An Anti-Invasive Role for Mdmx through the RhoA GTPase under the Control of the NEDD8 Pathway.

Mdmx (Mdm4) is established as an oncogene mainly through repression of the p53 tumour suppressor. On the other hand, anti-oncogenic functions for Mdmx have also been proposed, but the underlying regulatory pathways remain unknown. Investigations into the effect of inhibitors for the NEDD8 pathway in p53 activation, human cell morphology, and in cell motility during gastrulation in Xenopus embryos revealed an anti-invasive function of Mdmx. Through stabilisation and activation of the RhoA GTPase, Mdmx is required for the anti-invasive effects of NEDDylation inhibitors. Mechanistically, through its Zn finger domain, Mdmx preferentially interacts with the inactive GDP-form of RhoA. This protects RhoA from degradation and allows for RhoA targeting to the plasma membrane for its subsequent activation. The effect is transient, as prolonged NEDDylation inhibition targets Mdmx for degradation, which subsequently leads to RhoA destabilisation. Surprisingly, Mdmx degradation requires non-NEDDylated (inactive) Culin4A and the Mdm2 E3-ligase. This study reveals that Mdmx can control cell invasion through RhoA stabilisation/activation, which is potentially linked to the reported anti-oncogenic functions of Mdmx. As inhibitors of the NEDD8 pathway are in clinical trials, the status of Mdmx may be a critical determinant for the anti-tumour effects of these inhibitors.

Differences in boundary behavior in the 3D vertex and Voronoi models.

An important open question in the modeling of biological tissues is how to identify the right scale for coarse-graining, or equivalently, the right number of degrees of freedom. For confluent biological tissues, both vertex and Voronoi models, which differ only in their representation of the degrees of freedom, have effectively been used to predict behavior, including fluid-solid transitions and cell tissue compartmentalization, which are important for biological function. However, recent work in 2D has hinted that there may be differences between the two models in systems with heterotypic interfaces between two tissue types, and there is a burgeoning interest in 3D tissue models. Therefore, we compare the geometric structure and dynamic sorting behavior in mixtures of two cell types in both 3D vertex and Voronoi models. We find that while the cell shape indices exhibit similar trends in both models, the registration between cell centers and cell orientation at the boundary are significantly different between the two models. We demonstrate that these macroscopic differences are caused by changes to the cusp-like restoring forces introduced by the different representations of the degrees of freedom at the boundary, and that the Voronoi model is more strongly constrained by forces that are an artifact of the way the degrees of freedom are represented. This suggests that vertex models may be more appropriate for 3D simulations of tissues with heterotypic contacts.

Getting reshaped by the building of your substrate: Extracellular matrix assembly joins the morphogenesis toolkit.

In this issue of Developmental Cell, Serna-Morales et al. show evidence that autonomous assembly of the extracellular matrix drives remodeling of the Drosophila embryonic nervous system. This finding is consistent with the notion that self-assembly of the matrix generates stress that can be exploited by morphogenetic processes.

Ectoderm to mesoderm transition by down-regulation of actomyosin contractility.

Collective migration of cohesive tissues is a fundamental process in morphogenesis and is particularly well illustrated during gastrulation by the rapid and massive internalization of the mesoderm, which contrasts with the much more modest movements of the ectoderm. In the Xenopus embryo, the differences in morphogenetic capabilities of ectoderm and mesoderm can be connected to the intrinsic motility of individual cells, very low for ectoderm, high for mesoderm. Surprisingly, we find that these seemingly deep differences can be accounted for simply by differences in Rho-kinases (Rock)-dependent actomyosin contractility. We show that Rock inhibition is sufficient to rapidly unleash motility in the ectoderm and confer it with mesoderm-like properties. In the mesoderm, this motility is dependent on two negative regulators of RhoA, the small GTPase Rnd1 and the RhoGAP Shirin/Dlc2/ArhGAP37. Both are absolutely essential for gastrulation. At the cellular and tissue level, the two regulators show overlapping yet distinct functions. They both contribute to decrease cortical tension and confer motility, but Shirin tends to increase tissue fluidity and stimulate dispersion, while Rnd1 tends to favor more compact collective migration. Thus, each is able to contribute to a specific property of the migratory behavior of the mesoderm. We propose that the "ectoderm to mesoderm transition" is a prototypic case of collective migration driven by a down-regulation of cellular tension, without the need for the complex changes traditionally associated with the epithelial-to-mesenchymal transition.

