Live 3D imaging and mapping of shear stresses within tissues using incompressible elastic beads.

To investigate the role of mechanical constraints in morphogenesis and development, we have developed a pipeline of techniques based on incompressible elastic sensors. These techniques combine the advantages of incompressible liquid droplets, which have been used as precise in situ shear stress sensors, and of elastic compressible beads, which are easier to tune and to use. Droplets of a polydimethylsiloxane mix, made fluorescent through specific covalent binding to a rhodamin dye, are produced by a microfluidics device. The elastomer rigidity after polymerization is adjusted to the tissue rigidity. Its mechanical properties are carefully calibrated in situ, for a sensor embedded in a cell aggregate submitted to uniaxial compression. The local shear stress tensor is retrieved from the sensor shape, accurately reconstructed through an active contour method. In vitro, within cell aggregates, and in vivo, in the prechordal plate of the zebrafish embryo during gastrulation, our pipeline of techniques demonstrates its efficiency to directly measure the three dimensional shear stress repartition within a tissue.

Real-time tracking of root hair nucleus morphodynamics using a microfluidic approach.

Root hairs (RHs) are tubular extensions of root epidermal cells that favour nutrient uptake and microbe interactions. RHs show a fast apical growth, constituting a unique single cell model system for analysing cellular morphodynamics. In this context, live cell imaging using microfluidics recently developed to analyze root development is appealing, although high-resolution imaging is still lacking to enable an investigation of the accurate spatiotemporal morphodynamics of organelles. Here, we provide a powerful coverslip based microfluidic device (CMD) that enables us to capture high resolution confocal imaging of Arabidopsis RH development with real-time monitoring of nuclear movement and shape changes. To validate the setup, we confirmed the typical RH growth rates and the mean nuclear positioning previously reported with classical methods. Moreover, to illustrate the possibilities offered by the CMD, we have compared the real-time variations in the circularity, area and aspect ratio of nuclei moving in growing and mature RHs. Interestingly, we observed higher aspect ratios in the nuclei of mature RHs, correlating with higher speeds of nuclear migration. This observation opens the way for further investigations of the effect of mechanical constraints on nuclear shape changes during RH growth and nuclear migration and its role in RH and plant development.

A comparison of methods to assess cell mechanical properties.

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

Transient cell stiffening triggered by magnetic nanoparticle exposure.

BACKGROUND: The interactions between nanoparticles and the biological environment have long been studied, with toxicological assays being the most common experimental route. In parallel, recent growing evidence has brought into light the important role that cell mechanics play in numerous cell biological processes. However, despite the prevalence of nanotechnology applications in biology, and in particular the increased use of magnetic nanoparticles for cell therapy and imaging, the impact of nanoparticles on the cells' mechanical properties remains poorly understood. RESULTS: Here, we used a parallel plate rheometer to measure the impact of magnetic nanoparticles on the viscoelastic modulus G*(f) of individual cells. We show how the active uptake of nanoparticles translates into cell stiffening in a short time scale (< 30 min), at the single cell level. The cell stiffening effect is however less marked at the cell population level, when the cells are pre-labeled under a longer incubation time (2 h) with nanoparticles. 24 h later, the stiffening effect is no more present. Imaging of the nanoparticle uptake reveals almost immediate (within minutes) nanoparticle aggregation at the cell membrane, triggering early endocytosis, whereas nanoparticles are almost all confined in late or lysosomal endosomes after 2 h of uptake. Remarkably, this correlates well with the imaging of the actin cytoskeleton, with actin bundling being highly prevalent at early time points into the exposure to the nanoparticles, an effect that renormalizes after longer periods. CONCLUSIONS: Overall, this work evidences that magnetic nanoparticle internalization, coupled to cytoskeleton remodeling, contributes to a change in the cell mechanical properties within minutes of their initial contact, leading to an increase in cell rigidity. This effect appears to be transient, reduced after hours and disappearing 24 h after the internalization has taken place.

Beta-arrestins operate an on/off control switch for focal adhesion kinase activity.

