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Extracellular matrix (ECM) elasticity is perceived by cells via focal adhesion structures, which transduce mechanical cues into chemical signalling to conform cell behavior. Although the contribution of ECM compliance to the control of cell migration or division is extensively studied, little is reported regarding infectious processes. We study this phenomenon with the extraintestinal Escherichia coli pathogen UTI89. We show that UTI89 takes advantage, via its CNF1 toxin, of integrin mechanoactivation to trigger its invasion into cells. We identify the HACE1 E3 ligase-interacting protein Optineurin (OPTN) as a protein regulated by ECM stiffness. Functional analysis establishes a role of OPTN in bacterial invasion and integrin mechanical coupling and for stimulation of HACE1 E3 ligase activity towards the Rac1 GTPase. Consistent with a role of OPTN in cell mechanics, OPTN knockdown cells display defective integrin-mediated traction force buildup, associated with limited cellular invasion by UTI89. Nevertheless, OPTN knockdown cells display strong mechanochemical adhesion signalling, enhanced Rac1 activation and increased cyclin D1 translation, together with enhanced cell proliferation independent of ECM stiffness. Together, our data ascribe a new function to OPTN in mechanobiology. Uropathogenic strains of Escherichia coli (UPEC) are a leading cause of urinary tract infections (UTIs) and invasion involves Rho GTPase members, notably Rac1, to drive actin cytoskeleton rearrangement leading to engulfment. Here, Petracchini et al. provide evidence of an ECM stiffnessmodulated role of Optineurin (OPTN), which regulates HACE1-dependant Rac1 activity and thus controls integrinmediated mechanotransduction and bacterial invasion.

The Shigella effector IpaA co-opts the focal adhesion protein vinculin to promote bacterial invasion. Here, we show that IpaA triggers an unreported mode of vinculin activation through the cooperative binding of its three vinculin-binding sites (VBSs) leading to vinculin oligomerization via its D1 and D2 head subdomains and highly stable adhesions resisting actin relaxing drugs. Using cross-linking mass spectrometry, we found that while IpaA VBSs1-2 bound to D1, IpaA VBS3 interacted with D2, a subdomain masked to other known VBSs. Structural modeling indicated that as opposed to canonical activation linked to interaction with D1, these combined VBSs interactions triggered major allosteric changes leading to D1D2 oligomerization. A cysteine-clamp preventing these changes and D1D2 oligomerization impaired growth of vinculin microclusters and cell adhesion. We propose that D1D2-mediated vinculin oligomerization occurs during the maturation of adhesion structures to enable the scaffolding of high-order vinculin complexes, and is triggered by Shigella IpaA to promote bacterial invasion in the absence of mechanotransduction.SummaryThe Shigella IpaA effector binds to cryptic vinculin sites leading to oligomerization via its head domain. This vinculin oligomerization mode appears required for the maturation and strengthening of cell adhesion but is co-opted by invasive bacteria independent of actomyosin contractility.

Cancer cell softening increases with the progression of the disease, suggesting that mechanical phenotyping could be used as a diagnostic or prognostic method. Here we investigate the cell mechanics of gliomas, brain tumors that originate from glial cells or glial progenitors. We use two microrheology techniques, a single cell parallel plates rheometer to probe whole cell mechanics and optical-tweezers to probe intracellular rheology. We show that cell mechanics discriminates human glioma cells of different grades. When probed globally, grade IV glioblastoma cells are softer than grade III astrocytoma cells, while they are surprisingly stiffer at the intracellular level. We explain this difference between global and local intracellular behaviours by changes in the composition and spatial organization of the cytoskeleton and by changes in nuclear mechanics. Our study highlights the need to combine rheology techniques for potential diagnostic or prognostic methods based on cancer cell mechanophenotyping.

