Organotypic 3D Cell Culture of the Embryonic Lacrimal Gland.

Ectodermal organ development, including lacrimal gland, is characterized by an interaction between an epithelium and a mesenchyme. Murine lacrimal gland is a good model to study non-stereotypical branching morphogenesis. In vitro cultures allow the study of morphogenesis events with easy access to high-resolution imaging. Particularly, embryonic lacrimal gland organotypic 3D cell cultures enable the follow-up of branching morphogenesis thanks to the analysis of territories organization by immunohistochemistry. In this chapter, we describe a method to culture primary epithelial fragments together with primary mesenchymal cells, isolated from embryonic day 17 lacrimal glands.

Hexanematic crossover in epithelial monolayers depends on cell adhesion and cell density.

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.

Extending the Range of SLIM-Labeling Applications: From Human Cell Lines in Culture to Caenorhabditis elegans Whole-Organism Labeling.

The simple light isotope metabolic-labeling technique relies on the in vivo biosynthesis of amino acids from U-[12C]-labeled molecules provided as the sole carbon source. The incorporation of the resulting U-[12C]-amino acids into proteins presents several key advantages for mass-spectrometry-based proteomics analysis, as it results in more intense monoisotopic ions, with a better signal-to-noise ratio in bottom-up analysis. In our initial studies, we developed the simple light isotope metabolic (SLIM)-labeling strategy using prototrophic eukaryotic microorganisms, the yeasts Candida albicans and Saccharomyces cerevisiae, as well as strains with genetic markers that lead to amino-acid auxotrophy. To extend the range of SLIM-labeling applications, we evaluated (i) the incorporation of U-[12C]-glucose into proteins of human cells grown in a complex RPMI-based medium containing the labeled molecule, considering that human cell lines require a large number of essential amino-acids to support their growth, and (ii) an indirect labeling strategy in which the nematode Caenorhabditis elegans grown on plates was fed U-[12C]-labeled bacteria (Escherichia coli) and the worm proteome analyzed for 12C incorporation into proteins. In both cases, we were able to demonstrate efficient incorporation of 12C into the newly synthesized proteins, opening the way for original approaches in quantitative proteomics.

Mechanical stress driven by rigidity sensing governs epithelial stability.

Epithelia act as a barrier against environmental stress and abrasion and in vivo they are continuously exposed to environments of various mechanical properties. The impact of this environment on epithelial integrity remains elusive. By culturing epithelial cells on 2D hydrogels, we observe a loss of epithelial monolayer integrity through spontaneous hole formation when grown on soft substrates. Substrate stiffness triggers an unanticipated mechanical switch of epithelial monolayers from tensile on soft to compressive on stiff substrates. Through active nematic modelling, we find that spontaneous half-integer defect formation underpinning large isotropic stress fluctuations initiate hole opening events. Our data show that monolayer rupture due to high tensile stress is promoted by the weakening of cell-cell junctions that could be induced by cell division events or local cellular stretching. Our results show that substrate stiffness provides feedback on monolayer mechanical state and that topological defects can trigger stochastic mechanical failure, with potential application towards a mechanistic understanding of compromised epithelial integrity during immune response and morphogenesis.

SETDB1 fuels the lung cancer phenotype by modulating epigenome, 3D genome organization and chromatin mechanical properties.

Imbalance in the finely orchestrated system of chromatin-modifying enzymes is a hallmark of many pathologies such as cancers, since causing the affection of the epigenome and transcriptional reprogramming. Here, we demonstrate that a loss-of-function mutation (LOF) of the major histone lysine methyltransferase SETDB1 possessing oncogenic activity in lung cancer cells leads to broad changes in the overall architecture and mechanical properties of the nucleus through genome-wide redistribution of heterochromatin, which perturbs chromatin spatial compartmentalization. Together with the enforced activation of the epithelial expression program, cytoskeleton remodeling, reduced proliferation rate and restricted cellular migration, this leads to the reversed oncogenic potential of lung adenocarcinoma cells. These results emphasize an essential role of chromatin architecture in the determination of oncogenic programs and illustrate a relationship between gene expression, epigenome, 3D genome and nuclear mechanics.