EpCAM cellular functions in adhesion and migration, and potential impact on invasion: A critical review.

EpCAM has long been known as a cell surface protein highly expressed in carcinomas. It has since become one of the key cancer biomarkers. Despite its high fame, its actual role in cancer development is still controversial. Beyond a flurry of correlative studies, which point either to a positive or a negative link with tumour progression, there has been surprisingly few studies on the actual cellular mechanisms of EpCAM and on their functional consequences. Clearly, EpCAM plays multiple important roles, in cell proliferation as well as in cell adhesion and migration. The two latter functions, directly relevant for metastasis, are the focus of this review. We attempt here to bring together the available experimental data to build a global coherent view of EpCAM functions. We also include in this overview EpCAM2/Trop2, the close relative of EpCAM. At the core of EpCAM (and EpCAM2/Trop2) function stands the ability to repress contractility of the actomyosin cell cortex. This activity appears to involve direct inhibition by EpCAM of members of the novel PKC family and of a specific downstream PKD-Erk cascade. We will discuss how this activity can result in a variety of adhesive and migratory phenotypes, thus potentially explaining at least part of the apparent inconsistencies between different studies. The picture remains fragmented, and we will highlight some of the conflicting evidence and the many unsolved issues, starting with the controversy around its original description as a cell-cell adhesion molecule.

EpCAM as Modulator of Tissue Plasticity.

The Epithelial Cell Adhesion Molecule or EpCAM is a well-known marker highly expressed in carcinomas and showing a strong correlation with poor cancer prognosis. While its name relates to its proposed function as a cell adhesion molecule, EpCAM has been shown to have various signalling functions. In particular, it has been identified as an important positive regulator of cell adhesion and migration, playing an essential role in embryonic morphogenesis as well as intestinal homeostasis. This activity is not due to its putative adhesive function, but rather to its ability to repress myosin contractility by impinging on a PKC signalling cascade. This mechanism confers EpCAM the unique property of favouring tissue plasticity. I review here the currently available data, comment on possible connections with other properties of EpCAM, and discuss the potential significance in the context of cancer invasion.

Tissue segregation in the early vertebrate embryo.

This chapter discusses our current knowledge on the major segregation events that lead to the individualization of the building blocks of vertebrate organisms, starting with the segregation between "outer" and "inner" cells, the separation of the germ layers and the maintenance of their boundaries during gastrulation, and finally the emergence of the primary axial structure, the notochord. The amphibian embryo is used as the prototypical model, to which fish and mouse development are compared. This comparison highlights a striking conservation of the basic processes. It suggests that simple principles may account for the formation of divergent structures. One of them is based on the non-adhesive nature of the apical domain of epithelial cells, exploited to segregate superficial and deep cell populations as a result of asymmetric division. The other principle involves differential expression of contact cues, such as ephrins and protocadherins, to build up high tension along adhesive interfaces, which efficiently creates sharp boundaries.

Sorting at embryonic boundaries requires high heterotypic interfacial tension.

The establishment of sharp boundaries is essential for segregation of embryonic tissues during development, but the underlying mechanism of cell sorting has remained unclear. Opposing hypotheses have been proposed, either based on global tissue adhesive or contractile properties or on local signalling through cell contact cues. Here we use ectoderm-mesoderm separation in Xenopus to directly evaluate the role of these various parameters. We find that ephrin-Eph-based repulsion is very effective at inducing and maintaining separation, whereas differences in adhesion or contractility have surprisingly little impact. Computer simulations support and generalise our experimental results, showing that a high heterotypic interfacial tension between tissues is key to their segregation. We propose a unifying model, in which conditions of sorting previously considered as driven by differential adhesion/tension should be viewed as suboptimal cases of heterotypic interfacial tension.The mechanisms that cause different cells to segregate into distinct tissues are unclear. Here the authors show in Xenopus that formation of a boundary between two tissues is driven by local tension along the interface rather than by global differences in adhesion or cortical contractility.

