Basal actomyosin pulses expand epithelium coordinating cell flattening and tissue elongation.

Actomyosin networks constrict cell area and junctions to alter cell and tissue shape. However, during cell expansion under mechanical stress, actomyosin networks are strengthened and polarized to relax stress. Thus, cells face a conflicting situation between the enhanced actomyosin contractile properties and the expansion behaviour of the cell or tissue. To address this paradoxical situation, we study late Drosophila oogenesis and reveal an unusual epithelial expansion wave behaviour. Mechanistically, Rac1 and Rho1 integrate basal pulsatile actomyosin networks with ruffles and focal adhesions to increase and then stabilize basal area of epithelial cells allowing their flattening and elongation. This epithelial expansion behaviour bridges cell changes to oocyte growth and extension, while oocyte growth in turn deforms the epithelium to drive cell spreading. Basal pulsatile actomyosin networks exhibit non-contractile mechanics, non-linear structures and F-actin/Myosin-II spatiotemporal signal separation, implicating unreported expanding properties. Biophysical modelling incorporating these expanding properties well simulates epithelial cell expansion waves. Our work thus highlights actomyosin expanding properties as a key mechanism driving tissue morphogenesis.

Interfacial Organization and Forces Arising from Epithelial-Cancerous Monolayer Interactions.

The interfacial interactions between epithelia and cancer cells have profound relevance for tumor development and metastasis. Through monolayer confrontation of MCF10A (nontumorigenic human breast epithelial cells) and MDA-MB-231 (human epithelial breast cancer cells) cells, we investigate the epithelial-cancerous interfacial interactions at the tissue level. We show that the monolayer interaction leads to competitive interfacial morphodynamics and drives an intricate spatial organization of MCF10A cells into multicellular finger-like structures, which further branch into multiple subfinger-like structures. These hierarchical interfacial structures penetrate the cancer monolayer and can spontaneously segregate or even envelop cancer cell clusters, consistent with our theoretical prediction. By tracking the substrate displacements via embedded fluorescent nanobeads and implementing nanomechanical modeling that combines atomic force microscopy and finite element simulations, we computed mechanical force patterns, including traction forces and monolayer stresses, caused by the monolayer interaction. It is found that the heterogeneous mechanical forces accumulated in the monolayers are able to squeeze cancer cells, leading to three-dimensional interfacial bulges or cell extrusion, initiating the p53 apoptosis signaling pathways of cancer cells. We reveal that intercellular E-cadherin and P-cadherin of epithelial cells differentially regulate the interfacial organization including migration speed, directionality, spatial correlation, F-actin alignment, and subcellular protrusions of MCF10A cells; whereas E-cadherin governs interfacial geometry that is relevant to force localization and cancer cell extrusion, P-cadherin maintains interfacial integrity that enables long-range force transmission. Our findings suggest that the collaborative molecular and mechanical behaviors are crucial for preventing epithelial tissues from undergoing tumor invasion.

Substrate nesting guides cyst morphogenesis of human pluripotent stem cells without 3D extracellular matrix overlay.

Understanding the principles underlying the self-organization of stem cells into tissues is fundamental for deciphering human embryo development. Here, we report that, without three-dimensional (3D) extracellular matrix (ECM) overlay, human pluripotent stem cells (hPSCs) cultured on two-dimensional soft elastic substrates can self-organize into 3D cysts resembling the human epiblast sac in a stiffness-dependent manner. Our theoretical modeling predicts that this cyst organization is facilitated and guided by the spontaneous nesting of the soft substrate, which results from the adhesion-dependent mechanical interaction between cells and substrate. Such substrate nesting is sufficient for the 3D assembly and polarization of hPSCs required for cyst organization, even without 3D ECM overlay. Furthermore, we identify that the reversible substrate nesting and cyst morphogenesis also require appropriate activation of ROCK-Myosin II pathway. This indicates a unique set of tissue morphomechanical signaling mechanisms that clearly differ from the canonical cystogenic mechanism previously reported in 3D ECM. Our findings highlight an unanticipated synergy between mechanical microenvironment and mechanotransduction in controlling tissue morphogenesis and suggest a mechanics-based strategy for generation of hPSCs-derived models for early human embryogenesis. STATEMENT OF SIGNIFICANCE: Soft substrates can induce the self-organization of human pluripotent stem cells (hPSCs) into cysts without three-dimensional (3D) extracellular matrix (ECM) overlay. However, the underlying mechanisms by which soft substrate guides cystogenesis are largely unknown. This study shows that substrate nesting, resulting from cell-substrate interaction, plays an important role in cyst organization, including 3D assembly and apical-basal polarization. Additionally, actomyosin contractility mediated by the ROCK-Myosin II pathway also contributes to the substrate deformation and cyst morphology. These findings demonstrate the interplay between the mechanical microenvironment and cells in tissue morphogenesis, suggesting a mechanics-based strategy in building hPSC-derived models for early human embryo development.

