Whether at the molecular or cellular scale in organisms, cell-cell adhesion adapts to external mechanical cues arising from the static environment of cells and from dynamic interactions between neighboring cells. Cell-cell adhesion needs to resist detachment forces to secure the integrity and internal organization of organisms. In the past, various techniques have been developed to characterize adhesion properties of molecules and cells in vitro and to understand how cells sense and probe their environment. Atomic force microscopy and dual-pipette aspiration, where cells are mainly present in suspension, are common methods for studying detachment forces of cell-cell adhesion. How cell-cell adhesion forces are developed for adherent and environment-adapted cells, however, is less clear. Here, we designed the Cell-Cell Separation Device (CC-SD), a microstructured substrate that provides a step toward measuring both the intercellular forces and external stresses of cells toward the substrate. The device is based on micropillar arrays, originally developed for cell traction-force measurements. We designed PDMS micropillar-blocks, to which cells could adhere and be able to connect to each other across the gap. Controlled stretching of the whole substrate changed the distance between blocks and increased the gap size. This allowed us to apply strains to cell-cell contacts, eventually leading to cell-cell adhesion detachment, which was measured by pillar deflections. The CC-SD provided an increase in the gap between the blocks of up to 2.4-fold, which was sufficient to separate substrate-attached cells with a fully developed F-actin network. Simultaneously measured pillar deflections allowed us to address cellular response to the intercellular strain applied. The CC-SD thus opens up possibilities for the analysis of intercellular detachment forces and sheds light on the robustness of cell-cell adhesion against rupture in dynamic processes during tissue development.

It is unresolved how morphogenetic cell movements and Nodal signalling are involved in the formation of the embryonic body during gastrulation. We address this question by using the zebrafish embryoid model system, which consists of cultured blastula cells lacking yolk and signalling derived from the yolk syncytial layer (YSL). Zebrafish embryoids form a cylindrical-like structure under serum-free growth culture conditions. We find that embryoids elongate without directed convergence and maintain their proliferation rate and cell mixing behaviour. Surprisingly, they undergo budding activity that has not been reported in zebrafish embryos yet. Here, the constitutively active Nodal receptor type I promotes internalisation-like gastrulation movements and induces the expression of shield genetic markers in embryoids. This work demonstrates that the absence of the yolk and signalling derived from the YSL does not affect the cell behaviour, while the constitutively active Nodal receptor type I impacts budding, internalisation movement and the emergence of the second shield. Summary statement We demonstrate that the cell behaviour during gastrulation is robust to the deficiency of yolk topology and signalling originating from the yolk syncytial layer. ### Competing Interest Statement The authors have declared no competing interest.

A leading paradigm for understanding the large-scale behavior of tissues is via generalizations of liquid crystal physics; much like liquid crystals, tissues combine fluid-like, viscoelastic behaviors with local orientational order, such as nematic symmetry. Whilst aspects of quantitative agreement have been achieved for flat monolayers, the most striking features of tissue morphogenesis-including symmetry breaking, folding and invagination-concern surfaces with complex curved geometries in three dimensions. As yet, however, characterizing such behaviors has been frustrated due to the absence of proper image analysis methods; current state-of-the-art methods almost exclusively rely on two-dimensional intensity projections of multiple image planes, which superimpose data and lose geometric information that can be crucial. Here, we describe an analysis pipeline that properly captures the nematic order and topological defects associated with tissue surfaces of arbitrary geometry, which we demonstrate in the context of in vitro multicellular aggregates, and in vivo zebrafish hearts.

Whether at the intramolecular or cellular scale in organisms, cell-cell adhesion adapt to external mechanical cues arising from the static environment of cells and from dynamic interactions between neighboring cells. Cell-cell adhesions need to resist detachment forces to secure the integrity and internal organization of organisms. In the past, various techniques have been developed to characterize adhesion properties of molecules and cells in vitro, and to understand how cells sense and probe their environment. Atomic force microscopy and dual-pipette aspiration, where cells are mainly present in suspension, are common methods for studying detachment forces of cell-cell adhesions. How cell-cell adhesion forces are developed for adherent and environment-adapted cells, however, is less clear. Here, we designed the Cell-Cell Separation Device (CC-SD), a microstructured substrate that measures both intercellular forces and external stresses of cells towards the matrix. The design is based on micropillar arrays originally designed for cell traction-force measurements. We designed PDMS micropillar-blocks, to which cells could adhere and be able to connect to each other across the gap. Controlled stretching of the whole substrate changed the distance between blocks and increased gap size. That allowed us to apply strains to cell-cell contacts, eventually leading to cell-cell adhesion detachment, which was measured by pillar deflections. The CC-SD provided an increase of the gap between the blocks of up to 2.4-fold, which was sufficient to separate substrate-attached cells with fully developed F-actin network. Simultaneously measured pillar deflections allowed us to address cellular response to the intercellular strain applied. The CC-SD thus opens up possibilities for the analysis of intercellular force detachments and sheds light on the robustness of cell-cell adhesions in dynamic processes in tissue development.