Nuclear ß-Catenin under Control.

During Wnt stimulation, ß-catenin accumulates in the nucleus, where it regulates gene transcription. In this issue of Developmental Cell, Lu et al. (2015) report a mechanism that specifically targets this nuclear pool for degradation, using lysine demethylation as ubiquitination signal.

Regulation of cell adhesion and cell sorting at embryonic boundaries.

Embryonic boundaries are sharp delimitations that prevent intermingling between different cell populations. They are essential for the development of well-organized structures and ultimately a functional organism. It has been long believed that this process was driven by global differences in cell adhesion strength, or expression of different types of adhesion molecules. The actual picture turns out to be quite different: Boundaries should be viewed as abrupt discontinuities, where cortical contractility is acutely upregulated in response to specific cell surface contact receptors which act as repulsive cues. Cell adhesion is also modulated along the interface, in different ways depending on the type of boundary, but in all cases the process is subordinated to the function of the cortical actomyosin cytoskeleton.

Ephrin-Eph signaling in embryonic tissue separation.

The physical separation of the embryonic regions that give rise to the tissues and organs of multicellular organisms is a fundamental aspect of morphogenesis. Pioneer experiments by Holtfreter had shown that embryonic cells can sort based on "tissue affinities," which have long been considered to rely on differences in cell-cell adhesion. However, vertebrate embryonic tissues also express a variety of cell surface cues, in particular ephrins and Eph receptors, and there is now firm evidence that these molecules are systematically used to induce local repulsion at contacts between different cell types, efficiently preventing mixing of adjacent cell populations.

Variable combinations of specific ephrin ligand/Eph receptor pairs control embryonic tissue separation.

Ephrins and Eph receptors are involved in the establishment of vertebrate tissue boundaries. The complexity of the system is puzzling, however in many instances, tissues express multiple ephrins and Ephs on both sides of the boundary, a situation that should in principle cause repulsion between cells within each tissue. Although co-expression of ephrins and Eph receptors is widespread in embryonic tissues, neurons, and cancer cells, it is still unresolved how the respective signals are integrated into a coherent output. We present a simple explanation for the confinement of repulsion to the tissue interface: Using the dorsal ectoderm-mesoderm boundary of the Xenopus embryo as a model, we identify selective functional interactions between ephrin-Eph pairs that are expressed in partial complementary patterns. The combined repulsive signals add up to be strongest across the boundary, where they reach sufficient intensity to trigger cell detachments. The process can be largely explained using a simple model based exclusively on relative ephrin and Eph concentrations and binding affinities. We generalize these findings for the ventral ectoderm-mesoderm boundary and the notochord boundary, both of which appear to function on the same principles. These results provide a paradigm for how developmental systems may integrate multiple cues to generate discrete local outcomes.

The cellular basis of tissue separation.

The subdivision of the embryo into physically distinct regions is one of the most fundamental processes in development. General hypotheses for tissue separation based on differential adhesion or tension have been proposed in the past, but with little experimental support. During the last decade, the field has experienced a strong revival, largely driven by renewed interest in biophysical modeling of development. Here, I will discuss the various models of boundary formation and summarize recent studies that have shifted our understanding of the process from the simple juxtaposition of global tissue properties to the characterization of local cellular reactions. Current evidence favors a model whereby separation is controlled by cell surface cues, which, upon cell-cell contact, generate acute changes in cytoskeletal and adhesive properties to inhibit cell mixing, and whereby the integration of multiple local cues may dictate both the global morphogenetic properties of a tissue and its separation from adjacent cell populations.

Regulation of the phosphorylation and nuclear import and export of ß-catenin by APC and its cancer-related truncated form.