Modulation of designer biomimetic matrices for optimized differentiated intestinal epithelial cultures.

The field of intestinal biology is thirstily searching for different culture methods that complement the limitations of organoids, particularly the lack of a differentiated intestinal compartment. While being recognized as an important milestone for basic and translational biological studies, many primary cultures of intestinal epithelium (IE) rely on empirical trials using hydrogels of various stiffness, whose mechanical impact on epithelial organization remains vague until now. Here, we report the development of hydrogel scaffolds with a range of elasticities and their influence on IE expansion, organization, and differentiation. On stiff substrates (>5 kPa), mouse IE cells adopt a flat cell shape and detach in the short-term. In contrast, on soft substrates (80-500 Pa), they sustain for a long-term, pack into high density, develop columnar shape with improved apical-basal polarity and differentiation marker expression, a phenotype reminiscent of features in vivo mouse IE. We then developed a soft gel molding process to produce 3D Matrigel scaffolds of close-to-nature stiffness, which support and maintain a culture of mouse IE into crypt-villus architecture. Thus, the present work is up-to-date informative for the design of biomaterials for ex vivo intestinal models, offering self-renewal in vitro culture that emulates the mouse IE.

Active nematics across scales from cytoskeleton organization to tissue morphogenesis.

Biological tissues are composed of various cell types working cooperatively to perform their respective function within organs and the whole body. During development, embryogenesis followed by histogenesis relies on orchestrated division, death, differentiation and collective movements of cellular constituents. These cells are anchored to each other and/or the underlying substrate through adhesion complexes and they regulate force generation by active cytoskeleton remodelling. The resulting contractility related changes at the level of each single cell impact tissue architecture by triggering changes in cell shape, cell movement and remodelling of the surrounding environment. These out of equilibrium processes occur through the consumption of energy, allowing biological systems to be described by active matter physics. 'Active nematics' a subclass of active matter encompasses cytoskeleton filaments, bacterial and eukaryotic cells allowing them to be modelled as rod-like elements to which nematic liquid crystal theories can be applied. In this review, we will discuss the concept of active nematics to understand biological processes across subcellular and multicellular scales, from single cell organization to cell extrusion, collective cell movements, differentiation and morphogenesis.

Local contractions regulate E-cadherin rigidity sensing.

E-cadherin is a major cell-cell adhesion molecule involved in mechanotransduction at cell-cell contacts in tissues. Because epithelial cells respond to rigidity and tension in tissue through E-cadherin, there must be active processes that test and respond to the mechanical properties of these adhesive contacts. Using submicrometer, E-cadherin-coated polydimethylsiloxane pillars, we find that cells generate local contractions between E-cadherin adhesions and pull to a constant distance for a constant duration, irrespective of pillar rigidity. These cadherin contractions require nonmuscle myosin IIB, tropomyosin 2.1, α-catenin, and binding of vinculin to α-catenin. Cells spread to different areas on soft and rigid surfaces with contractions, but spread equally on soft and rigid without. We further observe that cadherin contractions enable cells to test myosin IIA-mediated tension of neighboring cells and sort out myosin IIA-depleted cells. Thus, we suggest that epithelial cells test and respond to the mechanical characteristics of neighboring cells through cadherin contractions.

Direct measurement of near-nano-Newton forces developed by self-organizing actomyosin fibers bound α-catenin.