Polymerization force-regulated actin filament-Arp2/3 complex interaction dominates self-adaptive cell migrations.

Cells migrate by adapting their leading-edge behaviors to heterogeneous extracellular microenvironments (ECMs) during cancer invasions and immune responses. Yet it remains poorly understood how such complicated dynamic behaviors emerge from millisecond-scale assembling activities of protein molecules, which are hard to probe experimentally. To address this gap, we establish a spatiotemporal "resistance-adaptive propulsion" theory based on the interactions between Arp2/3 complexes and polymerizing actin filaments and a multiscale dynamic modeling system spanning from molecular proteins to the cell. We quantitatively find that cells can accurately self-adapt propulsive forces to overcome heterogeneous ECMs via a resistance-triggered positive feedback mechanism, dominated by polymerization-induced actin filament bending and the bending-regulated actin-Arp2/3 binding. However, for high resistance regions, resistance triggers a negative feedback, hindering branched filament assembly, which adapts cellular morphologies to circumnavigate the obstacles. Strikingly, the synergy of the two opposite feedbacks not only empowers the cell with both powerful and flexible migratory capabilities to deal with complex ECMs but also enables efficient utilization of intracellular proteins by the cell. In addition, we identify that the nature of cell migration velocity depending on ECM history stems from the inherent temporal hysteresis of cytoskeleton remodeling. We also show that directional cell migration is dictated by the competition between the local stiffness of ECMs and the local polymerizing rate of actin network caused by chemotactic cues. Our results reveal that it is the polymerization force-regulated actin filament-Arp2/3 complex binding interaction that dominates self-adaptive cell migrations in complex ECMs, and we provide a predictive theory and a spatiotemporal multiscale modeling system at the protein level.

Unified multiscale theory of cellular mechanical adaptations to substrate stiffness.

Rigidity of the extracellular matrix markedly regulates many cellular processes. However, how cells detect and respond to matrix rigidity remains incompletely understood. Here, we propose a unified two-dimensional multiscale framework accounting for the chemomechanical feedback to explore the interrelated cellular mechanosensing, polarization, and migration, which constitute the dynamic cascade in cellular response to matrix stiffness but are often modeled separately in previous theories. By combining integrin dynamics and intracellular force transduction, we show that substrate stiffness can act as a switch to activate or deactivate cell polarization. Our theory quantitatively reproduces rich stiffness-dependent cellular dynamics, including spreading, polarity selection, migration pattern, durotaxis, and even negative durotaxis, reported in a wide spectrum of cell types, and reconciles some inconsistent experimental observations. We find that a specific bipolarized mode can determine the optimal substrate stiffness, which enables the fastest cell migration rather than the largest traction forces that cells apply on the substrate. We identify that such a mechanical adaptation stems from the force balance across the whole cell. These findings could yield universal insights into various stiffness-mediated cellular processes within the context of tissue morphogenesis, wound healing, and cancer invasion.

AFM-based indentation method for measuring the relaxation property of living cells.

