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Animal cells are active, contractile objects. While bioassays address the molecular characterization of cell contractility, the mechanical characterization of the active forces in cells remains challenging. Here by confronting theoretical analysis and experiments, we calculated both the resistive and the active components of the intracellular stresses that build up following cell adhesion. We obtained a linear relationship between the divergence of the passive stress and the traction forces, which we show is the consequence of the cell adhering and applying forces on the surface only through very localized adhesion points (whose size is inferior to our best resolution, of 400 nm). This entails that there are no measurable forces outside of these active point sources, and also that the passive stresses and active stresses inside cells are proportional.
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Estrés Mecánico , Animales , Adhesión CelularRESUMEN
We present an in-depth investigation of a fully automated Fourier-based analysis to determine the cell size and the width of its distribution in 3D biological tissues. The results are thoroughly tested using generated images, and we offer valuable criteria for image acquisition settings to optimize accuracy. We demonstrate that the most important parameter is the number of cells in the field of view, and we show that accurate measurements can already be made on volume only containing [Formula: see text] cells. The resolution in z is also not so important, and a reduced number of in-depth images, of order of one per cell, already provides a measure of the mean cell size with less than 5% error. The technique thus appears to be a very promising tool for very fast live local volume cell measurement in 3D tissues in vivo while strongly limiting photobleaching and phototoxicity issues.
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Procesamiento de Imagen Asistido por Computador , Tamaño de la Célula , Análisis de Fourier , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
It is now well established that the mechanical environment of the cells in tissues deeply impacts cellular fate, including life cycle, differentiation and tumor progression. Designs of biomaterials already include the control of mechanical parameters, and in general, their main focus is to control the rheological properties of the biomaterials at a macroscopic scale. However, recent studies have demonstrated that cells can stress their environment below the micron scale, and therefore could possibly respond to the rheological properties of their environment at this micron scale. In this context, probing the mechanical properties of physiological cellular environments at subcellular scales is becoming critical. To this aim, we performed in vitro indentation measurements using AFM on sliced human pituitary gland tissues. A robust methodology was implemented using elasto-adhesive models, which shows that accounting for the adhesion of the probe on the tissue is critical for the reliability of the measurement. In addition to quantifying for the first time the rigidity of normal pituitary gland tissue, with a geometric mean of 9.5 kPa, our measurements demonstrated that the mechanical properties of this tissue are far from uniform at subcellular scales. Gradients of rigidity as large as 12 kPa µm(-1) were observed. This observation suggests that physiological rigidity can be highly non-uniform at the micron-scale.
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Encéfalo/fisiología , Módulo de Elasticidad , Microscopía de Fuerza Atómica , Hipófisis/fisiología , HumanosRESUMEN
Neurons are sensitive to topographical cues provided either by in vivo or in vitro environments on the micrometric scale. We have explored the role of randomly distributed silicon nanopillars on primary hippocampal neurite elongation and axonal differentiation. We observed that neurons adhere on the upper part of nanopillars with a typical distance between adhesion points of about 500 nm. These neurons produce fewer neurites, elongate faster, and differentiate an axon earlier than those grown on flat silicon surfaces. Moreover, when confronted with a differential surface topography, neurons specify an axon preferentially on nanopillars. As a whole, these results highlight the influence of the physical environment in many aspects of neuronal growth.
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Confining cells on adhesive patterns allows performing robust, weakly dispersed, statistical analysis. A priori, adhesive patterns could be efficient tools to analyze intracellular cell stress fields, in particular when patterns are used to force the geometry of the cytoskeleton. This tool could then be very helpful in deciphering the relationship between the internal architecture of the cells and the mechanical, intracellular stresses. However, the quantification of the intracellular stresses is still something delicate to perform. Here we first propose a new, very simple and original method to quantify the intracellular stresses, which directly relates the strain the cells impose on the extracellular matrix to the intracellular stress field. This method is used to analyze how confinement influences the intracellular stress field. As a result, we show that the more confined the cells are, the more stressed they will be. The influence of the geometry of the adhesive patterns on the stress patterns is also discussed.
