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Mechanical cues from the cellular microenvironment are converted into biochemical signals controlling diverse cell behaviours, including growth and differentiation. But it is still unclear how mechanotransduction ultimately affects nuclear readouts, genome function and transcriptional programs. Key signaling pathways and transcription factors can be activated, and can relocalize to the nucleus, upon mechanosensing. Here, we tested the hypothesis that epigenetic regulators, such as methyltransferase enzymes, might also contribute to mechanotransduction. We found that the SMYD3 lysine methyltransferase is spatially redistributed dependent on cell geometry (cell shape and aspect ratio) in murine myoblasts. Specifically, elongated rectangles were less permissive than square shapes to SMYD3 nuclear accumulation, via reduced nuclear import. Notably, SMYD3 has both nuclear and cytoplasmic substrates. The distribution of SMYD3 in response to cell geometry correlated with cytoplasmic and nuclear lysine tri-methylation (Kme3) levels, but not Kme2. Moreover, drugs targeting cytoskeletal acto-myosin induced nuclear accumulation of Smyd3. We also observed that square vs rectangular geometry impacted the nuclear-cytoplasmic relocalisation of several mechano-sensitive proteins, notably YAP/TAZ proteins and the SETDB1 methyltransferase. Thus, mechanical cues from cellular geometric shapes are transduced by a combination of transcription factors and epigenetic regulators shuttling between the cell nucleus and cytoplasm.

In this work, we study in detail the distribution of stochastic forces generated by the molecular motors activity, in the actin cortex of pre-muscular cells. By combining active and passive rheology experiments, performed on the same micro-bead bound to the actin network through membrane adhesive receptors, we measure the auto-correlation function Cff(t) of the average force pulling on the bead. Like for any out-of-equilibrium system, the force distribution differs from the thermodynamical equilibrium one, especially at long time scale t>1sec where the bead motion becomes partially directed. Thus the fluctuation-dissipation theorem does not apply and one can measure the distance from equilibrium through its violation. This work focuses on the influence of various parameters (ligand density, temperature, ATP depletion, molecular motors activity) on the force distribution. In particular, it is shown that the amplitude of active forces increases when the bead is more tighly attached to the cortex: this is interpreted through a model which takes into account the number of bonds between the bead and the cytoskeleton and the viscoelastic properties of the medium. It also increases with temperature, consistently with a description of the cell metabolism in terms of thermally activated reactions. Last, but not least, ATP depletion in the cell, or partial inhibitition of the actomyosin activity, leads to a decrease of the amplitude of the force distribution. Altogether, we propose a consistent and quantitative description for the motion of a micrometric probe interacting with the actin network, and for the amplitude of the stochastic forces generated by molecular motors in the cortex surrounding this probe.

Cell shape affects proliferation and differentiation, which are processes known to depend on integrin-based focal adhesion (FA) signaling. Because shape results from force balance and FAs are mechanosensitive complexes transmitting tension from the cell structure to its mechanical environment, we investigated the interplay between 3D cell shape, traction forces generated through the cell body, and FA growth during early spreading. Combining measurements of cell-scale normal traction forces with FA monitoring, we show that the cell body contact angle controls the onset of force generation and, subsequently, the initiation of FA growth at the leading edge of the lamella. This suggests that, when the cell body switches from convex to concave, tension in the apical cortex is transmitted to the lamella where force-sensitive FAs start to grow. Along this line, increasing the stiffness resisting cell body contraction led to a decrease of the lag time between force generation and FA growth, indicating mechanical continuity of the cell structure and force transmission from the cell body to the leading edge. Remarkably, the overall normal force per unit area of FA increased with stiffness, and its values were similar to those reported for local tangential forces acting on individual FAs. These results reveal how the 3D cell shape feeds back on its internal organization and how it may control cell fate through FA-based signaling.

We investigated the role of low-amplitude magnetic pulse with low repetition frequency superimposed on the environmental electromagnetic field (EMF) on the secretion of anti-aggregant (Prostacyclin or PGI(2)) and pro-aggregant (Thromboxane A(2)) agents in endothelial cells of the human umbilical cord vein (HUVEC). We established that magnetic pulse exposure modulates both PGI(2) and TXA(2). These modulations depend on the frequency, width of the pulse, and intensity of the magnetic field. Moreover, we corroborated previous results obtained with an endothelial cell line (EaHy-926), concerning the increased thrombo-embolic risk for the 1 Hz frequency.