Probing the mechanical properties of cells is critical for understanding their deformation behaviors and biological functions. Although some methods have been proposed to characterize the elastic properties of cells, it is still difficult to measure their time-dependent properties. This paper investigates the use of atomic force microscope (AFM) to determine the reduced relaxation modulus of cells. In principle, AFM is hard to perform an indentation relaxation test that requires a constant indenter displacement during load relaxation, whereas the real AFM indenter displacement usually varies with time during relaxation due to the relatively small bending stiffness of its cantilever. We investigate this issue through a combined theoretical, computational, and experimental effort. A protocol relying on the choice of appropriate cantilever bending stiffness is proposed to perform an AFM-based indentation relaxation test of cells, which enables the measurement of reduced relaxation modulus with high accuracy. This protocol is first validated by performing nanoindentation relaxation tests on a soft material and by comparing the results with those from independent measurements. Then indentation tests of cartilage cells are conducted to demonstrate this method in determining time-dependent properties of living cells. Finally, the change in the viscoelasticity of MCF-7 cells under hyperthermia is investigated.

Ciona embryonic tail bending is driven by asymmetrical notochord contractility and coordinated by epithelial proliferation.

Ventral bending of the embryonic tail within the chorion is an evolutionarily conserved morphogenetic event in both invertebrates and vertebrates. However, the complexity of the anatomical structure of vertebrate embryos makes it difficult to experimentally identify the mechanisms underlying embryonic folding. This study investigated the mechanisms underlying embryonic tail bending in chordates. To further understand the mechanical role of each tissue, we also developed a physical model with experimentally measured parameters to simulate embryonic tail bending. Actomyosin asymmetrically accumulated at the ventral side of the notochord, and cell proliferation of the dorsal tail epidermis was faster than that in the ventral counterpart during embryonic tail bending. Genetic disruption of actomyosin activity and inhibition of cell proliferation dorsally caused abnormal tail bending, indicating that both asymmetrical actomyosin contractility in the notochord and the discrepancy of epidermis cell proliferation are required for tail bending. In addition, asymmetrical notochord contractility was sufficient to drive embryonic tail bending, whereas differential epidermis proliferation was a passive response to mechanical forces. These findings showed that asymmetrical notochord contractility coordinates with differential epidermis proliferation mechanisms to drive embryonic tail bending.This article has an associated 'The people behind the papers' interview.

Universal Statistical Laws for the Velocities of Collective Migrating Cells.

Migratory dynamics of collective cells is central to the morphogenesis of biological tissues. The statistical distribution of cell velocities in 2D confluent monolayers is measured through large-scale and long-term experiments of various cell types lying on different substrates. A linear relation is discovered between the variability and the mean of cell speeds during the jamming process of confluent cell monolayers, suggesting time-invariant distribution profile of cell velocities. It is further found that the probability density function of cell velocities obeys the non-canonical q-Gaussian statistics, regardless of cell types and substrate stiffness. It is the Tsallis entropy, instead of the classical Boltzmann-Gibbs entropy, that dictates the universal statistical laws of collective cell migration. The universal statistical law stems from cell-cell interactions, as demonstrated by the wound healing experiments. This previously unappreciated finding provides a linkage between cell-level heterogeneity and tissue-level ensembles in embryonic development and tumor growth.

Mesoscopic dynamic model of epithelial cell division with cell-cell junction effects.

Cell division is central for embryonic development, tissue morphogenesis, and tumor growth. Experiments have evidenced that mitotic cell division is manipulated by the intercellular cues such as cell-cell junctions. However, it still remains unclear how these cortical-associated cues mechanically affect the mitotic spindle machinery, which determines the position and orientation of the cell division. In this paper, a mesoscopic dynamic cell division model is established to explore the integrated regulations of cortical polarity, microtubule pulling forces, cell deformability, and internal osmotic pressure. We show that the distributed pulling forces of astral microtubules play a key role in encoding the instructive cortical cues to orient and position the spindle of a dividing cell. The present model can not only predict the spindle orientation and position, but also capture the morphological evolution of cell rounding. The theoretical results agree well with relevant experiments both qualitatively and quantitatively. This work sheds light on the mechanical linkage between cell cortex and mitotic spindle, and holds potential in regulating cell division and sculpting tissue morphology.