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Células Endoteliales de la Vena Umbilical Humana/fisiología , Modelos Biológicos , Estrés Mecánico , Adhesión Celular , Módulo de Elasticidad , HumanosRESUMEN
Cell culture on soft matrix, either in 2D and 3D, preserves the characteristics of progenitors. However, the mechanism by which the mechanical microenvironment determines progenitor phenotype, and its relevance to human biology, remains poorly described. Here we designed multi-well hydrogel plates with a high degree of physico-chemical uniformity to reliably address the molecular mechanism underlying cell state modification driven by physiological stiffness. Cell cycle, differentiation and metabolic activity could be studied in parallel assays, showing that the soft environment promotes an atypical S-phase quiescence and prevents cell drift, while preserving the differentiation capacities of human bronchoepithelial cells. These softness-sensitive responses are associated with calcium leakage from the endoplasmic reticulum (ER) and defects in proteostasis and enhanced basal ER stress. The analysis of available single cell data of the human lung also showed that this non-conventional state coming from the soft extracellular environment is indeed consistent with molecular feature of pulmonary basal cells. Overall, this study demonstrates that mechanical mimicry in 2D culture supports allows to maintain progenitor cells in a state of high physiological relevance for characterizing the molecular events that govern progenitor biology in human tissues. STATEMENT OF SIGNIFICANCE: This study focuses on the molecular mechanism behind the progenitor state induced by a soft environment. Using innovative hydrogel supports mimicking normal human lung stiffness, the data presented demonstrate that lung mechanics prevent drift while preserving the differentiation capabilities of lung epithelial cells. Furthermore, we show that the cells are positioned in a quiescent state in the atypical S phase. Mechanistically, we demonstrate that this quiescence: i) is driven by calcium leakage from the endoplasmic reticulum (ER) and basal activation of the PERK branch of ER stress signalling, and ii) protects cells from lethal ER stress caused by metabolic stress. Finally, we validate using human single-cell data that these molecular features identified on the soft matrix are found in basal lung cells. Our results reveal original and relevant molecular mechanisms orchestrating cell fate in a soft environment and resistance to exogenous stresses, thus providing new fundamental and clinical insights into basal cell biology.
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Estrés del Retículo Endoplásmico , Matriz Extracelular , Humanos , Matriz Extracelular/metabolismo , Pulmón/metabolismo , Diferenciación Celular , Hidrogeles/químicaRESUMEN
Adherens junctions are required for vascular endothelium integrity. These structures are formed by the clustering of the homophilic adhesive protein VE-cadherin, which recruits intracellular partners, such as ß- and α-catenins, vinculin, and actin filaments. The dogma according to which α-catenin bridges cadherin·ß-catenin complexes to the actin cytoskeleton has been challenged during the past few years, and the link between the VE-cadherin·catenin complex and the actin cytoskeleton remains unclear. Recently, epithelial protein lost in neoplasm (EPLIN) has been proposed as a possible bond between the E-cadherin·catenin complex and actin in epithelial cells. Herein, we show that EPLIN is expressed at similar levels in endothelial and epithelial cells and is located at interendothelial junctions in confluent cells. Co-immunoprecipitation and GST pulldown experiments provided evidence that EPLIN interacts directly with α-catenin and tethers the VE-cadherin·catenin complex to the actin cytoskeleton. In the absence of EPLIN, vinculin was delocalized from the junctions. Furthermore, suppression of actomyosin tension using blebbistatin triggered a similar vinculin delocalization from the junctions. In a Matrigel assay, EPLIN-depleted endothelial cells exhibited a reduced capacity to form pseudocapillary networks because of numerous breakage events. In conclusion, we propose a model in which EPLIN establishes a link between the cadherin·catenin complex and actin that is independent of actomyosin tension. This link acts as a mechanotransmitter, allowing vinculin binding to α-catenin and formation of a secondary molecular bond between the adherens complex and the cytoskeleton through vinculin. In addition, we provide evidence that the EPLIN clutch is necessary for stabilization of capillary structures in an angiogenesis model.
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Citoesqueleto de Actina/metabolismo , Capilares/metabolismo , Proteínas del Citoesqueleto/metabolismo , Células Endoteliales/metabolismo , Modelos Biológicos , Neovascularización Fisiológica/fisiología , alfa Catenina/metabolismo , Citoesqueleto de Actina/genética , Uniones Adherentes/genética , Uniones Adherentes/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Células CACO-2 , Cadherinas/genética , Cadherinas/metabolismo , Capilares/citología , Proteínas del Citoesqueleto/genética , Perros , Células Endoteliales/citología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Mecanotransducción Celular/fisiología , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Vinculina/genética , Vinculina/metabolismo , alfa Catenina/genéticaRESUMEN
Live staining of actin brings valuable information in the field of mechanobiology. Gene transfer of GFP-actin has been reported to disturb cell rheological properties while gene transfer of fluorescent actin binding proteins was not. However the influence of gene transfer on cellular forces in adhered cells has never been investigated. This would provide a more complete picture of mechanical disorders induced by actin live staining for mechanobiology studies. Indeed, most of these techniques were shown to alter cell morphology. Change in cell morphology may in itself be sufficient to perturb cellular forces. Here we focus on quantifying the alterations of cellular stresses that result from baculoviral transduction of GFP-actin in MDCK cell line. We report that GFP-actin transduction increases the proportion of cells with large intracellular or surface stresses, especially in epithelia with low cell density. We show that the enhancement of the mechanical stresses is accompanied by small perturbations of cell shape, but not by a significant change in cell size. We thus conclude that this live staining method enhances the cellular forces but only brings subtle shape alterations.
