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Studies of electric effects in biological systems, from the historical experiments of Galvani 1 and the ground-breaking work on action potential2 to studies on limb regeneration3 or wound healing4, share the common feature of being concerned with transitory behavior and not addressing the question of homeostasis. Here using a novel microfluidic device, we study how the homeostasis of confluent epithelial tissues is modified when a trans-epithelial electric potential (TEPD) different from the natural one is imposed on an epithelial layer. We show that epithelial fate is dependent on TEPD of few Volts/cm similar to the endogenous one. When the field direction matches the natural one, we can restore a perfect confluence in an epithelial layer turned defective either by E-cadherin knock-out or by weakening cell-substrate adhesion; additionally, the tissue pushes on the substrate with kilo-Pascals stress, inducing active cell response such as death and differentiation. When the field is opposite, homeostasis is destroyed by the perturbation of junctional actin and cell shapes, and the formation of dynamical mounds5, while the tissue pulls with similar strengths. Most of these observations can be quantitatively explained by an electro-hydrodynamic theory involving local electro-osmotic flows. We expect this work to motivate further studies on long time effects of electromechanical pathways with important tissue engineering applications.

Actomyosin machinery endows cells with contractility at a single cell level. However, at a tissue scale, cells can show either contractile or extensile behaviour based on the direction of pushing or pulling forces due to neighbour interactions or substrate interactions. Previous studies have shown that a monolayer of fibroblasts behaves as a contractile system1 while a monolayer of epithelial cells2,3 or neural crest cells behaves as an extensile system.4 How these two contradictory sources of force generation can coexist has remained unexplained. Through a combination of experiments using MDCK (Madin Darby Canine Kidney) cells, and in-silico modeling, we uncover the mechanism behind this switch in behaviour of epithelial cell monolayers from extensile to contractile as the weakening of intercellular contacts. We find that this switch in active behaviour also promotes the buildup of tension at the cell-substrate interface through an increase in actin stress fibers and higher traction forces. This in turn triggers a mechanotransductive response in vinculin translocation to focal adhesion sites and YAP (Yes-associated protein) transcription factor activation. Our studies also show that differences in extensility and contractility act to sort cells, thus determining a general mechanism for mechanobiological pattern formation during cell competition, morphogenesis and cancer progression.

Summary Cellular traction forces influence epithelial behavior, including wound healing and cell extrusion. Here, we describe a simple in vitro traction force microscopy (TFM) protocol using ECM protein-coated polydimethylsiloxane substrate and widefield fluorescence microscopy. We include detailed steps for analysis so readers can obtain traction forces to study the mechanobiology of epithelial cells. We also provide guidelines on when to adopt another common class of TFM protocols based on polyacrylamide hydrogels. For complete details on the use and execution of this protocol, please refer to Saw et al. (2017) and Teo et al. (2020)., Graphical Abstract, Highlights • A 2D, PDMS-based TFM experimental protocol that is easy to adopt • Detailed steps of analysis to obtain traction forces • A brief note and guidelines on other materials that can be used for TFM, Cellular traction forces influence epithelial behavior, including wound healing and cell extrusion. Here, we describe a simple in vitro traction force microscopy (TFM) protocol using ECM protein-coated polydimethylsiloxane substrate and widefield fluorescence microscopy. We include detailed steps for analysis so readers can obtain traction forces to study the mechanobiology of epithelial cells. We also provide guidelines on when to adopt another common class of TFM protocols based on polyacrylamide hydrogels.

Actomyosin machinery endows cells with contractility at a single-cell level. However, within a monolayer, cells can be contractile or extensile based on the direction of pushing or pulling forces exerted by their neighbours or on the substrate. It has been shown that a monolayer of fibroblasts behaves as a contractile system while epithelial or neural progentior monolayers behave as an extensile system. Through a combination of cell culture experiments and in silico modelling, we reveal the mechanism behind this switch in extensile to contractile as the weakening of intercellular contacts. This switch promotes the build-up of tension at the cell-substrate interface through an increase in actin stress fibres and traction forces. This is accompanied by mechanotransductive changes in vinculin and YAP activation. We further show that contractile and extensile differences in cell activity sort cells in mixtures, uncovering a generic mechanism for pattern formation during cell competition, and morphogenesis.It is now revealed, using cell cultures and in silico models, that weakening intercellular contacts is a fundamental process essential for switching from extensile to contractile tissue behaviour.