BACKGROUND INFORMATION: Actin cytoskeleton contractility plays a critical role in morphogenetic processes by generating forces that are then transmitted to cell-cell and cell-ECM adhesion complexes. In turn, mechanical properties of the environment are sensed and transmitted to the cytoskeleton at cell adhesion sites, influencing cellular processes such as cell migration, differentiation and survival. Anchoring of the actomyosin cytoskeleton to adhesion sites is mediated by adaptor proteins such as talin or α-catenin that link F-actin to transmembrane cell adhesion receptors, thereby allowing mechanical coupling between the intracellular and extracellular compartments. Thus, a key issue is to be able to measure the forces generated by actomyosin and transmitted to the adhesion complexes. Approaches developed in cells and those probing single molecule mechanical properties of α-catenin molecules allowed to identify α-catenin, an F-actin binding protein which binds to the cadherin complexes as a major player in cadherin-based mechanotransduction. However, it is still very difficult to bridge intercellular forces measured at cellular levels and those measured at the single-molecule level. RESULTS: Here, we applied an intermediate approach allowing reconstruction of the actomyosin-α-catenin complex in acellular conditions to probe directly the transmitted forces. For this, we combined micropatterning of purified α-catenin and spontaneous actomyosin network assembly in the presence of G-actin and Myosin II with microforce sensor arrays used so far to measure cell-generated forces. CONCLUSIONS: Using this method, we show that self-organizing actomyosin bundles bound to micrometric α-catenin patches can apply near-nano-Newton forces. SIGNIFICANCE: Our results pave the way for future studies on molecular/cellular mechanotransduction and mechanosensing.

Cell migration guided by long-lived spatial memory.

Living cells actively migrate in their environment to perform key biological functions-from unicellular organisms looking for food to single cells such as fibroblasts, leukocytes or cancer cells that can shape, patrol or invade tissues. Cell migration results from complex intracellular processes that enable cell self-propulsion, and has been shown to also integrate various chemical or physical extracellular signals. While it is established that cells can modify their environment by depositing biochemical signals or mechanically remodelling the extracellular matrix, the impact of such self-induced environmental perturbations on cell trajectories at various scales remains unexplored. Here, we show that cells can retrieve their path: by confining motile cells on 1D and 2D micropatterned surfaces, we demonstrate that they leave long-lived physicochemical footprints along their way, which determine their future path. On this basis, we argue that cell trajectories belong to the general class of self-interacting random walks, and show that self-interactions can rule large scale exploration by inducing long-lived ageing, subdiffusion and anomalous first-passage statistics. Altogether, our joint experimental and theoretical approach points to a generic coupling between motile cells and their environment, which endows cells with a spatial memory of their path and can dramatically change their space exploration.

Mechanical plasticity in collective cell migration.

Collective cell migration is crucial to maintain epithelium integrity during developmental and repair processes. It requires a tight regulation of mechanical coordination between neighboring cells. This coordination embraces different features including mechanical self-propulsion of individual cells within cellular colonies and large-scale force transmission through cell-cell junctions. This review discusses how the plasticity of biomechanical interactions at cell-cell contacts could help cellular systems to perform coordinated motions and adapt to the properties of the external environment.

Ankyrin G organizes membrane components to promote coupling of cell mechanics and glucose uptake.

The response of cells to forces is critical for their function and occurs via rearrangement of the actin cytoskeleton1. Cytoskeletal remodelling is energetically costly2,3, yet how cells signal for nutrient uptake remains undefined. Here we present evidence that force transmission increases glucose uptake by stimulating glucose transporter 1 (GLUT1). GLUT1 recruitment to and retention at sites of force transmission requires non-muscle myosin IIA-mediated contractility and ankyrin G. Ankyrin G forms a bridge between the force-transducing receptors and GLUT1. This bridge is critical for enabling cells under tension to tune glucose uptake to support remodelling of the actin cytoskeleton and formation of an epithelial barrier. Collectively, these data reveal an unexpected mechanism for how cells under tension take up nutrients and provide insight into how defects in glucose transport and mechanics might be linked.

Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers.

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.

Adhesion-mediated heterogeneous actin organization governs apoptotic cell extrusion.