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Actinas , Citoesqueleto , Actinas/metabolismo , Citoesqueleto/metabolismo , Línea Celular , Forma de la CélulaRESUMEN
Integrin endocytosis is essential for many fundamental cellular processes. Whether and how the internalization impacts cellular mechanics remains elusive. Whereas previous studies reported the contribution of the integrin activator, talin, in force development, the involvement of inhibitors is less documented. We identified ICAP-1 as an integrin inhibitor involved in mechanotransduction by co-working with NME2 to control clathrin-mediated endocytosis of integrins at the edge of focal adhesions (FA). Loss of ICAP-1 enables ß3-integrin-mediated force generation independently of ß1 integrin. ß3-integrin-mediated forces were associated with a decrease in ß3 integrin dynamics stemming from their reduced diffusion within adhesion sites and slow turnover of FA. The decrease in ß3 integrin dynamics correlated with a defect in integrin endocytosis. ICAP-1 acts as an adaptor for clathrin-dependent endocytosis of integrins. ICAP-1 controls integrin endocytosis by interacting with NME2, a key regulator of dynamin-dependent clathrin-coated pits fission. Control of clathrin-mediated integrin endocytosis by an inhibitor is an unprecedented mechanism to tune forces at FA.
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Clatrina , Endocitosis , Adhesiones Focales , Integrina beta1 , Integrina beta3 , Clatrina/metabolismo , Endocitosis/fisiología , Integrina beta1/genética , Mecanotransducción Celular , Talina/genéticaRESUMEN
The minimal structural unit that defines living organisms is a single cell. By proliferating and mechanically interacting with each other, cells can build complex organization such as tissues that ultimately organize into even more complex multicellular living organisms, such as mammals, composed of billions of single cells interacting with each other. As opposed to passive materials, living cells actively respond to the mechanical perturbations occurring in their environment. Tissue cell adhesion to its surrounding extracellular matrix or to neighbors is an example of a biological process that adapts to physical cues. The adhesion of tissue cells to their surrounding medium induces the generation of intracellular contraction forces whose amplitude adapts to the mechanical properties of the environment. In turn, solicitation of adhering cells with physical forces, such as blood flow shearing the layer of endothelial cells in the lumen of arteries, reinforces cell adhesion and impacts cell contractility. In biological terms, the sensing of physical signals is transduced into biochemical signaling events that guide cellular responses such as cell differentiation, cell growth and cell death. Regarding the biological and developmental consequences of cell adaptation to mechanical perturbations, understanding mechanotransduction in tissue cell adhesion appears as an important step in numerous fields of biology, such as cancer, regenerative medicine or tissue bioengineering for instance. Physicists were first tempted to view cell adhesion as the wetting transition of a soft bag having a complex, adhesive interaction with the surface. But surprising responses of tissue cell adhesion to mechanical cues challenged this view. This, however, did not exclude that cell adhesion could be understood in physical terms. It meant that new models and descriptions had to be created specifically for these biological issues, and could not straightforwardly be adapted from dead matter. In this review, we present physical concepts of tissue cell adhesion and the unexpected cellular responses to mechanical cues such as external forces and stiffness sensing. We show how biophysical approaches, both experimentally and theoretically, have contributed to our understanding of the regulation of cellular functions through physical force sensing mechanisms. Finally, we discuss the different physical models that could explain how tissue cell adhesion and force sensing can be coupled to internal mechanosensitive processes within the cell body.
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Fenómenos Mecánicos , Fenómenos Físicos , Animales , Fenómenos Biomecánicos , Adhesión Celular , Humanos , Modelos BiológicosRESUMEN
To understand cell migration, it is crucial to gain knowledge on how cells exert and integrate forces on and from their environment. A quantity of prime interest for biophysicists interested in cell movements modeling is the intracellular stresses. Up to now, three different methods have been proposed to calculate it, they are all in the regime of the thin plate approximation. Two are based on solving the mechanical equilibrium equation inside the cell material (monolayer stress microscopy and Bayesian inference stress microscopy) and one is based on the continuity of displacement at the cell-substrate interface (intracellular stress microscopy). We show here using 3D FEM modeling that these techniques do not calculate the same quantities (as was previously assumed), the first techniques calculate the sum of the active and resistive stresses within the cell, whereas the last one only calculates the resistive component. Combining these techniques should, in principle, permit access to the active stress alone.