A function of fascin1 in the colony formation of mouse embryonic stem cells.

Fascin1 is known to participate in the migration of cancer cells by binding to actin filaments. Recent studies evidenced that fascin1 also modulates processes such as the tumorigenesis and maintenance of pluripotency genes in cancer stem cells. However, the function of fascin1 in embryonic stem cells remains unclear. In this article, we report that fascin1 is highly expressed and widely distributed in mouse embryonic stem cells (mESCs), which are regulated by JAK-STAT3 and ß-catenin. We found that the overexpression of fascin1 impairs the formation of mESC colonies via the downregulation of intercellular adhesion molecules, and that mimicking the dephosphorylated mutation of fascin1 or inhibiting phosphorylation with Gö6983 significantly enhances colony formation. Hyperphosphorylated fascin1 can promote the maintenance of pluripotency in mESCs via nuclear localization and suppressing DNA methyltransferase expression. Our findings demonstrate a novel function of fascin1, as a vital regulator, in the colony formation and pluripotency of mESCs and provide insights into the molecular mechanisms underlying embryonic stem cell self-organization and development in vitro.

Geometric Confinement Guides the Expression of Cancer Stem Cell Molecular Markers CD44 via Cell Traction Forces.

Cancer stem cells (CSCs) play a critical role in the cancer metastasis and account for tumor heterogeneity. Growing evidence indicates that the CSC phenotypes are related to the tumor microenvironment. In this study, we report that the gradient of mechanical stresses guides the spatial patterning of the expression of CD44 and Yes-associated protein (YAP) in the geometrically confined multicellular sheets. Our study shows that the cytoskeletal contraction regulates the expression of CD44 through the translocation of YAP into the nucleus. The results demonstrate that geometric confinement and mechanical stresses are the regulators in the spatial patterning of CSC. It may help to understand the relationship between the tumor microenvironment and oncogenesis.

Heat Stress-Induced Multiple Multipolar Divisions of Human Cancer Cells.

Multipolar divisions of heated cells has long been thought to stem from centrosome aberrations of cells directly caused by heat stress. In this paper, through long-term live-cell imaging, we provide direct cellular evidences to demonstrate that heat stress can promote multiple multipolar divisions of MGC-803 and MCF-7 cells. Our results show that, besides facilitating centrosome aberration, polyploidy induced by heat stress is another mechanism that causes multipolar cell divisions, in which polyploid cancer cells engendered by mitotic slippage, cytokinesis failure, and cell fusion. Furthermore, we also find that the fates of theses polyploid cells depend on their origins, in the sense that the polyploid cells generated by mitotic slippage experience bipolar divisions with a higher rate than multipolar divisions, while those polyploid cells induced by both cytokinesis failure and cell fusion have a higher frequency of multipolar divisions compared with bipolar divisions. This work indicates that heat stress-induced multiple multipolar divisions of cancer cells usually produce aneuploid daughter cells, and might lead to genetically unstable cancer cells and facilitate tumor heterogeneity.

Dynamic instability and migration modes of collective cells in channels.

Migrating cells constantly experience geometrical confinements in vivo, as exemplified by cancer invasion and embryo development. In this paper, we investigate how intrinsic cellular properties and extrinsic channel confinements jointly regulate the two-dimensional migratory dynamics of collective cells. We find that besides external confinement, active cell motility and cell crowdedness also shape the migration modes of collective cells. Furthermore, the effects of active cell motility, cell crowdedness and confinement size on collective cell migration can be integrated into a unified dimensionless parameter, defined as the cellular motility number (CMN), which mirrors the competition between active motile force and passive elastic restoring force of cells. A low CMN favours laminar-like cell flows, while a high CMN destabilizes cell motions, resulting in a series of mode transitions from a laminar phase to an ordered vortex chain, and further to a mesoscale turbulent phase. These findings not only explain recent experiments but also predict dynamic behaviours of cell collectives, such as the existence of an ordered vortex chain mode and the mode selection under non-straight confinements, which are experimentally testable across different epithelial cell lines.

