Multifunctional role of GPCR signaling in epithelial tube formation

Epithelial tube formation requires Rho1-dependent actomyosin contractility to generate the cellular forces that drive cell shape changes and rearrangement. Rho1 signaling is activated by G protein-coupled receptor (GPCR) signaling at the cell surface. During Drosophila embryonic salivary gland (SG) invagination, the GPCR ligand Folded gastrulation (Fog) activates Rho1 signaling to drive apical constriction. The SG receptor that transduces the Fog signal into Rho1-dependent myosin activation has not been identified. Here, we reveal that the Smog GPCR transduces Fog signal to regulate Rho kinase accumulation and myosin activation in the apicomedial region of cells to control apical constriction during SG invagination. We also report on unexpected Fog-independent roles for Smog in maintaining epithelial integrity and organizing cortical actin. Our data supports a model wherein Smog regulates distinct myosin pools and actin cytoskeleton in a ligand-dependent manner during epithelial tube formation.

Soft Polymer-Based Technique for Cellular Force Sensing

Soft polymers have emerged as a vital type of material adopted in biomedical engineering to perform various biomechanical characterisations such as sensing cellular forces. Distinct advantages of these materials used in cellular force sensing include maintaining normal functions of cells, resembling in vivo mechanical characteristics, and adapting to the customised functionality demanded in individual applications. A wide range of techniques has been developed with various designs and fabrication processes for the desired soft polymeric structures, as well as measurement methodologies in sensing cellular forces. This review highlights the merits and demerits of these soft polymer-based techniques for measuring cellular contraction force with emphasis on their quantitativeness and cell-friendliness. Moreover, how the viscoelastic properties of soft polymers influence the force measurement is addressed. More importantly, the future trends and advancements of soft polymer-based techniques, such as new designs and fabrication processes for cellular force sensing, are also addressed in this review.

In Vitro Measurements of Cellular Forces and their Importance in the Lung—From the Sub- to the Multicellular Scale

Throughout life, the body is subjected to various mechanical forces on the organ, tissue, and cellular level. Mechanical stimuli are essential for organ development and function. One organ whose function depends on the tightly connected interplay between mechanical cell properties, biochemical signaling, and external forces is the lung. However, altered mechanical properties or excessive mechanical forces can also drive the onset and progression of severe pulmonary diseases. Characterizing the mechanical properties and forces that affect cell and tissue function is therefore necessary for understanding physiological and pathophysiological mechanisms. In recent years, multiple methods have been developed for cellular force measurements at multiple length scales, from subcellular forces to measuring the collective behavior of heterogeneous cellular networks. In this short review, we give a brief overview of the mechanical forces at play on the cellular level in the lung. We then focus on the technological aspects of measuring cellular forces at many length scales. We describe tools with a subcellular resolution and elaborate measurement techniques for collective multicellular units. Many of the technologies described are by no means restricted to lung research and have already been applied successfully to cells from various other tissues. However, integrating the knowledge gained from these multi-scale measurements in a unifying framework is still a major future challenge.

Real-time imaging of cellular forces using optical interference

Important dynamic processes in mechanobiology remain elusive due to a lack of tools to image the small cellular forces at play with sufficient speed and throughput. Here, we introduce a fast, interference-based force imaging method that uses the illumination of an elastic deformable microcavity with two rapidly alternating wavelengths to map forces. We show real-time acquisition and processing of data, obtain images of mechanical activity while scanning across a cell culture, and investigate sub-second fluctuations of the piconewton forces exerted by macrophage podosomes. We also demonstrate force imaging of beating neonatal cardiomyocytes at 100 fps which reveals mechanical aspects of spontaneous oscillatory contraction waves in between the main contraction cycles. These examples illustrate the wider potential of our technique for monitoring cellular forces with high throughput and excellent temporal resolution.

Regulation of apical constriction via microtubule- and Rab11-dependent apical transport during tissue invagination

During tissue invagination, contractile actomyosin structures generate the cellular forces that drive apical constriction. Using the Drosophila embryonic salivary gland as a model for epithelial tube formation, we show that microtubule- and Rab11-dependent apical transport is critical for regulating actomyosin networks during invagination.

Optogenetic control of apical constriction induces synthetic morphogenesis in mammalian tissues

During embryonic development, cellular forces synchronize in space and time to generate functional tissue shapes. Apical constriction is one of these force-generating processes, and it is necessary to modulate epithelial curvature in fundamental morphogenetic events, such as neural tube folding. The emerging field of synthetic developmental biology proposes bottom-up approaches to examine the contribution of each cellular process to complex morphogenesis. However, the shortage of tools to manipulate three-dimensional (3D) shapes of mammalian tissues currently hinders the progress of the field. Here we report the development of 'OptoShroom3', a new optogenetic tool that achieves fast spatiotemporal control of apical constriction in mammalian epithelia. Activation of OptoShroom3 through illumination of individual cells in an epithelial cell sheet reduced their apical surface while illumination of groups of cells caused deformation in the adjacent regions. By using OptoShroom3, we further manipulated 3D tissue shapes. Light-induced apical constriction provoked the folding of epithelial cell colonies on soft gels. Its application to murine and human neural organoids led to thickening of neuroepithelia, apical lumen reduction in optic vesicles, and flattening in neuroectodermal tissues. These results show that spatiotemporal control of apical constriction can trigger several types of 3D deformation depending on the initial tissue context.