International audience; Beyond its biochemical nature, the nucleus is also a physical object. There is accumulating evidence that its mechanics plays a key role in gene expression, cytoskeleton organization, and more generally in cell and developmental biology. Building on data mainly obtained from the animal literature, we show how nuclear mechanics may orchestrate development and gene expression. In other words, the nucleus may play the additional role of a mechanical rheostat. Although data from plant systems are still scarce, we pinpoint recent advances and highlight some differences with animal systems. Building on this survey, we propose a list of prospects for future research in plant nuclear mechanotransduction and development.

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.

T cells are mechanosensitive but the effect of stiffness on their functions is still debated. We characterize herein how human primary CD4+ T cell functions are affected by stiffness within the physiological Young’s modulus range of 0.5 kPa to 100 kPa. Stiffness modulates T lymphocyte migration and morphological changes induced by TCR/CD3 triggering. Stiffness also increases TCR-induced immune system, metabolism and cell-cycle-related genes. Yet, upon TCR/CD3 stimulation, while cytokine production increases within a wide range of stiffness, from hundreds of Pa to hundreds of kPa, T cell metabolic properties and cell cycle progression are only increased by the highest stiffness tested (100 kPa). Finally, mechanical properties of adherent antigen-presenting cells modulate cytokine production by T cells. Together, these results reveal that T cells discriminate between the wide range of stiffness values found in the body and adapt their responses accordingly. DOI: http://dx.doi.org/10.7554/eLife.23190.001, eLife digest Our immune system contains many cells that play various roles in defending the body against infection, cancer and other threats. For example, T cells constantly patrol the body ready to detect and respond to dangers. They do so by gathering cues from their surroundings, which can be specific chemical signals or physical properties such as the stiffness of tissues. Once the T cells are active they respond in several different ways including releasing hormones and dividing to produce more T cells. Tissue stiffness varies considerably between different organs. Furthermore, disease can lead to changes in tissue stiffness. For example, tissues become more rigid when they are inflamed. The stiffness and other physical properties of the surfaces that T cells interact with affect how the cells respond when they detect a threat, but few details are known about exactly how these cues tune T cell responses. Saitakis et al. studied how human T cells respond to artificial surfaces of varying stiffness that mimic the range found in the body. The experiments show that T cells that interact with stiff surfaces become more active than T cells that interact with softer surfaces. However, some responses are more sensitive to the stiffness of the surface than others. For example, the ability of the T cells to release hormones was affected by the whole range of stiffnesses tested in the experiments, whereas only very stiff surfaces stimulated the T cells to divide. These findings show that T cells can detect the stiffness of surfaces in the body and use this to adapt how they respond to threats. Future challenges will be to find out how T cells sense the physical properties of their surroundings and investigate whether cell and tissue stiffness affects immune responses in the body. This will help us to understand how T cells fight infections and other threats, and could be used to develop new ways of boosting these cells to fight cancer and other diseases. DOI: http://dx.doi.org/10.7554/eLife.23190.002

While plant growth is well known to rely on turgor pressure, it is challenging to quantify the contribution of turgor pressure to plant cell rheology. Here we used a custom-made micro-rheometer to quantify the viscoelastic behavior of isolated plant cells while varying their internal turgor pressure. To get insight into how plant cells adapt their internal pressure to the osmolarity of their medium, we compared the mechanical behavior of single plant cells to that of a simple, passive, pressurized shell: a soccer ball. While both systems exhibited the same qualitative behavior, a simple mechanical model allowed us to quantify turgor pressure regulation at the single cell scale.

