Mechanoresponse of Curved Epithelial Monolayers Lining Bowl-Shaped 3D Microwells.

The optimal functioning of many organs relies on the curved architecture of their epithelial tissues. However, the mechanoresponse of epithelia to changes in curvature remains misunderstood. Here, bowl-shaped microwells in hydrogels are designed via photopolymerization to faithfully replicate the shape and dimensions of lobular structures. Leveraging these hydrogel-based microwells, curved epithelial monolayers are engineered, and how in-plane and Gaussian curvatures at the microwell entrance influence epithelial behavior is investigated. Cells and nuclei around the microwell edge display a more pronounced centripetal orientation as the in-plane curvature decreases, and enhanced cell straightness and speed. Moreover, cells reorganize their actin cytoskeleton by forming a supracellular actin cable at the microwell edge, with its size becoming more pronounced as the in-plane curvature decreases. The Gaussian curvature at the microwell entrance enhances the maturation of the supracellular actin cable architecture and leads to a vertical orientation of nuclei toward the bottom of the microwell. Increasing Gaussian curvature results in flattened and elongated nuclear morphologies characterized by highly compacted chromatin states. This approach provides better understanding of the mechanoresponse of curved epithelial monolayers curvatures lining lobular structures. In addition, bowl-shaped microwells offer a powerful platform to study curvature-dependent mechanotransduction pathways in anatomically relevant 3D structures.

Keratin filaments mediate the expansion of extra-embryonic membranes in the post-gastrulation mouse embryo.

Mesoderm arises at gastrulation and contributes to both the mouse embryo proper and its extra-embryonic membranes. Two-photon live imaging of embryos bearing a keratin reporter allowed recording filament nucleation and elongation in the extra-embryonic region. Upon separation of amniotic and exocoelomic cavities, keratin 8 formed apical cables co-aligned across multiple cells in the amnion, allantois, and blood islands. An influence of substrate rigidity and composition on cell behavior and keratin content was observed in mesoderm explants. Embryos lacking all keratin filaments displayed a deflated extra-embryonic cavity, a narrow thick amnion, and a short allantois. Single-cell RNA sequencing of sorted mesoderm cells and micro-dissected amnion, chorion, and allantois, provided an atlas of transcriptomes with germ layer and regional information. It defined the cytoskeleton and adhesion expression profile of mesoderm-derived keratin 8-enriched cells lining the exocoelomic cavity. Those findings indicate a novel role for keratin filaments in the expansion of extra-embryonic structures and suggest mechanisms of mesoderm adaptation to the environment.

Appreciating the role of cell shape changes in the mechanobiology of epithelial tissues.

The wide range of epithelial cell shapes reveals the complexity and diversity of the intracellular mechanisms that serve to construct their morphology and regulate their functions. Using mechanosensitive steps, epithelial cells can sense a variety of different mechanochemical stimuli and adapt their behavior by reshaping their morphology. These changes of cell shape rely on a structural reorganization in space and time that generates modifications of the tensional state and activates biochemical cascades. Recent studies have started to unveil how the cell shape maintenance is involved in mechanical homeostatic tasks to sustain epithelial tissue folding, identity, and self-renewal. Here, we review relevant works that integrated mechanobiology to elucidate some of the core principles of how cell shape may be conveyed into spatial information to guide collective processes such as epithelial morphogenesis. Among many other parameters, we show that the regulation of the cell shape can be understood as the result of the interplay between two counteracting mechanisms: actomyosin contractility and intercellular adhesions, and that both do not act independently but are functionally integrated to operate on molecular, cellular, and tissue scales. We highlight the role of cadherin-based adhesions in force-sensing and mechanotransduction, and we report recent developments that exploit physics of liquid crystals to connect cell shape changes to orientational order in cell aggregates. Finally, we emphasize that the further intermingling of different disciplines to develop new mechanobiology assays will lead the way toward a unified picture of the contribution of cell shape to the pathophysiological behavior of epithelial tissues.

Collective migration during a gap closure in a two-dimensional haptotactic model.

The ability of cells to respond to substrate-bound protein gradients is crucial for many physiological processes, such as immune response, neurogenesis and cancer cell migration. However, the difficulty to produce well-controlled protein gradients has long been a limitation to our understanding of collective cell migration in response to haptotaxis. Here we use a photopatterning technique to create circular, square and linear fibronectin (FN) gradients on two-dimensional (2D) culture substrates. We observed that epithelial cells spread preferentially on zones of higher FN density, creating rounded or elongated gaps within epithelial tissues over circular or linear FN gradients, respectively. Using time-lapse experiments, we demonstrated that the gap closure mechanism in a 2D haptotaxis model requires a significant increase of the leader cell area. In addition, we found that gap closures are slower on decreasing FN densities than on homogenous FN-coated substrate and that fresh closed gaps are characterized by a lower cell density. Interestingly, our results showed that cell proliferation increases in the closed gap region after maturation to restore the cell density, but that cell-cell adhesive junctions remain weaker in scarred epithelial zones. Taken together, our findings provide a better understanding of the wound healing process over protein gradients, which are reminiscent of haptotaxis.