Focal adhesion kinase (FAK) regulates key biological processes downstream of G protein-coupled receptors (GPCRs) in normal and cancer cells, but the modes of kinase activation by these receptors remain unclear. We report that after GPCR stimulation, FAK activation is controlled by a sequence of events depending on the scaffolding proteins ß-arrestins and G proteins. Depletion of ß-arrestins results in a marked increase in FAK autophosphorylation and focal adhesion number. We demonstrate that ß-arrestins interact directly with FAK and inhibit its autophosphorylation in resting cells. Both FAK-ß-arrestin interaction and FAK inhibition require the FERM domain of FAK. Following the stimulation of the angiotensin receptor AT1AR and subsequent translocation of the FAK-ß-arrestin complex to the plasma membrane, ß-arrestin interaction with the adaptor AP-2 releases inactive FAK from the inhibitory complex, allowing its activation by receptor-stimulated G proteins and activation of downstream FAK effectors. Release and activation of FAK in response to angiotensin are prevented by an AP-2-binding deficient ß-arrestin and by a specific inhibitor of ß-arrestin/AP-2 interaction; this inhibitor also prevents FAK activation in response to vasopressin. This previously unrecognized mechanism of FAK regulation involving a dual role of ß-arrestins, which inhibit FAK in resting cells while driving its activation at the plasma membrane by GPCR-stimulated G proteins, opens new potential therapeutic perspectives in cancers with up-regulated FAK.

The desmin network is a determinant of the cytoplasmic stiffness of myoblasts.

BACKGROUND INFORMATION: The mechanical properties of cells are essential to maintain their proper functions, and mainly rely on their cytoskeleton. A lot of attention has been paid to actin filaments, demonstrating their central role in the cells mechanical properties, but much less is known about the participation of intermediate filament (IF) networks. Indeed the contribution of IFs, such as vimentin, keratins and lamins, to cell mechanics has only been assessed recently. We study here the involvement of desmin, an IF specifically expressed in muscle cells, in the rheology of immature muscle cells. Desmin can carry mutations responsible for a class of muscle pathologies named desminopathies. RESULTS: In this study, using three types of cell rheometers, we assess the consequences of expressing wild-type (WT) or mutated desmin on the rheological properties of single myoblasts. We find that the mechanical properties of the cell cortex are not correlated to the quantity, nor the quality of desmin expressed. On the contrary, the overall cell stiffness increases when the amount of WT or mutated desmin polymerised in cytoplasmic networks increases. However, myoblasts become softer when the desmin network is partially depleted by the formation of aggregates induced by the expression of a desmin mutant. CONCLUSIONS: We demonstrate that desmin plays a negligible role in the mechanical properties of the cell cortex but is a determinant of the overall cell stiffness. More particularly, desmin participates to the cytoplasm viscoelasticity. SIGNIFICANCE: Desminopathies are associated with muscular weaknesses attributed to a disorganisation of the structure of striated muscle that impairs the active force generation. The present study evidences for the first time the key role of desmin in the rheological properties of myoblasts, raising the hypothesis that desmin mutations could also alter the passive mechanical properties of muscles, thus participating to the lack of force build up in muscle tissue.

Mapping intracellular mechanics on micropatterned substrates.

The mechanical properties of cells impact on their architecture, their migration, intracellular trafficking, and many other cellular functions and have been shown to be modified during cancer progression. We have developed an approach to map the intracellular mechanical properties of living cells by combining micropatterning and optical tweezers-based active microrheology. We optically trap micrometer-sized beads internalized in cells plated on crossbow-shaped adhesive micropatterns and track their displacement following a step displacement of the cell. The local intracellular complex shear modulus is measured from the relaxation of the bead position assuming that the intracellular microenvironment of the bead obeys power-law rheology. We also analyze the data with a standard viscoelastic model and compare with the power-law approach. We show that the shear modulus decreases from the cell center to the periphery and from the cell rear to the front along the polarity axis of the micropattern. We use a variety of inhibitors to quantify the spatial contribution of the cytoskeleton, intracellular membranes, and ATP-dependent active forces to intracellular mechanics and apply our technique to differentiate normal and cancer cells.

Cells as liquid motors: mechanosensitivity emerges from collective dynamics of actomyosin cortex.