Changes in tissue geometry during developmental processes are associated with collective migration of cells. Recent experimental and numerical results suggest that these changes could leverage on the coexistence of nematic and hexatic orientational order at different length scales. How this multiscale organization is affected by the material properties of the cells and their substrate is presently unknown. In this study, we address these questions in monolayers of Madin-Darby canine kidney cells having various cell densities and molecular repertoires. At small length scales, confluent monolayers are characterized by a prominent hexatic order, independent of the presence of E-cadherin, monolayer density, and underlying substrate stiffness. However, all three properties affect the meso-scale tissue organization. The length scale at which hexatic order transits to nematic order, the “hexanematic” crossover scale, strongly depends on cell-cell adhesions and correlates with monolayer density. Our study demonstrates how epithelial organization is affected by mechanical properties, and provides a robust description of tissue organization during developmental processes.

Biological processes such as embryogenesis, wound healing and cancer progression, crucially rely on the ability of epithelial cells to coordinate their mechanical activity over length scales order of magnitudes larger than the typical cellular size. While regulated by various signalling pathways, it has recently become evident that this behavior can additionally hinge on a minimal toolkit of physical mechanisms, of which liquid crystal order is the most prominent example. Yet, experimental and theoretical studies have given so far inconsistent results in this respect: whereas nematic order is often invoked in the interpretation of experimental data, computational models have instead suggested that hexatic order could serve as a linchpin for collective migration in confluent cell layers. In this article we resolve this dilemma. Using a combination of in vitro experiments on Madin-Darby canine kidney cells (MDCK), numerical simulations and analytical work, we demonstrate that both nematic and hexatic order are, in fact, present in epithelial layers, with the former being dominant at large length scales and the latter at small length scales. In MDCK GII cells on uncoated glass, these different types of liquid crystal order crossover at a length scale of the order of ten cell sizes. Our work sheds light on the emergent organization of living matter, provides a new framework for deciphering the structure of epithelia, and paves the way toward a comprehensive and predictive mesoscopic theory of tissues.

Growing experimental evidence indicates that topological defects could serve as organizing centers in the morphogenesis of tissues. Here, we provide a quantitative explanation for this phenomenon, rooted in the buckling theory of deformable active polar liquid crystals. Using a combination of linear stability analysis and computational fluid dynamics, we demonstrate that active layers, such as confined cell monolayers, are unstable to the formation of protrusions in the presence of disclinations. The instability originates from an interplay between the focusing of the elastic forces, mediated by defects, and the renormalization of the system's surface tension by the active flow. The posttransitional regime is also characterized by several complex morphodynamical processes, such as oscillatory deformations, droplet nucleation, and active turbulence. Our findings offer an explanation of recent observations on tissue morphogenesis and shed light on the dynamics of active surfaces in general.

High-resolution and super-resolution techniques become more frequently used in thick, inhomogeneous samples. In particular for imaging life cells and tissue in which one wishes to observe a biological process at minimal interference and in the natural environment, sample inhomogeneities are unavoidable. Yet sample-inhomogeneities are paralleled by refractive index variations, for example between the cell organelles and the surrounding medium, that will result in the refraction of light, and therefore lead to sample-induced astigmatism. Astigmatism in turn will result in positional inaccuracies of observations that are at the heart of all super-resolution techniques. Here we introduce a simple model and define a figure-of-merit that allows one to quickly assess the importance of astigmatism for a given experimental setting. We found that astigmatism caused by the cell’s nucleus can easily lead to aberrations up to hundreds of nanometers, well beyond the accuracy of all super-resolution techniques. The astigmatism generated by small objects, like bacteria or vesicles, appear to be small enough to be of any significance in typical super-resolution experimentation.

