Benedikt Sabass

Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed

How focal adhesions (FAs) convert retrograde filamentous actin (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined traction force and fluorescent speckle microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8–10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.

Cell-ECM traction force modulates endogenous tension at cell–cell contacts

Cells in tissues are mechanically coupled both to the ECM and neighboring cells, but the coordination and interdependency of forces sustained at cell-ECM and cell–cell adhesions are unknown. In this paper, we demonstrate that the endogenous force sustained at the cell–cell contact between a pair of epithelial cells is approximately 100 nN, directed perpendicular to the cell–cell interface and concentrated at the contact edges. This force is stably maintained over time despite significant fluctuations in cell–cell contact length and cell morphology. A direct relationship between the total cellular traction force on the ECM and the endogenous cell–cell force exists, indicating that the cell–cell tension is a constant fraction of the cell-ECM traction. Thus, modulation of ECM properties that impact cell-ECM traction alters cell–cell tension. Finally, we show in a minimal model of a tissue that all cells experience similar forces from the surrounding microenvironment, despite differences in the extent of cell-ECM and cell–cell adhesion. This interdependence of cell–cell and cell-ECM forces has significant implications for the maintenance of the mechanical integrity of tissues, mechanotransduction, and tumor mechanobiology.

Plasmodium sporozoite motility is modulated by the turnover of discrete adhesion sites.

Sporozoites are the highly motile stages of the malaria parasite injected into the hosts skin during a mosquito bite. In order to navigate inside of the host, sporozoites rely on actin-dependent gliding motility. Although the major components of the gliding machinery are known, the spatiotemporal dynamics of the proteins and the underlying mechanism powering forward locomotion remain unclear. Here, we show that sporozoite motility is characterized by a continuous sequence of stick-and-slip phases. Reflection interference contrast and traction force microscopy identified the repeated turnover of discrete adhesion sites as the underlying mechanism of this substrate-dependent type of motility. Transient forces correlated with the formation and rupture of distinct substrate contact sites and were dependent on actin dynamics. Further, we show that the essential sporozoite surface protein TRAP is critical for the regulated formation and rupture of adhesion sites but is dispensable for retrograde capping.

Extracellular rigidity sensing by talin isoform-specific mechanical linkages

The ability of cells to adhere and sense differences in tissue stiffness is crucial for organ development and function. The central mechanisms by which adherent cells detect extracellular matrix compliance, however, are still unknown. Using two single-molecule-calibrated biosensors that allow the analysis of a previously inaccessible but physiologically highly relevant force regime in cells, we demonstrate that the integrin activator talin establishes mechanical linkages following cell adhesion, which are indispensable for cells to probe tissue stiffness. Talin linkages are exposed to a range of piconewton forces and bear, on average, 7–10 pN during cell adhesion depending on their association with F-actin and vinculin. Disruption of talin’s mechanical engagement does not impair integrin activation and initial cell adhesion but prevents focal adhesion reinforcement and thus extracellular rigidity sensing. Intriguingly, talin mechanics are isoform specific so that expression of either talin-1 or talin-2 modulates extracellular rigidity sensing.

Optimization of traction force microscopy for micron-sized focal adhesions

To understand how adherent cells regulate traction forces on their surrounding extracellular matrix (ECM), quantitative techniques are needed to measure forces at the cell-ECM interface. Microcontact printing is used to create a substrate of 1 μm diameter circles of ECM ligand to experimentally study the reconstruction of traction stresses at constrained, point-like focal adhesions. Traction reconstruction with point forces (TRPF) and Fourier transform traction cytometry (FTTC) are used to calculate the traction forces and stress field, respectively, at isolated adhesions. We find that the stress field calculated with FTTC peaks near the center of individual adhesions but propagates several microns beyond the adhesion location. We find the optimal set of FTTC parameters that yield the highest stress magnitude, minimizing information lost from over-smoothing and sampling of the displacement or stress field. A positive correlation between the TRPF and FTTC measurements exists, but integrating the FTTC stress field over the adhesion area yields only a small fraction of the force calculated by TRPF. An effective area similar to that defined by the width of the stress distribution measured with FTTC is required to reconcile these measurements. These measurements set bounds on the spatial resolution and precision of FTTC measurements on micron-sized adhesions.

High-resolution traction force microscopy.