Apoptotic extrusion is crucial in maintaining epithelial homeostasis. Current literature supports that epithelia respond to extrusion by forming a supracellular actomyosin purse-string in the neighbors. However, whether other actin structures could contribute to extrusion and how forces generated by these structures can be integrated are unknown. Here, we found that during extrusion, a heterogeneous actin network composed of lamellipodia protrusions and discontinuous actomyosin cables, was reorganized in the neighboring cells. The early presence of basal lamellipodia protrusion participated in both basal sealing of the extrusion site and orienting the actomyosin purse-string. The co-existence of these two mechanisms is determined by the interplay between the cell-cell and cell-substrate adhesions. A theoretical model integrates these cellular mechanosensitive components to explain why a dual-mode mechanism, which combines lamellipodia protrusion and purse-string contractility, leads to more efficient extrusion than a single-mode mechanism. In this work, we provide mechanistic insight into extrusion, an essential epithelial homeostasis process.

Active forces modulate collective behaviour and cellular organization.

Biological tissues are composed of various cell types working cooperatively to perform their respective function within organs and the whole body. During development, embryogenesis followed by histogenesis relies on orchestrated division, death, differentiation and collective movements of cellular constituents. These cells are anchored to each other and/or the underlying substrate through adhesion complexes and they regulate force generation by active cytoskeleton remodeling. The resulting changes in contractility at the level of each single cell impact tissue architecture and remodeling by triggering changes in cell shape, cell movement and remodeling of the surrounding environment. These out of equilibrium processes occur through cellular energy consumption, allowing biological systems to be described by active matter physics. Cytoskeleton filaments, bacterial and eukaryotic cells can be considered as a sub-class of active matter termed "active nematics". These biological objects can be modelled as rod-like elements to which nematic liquid crystal theories can be applied. In this work, using an analogy from liquid crystal physics, we show that cell sorting and boundary formation can be explained using differences in nematic activity. This difference in nematic activity arises from a balance of inter- and intra-cellular activity.

Les tissus biologiques sont composés de différents types de cellules qui travaillent en coopération pour remplir leur fonction respective au sein des organes et du corps entier. Au cours du développement, l'embryogenèse puis l'histogenèse reposent sur la division, la mort, la différenciation et les mouvements collectifs orchestrés des constituants cellulaires. Ces cellules sont ancrées les unes aux autres et/ou au substrat sous-jacent par des complexes d'adhésion et elles régulent la génération de force par un remodelage actif du cytosquelette. Les changements de contractilité qui en résultent au niveau de chaque cellule ont un impact sur l'architecture et le remodelage des tissus en déclenchant des changements dans la forme et le mouvement des cellules et le remodelage du milieu environnant. Ces processus sont hors d'équilibre dans la mesure où ils consomment de l'énergie, ce qui classe ces systèmes biologiques au sein de la matière active. Les filaments du cytosquelette, les cellules bactériennes et eucaryotes peuvent être considérés comme une sous-classe de matière active appelée "nématique active". Ces systèmes biologiques peuvent être décrits comme un ensemble de particules allongées auxquelles les théories des cristaux liquides nématiques peuvent être appliquées. Dans ce travail, en utilisant une analogie avec la physique des cristaux liquides, nous montrons que le tri cellulaire et la formation de frontières peuvent être expliqués par des différences d'activité nématique. Cette différence d'activité nématique résulte d'un équilibre entre l'activité inter- et intra-cellulaire.

The role of single cell mechanical behavior and polarity in driving collective cell migration.