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Brain tissues demonstrate heterogeneous mechanical properties, which evolve with aging and pathologies. The observation in these tissues of smooth to sharp rigidity gradients raises the question of brain cell responses to both different values of rigidity and their spatial variations, in dependence on the surface chemistry they are exposed to. Here, we used recent techniques of hydrogel photopolymerization to achieve stiffness texturing down to micrometer resolution in polyacrylamide hydrogels. We investigated primary neuron adhesion and orientation as well as glial cell proliferative properties on these rigidity-textured hydrogels for two adhesive coatings: fibronectin or poly-l-lysine/laminin. Our main observation is that glial cell adhesion and proliferation is favored on the stiffer regions when the adhesive coating is fibronectin and on the softer ones when it consists of poly-l-lysine/laminin. This behavior was unchanged by the presence or the absence of neuronal cells. In addition, glial cells were not confined by sharp, micron-scaled gradients of rigidity. Our observations suggest that rigidity sensing could involve adhesion-related pathways that profoundly depend on surface chemistry.
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Hidrogeles , Laminina , Adhesivos , Fibronectinas/farmacología , Hidrogeles/farmacología , Laminina/farmacología , Neuroglía , Polilisina/farmacologíaRESUMEN
Cell rigidity sensing-a basic cellular process allowing cells to adapt to mechanical cues-involves cell capabilities exerting force on the extracellular environment. In vivo, cells are exposed to multi-scaled heterogeneities in the mechanical properties of the surroundings. Here, we investigate whether cells are able to sense micron-scaled stiffness textures by measuring the forces they transmit to the extracellular matrix. To this end, we propose an efficient photochemistry of polyacrylamide hydrogels to design micron-scale stiffness patterns with kPa/µm gradients. Additionally, we propose an original protocol for the surface coating of adhesion proteins, which allows tuning the surface density from fully coupled to fully independent of the stiffness pattern. This evidences that cells pull on their surroundings by adjusting the level of stress to the micron-scaled stiffness. This conclusion was achieved through improvements in the traction force microscopy technique, e.g., adapting to substrates with a non-uniform stiffness and achieving a submicron resolution thanks to the implementation of a pyramidal optical flow algorithm. These developments provide tools for enhancing the current understanding of the contribution of stiffness alterations in many pathologies, including cancer.
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The binding properties of biomolecules play a crucial role in many biological phenomena, especially cell adhesion. Whereas the attachment kinetics of soluble proteins is considered well known, complex behavior arises when protein molecules are bound to the cell membrane. We probe the hidden kinetics of ligand-receptor bond formation using single-molecule flow chamber assays and Brownian dynamics simulations. We show that, consistent with our recently proposed hypothesis, association requires a minimum duration of contact between the reactive species. In our experiments, ICAM-1 anchored on a flat substrate binds to anti-ICAM-1 coated onto flowing microbeads. The interaction potential between bead and substrate is measured by microinterferometry and is used as an ingredient to simulate bead movement. Our simulation calculates the duration of ligand-receptor contacts imposed by the bead movement. We quantitatively predict the reduction of adhesion probability measured for shorter tether length of the ligand or if a repulsive hyaluronan layer is added onto the surface. To account for our results, we propose that bond formation may occur in our system by crossing of a diffusive plateau in the energy landscape, on the timescale of 5 ms and an energy barrier of 5 k(B)T, before reaching the first detectable bound state. Our results show how to relate cell-scale behavior to the combined information of molecular reactivity and biomolecule submicron-scale environment.