Cellular forces generated by the actomyosin cytoskeleton and transmitted to the extracellular matrix (ECM) through discrete, integrin-based protein assemblies, that is, focal adhesions, are critical to developmental morphogenesis and tissue homeostasis, as well as disease progression in cancer. However, quantitative mapping of these forces has been difficult since there has been no experimental technique to visualize nanonewton forces at submicrometer spatial resolution. Here, we provide detailed protocols for measuring cellular forces exerted on two-dimensional elastic substrates with a high-resolution traction force microscopy (TFM) method. We describe fabrication of polyacrylamide substrates labeled with multiple colors of fiducial markers, functionalization of the substrates with ECM proteins, setting up the experiment, and imaging procedures. In addition, we provide the theoretical background of traction reconstruction and experimental considerations important to design a high-resolution TFM experiment. We describe the implementation of a new algorithm for processing of images of fiducial markers that are taken below the surface of the substrate, which significantly improves data quality. We demonstrate the application of the algorithm and explain how to choose a regularization parameter for suppression of the measurement error. A brief discussion of different ways to visualize and analyze the results serves to illustrate possible uses of high-resolution TFM in biomedical research.

Size-dependent control of colloid transport via solute gradients in dead-end channels

Significance Dead-end geometries are commonly found in many porous systems. Particle transport into such dead-end pores is often important, but is difficult to achieve owing to the confinement. It is natural to expect that Brownian motion is the sole mechanism to deliver the particles into the pores, but that mechanism is, unfortunately, slow and inefficient. Here, we introduce solute gradient to control the transport of particles in dead-end channels. We demonstrate a size effect such that larger particles tend to focus more and reside deeper in the channels. Our findings suggest a potential pathway to many useful applications that are difficult to achieve in dead-end geometries such as particle sorting and sample preconcentration, which are important in pharmaceuticals and healthcare industries. Transport of colloids in dead-end channels is involved in widespread applications including drug delivery and underground oil and gas recovery. In such geometries, Brownian motion may be considered as the sole mechanism that enables transport of colloidal particles into or out of the channels, but it is, unfortunately, an extremely inefficient transport mechanism for microscale particles. Here, we explore the possibility of diffusiophoresis as a means to control the colloid transport in dead-end channels by introducing a solute gradient. We demonstrate that the transport of colloidal particles into the dead-end channels can be either enhanced or completely prevented via diffusiophoresis. In addition, we show that size-dependent diffusiophoretic transport of particles can be achieved by considering a finite Debye layer thickness effect, which is commonly ignored. A combination of diffusiophoresis and Brownian motion leads to a strong size-dependent focusing effect such that the larger particles tend to concentrate more and reside deeper in the channel. Our findings have implications for all manners of controlled release processes, especially for site-specific delivery systems where localized targeting of particles with minimal dispersion to the nontarget area is essential.

Modeling cytoskeletal flow over adhesion sites: competition between stochastic bond dynamics and intracellular relaxation.

In migrating cells, retrograde flow of the actin cytoskeleton is related to traction at adhesion sites located at the base of the lamellipodium. The coupling between the moving cytoskeleton and the stationary adhesions is mediated by the continuous association and dissociation of molecular bonds. We introduce a simple model for the competition between the stochastic dynamics of elastic bonds at the moving interface and relaxation within the moving actin cytoskeleton represented by an internal viscous friction coefficient. Using exact stochastic simulations and an analytical mean field theory, we show that the stochastic bond dynamics lead to biphasic friction laws as observed experimentally. At low internal dissipation, stochastic bond dynamics lead to a regime of irregular stick-and-slip motion. High internal dissipation effectively suppresses cooperative effects among bonds and hence stabilizes the adhesion.

Nonlinear, electrocatalytic swimming in the presence of salt

A small, bimetallic particle in a hydrogen peroxide solution can propel itself by means of an electrocatalytic reaction. The swimming is driven by a flux of ions around the particle. We model this process for the presence of a monovalent salt, where reaction-driven proton currents induce salt ion currents. A theory for thin diffuse layers is employed, which yields nonlinear, coupled transport equations. The boundary conditions include a compact Stern layer of adsorbed ions. Electrochemical processes on the particle surface are modeled with a first order reaction of the Butler-Volmer type. The equations are solved numerically for the swimming speed. An analytical approximation is derived under the assumption that the decomposition of hydrogen peroxide occurs mainly without inducing an electric current. We find that the swimming speed increases linearly with hydrogen peroxide concentration for small concentrations. The influence of ion diffusion on the reaction rate can lead to a concave shape of the function of speed vs. hydrogen peroxide concentration. The compact layer of ions on the particle diminishes the reaction rate and consequently reduces the speed. Our results are consistent with published experimental data.

Efficiency of surface-driven motion: nanoswimmers beat microswimmers.

Surface interactions provide a class of mechanisms which can be employed for propulsion of micrometer and nanometer sized particles. We investigate the related efficiency of externally and self-propelled swimmers. A general scaling relation is derived showing that only swimmers whose size is comparable to, or smaller than, the interaction range can have appreciable efficiency. An upper bound for efficiency at maximum power is 1/2. Numerical calculations for the case of diffusiophoresis are found to be in good agreement with analytical expressions for the efficiency.