Genetic and Mechanical Regulation of Intestinal Smooth Muscle Development.

The gastrointestinal tract is enveloped by concentric and orthogonally aligned layers of smooth muscle; however, an understanding of the mechanisms by which these muscles become patterned and aligned in the embryo has been lacking. We find that Hedgehog acts through Bmp to delineate the position of the circumferentially oriented inner muscle layer, whereas localized Bmp inhibition is critical for allowing formation of the later-forming, longitudinally oriented outer layer. Because the layers form at different developmental stages, the muscle cells are exposed to unique mechanical stimuli that direct their alignments. Differential growth within the early gut tube generates residual strains that orient the first layer circumferentially, and when formed, the spontaneous contractions of this layer align the second layer longitudinally. Our data link morphogen-based patterning to mechanically controlled smooth muscle cell alignment and provide a mechanistic context for potentially understanding smooth muscle organization in a wide variety of tubular organs.

Spatiotemporally distinct responses to mechanical forces shape the developing seed of Arabidopsis.

Organ morphogenesis depends on mechanical interactions between cells and tissues. These interactions generate forces that can be sensed by cells and affect key cellular processes. However, how mechanical forces, together with biochemical signals, contribute to the shaping of complex organs is still largely unclear. We address this question using the seed of Arabidopsis as a model system. We show that seeds first experience a phase of rapid anisotropic growth that is dependent on the response of cortical microtubule (CMT) to forces, which guide cellulose deposition according to shape-driven stresses in the outermost layer of the seed coat. However, at later stages of development, we show that seed growth is isotropic and depends on the properties of an inner layer of the seed coat that stiffens its walls in response to tension but has isotropic material properties. Finally, we show that the transition from anisotropic to isotropic growth is due to the dampening of cortical microtubule responses to shape-driven stresses. Altogether, our work supports a model in which spatiotemporally distinct mechanical responses control the shape of developing seeds in Arabidopsis.

To squeeze or not: Regulation of cell size by mechanical forces in development and human diseases.

Physical constraints, such as compression, shear stress, stretching and tension play major roles during development and tissue homeostasis. Mechanics directly impact physiology, and their alteration is also recognized as having an active role in driving human diseases. Recently, growing evidence has accumulated on how mechanical forces are translated into a wide panel of biological responses, including metabolism and changes in cell morphology. The aim of this review is to summarize and discuss our knowledge on the impact of mechanical forces on cell size regulation. Other biological consequences of mechanical forces will not be covered by this review. Moreover, wherever possible, we also discuss mechanosensors and molecular and cellular signaling pathways upstream of cell size regulation. We finally highlight the relevance of mechanical forces acting on cell size in physiology and human diseases.

Mechanical coupling of supracellular stress amplification and tissue fluidization during exit from quiescence.

Cellular quiescence is a state of reversible cell cycle arrest that is associated with tissue dormancy. Timely regulated entry into and exit from quiescence is important for processes such as tissue homeostasis, tissue repair, stem cell maintenance, developmental processes, and immunity. However, little is known about processes that control the mechanical adaption to cell behavior changes during the transition from quiescence to proliferation. Here, we show that quiescent human keratinocyte monolayers sustain an actinomyosin-based system that facilitates global cell sheet displacements upon serum-stimulated exit from quiescence. Mechanistically, exposure of quiescent cells to serum-borne mitogens leads to rapid amplification of preexisting contractile sites, leading to a burst in monolayer tension that subsequently drives large-scale displacements of otherwise motility-restricted monolayers. The stress level after quiescence exit correlates with the level of quiescence depth at the time of activation, and a critical stress magnitude must be reached to overcome the cell sheet displacement barrier. The study shows that static quiescent cell monolayers are mechanically poised for motility, and it identifies global stress amplification as a mechanism for overcoming motility restrictions in confined confluent cell monolayers.

Mechanics of neural tube morphogenesis.