Tissue engineering strategies, such as cellularized scaffolds approaches, have been explored for cartilage replacement. The challenge, however, remains to produce a cartilaginous tissue incorporating functional chondrocytes and being large and thick enough to be compatible with the replacement of articular defects. Here, we achieved unprecedented cartilage tissue production into a porous polysaccharide scaffold by combining of efficient magnetic condensation of mesenchymal stem cells, and dynamic maturation in a bioreactor. In optimal conditions, all the hallmarks of chondrogenesis were enhanced with a 50-fold increase in collagen II expression compared to negative control, an overexpression of aggrecan and collagen XI, and a very low expression of collagen I and RUNX2. Histological staining showed a large number of cellular aggregates, as well as an increased proteoglycan synthesis by chondrocytes. Interestingly, electron microscopy showed larger chondrocytes and a more abundant extracellular matrix. In addition, the periodicity of the neosynthesized collagen fibers matched that of collagen II. These results represent a major step forward in replacement tissue for cartilage defects.A combination of several innovative technologies (magnetic cell seeding, polysaccharide porous scaffolds, and dynamic maturation in bioreactor) enabled unprecedented successful chondrogenesis within scaffolds.

We have determined the microrheological response of the actin meshwork for individual cells. We applied oscillating forces with an optical tweezer to a micrometric bead specifically bound to the actin meshwork of C2 myoblasts, and measured the amplitude and phase shift of the induced cell deformation. For a non-perturbed single cell, we have shown that the elastic and loss moduli G' and G'' behave as power laws f (alpha) and f (beta) of the frequency f (0.01f50 Hz), alpha and beta being in the range 0.15-0.35. This demonstrates that the dissipation mechanisms in a single cell involve a broad and continuous distribution of relaxation times. After adding blebbistatin, an inhibitor of myosin II activity, the exponent of G' decreases to about 0.10, and G'' becomes roughly constant for 0.01f10 Hz. The actin meshwork appears less rigid and less dissipative than in the control experiment. This is consistent with an inhibition of ATPase and reduction of the gliding mobility of myosin II on actin filaments. In this frequency range, the actomyosin activity appears as an essential mechanism allowing the cell to adapt to an external mechanical stress.

We investigated the role of a 1 Hz low-strength magnetic pulse superimposed on the environmental electromagnetic field (emf) on the secretion of anti-aggregant (prostacyclin or PGI2) and pro-aggregant (thromboxane A2 or TXA2) agents in the EaHy-926 endothelial cell line. We established that magnetic pulse exposure has opposite effects on the two secretions: PGI2 is decreased, whereas TXA2 is increased, with a PGI2/TXA2 ratio shifted toward thrombosis. We also show that the effect of the magnetic field depends on its orientation, normal or parallel, to the cell monolayer. Finally, we show that the amplitude of the effect does not increase with the magnitude of the magnetic pulse, particularly with PGI2 secretion, which is increased as the field magnitude is decreased, suggesting a new concept for defining a threshold for health hazards.

The area expansion and the shear moduli of the free spectrin skeleton, freshly extracted from the membrane of a human red blood cell (RBC), are measured by using optical tweezers micromanipulation. An RBC is trapped by three silica beads bound to its membrane. After extraction, the skeleton is deformed by applying calibrated forces to the beads. The area expansion modulus K(C) and shear modulus mu(C) of the two-dimensional spectrin network are inferred from the deformations measured as functions of the applied stress. In low hypotonic buffer (25 mOsm/kg), one finds K(C) = 4.8 +/- 2.7 microN/m, mu(C) = 2.4 +/- 0.7 microN/m, and K(C)/mu(C) = 1.9 +/- 1.0. In isotonic buffer, one measures higher values for K(C), mu(C), and K(C)/mu(C), partly because the skeleton collapses in a high-ionic-strength environment. Some data concerning the time evolution of the mechanical properties of the skeleton after extraction and the influence of ATP are also reported. In the Discussion, it is shown that the measured values are consistent with estimates deduced from experiments carried out on the intact membrane and agree with theoretical and numerical predictions concerning two-dimensional networks of entropic springs.