Collective epithelial behaviors are essential for the development of lumens in organs. However, conventional assays of planar systems fail to replicate cell cohorts of tubular structures that advance in concerted ways on out-of-plane curved and confined surfaces, such as ductal elongation in vivo. Here, we mimic such coordinated tissue migration by forming lumens of epithelial cell sheets inside microtubes of 1–10 cell lengths in diameter. We show that these cell tubes reproduce the physiological apical–basal polarity, and have actin alignment, cell orientation, tissue organization, and migration modes that depend on the extent of tubular confinement and/or curvature. In contrast to flat constraint, the cell sheets in a highly constricted smaller microtube demonstrate slow motion with periodic relaxation, but fast overall movement in large microtubes. Altogether, our findings provide insights into the emerging migratory modes for epithelial migration and growth under tubular confinement, which are reminiscent of the in vivo scenario., Collective epithelial behaviours are studied in vitro in the context of flat sheets but a system to mimic tubular systems is lacking. Here, the authors develop a method to study collective behaviour in lumenal structures and show that several features depend on the extent of tubular confinement and/or curvature.

Epithelial tissues (epithelia) remove excess cells through extrusion, preventing the accumulation of unnecessary or pathological cells. The extrusion process can be triggered by apoptotic signalling1, oncogenic transformation2, 3 and overcrowding of cells4, 5, 6. Despite the important linkage of cell extrusion to developmental7, homeostatic5 and pathological processes2, 8 such as cancer metastasis, its underlying mechanism and connections to the intrinsic mechanics of the epithelium are largely unexplored. We approach this problem by modelling the epithelium as an active nematic liquid crystal (that has a long range directional order), and comparing numerical simulations to strain rate and stress measurements within monolayers of MDCK (Madin Darby canine kidney) cells. Here we show that apoptotic cell extrusion is provoked by singularities in cell alignments9, 10 in the form of comet-shaped topological defects. We find a universal correlation between extrusion sites and positions of nematic defects in the cell orientation field in different epithelium types. The results confirm the active nematic nature of epithelia, and demonstrate that defect-induced isotropic stresses are the primary precursors of mechanotransductive responses in cells, including YAP (Yes-associated protein) transcription factor activity11, caspase-3-mediated cell death, and extrusions. Importantly, the defect-driven extrusion mechanism depends on intercellular junctions, because the weakening of cell–cell interactions in an α-catenin knockdown monolayer reduces the defect size and increases both the number of defects and extrusion rates, as is also predicted by our model. We further demonstrate the ability to control extrusion hotspots by geometrically inducing defects through microcontact printing of patterned monolayers. On the basis of these results, we propose a mechanism for apoptotic cell extrusion: spontaneously formed topological defects in epithelia govern cell fate. This will be important in predicting extrusion hotspots and dynamics in vivo, with potential applications to tissue regeneration and the suppression of metastasis. Moreover, we anticipate that the analogy between the epithelium and active nematic liquid crystals will trigger further investigations of the link between cellular processes and the material properties of epithelia.

Communities of epithelial cells communicate through intercellular interactions, allowing them to coordinate their motility, which plays a key role in homeostasis, morphogenesis and cancer metastasis. Each cell in the epithelium is a constitutive energy-consuming agent, which can generate forces and interact with other cells through cell–cell junctions. Forces applied through external stimuli or endogenous cellular events are balanced by the cells within the epithelium, resulting in the adjustment of internal tissue contractile stresses and tissue reorganization. Materials science and microengineering techniques can be combined to create controllable environments to study epithelial movement and mechanics. By modulating the cell–material interface and by applying principles of active matter, key aspects of epithelial dynamics and mechanosensing mechanisms can be investigated. In this Review, we discuss epithelial tissues as active materials with particular rheological properties and active behaviours at different length scales. We highlight 2D and 3D materials for the study of epithelial dynamics and summarize key methods for the probing of epithelial mechanics. Tissue responses to mechanical stimuli are examined from the molecular level to the tissue level, and the effects of the shape, architecture and stiffness of the microenvironment are discussed. The dynamics of epithelial tissues play a key role in tissue organization, both in health and disease. In this Review, the authors discuss materials and techniques for the study of epithelial movement and mechanics and investigate epithelia as active matter from a theoretical and experimental perspective.

This study reports the ultra-thin Parylene-C deposition on PDMS substrate for a controllable and reliable process of Parylene-C caulked PDMS (pcPDMS). This work covers the depositions on PDMS substrates with different prepolymer ratios (mass of base to curing agent, @ 10:1, 30: 1 and 50: 1). Stiffness could be modulated in a wide range via the proposed pcPDMS process. The prepared pcPDMS would find applications in biomedical studies, such as mechanobiology.