Engineering slit-like channels for studying the growth of epithelial tissues in 3D-confined spaces.

The development of epithelial lumens in ducts is essential to the functioning of various organs and in organogenesis. Ductal elongation requires the collective migration of cell cohorts in three-dimensional (3D) confined spaces, while maintaining their epithelial integrity. Epithelial lumens generally adopt circular morphologies, however abnormalities in complex physiological environments can lead to the narrowing of glandular spaces that adopt elongated and slit-like morphologies. Here, we describe a simple method to form epithelial tissues in microchannels of various widths (100-300 µm) with a constant height of 25 µm that mimic elongated geometries of glandular spaces. The significance of this biomimetic platform has been evidenced by studying the migration of epithelial cell sheets inside these narrow slits of varying dimensions. We show that the growth of epithelial tissues in 3D-confined slits leads to a gradient of cell density along the slit axis and that the migration cell velocity depends on the extent of the spatial confinement. Our findings indicate that nuclear orientation is higher for leader cells and depends on the slit width, whereas YAP protein was predominantly localized in the nucleus of leader cells. This method will pave the way to studies aiming at understanding how 3D-confined spaces, which are reminiscent of in vivo pathological conditions, can affect the growth and the homeostasis of epithelial tissues.

Producing Collagen Micro-stripes with Aligned Fibers for Cell Migration Assays.

INTRODUCTION: The orientation of collagen fibers in native tissues plays an important role in cell signaling and mediates the progression of tumor cells in breast cancer by a contact guidance mechanism. Understanding how migration of epithelial cells is directed by the alignment of collagen fibers requires in vitro assays with standardized orientations of collagen fibers. METHODS: To address this issue, we produced micro-stripes with aligned collagen fibers using an easy-to-use and versatile approach based on the aspiration of a collagen solution within a microchannel. Glass coverslips were functionalized with a (3-aminopropyl)triethoxysilane/glutaraldehyde linkage to covalently anchor micro-stripes of aligned collagen fibers, whereas microchannels were functionalized with a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) nonionic triblock polymer to prevent adhesion of the collagen micro-stripes. RESULTS: Using this strategy, microchannels can be peeled off to expose micro-stripes of aligned collagen fibers without affecting their mechanical integrity. We used time-lapse confocal reflection microscopy to characterize the polymerization kinetics of collagen networks for different concentrations and the orientation of collagen fibers as a function of the microchannel width. Our results indicate a non-linear concentration dependence of the area of fluorescence, suggesting that the architecture of collagen networks is sensitive to small changes in concentration. We show the possibility to influence the collagen fibril coverage by adjusting the concentration of the collagen solution. CONCLUSION: We applied this novel approach to study the migration of epithelial cells, demonstrating that collagen micro-stripes with aligned fibers represent a valuable in-vitro assay for studying cell contact guidance mechanisms.

Actomyosin contractility scales with myoblast elongation and enhances differentiation through YAP nuclear export.

Skeletal muscle fibers are formed by the fusion of mononucleated myoblasts into long linear myotubes, which differentiate and reorganize into multinucleated myofibers that assemble in bundles to form skeletal muscles. This fundamental process requires the elongation of myoblasts into a bipolar shape, although a complete understanding of the mechanisms governing skeletal muscle fusion is lacking. To address this question, we consider cell aspect ratio, actomyosin contractility and the Hippo pathway member YAP as potential regulators of the fusion of myoblasts into myotubes. Using fibronectin micropatterns of different geometries and traction force microscopy, we investigated how myoblast elongation affects actomyosin contractility. Our findings indicate that cell elongation enhances actomyosin contractility in myoblasts, which regulate their actin network to their spreading area. Interestingly, we found that the contractility of cell pairs increased after their fusion and raise on elongated morphologies. Furthermore, our findings indicate that myoblast elongation modulates nuclear orientation and triggers cytoplasmic localization of YAP, increasing evidence that YAP is a key regulator of mechanotransduction in myoblasts. Taken together, our findings support a mechanical model where actomyosin contractility scales with myoblast elongation and enhances the differentiation of myoblasts into myotubes through YAP nuclear export.

Innovative Tools for Mechanobiology: Unraveling Outside-In and Inside-Out Mechanotransduction.