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Molécula 1 de Adhesión Intercelular/inmunología , Adsorción , Animales , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Fenómenos Biomecánicos , Materiales Biomiméticos/química , Adhesión Celular , Glicocálix/química , Humanos , Molécula 1 de Adhesión Intercelular/química , Ligandos , Microesferas , Polímeros/química , Propiedades de Superficie , Factores de TiempoRESUMEN
In general, cells are cultured and adapted to the in vitro rigidities of plastic or glass ranging between 1 and 10 GPa, which is very far from physiological values that are mostly in the kilopascal range. Stem cells however show a high sensitivity to the rigidity of their culture environment, which impacts their differentiation program. Here, we address the impact of rigidity on the long-term maintenance of pluripotency in human induced pluripotent stem cells (hiPSCs) to determine whether soft substrates could provide a new standard for hiPSC expansion and maintenance. To do this, we set up a fabrication process of polyacrylamide-based culture supports with a rigidity-decoupled surface chemistry. Soft elastic substrates with uniform and reproducible physicochemical properties were designed. The maintenance of pluripotency of two hiPSCs lines on substrates with stiffnesses ranging from 3 to 25 kPa was studied with an identical chemical coating consisting of a truncated recombinant vitronectin with defined surface density. Based on the analysis of cellular adhesion, survival, growth kinetics, three-dimensional distribution, and gene and protein expressions, we demonstrate that below 25 kPa hiPSCs do not maintain pluripotency on long-term culture, while pluripotency and self-renewal capacities are maintained above 25 kPa. In contrast to previous studies, no drift toward a specific germ line lineage was revealed. On soft substrates, cell colonies started to grow in three-dimensional (3D), suggesting that softness allows cells to limit contact with the synthetic matrix and to build their own microenvironment. These observations drastically limit the benefit of using standardized soft substrates to expand hiPSCs, at least with the current culture conditions. The development of a robust technology for the design of soft substrates nevertheless opens up perspectives to fine-tune physicochemical properties of the culture environment in addition to or in replacement of soluble growth factors to finely direct cell fate.
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Resinas Acrílicas , Células Madre Pluripotentes Inducidas , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , HumanosRESUMEN
Indentation Type-AFM (IT-AFM) is very useful to analyze the local rheological properties of soft or biological materials. However, analysis of the force-indentation curves is very sensitive to the way the curves are fitted: fits with elastic models such as Hertz or Sneddon's models performed on parts of the curve that indeed correspond to nonlinear elastic regimes, or that result from significant adhesive interactions of the AFM tip with the material lead to results that can be as much as twice larger than fits performed with appropriate models (nonlinear or adhesive).Here, we propose a methodology to address rigidity measurements by fitting parts of the force-indentation curves that correspond to the linear elastic response of the material, even in the presence of adhesion. The major contribution of this methodology is to set up an easy-handling criterion to mark out the linear elastic response to indentation, valid either for purely elastic and elasto-adhesive models.
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Elasticidad , Fenómenos Mecánicos , Microscopía de Fuerza Atómica , Algoritmos , Módulo de Elasticidad , Microscopía de Fuerza Atómica/métodosRESUMEN
Cell focal adhesions are micrometer-sized aggregates of proteins that anchor the cell to the extracellular matrix. Within the cell, these adhesions are connected to the contractile, actin cytoskeleton; this allows the adhesions to transmit forces to the surrounding matrix and makes the adhesion assembly sensitive to the rigidity of their environment. In this article, we predict the dynamics of focal adhesions as a function of the rigidity of the substrate. We generalize previous theories and include the fact that the dynamics of proteins that adsorb to adhesions are also driven by their coupling to cell contractility and the deformation of the matrix. We predict that adhesions reach a finite size that is proportional to the elastic compliance of the substrate, on a timescale that also scales with the compliance: focal adhesions quickly reach a relatively small, steady-state size on soft materials. However, their apparent sliding is not sensitive to the rigidity of the substrate. We also suggest some experimental probes of these ideas and discuss the nature of information that can be extracted from cell force microscopy on deformable substrates.
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Moléculas de Adhesión Celular/fisiología , Adhesión Celular/fisiología , Matriz Extracelular/fisiología , Interpretación de Imagen Asistida por Computador/métodos , Mecanotransducción Celular/fisiología , Microscopía de Fuerza Atómica/métodos , Modelos Biológicos , Simulación por Computador , Estrés MecánicoAsunto(s)
Adhesión Celular , Mecanotransducción Celular , Fenómenos Biológicos , Biofisica , Humanos , Modelos Biológicos , Modelos TeóricosRESUMEN
Cell/matrix adhesions are modulated by cytoskeletal or external stresses and adapt to the mechanical properties of the extracellular matrix. We propose that this mechanosensitivity arises from the activation of a mechanosensor located within the adhesion itself. We show that this mechanism accounts for the observed directional growth of focal adhesions and the reduction or even cessation of their growth when cells adhere to a soft extracellular matrix. We predict quantitatively that both the elasticity and the thickness of the matrix play a key role in the dynamics of focal adhesions. Two different types of dynamics are expected depending on whether the thickness of the matrix is of order of or much larger than the adhesion size. In the latter situation, we predict that the adhesion region reaches a saturation size that can be tuned by the mechanical properties of the matrix.