International audience; Optical tweezers are used to apply calibrated forces to human erythrocytes, via small silica beads bound to their membrane. The shear modulus μ of the membrane is inferred from measurements of the cell deformation in the small strain linear regime. We find the same result μ = 2.5 ± 0.4 μN/m for both discotic and nearly spherical swollen cells. This value is smaller than the one deduced from micropipettes experiments. However the two methods do not operate in the same deformation regime and are not expected to lead to the same result.

The direct effect of two types of mechanical stress was measured through the prostacyclin (PGI2) and thromboxane A2 (TXA2) secretions by a confluent monolayer of cells from the EA.hy926 line. Eight values of constant pressure were applied in the gas phase above the culture medium, around atmospheric pressure taken as a control (0 mm Hg), from -500 to +760 mm Hg. Three amplitudes of sinewave modulated pressure (+/- 40; +/- 80; +/- 160 mm Hg) were explored at a frequency of 1 Hz. Modulated pressure (+/- 40 mm Hg) was also applied synergetically to a shear stress generated under steady state conditions by a rectilinear laminar motion of the medium. The cells remained adherent and exhibited unchanged morphology and viability. Constant pressure or depressure increased both PGI2 and TXA2 release but to an extent depending on the pressure value. Under pressure, the PGI2/TXA2 ratio was unchanged, but was higher under depressure, compared to the control. Pressure modulation strongly stimulated the secretion of PGI2 but had no effect on TXA2. Modulation strongly increased the PGI2/TXA2 ratio to a similar extent for the three amplitudes. Pressure-shear synergy enhanced secretion of PGI2 markedly more than shear stress alone, but the level reached was similar to the one induced by pressure modulation. No cumulative effect on the secretion of PGI2 was observed, whereas TXA2 synthesis undergoes a more than cumulative effect. The PGI2/TXA2 ratio remained unchanged under shear alone or under combined shear-pressure modulation but was higher with the modulated pressure alone. These results demonstrate that pressure has an outstanding effect on secretion that may be origin to local disturbances of the vascular system, thus inducing pathologies such as thrombosis or atherosclerosis.

Pressure effect on cultured islets was investigated to simulate the behavior of the endocrine pancreas when subjected to an anatomical change in its environment. Insulin secretion from islets seeded on glass was determined by radioimmunoassays after 12-h exposure to constant or sine-wave pressure perturbation, at 37 degrees C and pH 7.4, under well-defined conditions applied through the culture medium. A specially devised setup allowed control of the constant component Po, the amplitude A, and the frequency f of the pressure perturbation. Under constant conditions, three Po values (760, 760 + 110, 760 + 250 mm Hg) were explored. Under sine-wave conditions, two frequencies (f = 0.2 and 4.33 Hz) were investigated for Po = 760 + 110 mm Hg and A = 10 mm Hg, as well as three amplitude values (A = +/-10, +/-50, and +/-110 mm Hg) for Po = 760 mm Hg and f = 0.2 Hz. Constant pressure inhibits secretion, by a factor of up to 60%, as well as low frequency (also by approximately 60%). Low amplitudes enhance secretion (by 41 and 31%), whereas a higher amplitude has a marked inhibiting effect (by 70%). In all cases, no linearity was observed, showing the existence of a threshold pressure effect. These results suggest that pressure may be an important parameter in certain forms of endocrine pancreatic dysfunctions.

International audience; Plant and animals have evolved different strategies for their development. Whether this is linked to major differences in their cell mechanics remains unclear, mainly because measurements on plant and animal cells relied on independent experiments and setups, thus hindering any direct comparison. In this study we used the same micro-rheometer to compare animal and plant single cell rheology. We found that wall-less plant cells exhibit the same weak power law rheology as animal cells, with comparable values of elastic and loss moduli. Remarkably, microtubules primarily contributed to the rheological behavior of wall-less plant cells whereas rheology of animal cells was mainly dependent on the actin network. Thus, plant and animal cells evolved different molecular strategies to reach a comparable cytoplasmic mechanical core, suggesting that evolutionary convergence could include the internal biophysical properties of cells.

We have used the EaHy926 endothelial cell line, able to secrete both pro and anti-aggregant platelet agents, as a model for thrombo-embolic diseases. We experimentally established, by comparing these two secretions with or without a Faraday cage, that the environmental electromagnetic field significantly increases the thrombo-embolic risks in this endothelial cell line.