The neural tube is an important model system of morphogenesis representing the developmental module of out-of-plane epithelial deformation. As the embryonic precursor of the central nervous system, the neural tube also holds keys to many defects and diseases. Recent advances begin to reveal how genetic, cellular and environmental mechanisms work in concert to ensure correct neural tube shape. A physical model is emerging where these factors converge at the regulation of the mechanical forces and properties within and around the tissue that drive tube formation towards completion. Here we review the dynamics and mechanics of neural tube morphogenesis and discuss the underlying cellular behaviours from the viewpoint of tissue mechanics. We will also highlight some of the conceptual and technical next steps.

Mechanical control of tissue shape: Cell-extrinsic and -intrinsic mechanisms join forces to regulate morphogenesis.

During vertebrate development, cells must proliferate, move, and differentiate to form complex shapes. Elucidating the mechanisms underlying the molecular and cellular processes involved in tissue morphogenesis is essential to understanding developmental programmes. Mechanical stimuli act as a major contributor of morphogenetic processes and impact on cell behaviours to regulate tissue shape and size. Specifically, cell extrinsic physical forces are translated into biochemical signals within cells, through the process of mechanotransduction, activating multiple mechanosensitive pathways and defining cell behaviours. Physical forces generated by tissue mechanics and the extracellular matrix are crucial to orchestrate tissue patterning and cell fate specification. At the cell scale, the actomyosin network generates the cellular tension behind the tissue mechanics involved in building tissue. Thus, understanding the role of physical forces during morphogenetic processes requires the consideration of the contribution of cell intrinsic and cell extrinsic influences. The recent development of multidisciplinary approaches, as well as major advances in genetics, microscopy, and force-probing tools, have been key to push this field forward. With this review, we aim to discuss recent work on how tissue shape can be controlled by mechanical forces by focusing specifically on vertebrate organogenesis. We consider the influences of mechanical forces by discussing the cell-intrinsic forces (such as cell tension and proliferation) and cell-extrinsic forces (such as substrate stiffness and flow forces). We review recently described processes supporting the role of intratissue force generation and propagation in the context of shape emergence. Lastly, we discuss the emerging role of tissue-scale changes in tissue material properties, extrinsic forces, and shear stress on shape establishment.

Mechanical forces exerted on floral primordia with a novel experimental system modify floral development in Arabidopsis thaliana.

Mechanical forces play a crucial role in plant development, including floral development. We previously reported that the phyllotactic variation in the staminate flowers of Ceratophyllum demersum may be caused by mechanical forces on the adaxial side of floral primordia, which may be a common mechanism in angiosperms. On the basis of this result, we developed a novel experimental system for analysis of the effects of mechanical forces on the floral meristem of Arabidopsis thaliana, aiming to induce morphological changes in flowers. In this experimental system, a micromanipulator equipped with a micro device, which is shaped to conform with the contour of the abaxial side of the young floral primordium, is used to exert contact pressure on a floral primordium. In the present study, we conducted contact experiments using this system and successfully induced diverse morphological changes during floral primordial development. In several primordia, the tip of the abaxial sepal primordium was incised with two or three lobes. A different floral primordium developed an additional sepal on the abaxial side (i.e., two abaxial sepals). Additionally, we observed the fusion of sepals in some floral primordia. These results suggest that mechanical forces have multiple effects on floral development, and changes in the tensile stress pattern in the cells of floral primordia are induced by the mechanical forces exerted with the micro device. These effects, in turn, lead to morphological changes in the floral primordia.

Tissue folding at the organ-meristem boundary results in nuclear compression and chromatin compaction.

Artificial mechanical perturbations affect chromatin in animal cells in culture. Whether this is also relevant to growing tissues in living organisms remains debated. In plants, aerial organ emergence occurs through localized outgrowth at the periphery of the shoot apical meristem, which also contains a stem cell niche. Interestingly, organ outgrowth has been proposed to generate compression in the saddle-shaped organ-meristem boundary domain. Yet whether such growth-induced mechanical stress affects chromatin in plant tissues is unknown. Here, by imaging the nuclear envelope in vivo over time and quantifying nucleus deformation, we demonstrate the presence of active nuclear compression in that domain. We developed a quantitative pipeline amenable to identifying a subset of very deformed nuclei deep in the boundary and in which nuclei become gradually narrower and more elongated as the cell contracts transversely. In this domain, we find that the number of chromocenters is reduced, as shown by chromatin staining and labeling, and that the expression of linker histone H1.3 is induced. As further evidence of the role of forces on chromatin changes, artificial compression with a MicroVice could induce the ectopic expression of H1.3 in the rest of the meristem. Furthermore, while the methylation status of chromatin was correlated with nucleus deformation at the meristem boundary, such correlation was lost in the h1.3 mutant. Altogether, we reveal that organogenesis in plants generates compression that is able to have global effects on chromatin in individual cells.

