Interplay of adherens junctions and matrix proteolysis determines the invasive pattern and growth of squamous cell carcinoma.

Cancers, such as squamous cell carcinoma, frequently invade as multicellular units. However, these invading units can be organised in a variety of ways, ranging from thin discontinuous strands to thick 'pushing' collectives. Here we employ an integrated experimental and computational approach to identify the factors that determine the mode of collective cancer cell invasion. We find that matrix proteolysis is linked to the formation of wide strands but has little effect on the maximum extent of invasion. Cell-cell junctions also favour wide strands, but our analysis also reveals a requirement for cell-cell junctions for efficient invasion in response to uniform directional cues. Unexpectedly, the ability to generate wide invasive strands is coupled to the ability to grow effectively when surrounded by extracellular matrix in three-dimensional assays. Combinatorial perturbation of both matrix proteolysis and cell-cell adhesion demonstrates that the most aggressive cancer behaviour, both in terms of invasion and growth, is achieved at high levels of cell-cell adhesion and high levels of proteolysis. Contrary to expectation, cells with canonical mesenchymal traits - no cell-cell junctions and high proteolysis - exhibit reduced growth and lymph node metastasis. Thus, we conclude that the ability of squamous cell carcinoma cells to invade effectively is also linked to their ability to generate space for proliferation in confined contexts. These data provide an explanation for the apparent advantage of retaining cell-cell junctions in squamous cell carcinomas.

A combination of Notch signaling, preferential adhesion and endocytosis induces a slow mode of cell intercalation in the Drosophila retina.

Movement of epithelial cells in a tissue occurs through neighbor exchange and drives tissue shape changes. It requires intercellular junction remodeling, a process typically powered by the contractile actomyosin cytoskeleton. This has been investigated mainly in homogeneous epithelia, where intercalation takes minutes. However, in some tissues, intercalation involves different cell types and can take hours. Whether slow and fast intercalation share the same mechanisms remains to be examined. To address this issue, we used the fly eye, where the cone cells exchange neighbors over ∼10 h to shape the lens. We uncovered three pathways regulating this slow mode of cell intercalation. First, we found a limited requirement for MyosinII. In this case, mathematical modeling predicts an adhesion-dominant intercalation mechanism. Genetic experiments support this prediction, revealing a role for adhesion through the Nephrin proteins Roughest and Hibris. Second, we found that cone cell intercalation is regulated by the Notch pathway. Third, we show that endocytosis is required for membrane removal and Notch activation. Taken together, our work indicates that adhesion, endocytosis and Notch can direct slow cell intercalation during tissue morphogenesis.

Tissue Mechanics Regulate Mitotic Nuclear Dynamics during Epithelial Development.

Cell divisions are essential for tissue growth. In pseudostratified epithelia, where nuclei are staggered across the tissue, each nucleus migrates apically before undergoing mitosis. Successful apical nuclear migration is critical for planar-orientated cell divisions in densely packed epithelia. Most previous investigations have focused on the local cellular mechanisms controlling nuclear migration. Inter-species and inter-organ comparisons of different pseudostratified epithelia suggest global tissue architecture may influence nuclear dynamics, but the underlying mechanisms remain elusive. Here, we use the developing Drosophila wing disc to systematically investigate, in a single epithelial type, how changes in tissue architecture during growth influence mitotic nuclear migration. We observe distinct nuclear dynamics at discrete developmental stages, as epithelial morphology changes. We use genetic and physical perturbations to show a direct effect of cell density on mitotic nuclear positioning. We find Rho kinase and Diaphanous, which facilitate mitotic cell rounding in confined cell conditions, are essential for efficient apical nuclear movement. Perturbation of Diaphanous causes increasing defects in apical nuclear migration as the tissue grows and cell density increases, and these defects can be reversed by acute physical reduction of cell density. Our findings reveal how the mechanical environment imposed on cells within a tissue alters the molecular and cellular mechanisms adopted by single cells for mitosis.

EpiGraph: an open-source platform to quantify epithelial organization.

SUMMARY: Here we present EpiGraph, an image analysis tool that quantifies epithelial organization. Our method combines computational geometry and graph theory to measure the degree of order of any packed tissue. EpiGraph goes beyond the traditional polygon distribution analysis, capturing other organizational traits that improve the characterization of epithelia. EpiGraph can objectively compare the rearrangements of epithelial cells during development and homeostasis to quantify how the global ensemble is affected. Importantly, it has been implemented in the open-access platform Fiji. This makes EpiGraph very user friendly, with no programming skills required. AVAILABILITY AND IMPLEMENTATION: EpiGraph is available at https://imagej.net/EpiGraph and the code is accessible (https://github.com/ComplexOrganizationOfLivingMatter/Epigraph) under GPLv3 license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Polarization of Myosin II Refines Tissue Material Properties to Buffer Mechanical Stress.