Living cells adapt and respond actively to the mechanical properties of their environment. In addition to biochemical mechanotransduction, evidence exists for a myosin-dependent purely mechanical sensitivity to the stiffness of the surroundings at the scale of the whole cell. Using a minimal model of the dynamics of actomyosin cortex, we show that the interplay of myosin power strokes with the rapidly remodeling actin network results in a regulation of force and cell shape that adapts to the stiffness of the environment. Instantaneous changes of the environment stiffness are found to trigger an intrinsic mechanical response of the actomyosin cortex. Cortical retrograde flow resulting from actin polymerization at the edges is shown to be modulated by the stress resulting from myosin contractility, which in turn, regulates the cell length in a force-dependent manner. The model describes the maximum force that cells can exert and the maximum speed at which they can contract, which are measured experimentally. These limiting cases are found to be associated with energy dissipation phenomena, which are of the same nature as those taking place during the contraction of a whole muscle. This similarity explains the fact that single nonmuscle cell and whole-muscle contraction both follow a Hill-like force-velocity relationship.

Three-dimensional cell body shape dictates the onset of traction force generation and growth of focal adhesions.

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.

Desmin Mutation in the C-Terminal Domain Impairs Traction Force Generation in Myoblasts.

The cytoskeleton plays a key role in the ability of cells to both resist mechanical stress and generate force, but the precise involvement of intermediate filaments in these processes remains unclear. We focus here on desmin, a type III intermediate filament, which is specifically expressed in muscle cells and serves as a skeletal muscle differentiation marker. By using several complementary experimental techniques, we have investigated the impact of overexpressing desmin and expressing a mutant desmin on the passive and active mechanical properties of C2C12 myoblasts. We first show that the overexpression of wild-type-desmin increases the overall rigidity of the cells, whereas the expression of a mutated E413K desmin does not. This mutation in the desmin gene is one of those leading to desminopathies, a subgroup of myopathies associated with progressive muscular weakness that are characterized by the presence of desmin aggregates and a disorganization of sarcomeres. We show that the expression of this mutant desmin in C2C12 myoblasts induces desmin network disorganization, desmin aggregate formation, and a small decrease in the number and total length of stress fibers. We finally demonstrate that expression of the E413K mutant desmin also alters the traction forces generation of single myoblasts lacking organized sarcomeres.

Human Primary Immune Cells Exhibit Distinct Mechanical Properties that Are Modified by Inflammation.

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.

A comparative mechanical analysis of plant and animal cells reveals convergence across kingdoms.

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.

Mechanical resistance of the environment affects root hair growth and nucleus dynamics.

Root hair (RH) cells are important for the growth and survival of seedlings. They favor plant-microbe interactions and nutrients uptake. When invading the soil, RH cells have to penetrate a dense medium exhibiting a variety of physical properties, such as mechanical resistance, that impact the growth and survival of plants. Here we investigate the effect of the mechanical resistance of the culture medium on RH-physical and phenotypical parameters such as length, time, and speed of growth. We also analyze the impact of the environment on nuclear dynamics. We show that the RH growth rate and the nucleus speed decrease similarly as mechanical resistance increases while the time of growth of RH cells is invariable. Moreover, during RH growth, the nucleus-to-tip distance was found to decrease when the stiffness of the environment was increased. Along this line, using Latrunculin B treatment in liquid growth media, we could internally slow down RH growth to reach speeds similar to those observed in stiff solid media while the nucleus-to-tip distance was only slightly affected, supporting thus the idea of a specific effect of mechanical resistance of the environment on nucleus dynamics.

Compressive stress triggers fibroblasts spreading over cancer cells to generate carcinoma in situ organization.

At the early stage of tumor progression, fibroblasts are located at the outer edges of the tumor, forming an encasing layer around it. In this work, we have developed a 3D in vitro model where fibroblasts' layout resembles the structure seen in carcinoma in situ. We use a microfluidic encapsulation technology to co-culture fibroblasts and cancer cells within hollow, permeable, and elastic alginate shells. We find that in the absence of spatial constraint, fibroblasts and cancer cells do not mix but segregate into distinct aggregates composed of individual cell types. However, upon confinement, fibroblasts enwrap cancer cell spheroid. Using a combination of biophysical methods and live imaging, we find that buildup of compressive stress is required to induce fibroblasts spreading over the aggregates of tumor cells. We propose that compressive stress generated by the tumor growth might be a mechanism that prompts fibroblasts to form a capsule around the tumor.

Real-time single-cell response to stiffness.

