Olivier Thoumine

Contribution of the nucleus to the mechanical properties of endothelial cells

The cell nucleus plays a central role in the response of the endothelium to mechanical forces, possibly by deforming during cellular adaptation. The goal of this work was to precisely quantify the mechanical properties of the nucleus. Individual endothelial cells were subjected to compression between glass microplates. This technique allows measurement of the uniaxial force applied to the cell and the resulting deformation. Measurements were made on round and spread cells to rule out the influence of cell morphology on the nucleus mechanical properties. Tests were also carried out with nuclei isolated from cell cultures by a chemical treatment. The non-linear force-deformation curves indicate that round cells deform at lower forces than spread cells and nuclei. Finite-element models were also built with geometries adapted to actual morphometric measurements of round cells, spread cells and isolated nuclei. The nucleus and the cytoplasm were modeled as separate homogeneous hyperelastic materials. The models simulate the compression and yield the force-deformation curve for a given set of elastic moduli. These parameters are varied to obtain a best fit between the theoretical and experimental data. The elastic modulus of the cytoplasm is found to be on the order of 500N/m(2) for spread and round cells. The elastic modulus of the endothelial nucleus is on the order of 5000N/m(2) for nuclei in the cell and on the order of 8000N/m(2) for isolated nuclei. These results represent an unambiguous measurement of the nucleus mechanical properties and will be important in understanding how cells perceive mechanical forces and respond to them.

Microplates: a new tool for manipulation and mechanical perturbation of individual cells

We present a new type of microinstrument allowing manipulation and mechanical perturbation of individual cells under an optical microscope. These instruments, which we call microplates, are pulled from rectangular glass bars. They have flat tips, typically 2 microm thick x 20 microm wide, whose specific shape and stiffness can be adjusted through the pulling protocol. After appropriate chemical treatment, microplates can support cell adhesion and/or spreading. Rigid microplates are used to hold cells, whereas more flexible ones serve as stress sensors, i.e. their deflexion is used to probe forces in the range of 1-1000 nN. The main advantages of microplates are their simple geometry and surface properties, and their ability to provide mechanical measurements. In this methodological paper, we give details about microplate preparation and adhesiveness, manipulation set-up, force calibration, and image analysis. Several manipulations have already been carried out on fibroblasts, including uniaxial deformation, micropipet aspiration of adherent cells, and cell-substrate separation. Our results to date provide new insights into the morphology, mechanical properties, and adhesive resistance of cells. Many future applications can be envisaged.

Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis

Abstract The biophysical properties of the interaction between fibronectin and its membrane receptor were inferred from adhesion tests on living cells. Individual fibroblasts were maintained on fibronectin-coated glass for short time periods (1–16 s) using optical tweezers. After contact, the trap was removed quickly, leading to either adhesion or detachment of the fibroblast. Through a stochastic analysis of bond kinetics, we derived equations of adhesion probability versus time, which fit the experimental data well and were used to compute association and dissociation rates (k+=0.3–1.4 s−1 and koff=0.05–0.25 s−1, respectively). The bond distribution is binomial, with an average bond number ≤10 at these time scales. Increasing the fibronectin density (100–3000 molecules/μm2) raised k+ in a diffusion-dependent manner, leaving koff relatively unchanged. Increasing the temperature (23–37 °C) raised both k+ and koff, allowing calculation of the activation energy of the chemical reaction (around 20 kBT). Increasing the compressive force on the cell during contact (up to 60 pN) raised k+ in a logarithmic manner, probably through an increase in the contact area, whereas koff was unaffected. Finally, by varying the pulling force to detach the cell, we could distinguish between two adhesive regimes, one corresponding to one bond, the other to at least two bonds. This transition occurred at a force around 20 pN, interpreted as the strength of a single bond.