Mechanotransduction, the ability of cells to sense and respond to the mechanical cues from their microenvironment, plays an important role in numerous cellular processes, ranging from cell migration to differentiation. Several techniques have been developed to investigate the underlying mechanisms of mechanotransduction, in particular, force measurement-based techniques. However, we still lack basic single cell quantitative comparison on the mechanical properties of commonly used cell types, such as endothelial and fibroblast cells. Such information is critical to provide a precedent for studying complex tissues and organs that consist of various cell types. In this short communication, we report on the mechanical characterization of the commonly used endothelial and fibroblast cells at the single cell level. Using a micropillar-based assay, we measured the traction force profiles of these cells. Our study showcases differences between the two cell types in their traction force distribution and morphology. The results reported can be used as a reference and to lay the groundwork for future analysis of numerous disease models involving these cells.

Cells sense and react on changes of the mechanical properties of their environment and, likewise, respond to external mechanical stress applied to them. However, whether the gravitational field as overall body force modulates cellular behavior is unclear. Different studies demonstrated that micro- and hypergravity influences the shape and elasticity of cells, initiate cytoskeleton reorganization, and influence cell motility. All these cellular properties are interconnected and contribute to forces that cells apply on their surrounding microenvironment. Yet, studies that investigated changes of cell traction forces under hypergravity conditions are scarce. Here, we performed hypergravity experiments on 3T3 fibroblast cells using the large-diameter centrifuge at the European Space Agency - European Space Research and Technology Centre. Cells were exposed to hypergravity of up to 19.5 g for 16 h in both the upright and the inverted orientation with respect to the g-force vector. We observed a decrease in cellular traction forces when the gravitational field was increased up to 5.4 g, followed by an increase of traction forces for higher gravity fields up to 19.5 g independent of the orientation of the gravity vector. We attribute the switch in cellular response to shear thinning at low g-forces, followed by significant rearrangement and enforcement of the cytoskeleton at high g-forces.

Intercellular adhesion is the key to multicellularity, and its malfunction plays an important role in various developmental and disease-related processes. Although it has been intensively studied by both biologists and physicists, a commonly accepted definition of cell-cell adhesion is still being debated. Cell-cell adhesion has been described at the molecular scale as a function of adhesion receptors controlling binding affinity, at the cellular scale as resistance to detachment forces or modulation of surface tension, and at the tissue scale as a regulator of cellular rearrangements and morphogenesis. In this review, we aim to summarize and discuss recent advances in the molecular, cellular, and theoretical description of cell-cell adhesion, ranging from biomimetic models to the complexity of cells and tissues in an organismal context. In particular, we will focus on cadherin-mediated cell-cell adhesion and the role of adhesion signaling and mechanosensation therein, two processes central for understanding the biological and physical basis of cell-cell adhesion.

Cell-cell junctions and extracellular matrix adhesions are important for communication and coordination within tissues. Thereby, cell-cell junctions couple the actomyosin network of cells to generate a mechanical tissue entity. At the cell level this network generates an internal tension by actin fiber contraction. The role of intracellular stress and the specific involvement of each individual type of cell-cell junction in tissue mechanics is less known. We have developed a technology to measure the tensile strength of cell-cell junctions and the respective internal stress between cells. Based on elastic micropillar arrays for cell traction force measurements, we produced array blocks of single cell size and permitting those to connect by a defined distance to each other. Cells adhere on their blocks and connect over the gaps. Via a controlled stretch, it is possible to change the distance between the blocks and to study the breaking stress of the cell-cell junctions. We were simultaneously able to measure changes in traction forces which cells apply on the micropillar arrays. The displaced pillar positions were used to reconstruct the internal stress between the cells. This technology allows us to study the influence of different cell junctions on cell-cell adhesions between the same and different cell types. These measurements are used to investigate the early state of metastasis, where cancer cells detach from the primary tumor and start invading the surrounding tissue.

Gravity-sensitive cellular responses are regularly observed in both specialized and nonspecialized cells. One potential mechanism for this sensitivity is a changing viscosity of the intracellular organelles. Here, we report a novel, to our knowledge, viscosity-sensitive molecular rotor based on mesosubstituted boron-dipyrrin used to investigate the response of viscosity of cellular membranes to hypergravity conditions created at the large diameter centrifuge at the European Space Agency Technology Centre. Mouse osteoblastic (MC3T3-E1) and endothelial (human umbilical vein endothelial cell) cell lines were tested, and an increase in viscosity was found with increasing hypergravity loading. This response is thought to be primarily biologically driven, with the potential for a small, instantaneous physical mechanism also contributing to the observed effect. This work provides the first, to our knowledge, quantitative data for cellular viscosity changes under hypergravity, up to 15 × g.