Living cells adapt to the stiffness of their environment. However, cell response to stiffness is mainly thought to be initiated by the deformation of adhesion complexes under applied force. In order to determine whether cell response was triggered by stiffness or force, we have developed a unique method allowing us to tune, in real time, the effective stiffness experienced by a single living cell in a uniaxial traction geometry. In these conditions, the rate of traction force buildup dF/dt was adapted to stiffness in less than 0.1 s. This integrated fast response was unambiguously triggered by stiffness, and not by force. It suggests that early cell response could be mechanical in nature. In fact, local force-dependent signaling through adhesion complexes could be triggered and coordinated by the instantaneous cell-scale adaptation of dF/dt to stiffness. Remarkably, the effective stiffness method presented here can be implemented on any mechanical setup. Thus, beyond single-cell mechanosensing, this method should be useful to determine the role of rigidity in many fundamental phenomena such as morphogenesis and development.

In vivo measurement of the Young's modulus of the cell wall of single root hairs.

Root hairs are cells from the root epidermis that grow as long tubular bulges perpendicular to the root. They can grow in a variety of mechanical or chemical environments. Their mechanical properties are mainly due to their stiff cell wall which also constitutes a physical barrier between the cell and its environment. Thus, it is essential to be able to quantify the cell wall mechanical properties and their adaptation to environmental cues. Here, we present a technique we developed to measure the Young's (elastic) modulus of the root hair cell wall. In essence, using custom-made glass microplates as cantilevers of calibrated stiffness, we are able to measure the force necessary to bend a single living root hair. From these experiments one can determine the stiffness and Young's modulus of the root hair cell wall.

A Microfluidic-Like System (MLS) to Grow, Image, and Quantitatively Characterize Rigidity Sensing by Plant's Roots and Root Hair Cells.

Plant's roots grow in soils of different rigidities. Understanding how the stiffness of the surrounding environment impacts growth and cell fate of roots and root hair cells is an important and open question. Here, we describe a simple method to setup a microfluidic-like system (MLS) to tackle this question. This system enables to grow plantlets during weeks in microfluidic chips filled with gels of controlled stiffness and to image them under a microscope from a few minutes up to a few days. Furthermore, MLS keeps the numerous benefits of microfluidic chips, such as high-resolution imaging, precise control of the geometry of growth, and standardization of the measurements. In sum, MLS enables one to quantitatively test, even on long time scales, the effect of the rigidity and the geometry of the environment on the growth of roots and root hair cells, including mechanotransduction to the nucleus.

Shigella IpaA mediates actin bundling through diffusible vinculin oligomers with activation imprint.

Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin head and tail domains that bind to actin filaments. Here, we show that Shigella IpaA triggers major allosteric changes in the head domain, leading to vinculin homo-oligomerization. Through the cooperative binding of its three vinculin-binding sites (VBSs), IpaA induces a striking reorientation of the D1 and D2 head subdomains associated with vinculin oligomerization. IpaA thus acts as a catalyst producing vinculin clusters that bundle actin at a distance from the activation site and trigger the formation of highly stable adhesions resisting the action of actin relaxing drugs. Unlike canonical activation, vinculin homo-oligomers induced by IpaA appear to keep a persistent imprint of the activated state in addition to their bundling activity, accounting for stable cell adhesion independent of force transduction and relevant to bacterial invasion.

Single-cell response to stiffness exhibits muscle-like behavior.

Living cells sense the rigidity of their environment and adapt their activity to it. In particular, cells cultured on elastic substrates align their shape and their traction forces along the direction of highest stiffness and preferably migrate towards stiffer regions. Although numerous studies investigated the role of adhesion complexes in rigidity sensing, less is known about the specific contribution of acto-myosin based contractility. Here we used a custom-made single-cell technique to measure the traction force as well as the speed of shortening of isolated myoblasts deflecting microplates of variable stiffness. The rate of force generation increased with increasing stiffness and followed a Hill force-velocity relationship. Hence, cell response to stiffness was similar to muscle adaptation to load, reflecting the force-dependent kinetics of myosin binding to actin. These results reveal an unexpected mechanism of rigidity sensing, whereby the contractile acto-myosin units themselves can act as sensors. This mechanism may translate anisotropy in substrate rigidity into anisotropy in cytoskeletal tension, and could thus coordinate local activity of adhesion complexes and guide cell migration along rigidity gradients.