Changes in the mechanical properties of fibroblasts during spreading: a micromanipulation study

Abstract Cell morphology is controlled in part by physical forces. If the main mechanical properties of cells have been identified and quantitated, the question remains of how the cell structure specifically contributes to these properties. In this context, we addressed the issue of whether cell rheology was altered during cell spreading, taken as a fundamental morphological change. On the experimental side, we used a novel dual micromanipulation system. Individual chick fibroblasts were allowed to spread for varying amounts of time on glass microplates, then their free extremity was aspirated into a micropipet at given pressure levels. Control experiments were also done on suspended cells. On the theoretical side, the cell was modeled as a fluid drop of viscosity μ, bounded by a contractile cortex whose tension above a resting value was taken to be linearly dependent on surface area expansion. The pipet negative pressure was first adjusted to an equilibrium value, corresponding to formation of a static hemispherical cap into the pipet. This allowed computation, through Laplaces law, of the resting tension (τ0), on the order of 3×10–4 N/m. No difference in τ0 was found between the different groups of cells studied (suspended, adherent for 5 min, spread for 0.5 h, and spread for 3 h). However, τ0 was significantly decreased upon treatment of fibroblasts with inhibitors of actin polymerization or myosin function. Then, the pressure was set at 30 mmH2O above the equilibrium pressure. All cells showed a biphasic behavior: (1) a rapid initial entrance corresponding to an increase in surface area, which was used to extract an area expansion elastic modulus (K), in the range of 10–2 N/m; this coefficient was found to increase up to 40% with cell spreading; (2) a more progressive penetration into the pipet, linear with time; this phase, attributed to viscous behavior of the cytoplasm, was used to compute the apparent viscosity (μ, in the range of 2–5×104 Pa s) which was found to increase by as much as twofold with cell spreading. In some experiments the basal force at the cell-microplate interface was quantitated with flexible microplates and found to be around 1 nN, in agreement with values calculated from the model. Taken together, our results indicate a stiffening of fibroblasts upon spreading, possibly correlated with structural organization of the cytoskeleton during this process. This study may help understand better the morphology of fibroblasts and their mechanical role in connective tissue integrity.

COMPARISON OF THE MECHANICAL PROPERTIES OF NORMAL AND TRANSFORMED FIBROBLASTS

In order to achieve coordinated migration through extracellular matrix and endothelial barriers during metastasis, cancer cells must be endowed with specific structural and adhesive properties. In this context, comparison of the mechanical properties of transformed versus normal cells, on which little quantitative information is available, was the focus of this study. Normal human dermal fibroblasts and their SV40-transformed counterparts were analyzed using various manipulations. First, micropipet aspiration of suspended cells allowed calculation of a cortical tension (similar for normal and transformed cells), and an apparent viscosity (30% lower for transformed than for normal fibroblasts); in addition, transformed fibroblasts exhibited a more fragile surface than their normal counterparts. Second, tangential ultracentrifugation of adherent cells demonstrated cellular elongation in the direction of the centrifugal field and the existence of critical forces for cell detachment, around 10(-7) N: these were 1.6-fold greater for normal than for transformed cells. Finally, examination of the wrinkle patterns formed by cells plated on a deformable polydimethylsiloxane substrate, plus analysis of cell retraction caused by ATP treatment following detergent permeabilization showed that normal fibroblasts exhibited much more contractility than their transformed counterparts, which we characterized by a cell contraction rate. Such quantitative parameters which reveal differences in the mechanical behavior of normal and transformed cells may be used in the future as new markers of oncogenic transformation.

Critical centrifugal forces induce adhesion rupture or structural reorganization in cultured cells

Cultured epithelial cells were exposed to accelerations ranging from 9,000 to 70,000g for time periods of 5, 15, or 60 min, by centrifugation in a direction tangential to their plastic substrate. Three regimes describe the cellular response: (1) Cell morphology and density remain unaltered at forces below a threshold of about 10(-7) N; (2) Between this critical force and a second threshold of about 1.5 10(-7)N, the number of adherent cells decreases exponentially with time and acceleration, with no alteration of cell morphology. This behavior can be modeled by a constant probability of detaching and by an exponential distribution of cell-to-substrate adhesive forces; (3) Past the second threshold, cells that are still adherent exhibit elongated morphologies, the degree of elongation increasing linearly with the force. The fact that cells lose their vinculin-rich focal contacts past the first threshold and that cells cultured on gelatin-coated plastic show an increased resistance to detachment suggests a rupture of cell-to-substrate adhesions upon centrifugation. Immunofluorescent labeling of cells for actin and tubulin shows a reorganization of the cytoskeleton upon centrifugation, and treatment of cells with the drugs cytochalasin D and nocodazole demonstrates that cytoskeletal elements are actively involved in the structural deformation of cells past the second acceleration threshold, microtubules and microfilaments paying antagonistic roles.