As tissues develop, they are subjected to a variety of mechanical forces. Some of these forces are instrumental in the development of tissues, while others can result in tissue damage. Despite our extensive understanding of force-guided morphogenesis, we have only a limited understanding of how tissues prevent further morphogenesis once the shape is determined after development. Here, through the development of a tissue-stretching device, we uncover a mechanosensitive pathway that regulates tissue responses to mechanical stress through the polarization of actomyosin across the tissue. We show that stretch induces the formation of linear multicellular actomyosin cables, which depend on Diaphanous for their nucleation. These stiffen the epithelium, limiting further changes in shape, and prevent fractures from propagating across the tissue. Overall, this mechanism of force-induced changes in tissue mechanical properties provides a general model of force buffering that serves to preserve the shape of tissues under conditions of mechanical stress.

Fundamental physical cellular constraints drive self-organization of tissues.

Morphogenesis is driven by small cell shape changes that modulate tissue organization. Apical surfaces of proliferating epithelial sheets have been particularly well studied. Currently, it is accepted that a stereotyped distribution of cellular polygons is conserved in proliferating tissues among metazoans. In this work, we challenge these previous findings showing that diverse natural packed tissues have very different polygon distributions. We use Voronoi tessellations as a mathematical framework that predicts this diversity. We demonstrate that Voronoi tessellations and the very different tissues analysed share an overriding restriction: the frequency of polygon types correlates with the distribution of cell areas. By altering the balance of tensions and pressures within the packed tissues using disease, genetic or computer model perturbations, we show that as long as packed cells present a balance of forces within tissue, they will be under a physical constraint that limits its organization. Our discoveries establish a new framework to understand tissue architecture in development and disease.

Cost-benefit analysis of the mechanisms that enable migrating cells to sustain motility upon changes in matrix environments.

Cells can move through extracellular environments with varying geometries and adhesive properties. Adaptation to these differences is achieved by switching between different modes of motility, including lamellipod-driven and blebbing motility. Further, cells can modulate their level of adhesion to the extracellular matrix (ECM) depending on both the level of force applied to the adhesions and cell intrinsic biochemical properties. We have constructed a computational model of cell motility to investigate how motile cells transition between extracellular environments with varying surface continuity, confinement and adhesion. Changes in migration strategy are an emergent property of cells as the ECM geometry and adhesion changes. The transition into confined environments with discontinuous ECM fibres is sufficient to induce shifts from lamellipod-based to blebbing motility, while changes in confinement alone within a continuous geometry are not. The geometry of the ECM facilitates plasticity, by inducing shifts where the cell has high marginal gain from a mode change, and conserving persistency where the cell can continue movement regardless of the motility mode. This regulation of cell motility is independent of global changes in cytoskeletal properties, but requires locally higher linkage between the actin network and the plasma membrane at the cell rear, and changes in internal cell pressure. In addition to matrix geometry, we consider how cells might transition between ECM of different adhesiveness. We find that this requires positive feedback between the forces cells apply on the adhesion points, and the strength of the cell-ECM adhesions on those sites. This positive feedback leads to the emergence of a small number of highly adhesive cores, similar to focal adhesions. While the range of ECM adhesion levels the cell can invade is expanded with this feedback mechanism; the velocities are lowered for conditions where the positive feedback is not vital. Thus, plasticity of cell motility sacrifices the benefits of specialization, for robustness.

STRIPAK components determine mode of cancer cell migration and metastasis.

The contractile actomyosin cytoskeleton and its connection to the plasma membrane are critical for control of cell shape and migration. We identify three STRIPAK complex components, FAM40A, FAM40B and STRN3, as regulators of the actomyosin cortex. We show that FAM40A negatively regulates the MST3 and MST4 kinases, which promote the co-localization of the contractile actomyosin machinery with the Ezrin/Radixin/Moesin family proteins by phosphorylating the inhibitors of PPP1CB, PPP1R14A-D. Using computational modelling, in vitro cell migration assays and in vivo breast cancer metastasis assays we demonstrate that co-localization of contractile activity and actin-plasma membrane linkage reduces cell speed on planar surfaces, but favours migration in confined environments similar to those observed in vivo. We further show that FAM40B mutations found in human tumours uncouple it from PP2A and enable it to drive a contractile phenotype, which may underlie its role in human cancer.

Matrix geometry determines optimal cancer cell migration strategy and modulates response to interventions.

