Mechanical response analysis and power generation by single-cell stretching

To harvest useful information about cell response due to mechanical perturbations under physiological conditions, a cantilever-based technique was designed, which allowed precise application of arbitrary forces or deformation histories on a single cell in vitro. Essential requirements for these investigations are a mechanism for applying an automated cell force and an induced-deformation detection system based on fiber-optical force sensing and closed loop control. The required mechanical stability of the setup can persist for several hours since mechanical drifts due to thermal gradients can be eliminated sufficiently (these gradients are caused by local heating of the cell observation chamber to 37 °C). During mechanical characterization, the cell is visualized with an optical microscope, which enables the simultaneous observation of cell shape and intracellular morphological changes. Either the cell elongation is observed as a reaction against a constant load or the cell force is measured as a response to constant deformation. Passive viscoelastic deformation and active cell response can be discriminated. The active power generated during contraction is in the range of Pmax=10−16Watts, which corresponds to 2500 ATP molecules s−1at 10 kBT/molecule. The ratio of contractive to dissipative power is estimated to be in the range of 10−2. The highest forces supported by the cell suggest that about 104molecular motors must be involved in contraction. This indicates an energy-conversion efficiency of approximately 0.5. Our findings propose that, in addition to the recruitment of cell-contractile elements upon mechanical stimulation, the cell cytoskeleton becomes increasingly crosslinked in response to a mechanical pull. Quantitative stress–strain data, such as those presented here, may be employed to test physical models that describe cellular responses to mechanical stimuli

Direct assessment of living cell mechanical responses during deformation inside microchannel restrictions

The deformation of suspended cells inside microchannel restrictions mimics passive cell transportation in the blood circulation system of the body. The cells traverse or get stuck in narrow vessels, as, e.g., during the metastasis of tumor cells. In this work, the mechanical responses of suspended pancreatic cancer cells as they move through and deform inside microchannel restrictions are assessed with a cantilever-based polydimethylsiloxane (PDMS) force sensor. Incorporated into a flow cell chip, the PDMS cantilever is integrated into the boundary wall of a narrow microrestriction. Upon being forced to enter the restriction by an applied flow, the cell exerts pressure on the cantilever, which then bends. By assuming a uniformly loaded cantilever, the total force and pressure on the cantilever can be calculated using elastic beam theory. This technique has the advantage of presenting an absolute and direct measure, which is independent of the applied flow and frictional processes at the channel-cell interface; in contrast to, e.g., measuring cell mechanics indirectly via cell sliding velocities. Furthermore, a high number of cells can be examined in a short time compared to other single cell mechanical testing devices

3D finite element analysis of uniaxial cell stretching: from image to insight

Mechanical forces play an important role in many microbiological phenomena such as embryogenesis, regeneration, cell proliferation and differentiation. Micromanipulation of cells in a controlled environment is a widely used approach for understanding cellular responses with respect to external mechanical forces. While modern micromanipulation and imaging techniques provide useful optical information about the change of overall cell contours under the impact of external loads, the intrinsic mechanisms of energy and signal propagation throughout the cell structure are usually not accessible by direct observation. This work deals with the computational modelling and simulation of intracellular strain state of uniaxially stretched cells captured in a series of images. A nonlinear elastic finite element method on tetrahedral grids was applied for numerical analysis of inhomogeneous stretching of a rat embryonic fibroblast 52 (REF 52) using a simplified two-component model of a eukaryotic cell consisting of a stiffer nucleus surrounded by a softer cytoplasm. The difference between simulated and experimentally observed cell contours is used as a feedback criterion for iterative estimation of canonical material parameters of the two-component model such as stiffness and compressibility. Analysis of comparative simulations with varying material parameters shows that (i) the ratio between the stiffness of cell nucleus and cytoplasm determines intracellular strain distribution and (ii) large deformations result in increased stiffness and decreased compressibility of the cell cytoplasm. The proposed model is able to reproduce the evolution of the cellular shape over a sequence of observed deformations and provides complementary information for a better understanding of mechanical cell response

Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly

Cell-extracellular matrix (cell-ECM) interactions mediated by integrin receptors are essential for providing positional and environmental information necessary for many cell functions, such as proliferation, differentiation and survival. In vitro studies on cell adhesion to randomly adsorbed molecules on substrates have been limited to sub-micrometer patches, thus preventing the detailed study of structural arrangement of integrins and their ligands. In this article, we illustrate the role of the distance between integrin ligands, namely the RGD (arginine-glycine-aspartate) sequence present in ECM proteins, in the control of cell adhesion. By using substrates, which carry cyclic RGD peptides arranged in highly defined nanopatterns, we investigated the dynamics of cell spreading and the molecular composition of adhesion sites in relation to a fixed spacing between the peptides on the surface. Our novel approach for in vitro studies on cell adhesion indicates that not only the composition, but also the spatial organization of the extracellular environment is important in regulating cell-ECM interactions

Force-induced growth of adhesion domains is controlled by receptor mobility

In living cells, adhesion structures have the astonishing ability to grow and strengthen under force. Despite the rising evidence of the importance of this phenomenon, little is known about the underlying mechanism. Here, we show that force-induced adhesion-strengthening can occur purely because of the thermodynamic response to the elastic deformation of the membrane, even in the absence of the actively regulated cytoskeleton of the cell, which was hitherto deemed necessary. We impose pN-forces on two fluid membranes, locally pre-adhered by RGD-integrin binding. One of the binding partners is always mobile whereas the mobility of the other can be switched on or off. Immediate passive strengthening of adhesion structures occurs in both cases. When both binding partners are mobile, strengthening is aided by lateral movement of intact bonds as a transient response to force-induced membrane-deformation. By extending our microinterferometric technique to the suboptical regime, we show that the adhesion, as well as the resistance to force-induced de-adhesion, is greatly enhanced when both, rather than only one, of the binding partners are mobile. We formulate a theory that explains our observations by linking the macroscopic shape deformation with the microscopic formation of bonds, which further elucidates the importance of receptor mobility. We propose this fast passive response to be the first-recognition that triggers signaling events leading to mechanosensing in living cells

Segregation Versus Colocalization: Orthogonally Functionalized Binary Micropatterned Substrates Regulate the Molecular Distribution in Focal Adhesions

Orthogonally functionalized binary micropatterned substrates are produced using a novel protocol. The use of adequate peptido-mimetics enables an unprecedented segregation of purified αvβ3 and α5β1 integrins in adjacent microislands and evidences the preference of U2OS cells to colocalize such receptors. Moreover, this tendency can be altered by varying the geometry and composition of the micropatterns