Influence of adhesion and cytoskeletal integrity on fibroblast traction

Cellular contractility plays an important role in cell morphogenesis and tissue pattern formation. In this context, we examined how the expression of cell traction depends on cell-to-substrate contacts and cytoskeletal organization. Qualitative observation of chick fibroblasts cultured on an elastic film of polydimethylsiloxane indicated a strong spatial relationship between wrinkle pattern and distribution of actin stress fibers and focal contacts. In order to further characterize cell contractility, the projected area of Triton-permeabilized fibroblasts upon ATP-induced retraction was measured in various conditions of substrate adhesivity, cytoskeletal perturbation, and temperature. In all conditions, the relationship between degree of cell retraction and ATP concentration was well described by the laws of enzyme kinetics. Culturing cells on a gelatin-coated substrate, decreasing the temperature, using phosphate ribonucleotides other than ATP, and treating cells with cytochalasin D all diminished the rate of cell retraction, indicating that fibroblast traction is generated by a temperature- and ATP-dependent actin/myosin stress fiber sliding mechanism, transmitted to the substrate through focal adhesions. Treatment of cells with either nocodazole or taxol did not affect retraction of permeabilized fibroblasts upon stimulation with ATP, suggesting that microtubules do not directly resist cell traction. Treatment of cells with vanadate increased cell retraction, suggesting that intermediate filaments help transmit tension.

Dynamics of adhesive rupture between fibroblasts and fibronectin: microplate manipulations and deterministic model

Abstract To characterize the dynamics of cell-substrate adhesive rupture, we used a novel micromanipulation technique, in which individual fibroblasts seized on a rigid microplate were placed into contact with a fibronectin-coated flexible microplate, then pulled away. The fibronectin density (0–3000 molecules/μm2) and the pulling rate (1–10 μm/s) were controlled. The extent of the contact zone decreased to zero at a time threshold corresponding to adhesive rupture. The uniaxial force at the interface, computed from the deflection of the microplate, increased linearly with time and reached a maximum before dropping to zero. A deterministic model, focusing on the mean number of bonds between fibronectin and its membrane receptor on the cell surface, shows rapid rupture when the force reaches a critical value, in agreement with experimental observations. Increasing the ligand density and the rate of load raises the maximal force (30–200 nN), in reasonably good agreement with the model predictions. Minimization of error between experimental and simulated forces allowed identification of two physicochemical properties of the bond, i.e. its association rate constant (k2Don=3 × 10−4 μm2/s) and structural length (d=3 nm). These results may help understand better fibroblast locomotion and interaction with the extracellular matrix.

Biomechanical Properties of Fibroblasts

Cells are a complex topic of study for materials scientists. They are the fundamental building blocks of living organisms, able to sense their environment and act in response to it. In addition to their many biochemical functions, cells also play a mechanical role: They hold organs in place and move to the locations where they are needed in processes like wound healing, metastasis, or embryogenesis. Their mechanical behavior is mostly determined by a meshwork of three types of connected biopolymers (actin microfilaments, microtubules, and intermediate filaments) that compose a structural framework called the cytoskeleton, surrounded by a lipid membrane (Figure 1). In contrast to this simple picture, cells are very different from polymer gels or liposomes: They are active materials, powered by chemically stored energy. Their mechanical condition is closely linked to their biochemical function; for example, they may “commit suicide,” following a well-defined protocol known as apoptosis, which can be triggered by their mechanical state. The enormous progress of modern cell biology combined with new micromanipulation techniques is leading researchers toward a more global understanding of the mechanical properties of cells and toward finding a functional link between biochemistry, chemical signaling, and cell mechanics, thus crossing the boundaries between these subjects. The characterization of cell mechanical behavior has been the object of numerous studies. Red blood cells are a simple model system; if deprived of a nucleus while retaining a constant surface area, they have properties reminiscent of lipid vesicles.