tension on rate reveals hole edge energy and frequency scale for thermal fluctuations in size. To cite this article: E.xa0Evans, V. Heinrich, C.xa0R. Physique 4 (2003).

Exploring Reaction Pathways of Single-Molecule Interactions through the Manipulation and Tracking of a Potential-Confined Microsphere in Three Dimensions

Weak non-covalent interactions between single molecules govern many aspects of microscopic biological structure and function, e.g. cell adhesion, protein folding, molecular motors and mechanical enzymes. The dynamics of a weak biomolecular bond are suitably characterized by the kinetic transport of molecular states over an effective energy landscape defined along one or more optimal reaction pathways. Motivated by earlier developments [1,2], we present a novel method to quantify subtle features of weak chemical transitions by analyzing the 3D Brownian fluctuations of a functionalized microsphere held near a reactive substrate. A weak optical-trapping potential is used to confine motion of the bead to a nanoscale domain, and to apply a controlled bias field to the interaction. Stochastic interruptions in the monitored bead dynamics report formation and release of single molecular bonds. In addition, variations in the motion of a bead linked to the substrate via a biomolecule (a protein or nucleic acid) signal conformational changes in the molecule, such as the folding/unfolding of protein domains or the unzipping of DNA. Thus, energy landscapes of complex biomolecular interactions are mapped by identifying distinct fluctuation regimes in the 3D motion of a test microsphere, and by quantifying the rates of transition between these regimes as mediated by the applied confining potential. The 3D motion of the bead is tracked using a reflection interference technique combined with high-speed video microscopy. The position of the bead is measured over 100 times per second with a lateral resolution of ~3-5 nm and a vertical resolution of ~1-2 nm. Crucial to the interpretation of results, a Brownian Dynamics simulation has been developed to relate the statistics of bead displacements to molecular-scale kinetics of chemical interactions and structural transitions. The experimental approach is designed to enlarge the scope of current techniques (e.g. dynamic force spectroscopy [3]) to encompass near-equilibrium forward/reverse transitions of weak-complex interactions with multiple binding configurations and more than one transition pathway.

Using a Maxwell's demon to orient a microsphere in a laser trap and initiate thermodynamic assays of photonic nanofields

Seeking to control free rotations of a microsphere in a laser trap, we have created a Maxwells demon that identifies and captures a preferred up-or-down polarity of the microsphere. Breaking rotational symmetry, we attach a single Raleigh-size nanoparticle to a micron-size sphere, which establishes a nanodirector defining microsphere orientations in a trap. With radius <10% of the NIR trapping wavelength (1.064 μm), a polystyrene nanoparticle appended to a ∼1.3 μm glass sphere adds negligibly to scattering of the trapping beam and imperceptibly to forces trapping a doublet probe. Yet, constrained to a large orbit (∼1.5 μm diameter), the weak Raleigh dipole force induced in the nanoparticle imparts significant pole-attracting torques to the probe. At the same time, Brownian-thermal excitations contribute torque fluctuations to the probe randomizing orientations. Thus, we have combined demon control and Boltzmann thermodynamics to examine the intense competition between photonic torques aligning the nanodirector to the optical axis and the entropy confinement opposing alignment when equilibrated over long times for an order of magnitude span in laser powers. To reveal orientation, we developed novel multistep pattern-processing software to expose and enhance weak-diffuse visible light scattered from the nanoparticle. Processing a continuous stream of doublet images offline at ∼700 fps, the final step is to super resolve the transverse XY origin of the scattering pattern relative to the synchronous probe center, albeit limited to up state segments because of intensity. Transforming the dense histograms (∼104-105) of radial positions to polar angle (θ) distributions, we plot the results on a natural log scale versus sin(θ) to quantify the photonic potentials aligning the nanodirector to the optical axis. Then guided by principles of canonical thermodynamics, we invoke self-consistent methodology to reveal photonic potentials in the down state.