The molecular requirements and morphology of migrating cells can vary depending on matrix geometry; therefore, predicting the optimal migration strategy or the effect of experimental perturbation is difficult. We present a model of cell motility that encompasses actin-polymerization-based protrusions, actomyosin contractility, variable actin-plasma membrane linkage leading to membrane blebbing, cell-extracellular-matrix adhesion and varying extracellular matrix geometries. This is used to explore the theoretical requirements for rapid migration in different matrix geometries. Confined matrix geometries cause profound shifts in the relationship of adhesion and contractility to cell velocity; indeed, cell-matrix adhesion is dispensable for migration in discontinuous confined environments. The model is challenged to predict the effect of different combinations of kinase inhibitors and integrin depletion in vivo, and in confined matrices based on in vitro two-dimensional measurements. Intravital imaging is used to verify bleb-driven migration at tumour margins, and the predicted response to single and combinatorial manipulations.

Alternative allosteric mechanisms can regulate the substrate and E2 in SUMO conjugation.

Sumoylation is the covalent attachment of small ubiquitin-like modifier (SUMO) to a target protein. Similar to other ubiquitin-like pathways, three enzyme types are involved that act in succession: an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). To date, unlike other ubiquitin-like mechanisms, sumoylation of the target RanGAP1 (Target(RanGAP1)) does not absolutely require the E3 of the system, RanBP2 (E3(RanBP2)), since the presence of E2 (E2(Ubc9)) is enough to sumoylate Target(RanGAP1). However, in the presence of E3, sumoylation is more efficient. To understand the role of the target specificity of E3(RanBP2) and E2(Ubc9), we carried out molecular dynamics simulations for the structure of E2(Ubc9)-SUMO-Target(RanGAP1) with and without the E3(RanBP2) ligase. Analysis of the dynamics of E2(Ubc9)-SUMO-Target(RanGAP1) in the absence and presence of E3(RanBP2) revealed that two different allosteric sites regulate the ligase activity: (i) in the presence of E3(RanBP2), the E2(Ubc9)'s loop 2; (ii) in the absence of E3(RanBP2), the Leu65-Arg70 region of SUMO. These results provide a first insight into the question of how E3(RanBP2) can act as an intrinsic E3 for E2(Ubc9) and why, in its absence, the activity of E2(Ubc9)-SUMO-Target(RanGAP1) could still be maintained, albeit at lower efficiency.

A mechanistic view of the role of E3 in sumoylation.

Sumoylation, the covalent attachment of SUMO (Small Ubiquitin-Like Modifier) to proteins, differs from other Ubl (Ubiquitin-like) pathways. In sumoylation, E2 ligase Ubc9 can function without E3 enzymes, albeit with lower reaction efficiency. Here, we study the mechanism through which E3 ligase RanBP2 triggers target recognition and catalysis by E2 Ubc9. Two mechanisms were proposed for sumoylation. While in both the first step involves Ubc9 conjugation to SUMO, the subsequent sequence of events differs: in the first E2-SUMO forms a complex with the target and E3, followed by SUMO transfer to the target. In the second, Ubc9-SUMO binds to the target and facilitates SUMO transfer without E3. Using dynamic correlations obtained from explicit solvent molecular dynamic simulations we illustrate the key roles played by allostery in both mechanisms. Pre-existence of conformational states explains the experimental observations that sumoylation can occur without E3, even though at a reduced rate. Furthermore, we propose a mechanism for enhancement of sumoylation by E3. Analysis of the conformational ensembles of the complex of E2 conjugated to SUMO illustrates that the E2 enzyme is already largely pre-organized for target binding and catalysis; E3 binding shifts the equilibrium and enhances these pre-existing populations. We further observe that E3 binding regulates allosterically the key residues in E2, Ubc9 Asp100/Lys101 E2, for the target recognition.

Cataloging and organizing p73 interactions in cell cycle arrest and apoptosis.

We have compiled the p73-mediated cell cycle arrest and apoptosis pathways. p73 is a member of the p53 family, consisting of p53, p63 and p73. p73 exists in several isoforms, presenting different domain structures. p73 functions not only as a tumor suppressor in apoptosis but also as differentiator in embryo development. p53 mutations are responsible for half of the human cancers; p73 can partially substitute mutant p53 as tumor suppressor. The pathways we assembled create a p73-centered network consisting of 53 proteins and 176 interactions. We clustered our network into five functional categories: Upregulation, Activation, Suppression, Transcriptional Activity and Degradation. Our literature searches led to discovering proteins (c-Jun and pRb) with apparent opposing functional effects; these indicate either currently missing proteins and interactions or experimental misidentification or functional annotation. For convenience, here we present the p73 network using the molecular interaction map (MIM) notation. The p73 MIM is unique amongst MIMs, since it further implements detailed domain features. We highlight shared pathways between p53 and p73. We expect that the compiled and organized network would be useful to p53 family-based studies.