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The motility of cancer cells in 3D matrices is of two types: mesenchymal motility, in which the cells are elongated and amoeboid motility, in which the cells are round. Amoeboid motility is driven by an actomyosin-based contractile force, which is regulated by the Rho/ROCK pathway. However, the molecular mechanisms underlying the motility of elongated cells remain unknown. Here, we show that the motility of elongated cells is regulated by Rac signaling through the WAVE2/Arp2/3-dependent formation of elongated pseudopodia and cell-substrate adhesion in 3D substrates. The involvement of Rac signaling in cell motility was different in cell lines that displayed an elongated morphology in 3D substrates. In U87MG glioblastoma cells, most of which exhibit mesenchymal motility, inhibition of Rac signaling blocked the invasion of these cells in 3D substrates. In HT1080 fibrosarcoma cells, which display mixed cell motility involving both elongated and rounded cells, inhibition of Rac1 signaling not only blocked mesenchymal motility but also caused a mesenchymal–amoeboid transition. Additionally, Rac1 and RhoA signaling regulated the mesenchymal and amoeboid motility in these cells, respectively, and the inhibition of both pathways dramatically decreased cell invasion. Hence, we could conclude that Rac1 and RhoA signaling simultaneously regulate cell invasion in 3D matrices.

Actomyosin contractility affects cellular organization within tissues in part through the generation of mechanical forces at sites of cell-matrix and cell-cell contact. While increased mechanical loading at cell-matrix adhesions results in focal adhesion growth, whether forces drive changes in the size of cell-cell adhesions remains an open question. To investigate the responsiveness of adherens junctions (AJ) to force, we adapted a system of microfabricated force sensors to quantitatively report cell-cell tugging force and AJ size. We observed that AJ size was modulated by endothelial cell-cell tugging forces: AJs and tugging force grew or decayed with myosin activation or inhibition, respectively. Myosin-dependent regulation of AJs operated in concert with a Rac1, and this coordinated regulation was illustrated by showing that the effects of vascular permeability agents (S1P, thrombin) on junctional stability were reversed by changing the extent to which these agents coupled to the Rac and myosin-dependent pathways. Furthermore, direct application of mechanical tugging force, rather than myosin activity per se, was sufficient to trigger AJ growth. These findings demonstrate that the dynamic coordination of mechanical forces and cell-cell adhesive interactions likely is critical to the maintenance of multicellular integrity and highlight the need for new approaches to study tugging forces.

The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells. Silberzan and colleagues demonstrate that local RhoA activity and mechanical forces control the formation of 'migration fingers', cell protrusions involved in the leader-cell-driven collective migration of epithelial cell monolayers.

Cells respond and adapt to their physical environments and to the mechanical forces that they experience. The translation of physical forces into biochemical signalling pathways is known as mechanotransduction. In this review, we focus on two aspects of mechanotransduction. First, we consider how forces exerted on cell adhesion molecules at the cell surface regulate the RhoA signalling pathway by controlling the activities of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). In the second part of the review, we discuss how the nucleus contributes to mechanotransduction as a physical structure connected to the cytoskeleton. We focus on recent studies that have either severed the connections between the nucleus and the cytoskeleton, or that have entirely removed the nucleus from cells. These actions reduce the levels of active RhoA, thereby altering the mechanical properties of cells and decreasing their ability to generate tension and respond to external mechanical forces. This article is part of a discussion meeting issue ‘Forces in cancer: interdisciplinary approaches in tumour mechanobiology’.

Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive‐age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial‐mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial‐mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up‐regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up‐regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.

The removal of unwanted or damaged cells by phagocytes is achieved via a finely regulated cleaning process called efferocytosis. To characterize the mechanisms through which phagocytes control the intake of apoptotic cells, we investigated how the phagocyte's appetite for engulfed cells may be coordinated by RhoA and Rac1 in the phagocytic cup. We used FRET biosensors to visualize the spatiotemporal dynamics of Rho-family GTPases, and found that RhoA, which is known to be downregulated during phagocytosis, was transiently upregulated at the phagocytic cup immediately prior to ingestion. Conversely, Rac1 was upregulated during the engulfment process and then downregulated prior to phagosomal maturation. Moreover, disturbance of the dynamic activities of RhoA led to uncontrolled engulfment, such as fast and undiscerning eating. Our results reveal that the temporal activity of RhoA GTPase alters the Rac1/RhoA balance at the phagocytic cup prior to ingestion, and that this plays a distinct role in orchestrating efferocytosis, with RhoA modulating the rate of engulfment to ensure that the phagocyte engulfs an appropriate amount of the correct material.

Epithelial bending is a central feature of morphogenesis in animals. Here we show that mutual antagonism by the small Rho GTPases Rac1 and RhoA determines cell shape, tissue curvature, and invagination activity in the model epithelium of the developing mouse lens. The epithelial cells of the invaginating lens placode normally elongate and change from a cylindrical to an apically constricted, conical shape. RhoA mutant lens placode cells are both longer and less apically constricted than control cells, thereby reducing epithelial curvature and invagination. By contrast, Rac1 mutant lens placode cells are shorter and more apically restricted than controls, resulting in increased epithelial curvature and precocious lens vesicle closure. Quantification of RhoA- and Rac1-dependent pathway markers over the apical–basal axis of lens pit cells showed that in RhoA mutant epithelial cells there was a Rac1 pathway gain of function and vice versa. These findings suggest that mutual antagonism produces balanced activities of RhoA-generated apical constriction and Rac1-dependent cell elongation that controls cell shape and thus curvature of the invaginating epithelium. The ubiquity of the Rho family GTPases suggests that these mechanisms are likely to apply generally where epithelial morphogenesis occurs.