Cells and tissues can sense and react to the modifications of the physico-chemical properties of the extracellular environment (ECM) through integrin-based adhesion sites and adapt their physiological response in a process called mechanotransduction. Due to their critical localization at the cell-ECM interface, transmembrane integrins are mediators of bidirectional signaling, playing a key role in "outside-in" and "inside-out" signal transduction. After presenting the basic conceptual fundamentals related to cell mechanobiology, we review the current state-of-the-art technologies that facilitate the understanding of mechanotransduction signaling pathways. Finally, we highlight innovative technological developments that can help to advance our understanding of the mechanisms underlying nuclear mechanotransduction.

Probing cytoskeletal pre-stress and nuclear mechanics in endothelial cells with spatiotemporally controlled (de-)adhesion kinetics on micropatterned substrates.

The mechanical properties of living cells reflect their propensity to migrate and respond to external forces. Both cellular and nuclear stiffnesses are strongly influenced by the rigidity of the extracellular matrix (ECM) through reorganization of the cyto- and nucleoskeletal protein connections. Changes in this architectural continuum affect cell mechanics and underlie many pathological conditions. In this context, an accurate and combined quantification of the mechanical properties of both cells and nuclei can contribute to a better understanding of cellular (dys-)function. To address this challenge, we have established a robust method for probing cellular and nuclear deformation during spreading and detachment from micropatterned substrates. We show that (de-)adhesion kinetics of endothelial cells are modulated by substrate stiffness and rely on the actomyosin network. We combined this approach with measurements of cell stiffness by magnetic tweezers to show that relaxation dynamics can be considered as a reliable parameter of cellular pre-stress in adherent cells. During the adhesion stage, large cellular and nuclear deformations occur over a long time span (>60 min). Conversely, nuclear deformation and condensed chromatin are relaxed in a few seconds after detachment. Finally, our results show that accumulation of farnesylated prelamin leads to modifications of the nuclear viscoelastic properties, as reflected by increased nuclear relaxation times. Our method offers an original and non-intrusive way of simultaneously gauging cellular and nuclear mechanics, which can be extended to high-throughput screens of pathological conditions and potential countermeasures.

Persistence of fan-shaped keratocytes is a matrix-rigidity-dependent mechanism that requires α5ß1 integrin engagement.

Despite the importance of matrix rigidity on cell functions, many aspects of the mechanosensing process in highly migratory cells remain elusive. Here, we studied the migration of highly motile keratocytes on culture substrates with similar biochemical properties and rigidities spanning the range between soft tissues (~kPa) and stiff culture substrates (~GPa). We show that morphology, polarization and persistence of motile keratocytes are regulated by the matrix stiffness over seven orders of magnitude, without changing the cell spreading area. Increasing the matrix rigidity leads to more F-actin in the lamellipodia and to the formation of mature contractile actomyosin fibers that control the cell rear retraction. Keratocytes remain rounded and form nascent adhesions on compliant substrates, whereas large and uniformly distributed focal adhesions are formed on fan-shaped keratocytes migrating on rigid surfaces. By combining poly-L-lysine, fibronectin and vitronectin coatings with selective blocking of αvß3 or α5ß1 integrins, we show that αVß3 integrins permit the spreading of keratocytes but are not sufficient for polarization and rigidity sensing that require the engagement of α5ß1 integrins. Our study demonstrates a matrix rigidity-dependent regulation of the directional persistence in motile keratocytes and refines the role of αvß3 and α5ß1 integrins in the molecular clutch model.

Matrix stiffness modulates formation and activity of neuronal networks of controlled architectures.

The ability to construct easily in vitro networks of primary neurons organized with imposed topologies is required for neural tissue engineering as well as for the development of neuronal interfaces with desirable characteristics. However, accumulating evidence suggests that the mechanical properties of the culture matrix can modulate important neuronal functions such as growth, extension, branching and activity. Here we designed robust and reproducible laminin-polylysine grid micropatterns on cell culture substrates that have similar biochemical properties but a 100-fold difference in Young's modulus to investigate the role of the matrix rigidity on the formation and activity of cortical neuronal networks. We found that cell bodies of primary cortical neurons gradually accumulate in circular islands, whereas axonal extensions spread on linear tracks to connect circular islands. Our findings indicate that migration of cortical neurons is enhanced on soft substrates, leading to a faster formation of neuronal networks. Furthermore, the pre-synaptic density was two times higher on stiff substrates and consistently the number of action potentials and miniature synaptic currents was enhanced on stiff substrates. Taken together, our results provide compelling evidence to indicate that matrix stiffness is a key parameter to modulate the growth dynamics, synaptic density and electrophysiological activity of cortical neuronal networks, thus providing useful information on scaffold design for neural tissue engineering.

Super-resolution microscopy reveals LINC complex recruitment at nuclear indentation sites.