We report the first direct analysis of the endogenous ß-catenin phosphorylation activity in colon cancer SW480 cells. By comparing parental SW480 cells that harbor a typical truncated adenomatous polyposis coli (APC) form, cells expressing full-length APC and APC-depleted cells, we provide the formal demonstration that APC is necessary for ß-catenin phosphorylation, both for priming of the protein at residue serine 45 and for the subsequent phosphorylation of residues 33, 37 and 41. Truncated APC still sustains a surprisingly high phosphorylation activity, which requires the protein to bind to ß-catenin through the APC 20-amino-acid (20AA) repeats, thus providing a biochemical explanation for the precise truncations found in cancer cells. We also show that most of the ß-catenin phosphorylation activity is associated with a dense insoluble fraction. We finally examine the impact of full-length and truncated APC on ß-catenin nuclear transport. We observe that ß-catenin is transported much faster than previously thought. Although this fast translocation is largely insensitive to the presence of wild-type or truncated APC, the two forms appear to limit the pool of ß-catenin that is available for transport, which could have an impact on ß-catenin nuclear activities in normal and cancer cells.

EpCAM controls actomyosin contractility and cell adhesion by direct inhibition of PKC.

Epithelial cell adhesion molecule (EpCAM) is a cell-surface protein highly expressed in embryonic tissues and in malignant carcinomas. We report that EpCAM acts as a potent inhibitor of novel protein kinase C (nPKC) in both embryos and cancer cells. We observed dramatic effects of loss of EpCAM on amphibian embryonic tissues, which include sequentially strong overstimulation of PKC activity and of the Erk pathway, leading to exacerbated myosin contractility, loss of cadherin-mediated adhesion, tissue dissociation, and, ultimately, cell death. We show that PKC inhibition is caused by a short segment of the EpCAM cytoplasmic tail. This motif resembles the pseudosubstrate inhibitory domains of PKCs and binds nPKCs with high affinity. A bioinformatics search reveals the existence of similar motifs in other plasma membrane proteins, most of which are cell-cell adhesion molecules. Thus, direct inhibition of PKC by EpCAM represents a general mode of regulation of signal transduction by cell-surface proteins.

A molecular base for cell sorting at embryonic boundaries: contact inhibition of cadherin adhesion by ephrin/ Eph-dependent contractility.

The mechanism responsible for subdividing the embryo into individual tissues is a fundamental, yet still poorly understood, question in developmental biology. Various general hypotheses have been proposed, involving differences in cell adhesion, contractility, or contact-mediated repulsion. However, the key parameter in tissue separation, i.e., the regulation of cadherin-based adhesion at the boundary, has not yet been investigated. We show that cadherin clustering is specifically inhibited at the vertebrate notochord-presomitic mesoderm boundary, preventing formation of adhesive bonds between cells of the two different types. This local regulation depends on differentially expressed ephrins and Eph receptors, which increase cell contractility and generate a membrane blebbing-like behavior along the boundary. Inhibiting myosin activity is sufficient to induce cadherin clustering and formation of stable contacts across the boundary, causing notochord and presomitic tissues to fuse. Local inhibition of cadherin adhesion explains how sharp separation can be achieved in response to cell-cell contact signals.

Looking beyond the Wnt pathway for the deep nature of ß-catenin.

After two decades of stardom, one would think that ß-catenin has revealed all of its most intimate details. Yet the essence of its duality has remained mysterious--how can a single protein both be the core link between cadherins and the cytoskeleton, and the nuclear messenger for Wnt signalling? On the basis of the available evidence and on molecular and evolutionary considerations, I propose that ß-catenin was a born nuclear transport receptor, which by interacting with adhesion molecules acquired the property to coordinate nuclear functions with cell-cell adhesion. While Wnt signalling diverted this activity, the original pathway might still function in modern eukaryotes.

Proteomic analysis of differences in ectoderm and mesoderm membranes by DiGE.

Ectoderm and mesoderm can be considered as prototypes for epithelial and mesenchymal cell types. These two embryonic tissues display clear differences in adhesive and motility properties, which are phenomenologically well characterized but remain largely unexplored at the molecular level. Because the key downstream regulations must occur at the plasma membrane and in the underlying actin cortical structures, we have set out to compare the protein content of membrane fractions from Xenopus ectoderm and mesoderm tissues using 2-dimensional difference gel electrophoresis (DiGE). We have thus identified several proteins that are enriched in one or the other tissues, including regulators of the cytoskeleton and of cell signaling. This study represents to our knowledge the first attempt to use proteomics specifically targeted to the membrane-cortex compartment of embryonic tissues. The identified components should help unraveling a variety of tissue-specific functions in the embryo.