In various physiological processes, the cell collective is organized in a monolayer, such as seen in a simple epithelium. The advances in the understanding of mechanical behavior of the monolayer and its underlying cellular and molecular mechanisms will help to elucidate the properties of cell collectives. In this Review, we discuss recent in vitro studies on monolayer mechanics and their implications on collective dynamics, regulation of monolayer mechanics by physical confinement and geometrical cues and the effect of tissue mechanics on biological processes, such as cell division and extrusion. In particular, we focus on the active nematic property of cell monolayers and the emerging approach to view biological systems in the light of liquid crystal theory. We also highlight the mechanosensing and mechanotransduction mechanisms at the sub-cellular and molecular level that are mediated by the contractile actomyosin cytoskeleton and cell–cell adhesion proteins, such as E-cadherin and α-catenin. To conclude, we argue that, in order to have a holistic understanding of the cellular response to biophysical environments, interdisciplinary approaches and multiple techniques – from large-scale traction force measurements to molecular force protein sensors – must be employed.

In the version of this Article originally published, the captions for Extended Data Figs. 1, 2 and 3 were in the wrong order and did not correspond to their associated figures. The correct captions are listed below and the Article has been corrected accordingly. In addition, the cell line MCF7 was mistakenly written as ‘MCF7A’ in seven instances in the main text, Methods and Extended Data Fig. 4 caption, and as ‘MCF10A’ in one instance in the ‘Author contributions’ section; these errors have now been corrected.

International audience; The control of tissue growth, which is a key to maintain the protective barrier function of the epithelium, depends on the balance between cell division and cell extrusion rates [1, 2]. Cells within confluent epithelial layers undergo cell extrusion, which relies on cell-cell interactions [3] and actomyosin contractility [4, 5]. Although it has been reported that cell extrusion is also dependent on cell density [6, 7], the contribution of tissue mechanics, which is tightly regulated by cell density [8-12], to cell extrusion is still poorly understood. By measuring the multicellular dynamics and traction forces, we show that changes in epithelial packing density lead to the emergence of distinct modes of cell extrusion. In confluent epithelia with low cell density, cell extrusion is mainly driven by the lamellipodia-based crawling mechanism in the neighbor non-dying cells in connection with large-scale collective movements. As cell density increases, cell motion is shown to slow down, and the role of a supracellular actomyosin cable formation and its contraction in the neighboring cells becomes the preponderant mechanism to locally promote cell extrusion. We propose that these two distinct mechanisms complement each other to ensure proper cell extrusion depending on the cellular environment. Our study provides a quantitative and robust framework to explain how cell density can influence tissue mechanics and in turn regulate cell extrusion mechanisms.

Collective cell migration (CCM) can be described as a large scale coordinated movement of cells that are in close proximity with each other. It is a phenomenon that is observed not only in physiological processes such as that found in embryogenesis and wound healing but also in pathophysiological processes such as cancer metastasis. Some of the factors influencing this concerted process include cell–cell adhesion, cell–substrate interaction and mechanical cues such as geometrical constraints among others. Here, we review recent research work done to investigate CCM of adherent cells. We highlight the classical example of an in vitro cell monolayer to illustrate our current understanding of the different mechanobiological mechanisms involved as these cells respond to the mechanical cues present in their environment. It is hoped that such understanding may potentially lead to better therapeutic strategies for diseases such as cancer and for tissue engineering and repair.

Live tissues can self-organize and be described as active materials composed of cells that generate active stresses through continuous injection of energy. In vitro reconstituted molecular networks, as well as single-cell cytoskeletons show that their filamentous structures can portray nematic liquid crystalline properties and can promote nonequilibrium processes induced by active processes at the microscale. The appearance of collective patterns, the formation of topological singularities, and spontaneous phase transition within the cell cytoskeleton are emergent properties that drive cellular functions. More integrated systems such as tissues have cells that can be seen as coarse-grained active nematic particles and their interaction can dictate many important tissue processes such as epithelial cell extrusion and migration as observed in vitro and in vivo. Here, a brief introduction to the concept of active nematics is provided, and the main focus is on the use of this framework in the systematic study of predominantly 2D tissue architectures and dynamics in vitro. In addition how the nematic state is important in tissue behavior, such as epithelial expansion, tissue homeostasis, and the atherosclerosis disease state, is discussed. Finally, how the nematic organization of cells can be controlled in vitro for tissue engineering purposes is briefly discussed.