Mechanical control of tissue shape and morphogenetic flows during vertebrate body axis elongation

Shaping embryonic tissues into their functional morphologies requires cells to control the physical state of the tissue in space and time. While regional variations in cellular forces or cell proliferation have been typically assumed to be the main physical factors controlling tissue morphogenesis, recent experiments have revealed that spatial variations in the tissue physical (fluid/solid) state play a key role in shaping embryonic tissues. Here we theoretically study how the regional control of fluid and solid tissue states guides morphogenetic flows to shape the extending vertebrate body axis. Our results show that both the existence of a fluid-to-solid tissue transition along the anteroposterior axis and the tissue surface tension determine the shape of the tissue and its ability to elongate unidirectionally, with large tissue tensions preventing unidirectional elongation and promoting blob-like tissue expansions. We predict both the tissue morphogenetic flows and stresses that enable unidirectional axis elongation. Our results show the existence of a sharp transition in the structure of morphogenetic flows, from a flow with no vortices to a flow with two counter-rotating vortices, caused by a transition in the number and location of topological defects in the flow field. Finally, comparing the theoretical predictions to quantitative measurements of both tissue flows and shape during zebrafish body axis elongation, we show that the observed morphogenetic events can be explained by the existence of a fluid-to-solid tissue transition along the anteroposterior axis. These results highlight the role of spatiotemporally-controlled fluid-to-solid transitions in the tissue state as a physical mechanism of embryonic morphogenesis.

Study on the Expansion Dynamics of MDCK Epithelium by Interstitial Flow Using a Traction Force-Measurable Microfluidic Chip

The movement of collective cells is affected through changes in physical interactions of cells in response to external mechanical stimuli, including fluid flow. Most tissues are affected by fluid flow at the interstitial level, but few studies have investigated the physical effects in collective cells affected by a low flow rate. In this study, collective cell migration of Madin–Darby canine kidney (MDCK) epithelial cells was investigated under static or interstitial flow (0, 0.1, and 1 μL/min) using a traction microfluidic device. The optimization of calculation of cellular traction forces was first achieved by changing interrogation window size from the fluorescent bead images. Migration analysis of cell collectives patterned with a 700 μm circular shape reveals that cells under the slow flow (0.1 and 1 μL/min) showed the inhibitory migration by decreasing cell island size and cellular speed compared to that of static condition. Analysis of cellular forces shows that level of traction forces was lower in the slow flow condition (~20 Pa) compared to that of static condition (~50 Pa). Interestingly, the standard deviation of traction force of cells was dramatically decreased as the flow rate increased from 0 to 1 μL/min, which indicates that flow affects the distribution of cellular traction forces among cell collectives. Cellular tension was increased by 50% in the cells under the fluid flow rate of 1 μL/min. Treatment of calcium blocker increased the migratory speed of cells under the flow condition, whereas there is little change of cellular forces. In conclusion, it has been shown that the interstitial flow inhibited the collective movement of epithelial cells by decreasing and re-distributing cellular forces. These findings provide insights into the study of the effect of interstitial flow on cellular behavior, such as development, regeneration, and morphogenesis.

Wrinkle force microscopy: a new machine learning based approach to predict cell mechanics from images

Combining experiments with artificial intelligence algorithms, we propose a new machine learning based approach to extract the cellular force distributions from the microscope images. The full process can be divided into three steps. First, we culture the cells on a special substrate allowing to measure both the cellular traction force on the substrate and the corresponding substrate wrinkles simultaneously. The cellular forces are obtained using the traction force microscopy (TFM), at the same time that cell-generated contractile forces wrinkle their underlying substrate. Second, the wrinkle positions are extracted from the microscope images. Third, we train the machine learning system with GAN (generative adversarial network) by using sets of corresponding two images, the traction field and the input images (raw microscope images or extracted wrinkle images), as the training data. The network understands the way to convert the input images of the substrate wrinkles to the traction distribution from the training. After sufficient training, the network is utilized to predict the cellular forces just from the input images. Our system provides a powerful tool to evaluate the cellular forces efficiently because the forces can be predicted just by observing the cells under the microscope, which is a way simpler method compared to the TFM experiment. Additionally, the machine learning based approach presented here has the profound potential for being applied to diverse cellular assays for studying mechanobiology of cells.Significance StatementCell-generated forces are indispensable determinants of fundamental cell functions such as motility and cell division. As such, quantifying how the forces change upon perturbations to the cells such as gene mutations and drug administration is of profound importance. Here we present a novel machine learning based system that allows for efficient estimations of the forces that are determined only by “observing” microscope images. Given that the cellular traction forces are regulated downstream of diverse signaling pathways, our system – that helps significantly improve the throughput of the measurements – presents a new, high throughput platform for real time analysis of the effects of a massive number of genetic and molecular perturbations on the forces and resulting cell mechanics.