Ulrich Schwarz

Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates

Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhesions. A novel approach was developed for real-time, high-resolution measurements of forces applied by cells at single adhesion sites. This method combines micropatterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFP-tagged vinculin. Local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 ± 2 nNμm-2. The dynamics of the force-dependent modulation of focal adhesions were characterized by blocking actomyosin contractility and were found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force.

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.

Calculation of forces at focal adhesions from elastic substrate data: the effect of localized force and the need for regularization.

Forces exerted by stationary cells have been investigated on the level of single focal adhesions by combining elastic substrates, fluorescence labeling of focal adhesions, and the assumption of localized force when solving the inverse problem of linear elasticity theory. Data simulation confirms that the inverse problem is ill-posed in the presence of noise and shows that in general a regularization scheme is needed to arrive at a reliable force estimate. Spatial and force resolution are restricted by the smoothing action of the elastic kernel, depend on the details of the force and displacement patterns, and are estimated by data simulation. Corrections arising from the spatial distribution of force and from finite substrate size are treated in the framework of a force multipolar expansion. Our method is computationally cheap and could be used to study mechanical activity of cells in real time.

Cell organization in soft media due to active mechanosensing

Adhering cells actively probe the mechanical properties of their environment and use the resulting information to position and orient themselves. We show that a large body of experimental observations can be consistently explained from one unifying principle, namely that cells strengthen contacts and cytoskeleton in the direction of large effective stiffness. Using linear elasticity theory to model the extracellular environment, we calculate optimal cell organization for several situations of interest and find excellent agreement with experiments for fibroblasts, both on elastic substrates and in collagen gels: cells orient in the direction of external tensile strain; they orient parallel and normal to free and clamped surfaces, respectively; and they interact elastically to form strings. Our method can be applied for rational design of tissue equivalents. Moreover, our results indicate that the concept of contact guidance has to be reevaluated. We also suggest that cell–matrix contacts are up-regulated by large effective stiffness in the environment because, in this way, build-up of force is more efficient.

Mechanosensing in actin stress fibers revealed by a close correlation between force and protein localization

The mechanics of the actin cytoskeleton have a central role in the regulation of cells and tissues, but the details of how molecular sensors recognize deformations and forces are elusive. By performing cytoskeleton laser nanosurgery in cultured epithelial cells and fibroblasts, we show that the retraction of stress fibers (SFs) is restricted to the proximity of the cut and that new adhesions form at the retracting end. This suggests that SFs are attached to the substrate. A new computational model for SFs confirms this hypothesis and predicts the distribution and propagation of contractile forces along the SF. We then analyzed the dynamics of zyxin, a focal adhesion protein present in SFs. Fluorescent redistribution after laser nanosurgery and drug treatment shows a high correlation between the experimentally measured localization of zyxin and the computed localization of forces along SFs. Correlative electron microscopy reveals that zyxin is recruited very fast to intermediate substrate anchor points that are highly tensed upon SF release. A similar acute localization response is found if SFs are mechanically perturbed with the cantilever of an atomic force microscope. If actin bundles are cut by nanosurgery in living Drosophila egg chambers, we also find that zyxin redistribution dynamics correlate to force propagation and that zyxin relocates at tensed SF anchor points, demonstrating that these processes also occur in living organisms. In summary, our quantitative analysis shows that force and protein localization are closely correlated in stress fibers, suggesting a very direct force-sensing mechanism along actin bundles.

United we stand – integrating the actin cytoskeleton and cell–matrix adhesions in cellular mechanotransduction

Summary Many essential cellular functions in health and disease are closely linked to the ability of cells to respond to mechanical forces. In the context of cell adhesion to the extracellular matrix, the forces that are generated within the actin cytoskeleton and transmitted through integrin-based focal adhesions are essential for the cellular response to environmental clues, such as the spatial distribution of adhesive ligands or matrix stiffness. Whereas substantial progress has been made in identifying mechanosensitive molecules that can transduce mechanical force into biochemical signals, much less is known about the nature of cytoskeletal force generation and transmission that regulates the magnitude, duration and spatial distribution of forces imposed on these mechanosensitive complexes. By focusing on cell-matrix adhesion to flat elastic substrates, on which traction forces can be measured with high temporal and spatial resolution, we discuss our current understanding of the physical mechanisms that integrate a large range of molecular mechanotransduction events on cellular scales. Physical limits of stability emerge as one important element of the cellular response that complements the structural changes affected by regulatory systems in response to mechanical processes.

Cell Adhesion Strength Is Controlled by Intermolecular Spacing of Adhesion Receptors

Spatial patterning of biochemical cues on the micro- and nanometer scale controls numerous cellular processes such as spreading, adhesion, migration, and proliferation. Using force microscopy we show that the lateral spacing of individual integrin receptor-ligand bonds determines the strength of cell adhesion. For spacings > or = 90 nm, focal contact formation was inhibited and the detachment forces as well as the stiffness of the cell body were significantly decreased compared to spacings < or = 50 nm. Analyzing cell detachment at the subcellular level revealed that rupture forces of focal contacts increase with loading rate as predicted by a theoretical model for adhesion clusters. Furthermore, we show that the weak link between the intra- and extracellular space is at the intracellular side of a focal contact. Our results show that cells can amplify small differences in adhesive cues to large differences in cell adhesion strength.

Superconductivity in Weyl semimetal candidate MoTe2

Transition metal dichalcogenides have attracted research interest over the last few decades due to their interesting structural chemistry, unusual electronic properties, rich intercalation chemistry and wide spectrum of potential applications. Despite the fact that the majority of related research focuses on semiconducting transition-metal dichalcogenides (for example, MoS2), recently discovered unexpected properties of WTe2 are provoking strong interest in semimetallic transition metal dichalcogenides featuring large magnetoresistance, pressure-driven superconductivity and Weyl semimetal states. We investigate the sister compound of WTe2, MoTe2, predicted to be a Weyl semimetal and a quantum spin Hall insulator in bulk and monolayer form, respectively. We find that bulk MoTe2 exhibits superconductivity with a transition temperature of 0.10 K. Application of external pressure dramatically enhances the transition temperature up to maximum value of 8.2 K at 11.7 GPa. The observed dome-shaped superconductivity phase diagram provides insights into the interplay between superconductivity and topological physics.

Physics of adherent cells

properties of cells and their physical environment. Here we review recent progress in our understanding of the role of forces in cell adhesion from the viewpoint of theoretical soft matter physics and in close relation to the relevant experiments.