Targeted cell elimination reveals an auxin-guided biphasic mode of lateral root initiation.

To sustain a lifelong ability to initiate organs, plants retain pools of undifferentiated cells with a preserved proliferation capacity. The root pericycle represents a unique tissue with conditional meristematic activity, and its tight control determines initiation of lateral organs. Here we show that the meristematic activity of the pericycle is constrained by the interaction with the adjacent endodermis. Release of these restraints by elimination of endodermal cells by single-cell ablation triggers the pericycle to re-enter the cell cycle. We found that endodermis removal substitutes for the phytohormone auxin-dependent initiation of the pericycle meristematic activity. However, auxin is indispensable to steer the cell division plane orientation of new organ-defining divisions. We propose a dual, spatiotemporally distinct role for auxin during lateral root initiation. In the endodermis, auxin releases constraints arising from cell-to-cell interactions that compromise the pericycle meristematic activity, whereas, in the pericycle, auxin defines the orientation of the cell division plane to initiate lateral roots.

Dominant Chemical Interactions Governing the Folding Mechanism of Oligopeptides.

The hydrophobic effect is the main factor that drives the folding of polypeptide chains. In this study, we have examined the influence of the hydrophobic effect in the context of the main mechanical forces approach, mainly in relation to the establishment of specific interplays, such as hydrophobic and CH-π cloud interactions. By adopting three oligopeptides as model systems to assess folding features, we demonstrate herein that these finely tuned interactions dominate over electrostatic interactions, including H-bonds and electrostatic attractions/repulsions. The folding mechanism analysed here demonstrates cooperation at the single-residue level, for which we propose the terminology of "single residues cooperative folding". Overall, hydrophobic and CH-π cloud interactions produce the main output of the hydrophobic effect and govern the folding mechanism, as demonstrated in this study with small polypeptide chains, which in turn represent the main secondary structures in proteins.

Forced into shape: Mechanical forces in Drosophila development and homeostasis.

Mechanical forces play a central role in shaping tissues during development and maintaining epithelial integrity in homeostasis. In this review, we discuss the roles of mechanical forces in Drosophila development and homeostasis, starting from the interplay of mechanics with cell growth and division. We then discuss several examples of morphogenetic processes where complex 3D structures are shaped by mechanical forces, followed by a closer look at patterning processes. We also review the role of forces in homeostatic processes, including cell elimination and wound healing. Finally, we look at the interplay of mechanics and developmental robustness and discuss open questions in the field, as well as novel approaches that will help tackle them in the future.

Mechanical forces in avian embryo development.

Research using avian embryos has led to major conceptual advances in developmental biology, virology, immunology, genetics and cell biology. The avian embryo has several significant advantages, including ready availability and ease of accessibility, rapid development with marked similarities to mammals and a high amenability to manipulation. As mechanical forces are increasingly recognised as key drivers of morphogenesis, this powerful model system is shedding new light on the mechanobiology of embryonic development. Here, we highlight progress in understanding how mechanical forces direct key morphogenetic processes in the early avian embryo. Recent advances in quantitative live imaging and modelling are elaborating upon traditional work using physical models and embryo manipulations to reveal cell dynamics and tissue forces in ever greater detail. The recent application of transgenic technologies further increases the strength of the avian model and is providing important insights about previously intractable developmental processes.

Mechanical forces suppress antiviral innate immune responses from asthmatic airway epithelial cells following rhinovirus infection.