We designed a micromanipulation device that allows the local application of a constant force on living cells, and the measurement of their stiffness. The force is applied through an Arg-Gly-Asp-coated bead adhering on the cell and trapped in optical tweezers controlled by a feedback loop. Epifluorescence observations of green fluorescent protein-actin in the cells are made during force application. We observe a stiffening of cells submitted to a constant force within a few minutes, coupled to actin recruitment both at the bead-cell contact and up to several micrometers from the stress application zone. Moreover, kinetics of stiffening and actin recruitment exhibit a strong correlation. This work presents the first quantification of the dynamics of cell mechanical reinforcement under stress, which is a novel insight into the elucidation of the more general phenomenon of cell adaptation to stress.

We used a novel uniaxial stretching rheometer to measure the creep function J(t) of an isolated living cell. We show, for the first time at the scale of the whole cell, that J(t) behaves as a power-law J(t) = At(alpha). For N = 43 mice myoblasts (C2-7), we find alpha = 0.24 +/- 0.01 and A = (2.4 +/- 0.3) 10(-3) Pa(-1) s(-alpha). Using Laplace Transforms, we compare A and alpha to the parameters G(0) and beta of the complex modulus G*(omega) = G(0)omega(beta) measured by other authors using magnetic twisting cytometry and atomic force microscopy. Excellent agreement between A and G(0) on the one hand, and between alpha and beta on the other hand, indicated that the power-law is an intrinsic feature of cell mechanics and not the signature of a particular technique. Moreover, the agreement between measurements at very different size scales, going from a few tens of nanometers to the scale of the whole cell, suggests that self-similarity could be a central feature of cell mechanical structure. Finally, we show that the power-law behavior could explain previous results first interpreted as instantaneous elasticity. Thus, we think that the living cell must definitely be thought of as a material with a large and continuous distribution of relaxation time constants which cannot be described by models with a finite number of springs and dash-pots.

We present an approach to fabricate an array of elastomer posts in order to dynamically measure the traction forces exerted by living cells on a surface with a micrometer lateral resolution. Arrays of closely spaced vertical microposts are made in silicone elastomer [poly(dimethylsiloxane) (PDMS)] by molding a Silicon substrate that has been machined by deep Si etching after standard photolithography. The surface of the micropillars was modified to allow cell culture. Deflections of the calibrated posts were dynamically followed by direct obervation with an optical microscope. By using this set-up, we could dynamically draw up a cartography of the local traction forces exerted by the cells.

Changing shape is changing structure. Deciphering the mechanical contribution of the structural elements of the cells in shape changes is thus crucial to link the mechanical control of growth with development. Many measurements on plant and animal cells rather stress the differences in mechanical properties between both kingdoms. However, this conclusion relies on independent measurements, with very different set-ups and tissues, thus impairing any quantitative comparison. Here we took advantage of a previously described micro-rheometer to compare animal and plant single cell rheology with the same set-up. Using this method, we were able to quantitatively assess the dominant elastic behavior of plant cells in different conditions, and compare it with the viscoelastic behavior of animal cells. Surprisingly, we found that wall-less plant cells exhibit the same rheology as animal cells. This suggests that, despite the main structural differences between animal and plant cells, they also share a common mechanical core. Further investigations revealed that microtubules were the main responsible for the rheological behavior of wall-less plant cells whereas the mechanical properties of animal cells were mainly dependent on the actin network. Thus, wall-less plant cells and animal cells may have developed different strategies to converge to the same mechanical behavior.

The effect of constant and modulated electrical charges applied through the culture support on endothelial cell (EC) morphology was investigated. Endothelial cells tolerated electrical charges for 48 hours, in a serum-free medium. The EC had normal morphology, except at high stationary charges, for which nucleus and cell size increased. The observed cell divisions and cellular secretions indicated that the cells rapidly adapted to the imposed stresses.