We derive and simulate a mathematical model for mechanotransduction related to the Rho GTPase signalling pathway. The model addresses the bidirectional coupling between signalling processes and cell mechanics. A numerical method based on bulk-surface finite elements is proposed for the approximation of the coupled system of nonlinear reaction-diffusion equations, defined inside the cell and on the cell membrane, and the equations of elasticity. Our simulation results illustrate novel emergent features such as the strong dependence of the dynamics on cell shape, a threshold-like response to changes in substrate stiffness, and the fact that coupling mechanics and signalling can lead to the robustness of cell deformation to larger changes in substrate stiffness, ensuring mechanical homeostasis in agreement with experiments.

Key Points The clearance of apoptotic cells occurs throughout life in multi-cellular organisms; the failure to properly engulf and remove these dying cells has been linked to a break in self tolerance and autoimmunity. Recent evidence suggests that phagocytes sense the presence of apoptotic cells at the earliest stages of programmed cell death. Some of this sensing involves the secretion of soluble factors that help to recruit phagocytic cells (such as monocytes) to the dying cells. Such prompt removal of apoptotic cells is necessary before they lose their membrane integrity in the later stages of apoptosis, and potentially release their intracellular contents. Phosphatidylserine (PtdSer) is one of the key 'eat-me' signals that is exposed on the surface of apoptotic cells. The exposed PtdSer can be recognized by bridging molecules that can in turn be bound by phagocytic receptors, or by receptors that can directly engage PtdSer. Although the identity of phagocyte receptors that can directly engage PtdSer has remained controversial, recent studies have begun to resolve the conflicting issues and identify new PtdSer recognition receptors. With respect to intracellular signalling within the phagocyte, the RAS homologue (RHO)-family GTPase RAC has a crucial role in the reorganization of the cytoskeleton during the engulfment of apoptotic cells. Upon recognition of apoptotic targets, the intracellular signalling proteins DOCK180 and ELMO1 (engulfment and cell motility 1) function together to activate RAC within the phagocyte and thereby facilitate the uptake of apoptotic cells. The signalling pathway involving DOCK180–ELMO1–RAC that facilitates the uptake of apoptotic cells is evolutionarily conserved from worms to humans. One of the hallmarks of the engulfment of apoptotic cells is that such uptake is immunologically silent, and the phagocytes that engulf apoptotic cells also produce anti-inflammatory cytokines. This is in contrast to the uptake of bacteria or other pathogens, which readily induces an inflammatory response. Defining the intracellular mechanisms within the phagocyte that are induced by the recognition of apoptotic cells that, in turn, inhibit inflammatory signalling, is one of the key areas of ongoing research. When a phagocyte engulfs one or more apoptotic cells, it essentially doubles its cellular contents, including phospholipids, cholesterol, ATP and so on. How the phagocyte maintains homeostasis is beginning to be addressed. Early studies suggest a crucial role for the PtdSer exposed on apoptotic cells in regulating cholesterol efflux from the engulfing macrophages. Future studies will probably focus on how the phagocyte responses are regulated and whether the mechanisms of such apoptotic-cell-mediated immunosuppression could be harnessed for treating chronic inflammatory diseases and autoimmunity. This Review describes the recent insights into the receptors and signalling pathways that are required for the engulfment of apoptotic cells, with particular emphasis on the 'find-me' and 'eat-me' signals expressed on apoptotic cells. The clearance of apoptotic cells by phagocytes is an integral component of normal life, and defects in this process can have significant implications for self tolerance and autoimmunity. Recent studies have provided new insights into the engulfment process, including how phagocytes seek apoptotic cells, how they recognize and ingest these targets and how they maintain cellular homeostasis after the 'meal'. Several new factors that regulate engulfment have been identified, whereas the roles of some of the older players require revision. This Review focuses on these recent developments and attempts to highlight some of the important questions in this field.

The role of exercise to prevent or reverse aging-induced cognitive decline has been widely reported. This neuroprotection is associated with changes in the synaptic structure plasticity. However, the mechanisms of exercise-induced synaptic plasticity in the aging brain are still unclear. Thus, the aim of the present study is to investigate the aging-related alterations of Rho-GTPase and the modulatory influences of exercise training. Young and old rats were used in this study. Old rats were subjected to different schedules of aerobic exercise (12 m/min, 60 min/d, 3d/w or 5d/w) or kept sedentary for 12 w. After 12 w of aerobic exercise, the synapse density in the cortex and hippocampus was detected with immunofluorescent staining using synaptophysin as a marker. The total protein levels of RhoA, Rac1, Cdc42 and cofilin in the cortex and hippocampus were detected with Western Blot. The activities of RhoA, Rac1 and Cdc42 were determined using a pull down assay. We found that synapse loss occurred in aging rats. However, the change of expression and activity of RhoA, Rac1 and Cdc42 was different in the cortex and hippocampus. In the cortex, the expression and activity of Rac1 and Cdc42 was greatly increased with aging, whereas there were no changes in the expression and activity of RhoA. In the hippocampus, the expression and activity of Rac1 and Cdc42 was greatly decreased and there were no changes in the expression and activity of RhoA. As a major downstream substrate of the Rho GTPase family, the increased expression of cofilin was only observed in the cortex. High frequency exercise ameliorated all aging-related changes in the cortex and hippocampus. These data suggest that aerobic exercise reverses synapse loss in the cortex and hippocampus in aging rats, which might be related to the regulation of Rho GTPases.