Collective migration of cells is of fundamental importance for a number of biological functions such as tissue development and regeneration, wound healing and cancer metastasis. The movement of cell groups consisting of multiple cells connected by cell-cell junctions depends on both extracellular and intercellular contacts. Epithelial cell assemblies are thus regulated by a cross-talk between cell-substrate and cell-cell interactions. Here, we investigated the onset of collective migration in groups of cells as they expand from a few cells into large colonies as a function of extracellular matrix (ECM) protein coating. By varying the amount of ECM presented to the cells, we observe that the mode of colony expansion, as well as their overall geometry, is strongly dependent on substrate adhesiveness. On high ECM protein coated surfaces, cells at the edges of the colonies are well spread exhibiting large outward-pointing protrusive activity, whereas cellular colonies display more circular and convex shapes on less adhesive surfaces. Actin structures at the edge of the colonies also show different organizations with the formation of lamellipodial structures on highly adhesive surfaces and a pluricellular actin cable on less adhesive ones. The analysis of traction forces and cell velocities within the cellular assemblies confirm these results. By increasing ECM protein density, cells exert higher traction forces together with a higher outward motility at the edges. Furthermore, tuning cell-cell adhesion of epithelial cells modified the mode of expansion of the colonies. Finally, we used a recently developed computational model to recapitulate the emergent experimental behaviors of expanding cell colonies and extract that the main effect of the different cell-substrate interactions is on the ability of edge cells to form outward lamellipodia-driven motility. Overall, our data suggest that switching behaviors of epithelial cell assemblies result in a tug-of-war between friction forces at the cell-substrate interface and cell-cell interactions.

We introduce the notion of cell division-induced activity and show that the cell division generates extensile forces and drives dynamical patterns in cell assemblies. Extending the hydrodynamic models of lyotropic active nematics we describe turbulent-like velocity fields that are generated by the cell division in a confluent monolayer of cells. We show that the experimentally measured flow field of dividing Madin-Darby Canine Kidney (MDCK) cells is reproduced by our modeling approach. Division-induced activity acts together with intrinsic activity of the cells in extensile and contractile cell assemblies to change the flow and director patterns and the density of topological defects. Finally we model the evolution of the boundary of a cellular colony and compare the fingering instabilities induced by cell division to experimental observations on the expansion of MDCK cell cultures.

Future quantum technologies rely heavily on good protection of quantum entanglement against environment-induced decoherence. A recent study showed that an extension of Uhrig's dynamical decoupling (UDD) sequence can (in theory) lock an arbitrary but known two-qubit entangled state to the $N$th order using a sequence of $N$ control pulses [Mukhtar et al., Phys. Rev. A 81, 012331 (2010)]. By nesting three layers of explicitly constructed UDD sequences, here we first consider the protection of unknown two-qubit states as superposition of two known basis states, without making assumptions of the system-environment coupling. It is found that the obtained decoherence suppression can be highly sensitive to the ordering of the three UDD layers and can be remarkably effective with the correct ordering. The detailed theoretical results are useful for general understanding of the nature of controlled quantum dynamics under nested UDD. As an extension of our three-layer UDD, it is finally pointed out that a completely unknown two-qubit state can be protected by nesting four layers of UDD sequences. This work indicates that when UDD is applicable (e.g., when environment has a sharp frequency cut-off and when control pulses can be taken as instantaneous pulses), dynamical decoupling using nested UDD sequences is a powerful approach for entanglement protection., 11 pages, 3 figures, published version

Uhrig's dynamical decoupling pulse sequence has emerged as one universal and highly promising approach to decoherence suppression. So far both the theoretical and experimental studies have examined single-qubit decoherence only. This work extends Uhrig's universal dynamical decoupling from one-qubit to two-qubit systems and even to general multi-level quantum systems. In particular, we show that by designing appropriate control Hamiltonians for a two-qubit or a multi-level system, Uhrig's pulse sequence can also preserve a generalized quantum coherence measure to the order of $1+O(T^{N+1})$, with only $N$ pulses. Our results lead to a very useful scheme for efficiently locking two-qubit entangled states. Future important applications of Uhrig's pulse sequence in preserving the quantum coherence of multi-level quantum systems can also be anticipated., Comment: 10 pages, 4 figures, minor changes made, submitted to PRA