International audience; 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.; Parmi les nombreuses énigmes posées par les cellules vivantes, leurcapacité à se déplacer et à changer de forme a attiré notre attention entant que physiciens. Une double approche de modélisation et d'expériencesnous conduit à expliquer deux observations troublantes : premièrement, lacellule adapte l'intensité des forces avec lesquelles elle tire sur sonenvironnement selon la rigidité de celui-ci. Et deuxièmement, pendantqu'une cellule progresse dans une direction en développant uneprotrusion, son squelette interne s'écoule en fait dans l'autre direction,dans un mouvement apparemment contre-productif, appelé écoulementrétrograde. Nous montrons que ces deux phénomènes émanent d'une mêmepropriété paradoxale du squelette interne de la cellule, qui est fait defilaments d'actine assemblés en un réseau. Cet assemblage est bâtipar des liens dont la durée de vie est très courte, ce qui en fait enréalité un liquide qui va lentement s'écouler. À première vue, cela paraîtincompatible avec l'observation, puisque la forme d'un liquide est dictéepar son environnement, alors que les cellules déforment activement leurmilieu. Cependant, parmi les liens formant le réseau d'actine se trouventdes moteurs moléculaires, appelés myosines, capables de tirer sur lesfilaments d'actine et ainsi d'engendrer un écoulement de l'intérieur. Nousmontrons que c'est l'interaction de ces myosines avec la rigidité del'extérieur qui détermine la forme que la cellule prendra. Cela impliqueune consommation continuelle d'énergie par les myosines même lorsque lacellule est globalement immobile, mais nous montrons que ce fonctionnementdote le squelette cellulaire de deux avantages cruciaux : l'agilité d'unliquide pour s'adapter et accomplir les multiples rôles physiologiques dela cellule, et simultanément la résistance d'un solide élastique qui répondinstantanément à des sollicitations mécaniques.

We describe here the parallel plates technique which enables quantifying single-cell mechanics, either passive (cell deformability) or active (whole-cell traction forces). Based on the bending of glass microplates of calibrated stiffness, it is easy to implement on any microscope, and benefits from protocols and equipment already used in biology labs (coating of glass slides, pipette pullers, micromanipulators, etc.). We first present the principle of the technique, the design and calibration of the microplates, and various surface coatings corresponding to different cell–substrate interactions. Then we detail the specific cell preparation for the assays, and the different mechanical assays that can be carried out. Finally, we discuss the possible technical simplifications and the specificities of each mechanical protocol, as well as the possibility of extending the use of the parallel plates to investigate the mechanics of cell aggregates or tissues.

The present study is an attempt to relate the multicomponent response of the cytoskeleton (CSK), evaluated in twisted living adherent cells, to the heterogeneity of the cytoskeletal structure - evaluated both experimentally by means of 3D reconstructions, and theoretically considering the predictions given by two tensegrity models composed of (four and six) compressive elements and (respectively 12 and 24) tensile elements. Using magnetic twisting cytometry in which beads are attached to integrin receptors linked to the actin CSK of living adherent epithelial cells, we specifically measured the elastic CSK response at quasi equilibrium state and partitioned this response in terms of cortical and cytosolic contributions with a two-component model (i.e., a series of two Voigt bodies). These two CSK components were found to be prestressed and exhibited a stress-hardening response which both characterize tensegrity behaviour with however significant differences: compared to the cytosolic component, the cortical cytoskeleton appears to be a faster responding component, being a less prestressed and easily deformable structure. The discrepancies in elastic behaviour between the cortical and cytosolic CSK components may be understood on the basis of prestress tensegrity model predictions, given that the length and number of constitutive actin elements are taken into account.

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.

International audience; The generation of cell polarity is one of the most intriguing symmetry-breaking events in biology. It is involved in almost all physiological and developmental processes and, despite the differences between plant and animal cell structures, cell polarity is generated by a similar core mechanism that comprises the extracellular matrix (ECM), Rho GTPase, the cytoskeleton, and the membranes. Several recent articles show that mechanical factors also contribute to the establishment and robustness of cell polarity, and the different molecular actors of cell polarity are now viewed as integrators of both biochemical and mechanical signals. Although cell polarity remains a complex process, some level of functional convergence between plants and animals is revealed. Following comparative presentation of cell polarity in plants and animals, we will discuss the theoretical background behind the role of mechanics in polarity and the relevant experimental tests, focusing on ECM anchorage, cytoskeleton behavior, and membrane tension.

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.

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