Three-dimensional collective cell motions in an acinus-like lumen.

Collective cell migration is an essential process in embryo development, wound healing, inflammatory response, and cancer invasion. Although cell motions in two-dimensional (2D) monolayers have been studied previously, three-dimensional (3D) collective cell migration, which constantly occurs during embryogenesis such as the establishment of ducts and acini in vivo, remains elusive. In this paper, we develop a cell-based model incorporating cell mechanics and cell motility to address coherent cell motions in a spherical acinus-like lumen with different cell populations. It is found that the interplays between cell persistence, random fluctuation, and geometrical confinement may engender rich and novel migratory modes. In a 3D spherical lumen, two cells may undergo stripe-like or cross-circular coherent rotations, whereas multiple cells can form dynamic twisting or circulating bands, leaving sparse cells at the center or even a hollow cavity in the cell aggregate. The cell density is found to profoundly influence the collective cell migration modes. Our model can reproduce the fundamental features observed in experiments and highlight the role of mechanics in steering 3D collective cell dynamics during mammary acinar morphogenesis.

Dynamic Migration Modes of Collective Cells.

Collective cell migration occurs in a diversity of physiological processes such as wound healing, cancer metastasis, and embryonic morphogenesis. In the collective context, cohesive cells may move as a translational solid, swirl as a fluid, or even rotate like a disk, with scales ranging from several to dozens of cells. In this work, an active vertex model is presented to explore the regulatory roles of social interactions of neighboring cells and environmental confinements in collective cell migration in a confluent monolayer. It is found that the competition between two kinds of intercellular social interactions-local alignment and contact inhibition of locomotion-drives the cells to self-organize into various dynamic coherent structures with a spatial correlation scale. The interplay between this intrinsic length scale and the external confinement dictates the migration modes of collective cells confined in a finite space. We also show that the local alignment-contact inhibition of locomotion coordination can induce giant density fluctuations in a confluent cell monolayer without gaps, which triggers the spontaneous breaking of orientational symmetry and leads to phase separation.

Micropipette aspiration method for characterizing biological materials with surface energy.

Many soft biological tissues possess a considerable surface stress, which plays a significant role in their biophysical functions, but most previous methods for characterizing their mechanical properties have neglected the effects of surface stress. In this work, we investigate the micropipette aspiration method to measure the mechanical properties of soft tissues and cells with surface effects. The neo-Hookean constitutive model is adopted to describe the hyperelasticity of the measured biological material, and the surface effect is taken into account by the finite element method. It is found that when the pipette radius or aspiration length is comparable to the elastocapillary length, surface energy may distinctly alter the aspiration response. Generally, both the aspiration length and the bulk normal stress decrease with increasing surface energy, and thus neglecting the surface energy would lead to an overestimation of elastic modulus. Through dimensional analysis and numerical simulations, we provide an explicit relation between the imposed pressure and the aspiration length. This method can be applied to determine the mechanical properties of soft biological tissues and organs, e.g., livers, tumors and embryos.

Swertia mussotii extracts induce mitochondria-dependent apoptosis in gastric cancer cells.

Swertia mussotii (Gentianaceae) is a traditional Chinese medicinal plant grown in the Qinghai-Tibet Plateau. Three fractions from S. mussotii extract, named SWF50, SWF 70 and SWF100, were screened for in vitro anti-proliferative activity on two gastric cancer cell lines, MGC-803 and BGC-823 cells using MTT assay. Our results demonstrated that SMF70 showed an anti-proliferative effect in MGC-803 cells and SMF100 showed an anti-proliferative effect in BGC-823 cells in vitro. Moreover, both two fractions induced apoptosis via depolymerization of cytoskeletal filaments, increased cytoplasmic levels of ROS and Ca2+ and disrupted mitochondrial transmembrane potential. In addition, flow cytometry analysis indicated that both two fractions could induce cell apoptosis and arrest the cell cycle at S phase. Our results indicate that SMF70 induces apoptosis of MGC-803 cells and SMF100 induces apoptosis of BGC-823 cells via a mitochondrial-dependent pathway. Meanwhile, we also investigated antitumor effect of SMF70 in vivo, and exhibited effective tumor growth inhibition. Our findings demonstrate that S. mussotii extracts could be a potential new alternative therapeutic agent gastric cancer.

