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Collective cell migration (CCM) is involved in multiple biological processes, including embryonic morphogenesis, angiogenesis, and cancer invasion. However, the molecular mechanisms underlying CCM, especially leader cell formation, are poorly understood. Here, we show that a signaling pathway regulating angiomotin (AMOT) cleavage plays a role in CCM, using mammalian epithelial cells and mouse models. In a confluent epithelial monolayer, full-length AMOT localizes at cell-cell junctions and limits cell motility. After cleavage, the C-terminal fragment of AMOT (AMOT-CT) translocates to the cell-matrix interface to promote the maturation of focal adhesions (FAs), generate traction force, and induce leader cell formation. Meanwhile, decreased full-length AMOT at cell-cell junctions leads to tissue fluidization and coherent migration of cell collectives. Hence, the cleavage of AMOT serves as a molecular switch to generate polarized contraction, promoting leader cell formation and CCM., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The forces that cells, tissues, and organisms exert on the surface of a soft substrate can be measured using Traction Force Microscopy (TFM), an important and well-established technique in Mechanobiology. The usual TFM technique (two-dimensional, 2D TFM) treats only the in-plane component of the traction forces and omits the out-of-plane forces at the substrate interfaces (2.5D) that turn out to be important in many biological processes such as tissue migration and tumour invasion. Here, we review the imaging, material, and analytical tools to perform "2.5D TFM" and explain how they are different from 2D TFM. Challenges in 2.5D TFM arise primarily from the need to work with a lower imaging resolution in the z-direction, track fiducial markers in three-dimensions, and reliably and efficiently reconstruct mechanical stress from substrate deformation fields. We also discuss how 2.5D TFM can be used to image, map, and understand the complete force vectors in various important biological events of various length-scales happening at two-dimensional interfaces, including focal adhesions forces, cell diapedesis across tissue monolayers, the formation of three-dimensional tissue structures, and the locomotion of large multicellular organisms. We close with future perspectives including the use of new materials, imaging and machine learning techniques to continuously improve the 2.5D TFM in terms of imaging resolution, speed, and faithfulness of the force reconstruction procedure., Competing Interests: Declaration of Competing Interest We declare that there is no conflict of interest regarding the preparation of this manuscript., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Three-dimensional collective epithelial rotation around a given axis represents a coordinated cellular movement driving tissue morphogenesis and transformation. Questions regarding these behaviors and their relationship with substrate curvatures are intimately linked to spontaneous active matter processes and to vital morphogenetic and embryonic processes. Here, using interdisciplinary approaches, we study the dynamics of epithelial layers lining different cylindrical surfaces. We observe large-scale, persistent, and circumferential rotation in both concavely and convexly curved cylindrical tissues. While epithelia of inverse curvature show an orthogonal switch in actomyosin network orientation and opposite apicobasal polarities, their rotational movements emerge and vary similarly within a common curvature window. We further reveal that this persisting rotation requires stable cell-cell adhesion and Rac-1-dependent cell polarity. Using an active polar gel model, we unveil the different relationships of collective cell polarity and actin alignment with curvatures, which lead to coordinated rotational behavior despite the inverted curvature and cytoskeleton order.

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., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

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)., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

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., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

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.