Increasing evidences show that the actin cytoskeleton is a key parameter of the nuclear remodeling process in response to the modifications of cellular morphology. However, detailed information on the interaction between the actin cytoskeleton and the nuclear lamina was still lacking. We addressed this question by constraining endothelial cells on rectangular fibronectin-coated micropatterns and then using Structured Illumination Microscopy (SIM) to observe the interactions between actin stress fibers, nuclear lamina and LINC complexes at a super-resolution scale. Our results show that tension in apical actin stress fibers leads to deep nuclear indentations that significantly deform the nuclear lamina. Interestingly, indented nuclear zones are characterized by a local enrichment of LINC complexes, which anchor apical actin fibers to the nuclear lamina. Moreover, our findings indicate that nuclear indentations induce the formation of segregated domains of condensed chromatin. However, nuclear indentations and condensed chromatin domains are not irreversible processes and both can relax in absence of tension in apical actin stress fibers.

Preparation of hydroxy-PAAm hydrogels for decoupling the effects of mechanotransduction cues.

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions. Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

Micropatterning hydroxy-PAAm hydrogels and Sylgard 184 silicone elastomers with tunable elastic moduli.

This protocol describes a simple method to deposit protein micropatterns over a wide range of culture substrate stiffness (three orders of magnitude) by using two complementary polymeric substrates. In the first part, we introduce a novel polyacrylamide hydrogel, called hydroxy-polyacrylamide (PAAm), that permits to surmount the intrinsically nonadhesive properties of polyacrylamide with minimal requirements in cost or expertize. We present a protocol for tuning easily the rigidity of "soft" hydroxy-PAAm hydrogels between ~0.5 and 50 kPa and a micropatterning method to locally deposit protein micropatterns on these hydrogels. In a second part, we describe a protocol for tuning the rigidity of "stiff" silicone elastomers between ~100 and 1000 kPa and printing efficiently proteins from the extracellular matrix. Finally, we investigate the effect of the matrix rigidity on the nucleus of primary endothelial cells by tuning the rigidity of both polymeric substrates. We envision that the complementarity of these two polymeric substrates, combined with an efficient microprinting technique, can be further developed in the future as a powerful mechanobiology platform to investigate in vitro the effect of mechanotransduction cues on cellular functions, gene expression, and stem cell differentiation.

A simple route to functionalize polyacrylamide hydrogels for the independent tuning of mechanotransduction cues.

Physico-chemical and biochemical factors in the local cellular microenvironment are known to impact on multiple aspects of cell behaviour through specific signal pathways. These mechanotransduction cues can couple each other to regulate cell fate, and it remains unclear whether mechanotransduction in different contexts shares common mechanisms. Undoubtedly, a challenge will involve the further characterization of such cooperative mechanisms, as well as clearly defining the individual role of each mechanical and biochemical parameter. To control these mechanotransduction cues in an independent manner, we developed a simple and efficient strategy to immobilize any desired nature of proteins on polyacrylamide hydrogels and independently control various parameters of the cell microenvironment, such as matrix stiffness, cell-binding ligand density and confined adhesiveness. This novel platform is validated by conducting single-cell experiments and opens a broad avenue for studying complex interplays involved in mechanotransduction with a facile and versatile approach.

Spatial coordination between cell and nuclear shape within micropatterned endothelial cells.

Growing evidence suggests that cytoplasmic actin filaments are essential factors in the modulation of nuclear shape and function. However, the mechanistic understanding of the internal orchestration between cell and nuclear shape is still lacking. Here we show that orientation and deformation of the nucleus are regulated by lateral compressive forces driven by tension in central actomyosin fibres. By using a combination of micro-manipulation tools, our study reveals that tension in central stress fibres is gradually generated by anisotropic force contraction dipoles, which expand as the cell elongates and spreads. Our findings indicate that large-scale cell shape changes induce a drastic condensation of chromatin and dramatically affect cell proliferation. On the basis of these findings, we propose a simple mechanical model that quantitatively accounts for our experimental data and provides a conceptual framework for the mechanistic coordination between cell and nuclear shape.

A simple microfluidic method to select, isolate, and manipulate single-cells in mechanical and biochemical assays.

This article describes a simple and low-tech microfluidic method for single-cell experimentation, which permits cell selection without stress, cell manipulation with fine control, and passive self-exclusion of all undesired super-micronic particles. The method requires only conventional soft lithography microfabrication techniques and is applicable to any microfluidic single-cell circuitry. The principle relies on a bypass plugged in parallel with a single-cell assay device and collecting 97% of the flow rate. Cell selection into the single cell device is performed by moving the cell of interest back and forth in the vicinity of the junction between the bypass and the analysis circuitry. Cell navigation is finely controlled by hydrostatic pressure via centimetre-scale actuation of external macroscopic reservoirs connected to the device. We provide successful examples of biomechanical and biochemical assays on living human leukocytes passing through 4 mum wide capillaries. The blebbing process dynamics are monitored by conventional 24 fps videomicroscopy and subcellular cytoskeleton organization is imaged by on-chip immunostaining.