Bronchoconstriction is the main physiological event in asthma, which leads to worsened clinical symptoms and generates mechanical stress within the airways. Virus infection is the primary cause of exacerbations in people with asthma, however, the impact that bronchoconstriction itself on host antiviral responses and viral replication is currently not well understood. Here we demonstrate how mechanical forces generated during bronchoconstriction may suppress antiviral responses at the airway epithelium without any difference in viral replication. Primary bronchial epithelial cells from donors with asthma were differentiated at the air-liquid interface. Differentiated cells were apically compressed (30 cmH2O) for 10 min every hour for 4 days to mimic bronchoconstriction. Two asthma disease models were developed with the application of compression, either before ("poor asthma control model," n = 7) or following ("exacerbation model," n = 4) rhinovirus (RV) infection. Samples were collected at 0, 24, 48, 72, and 96 h postinfection (hpi). Viral RNA, interferon (IFN)-ß, IFN-λ, and host defense antiviral peptide gene expressions were measured along with IFN-ß, IFN-λ, TGF-ß2, interleukin-6 (IL-6), and IL-8 protein expression. Apical compression significantly suppressed RV-induced IFN-ß protein from 48 hpi and IFN-λ from 72 hpi in the poor asthma control model. There was a nonsignificant reduction of both IFN-ß and IFN-λ proteins from 48 hpi in the exacerbation model. Despite reductions in antiviral proteins, there was no significant change in viral replication in either model. Compressive stress mimicking bronchoconstriction inhibits antiviral innate immune responses from asthmatic airway epithelial cells when applied before RV infection.NEW & NOTEWORTHY Bronchoconstriction is the main physiological event in asthma, which leads to worsened clinical symptoms and generates mechanical stress within the airways. Virus infection is the primary cause of exacerbations in people with asthma, however, the impact of bronchoconstriction on host antiviral responses and viral replication is unknown. We developed two disease models, in vitro, and found suppressed IFN response from cells following the application of compression and RV-A1 infection. This explains why people with asthma have deficient IFN response.

Links between autophagy and tissue mechanics.

Physical constraints, such as compression, shear stress, stretching and tension, play major roles during development, tissue homeostasis, immune responses and pathologies. Cells and organelles also face mechanical forces during migration and extravasation, and investigations into how mechanical forces are translated into a wide panel of biological responses, including changes in cell morphology, membrane transport, metabolism, energy production and gene expression, is a flourishing field. Recent studies demonstrate the role of macroautophagy in the integration of physical constraints. The aim of this Review is to summarize and discuss our knowledge of the role of macroautophagy in controlling a large panel of cell responses, from morphological and metabolic changes, to inflammation and senescence, for the integration of mechanical forces. Moreover, wherever possible, we also discuss the cell surface molecules and structures that sense mechanical forces upstream of macroautophagy.

On growth and force: mechanical forces in development.

The EMBO/EMBL Symposium 'Mechanical Forces in Development' was held in Heidelberg, Germany, on 3-6 July 2019. This interdisciplinary symposium brought together an impressive and diverse line-up of speakers seeking to address the origin and role of mechanical forces in development. Emphasising the importance of integrative approaches and theoretical simulations to obtain comprehensive mechanistic insights into complex morphogenetic processes, the meeting provided an ideal platform to discuss the concepts and methods of developmental mechanobiology in an era of fast technical and conceptual progress. Here, we summarise the concepts and findings discussed during the meeting, as well as the agenda it sets for the future of developmental mechanobiology.

Extracellular-matrix mechanics regulate cellular metabolism: A ninja warrior behind mechano-chemo signaling crosstalk.

Mechanical forces are the indispensable constituent of environmental cues, such as gravity, barometric pressure, vibration, and contact with bodies, which are involved in pattern and organogenesis, providing mechanical input to tissues and determining the ultimate fate of cells. Extracellular matrix (ECM) stiffness, the slow elastic force, carries the external physical force load onto the cell or outputs the internal force exerted by the cell and its neighbors into the environment. Accumulating evidence illustrates the pivotal role of ECM stiffness in the regulation of organogenesis, maintenance of tissue homeostasis, and the development of multiple diseases, which is largely fulfilled through its systematical impact on cellular metabolism. This review summarizes the establishment and regulation of ECM stiffness, the mechanisms underlying how ECM stiffness is sensed by cells and signals to modulate diverse cell metabolic pathways, and the physiological and pathological significance of the ECM stiffness-cell metabolism axis.