Determinative Surface-Wrinkling Microstructures on Polypyrrole Films by Laser Writing.

We report a simple and efficient laser-writing strategy to fabricate hierarchical nested wrinkling microstructures on conductive polypyrrole (PPy) films, which enables us to develop advanced functional surfaces with diverse applications. The present strategy adopts the photothermal effect of PPy films to mimick the formation of hierarchical nested wrinkles observed in nature and design controlled microscale wrinkling patterns. Here, the PPy film is grown on a poly(dimethylsiloxane) (PDMS) substrate via oxidation polymerization of pyrrole in an acidic solution, accompanied by in situ self-wrinkling with wavelengths of two different scales (i.e., λ1 and λ2). Subsequent laser exposure of the PPy/PDMS bilayer induces a new surface wrinkling with a larger wavelength (i.e., λ3). Owing to the retention of the initial λ1 wrinkles, we obtain hierarchical nested wrinkles with the smaller λ1 wrinkles nested in the larger λ3 ones. Importantly, we realize the large-scale path-determinative fabrication of complex oriented wrinkling microstructures by controlling the relative motion between the bilayer and the laser. Combined with the induced changes in surface color, surface-wrinkling microstructures, and conductivity in the PPy films, the laser-writing strategy can find broad applications, for example, in modulation of surface wetting properties and fabrication of microcircuits, as demonstrated in this work.

Orientations of Cells on Compliant Substrates under Biaxial Stretches: A Theoretical Study.

Mechanical cues from the microenvironments play a regulating role in many physiological and pathological processes, such as stem cell differentiation and cancer cell metastasis. Experiments showed that cells adhered on a compliant substrate may change orientation with an externally applied strain in the substrate. By accounting for actin polymerization, actin retrograde flow, and integrin binding dynamics, here we develop a mechanism-based tensegrity model to study the orientations of polarized cells on a compliant substrate under biaxial stretches. We show that the cell can actively regulate its mechanical state by generating different traction force levels along its polarized direction. Under static or ultralow-frequency cyclic stretches, stretching a softer substrate leads to a higher increase in the traction force and induces a narrower distribution of cell alignment. Compared to static loadings, high-frequency cyclic loadings have a more significant influence on cell reorientation on a stiff substrate. In addition, the width of the cellular angular distribution scales inversely with the stretch amplitude under both static and cyclic stretches. Our results are in agreement with a wide range of experimental observations, and provide fundamental insights into the functioning of cellular mechanosensing systems.

A nonlinear poroelastic theory of solid tumors with glycosaminoglycan swelling.

Mechanics plays a crucial role in the growth, development, and therapeutics of tumors. In this paper, a nonlinear poroelastic theory is established to describe the mechanical behaviors of solid tumors. The free-swollen state of a tumor is chosen as the reference state, which enables us to avoid pursuing a dry and stress-free state that is hard to achieve for living tissues. Our results reveal that the compression resistance of a tumor is primarily attributed to glycosaminoglycan (GAG) swelling, and the compactness of cell aggregates is found to affect tumor consolidation. Over-expressed GAGs and dense cell aggregates can stiffen the tumor, a remodeling mechanism that makes the tumor with higher elastic modulus than its surrounding host tissues. Glycosaminoglycan chains also influence the transient mechanical response of the tumor by modulating the tissue permeability. The theoretical results show good agreement with relevant experimental observations. This study may not only deepen our understanding of tumorigenesis but also provide cues for developing novel anticancer strategies.