Living cells exhibit an important out-of-equilibrium mechanical activity, mainly due to the forces generated by molecular motors. These motor proteins, acting individually or collectively on the cytoskeleton, contribute to the violation of the fluctuation-dissipation theorem in living systems. In this work we probe the cytoskeletal out-of-equilibrium dynamics by performing simultaneous active and passive microrheology experiments, using the same micron-sized probe specifically bound to the actin cortex. The free motion of the probe exhibits a constrained, subdiffusive behavior at short time scales (t < 2s), and a directed, superdiffusive behavior at larger time scales, while, in response to a step force, its creep function presents the usual weak power law dependence with time. Combining the results of both experiments, we precisely measure for the first time the power spectrum of the force fluctuations exerted on this probe, which lies more than one order of magnitude above the spectrum expected at equilibrium, and greatly depends on frequency. We retrieve an effective temperature Teff of the system, as an estimate of the departure from thermal equilibrium. This departure is especially pronounced on long time scales, where Teff bears the footprint of the cooperative activity of motors pulling on the actin network. ATP depletion reduces the fluctuating force amplitude and results in a sharp decrease of Teff towards equilibrium.

T lymphocytes are key modulators of the immune response. Their activation requires cell-cell interaction with different myeloid cell populations of the immune system called antigen-presenting cells (APCs). Although T lymphocytes have recently been shown to respond to mechanical cues, in particular to the stiffness of their environment, little is known about the rigidity of APCs. In this study, single-cell microplate assays were performed to measure the viscoelastic moduli of different human myeloid primary APCs, i.e., monocytes (Ms, storage modulus of 520 +90/−80 Pa), dendritic cells (DCs, 440 +110/−90 Pa), and macrophages (MPHs, 900 +110/−100 Pa). Inflammatory conditions modulated these properties, with storage moduli ranging from 190 Pa to 1450 Pa. The effect of inflammation on the mechanical properties was independent of the induction of expression of commonly used APC maturation markers, making myeloid APC rigidity an additional feature of inflammation. In addition, the rigidity of human T lymphocytes was lower than that of all myeloid cells tested and among the lowest reported (Young’s modulus of 85 ± 5 Pa). Finally, the viscoelastic properties of myeloid cells were dependent on both their filamentous actin content and myosin IIA activity, although the relative contribution of these parameters varied within cell types. These results indicate that T lymphocytes face different cell rigidities when interacting with myeloid APCs in vivo and that this mechanical landscape changes under inflammation.

In a three-dimensional environment, cells migrate through complex topographical features. Using microstructured substrates, we investigate the role of substrate topography in cell adhesion and migration. To do so, fibroblasts are plated on chemically identical substrates composed of microfabricated pillars. When the dimensions of the pillars (i.e., the diameter, length, and spacing) are varied, migrating cells encounter alternating flat and rough surfaces that depend on the spacing between the pillars. Consequently, we show that substrate topography affects cell shape and migration by modifying cell-to-substrate interactions. Cells on micropillar substrates exhibit more elongated and branched shapes with fewer actin stress fibers compared with cells on flat surfaces. By analyzing the migration paths in various environments, we observe different mechanisms of cell migration, including a persistent type of migration, that depend on the organization of the topographical features. These responses can be attributed to a spatial reorganization of the actin cytoskeleton due to physical constraints and a preferential formation of focal adhesions on the micropillars, with an increased lifetime compared to that observed on flat surfaces. By changing myosin II activity, we show that actomyosin contractility is essential in the cellular response to micron-scale topographic signals. Finally, the analysis of cell movements at the frontier between flat and micropillar substrates shows that cell transmigration through the micropillar substrates depends on the spacing between the pillars.

T cells are at the root of the immune response protecting living organisms against pathogens and tumor cells, and have recently been shown to respond to mechanical changes. The activation of T cells triggering the immune response requires the formation of cell-cell interaction between T lymphocytes and various antigen presenting cells (APC) (tumor cells, dendritic cells, macrophages, B lymphocytes...). Yet, little is known about the rigidity of these different APCs. In the present study we use the single-cell microplates assay to measure the mechanical properties of human monocyte derived APCs. We show that APCs present a wide range of stiffnesses, that are affected during differentiation, maturation and in response to inflammatory signals.Noteworthily, dendritic cells, the most efficient APC for T cell activation, present the closest rigidity to T cell rigidity. These results suggest that cell stiffness could be an important factor in immune cell-cell interactions and T cell mediated responses. We will then show preliminary studies on the role of rigidity in T cell activation ; involving laying T cells on substrates mimicking APCs rigidities and monitoring T cell activation and T cell / substrate interface organization.View Large Image | View Hi-Res Image | Download PowerPoint Slide