The Effect of Pulling and Twisting Forces on Chameleon Sequence Peptides.

Chameleon sequences are amino acid sequences found in several distinct configurations in experiment. They challenge our understanding of the link between sequence and structure, and provide insight into structural competition in proteins. Here, we study the energy landscapes for three such sequences, and interrogate how pulling and twisting forces impact the available structural ensembles. Chameleon sequences do not necessarily exhibit multiple structural ensembles on a multifunnel energy landscape when we consider them in isolation. The application of even small forces leads to drastic changes in the energy landscapes. For pulling forces, we observe transitions from helical to extended structures in a very small span of forces. For twisting forces, the picture is much more complex, and highly dependent on the magnitude and handedness of the applied force as well as the reference angle for the twist. Depending on these parameters, more complex and more simplistic energy landscapes are observed alongside more and less diverse structural ensembles. The impact of even small forces is significant, confirming their likely role in folding events. In addition, small forces exerted by the remaining scaffold of a protein may be sufficient to lead to the adoption of a specific structural ensemble by a chameleon sequence.

Real-Time Observation and Analysis of Magnetomechanical Actuation of Magnetic Nanoparticles in Cells.

The origin of cell death in the magnetomechanical actuation of cells induced by magnetic nanoparticle motion under low-frequency magnetic fields is still elusive. Here, a miniaturized electromagnet fitted under a confocal microscope is used to observe in real time cells specifically targeted by superparamagnetic nanoparticles and exposed to a low-frequency rotating magnetic field. Our analysis reveals that the lysosome membrane is permeabilized in only a few minutes after the start of magnetic field application, concomitant with lysosome movements toward the nucleus. Those events are associated with disorganization of the tubulin microtubule network and a change in cell morphology. This miniaturized electromagnet will allow a deeper insight into the physical, molecular, and biological process occurring during the magnetomechanical actuation of magnetic nanoparticles.

Hippo/yes-associated protein signaling functions as a mechanotransducer in regulating vascular homeostasis.

Cells are constantly exposed to various mechanical forces, including hydrostatic pressure, cyclic stretch, fluid shear stress, and extracellular matrix stiffness. Mechanical cues can be translated into the cell-specific transcriptional process by a cellular mechanic-transducer. Evidence suggests that mechanical signals assist activated intracellular signal transduction pathways and the relative phenotypic adaptation to coordinate cell behavior and disease appropriately. The Hippo/yes-associated protein (YAP) signaling pathway is regulated in response to numerous mechanical stimuli. It plays an important role in the mechanotransduction mechanism, which converts mechanical forces to cascades of molecular signaling to modulate gene expression. This review summarizes the recent findings relevant to the Hippo/YAP pathway-based mechanotransduction in cell behavior and maintaining blood vessels, as well as cardiovascular disease.

Langerhans cells and SFRP2/Wnt/beta-catenin signalling control adaptation of skin epidermis to mechanical stretching.

Skin can be mechanically stimulated to grow through a clinical procedure called tissue expansion (TE). Using a porcine TE model, we determined that expansion promptly activates transcription of SFRP2 in skin and we revealed that in the epidermis, this protein is secreted by Langerhans cells (LCs). Similar to well-known mechanosensitive genes, the increase in SFRP2 expression was proportional to the magnitude of tension, showing a spike at the apex of the expanded skin. This implies that SFRP2 might be a newly discovered effector of mechanotransduction pathways. In addition, we found that acute stretching induces accumulation of b-catenin in the nuclei of basal keratinocytes (KCs) and LCs, indicating Wnt signalling activation, followed by cell proliferation. Moreover, TE-activated LCs proliferate and migrate into the suprabasal layer of skin, suggesting that LCs rebuild their steady network within the growing epidermis. We demonstrated that in vitro hrSFRP2 treatment on KCs inhibits Wnt/b-catenin signalling and stimulates KC differentiation. In parallel, we observed an accumulation of KRT10 in vivo in the expanded skin, pointing to TE-induced, SFRP2-augmented KC maturation. Overall, our results reveal that a network of LCs delivers SFRP2 across the epidermis to fine-tune Wnt/b-catenin signalling to restore epidermal homeostasis disrupted by TE.