The directed migration of cell collectives is essential in various physiological processes, such as epiboly, intestinal epithelial turnover, and convergent extension during morphogenesis as well as during pathological events like wound healing and cancer metastasis. Collective cell migration leads to the emergence of coordinated movements over multiple cells. Our current understanding emphasizes that these movements are mainly driven by large-scale transmission of signals through adherens junctions. In this study, we show that collective movements of epithelial cells can be triggered by polarity signals at the single cell level through the establishment of coordinated lamellipodial protrusions. We designed a minimalistic model system to generate one-dimensional epithelial trains confined in ring shaped patterns that recapitulate rotational movements observed in vitro in cellular monolayers and in vivo in genitalia or follicular cell rotation. Using our system, we demonstrated that cells follow coordinated rotational movements after the establishment of directed Rac1-dependent polarity over the entire monolayer. Our experimental and numerical approaches show that the maintenance of coordinated migration requires the acquisition of a front-back polarity within each single cell but does not require the maintenance of cell-cell junctions. Taken together, these unexpected findings demonstrate that collective cell dynamics in closed environments as observed in multiple in vitro and in vivo situations can arise from single cell behavior through a sustained memory of cell polarity.

Cell response to substrate rigidity is regulated by active and passive cytoskeletal stress.

Morphogenesis, tumor formation, and wound healing are regulated by tissue rigidity. Focal adhesion behavior is locally regulated by stiffness; however, how cells globally adapt, detect, and respond to rigidity remains unknown. Here, we studied the interplay between the rheological properties of the cytoskeleton and matrix rigidity. We seeded fibroblasts onto flexible microfabricated pillar arrays with varying stiffness and simultaneously measured the cytoskeleton organization, traction forces, and cell-rigidity responses at both the adhesion and cell scale. Cells adopted a rigidity-dependent phenotype whereby the actin cytoskeleton polarized on stiff substrates but not on soft. We further showed a crucial role of active and passive cross-linkers in rigidity-sensing responses. By reducing myosin II activity or knocking down α-actinin, we found that both promoted cell polarization on soft substrates, whereas α-actinin overexpression prevented polarization on stiff substrates. Atomic force microscopy indentation experiments showed that this polarization response correlated with cell stiffness, whereby cell stiffness decreased when active or passive cross-linking was reduced and softer cells polarized on softer matrices. Theoretical modeling of the actin network as an active gel suggests that adaptation to matrix rigidity is controlled by internal mechanical properties of the cytoskeleton and puts forward a universal scaling between nematic order of the actin cytoskeleton and the substrate-to-cell elastic modulus ratio. Altogether, our study demonstrates the implication of cell-scale mechanosensing through the internal stress within the actomyosin cytoskeleton and its coupling with local rigidity sensing at focal adhesions in the regulation of cell shape changes and polarity.

A subtle relationship between substrate stiffness and collective migration of cell clusters.

The physical cues from the extracellular environment mediates cell signaling spatially and temporally. Cells respond to physical cues from their environment in a non-monotonic fashion. Despite our understanding of the role of substrate rigidity on single cell migration, how cells respond collectively to increasing extracellular matrix stiffness is not well established. Here we patterned multicellular epithelial Madin-Darby canine kidney (MDCK) islands on polyacrylamide gels of varying stiffness and studied their expansion. Our findings show that the MDCK islands expanded faster with increasing stiffness only up to an optimum stiffness, over which the expansion plateaued. We then focused on the expansion of the front of the assemblies and the formation of leader cells. We observed cell front destabilization only above substrate stiffness of a few kPa. The extension of multicellular finger-like structures at the edges of the colonies for intermediate and high stiffnesses from 6 to 60 kPa responded to higher substrate stiffness by increasing focal adhesion areas and actin cable assembly. Additionally, the number of leader cells at the finger-like protrusions increased with stiffness in correlation with an increase of the area of these multicellular protrusions. Consequently, the force profile along the epithelial fingers in the parallel and transverse directions of migration showed an unexpected relationship leading to a global force decrease with the increase of stiffness. Taken together, our findings show that epithelial cell colonies respond to substrate stiffness but in a non-trivial manner that may be of importance to understand morphogenesis and collective cell invasion during tumour progression.