PCP and Septins govern the polarized organization of the actin cytoskeleton during convergent extension.

Convergent extension (CE) requires the coordinated action of the planar cell polarity (PCP) proteins1,2 and the actin cytoskeleton,3,4,5,6 but this relationship remains incompletely understood. For example, PCP signaling orients actomyosin contractions, yet actomyosin is also required for the polarized localization of PCP proteins.7,8 Moreover, the actin-regulating Septins play key roles in actin organization9 and are implicated in PCP and CE in frogs, mice, and fish5,6,10,11,12 but execute only a subset of PCP-dependent cell behaviors. Septin loss recapitulates the severe tissue-level CE defects seen after core PCP disruption yet leaves overt cell polarity intact.5 Together, these results highlight the general fact that cell movement requires coordinated action by distinct but integrated actin populations, such as lamella and lamellipodia in migrating cells13 or medial and junctional actin populations in cells engaged in apical constriction.14,15 In the context of Xenopus mesoderm CE, three such actin populations are important, a superficial meshwork known as the "node-and-cable" system,4,16,17,18 a contractile network at deep cell-cell junctions,6,19 and mediolaterally oriented actin-rich protrusions, which are present both superficially and deeply.4,19,20,21 Here, we exploited the amenability of the uniquely "two-dimensional" node and cable system to probe the relationship between PCP proteins, Septins, and the polarization of this actin network. We find that the PCP proteins Vangl2 and Prickle2 and Septins co-localize at nodes, and that the node and cable system displays a cryptic, PCP- and Septin-dependent anteroposterior (AP) polarity in its organization and dynamics.

New tools reveal PCP-dependent polarized mechanics in the cortex and cytoplasm of single cells during convergent extension.

Understanding biomechanics of biological systems is crucial for unraveling complex processes like tissue morphogenesis. However, current methods for studying cellular mechanics in vivo are limited by the need for specialized equipment and often provide limited spatiotemporal resolution. Here we introduce two new techniques, Tension by Transverse Fluctuation (TFlux) and in vivo microrheology, that overcome these limitations. They both offer time-resolved, subcellular biomechanical analysis using only fluorescent reporters and widely available microscopes. Employing these two techniques, we have revealed a planar cell polarity (PCP)-dependent mechanical gradient both in the cell cortex and the cytoplasm of individual cells engaged in convergent extension. Importantly, the non-invasive nature of these methods holds great promise for its application for uncovering subcellular mechanical variations across a wide array of biological contexts. Summary: Non-invasive imaging-based techniques providing time-resolved biomechanical analysis at subcellular scales in developing vertebrate embryos.

Inhibition of ribosome biogenesis in the epidermis is sufficient to trigger organism-wide growth quiescence independently of nutritional status in C. elegans.

Interorgan communication is crucial for multicellular organismal growth, development, and homeostasis. Cell nonautonomous inhibitory cues, which limit tissue-specific growth alterations, are not well characterized due to cell ablation approach limitations. In this study, we employed the auxin-inducible degradation system in C. elegans to temporally and spatially modulate ribosome biogenesis, through depletion of essential factors (RPOA-2, GRWD-1, or TSR-2). Our findings reveal that embryo-wide inhibition of ribosome biogenesis induces a reversible early larval growth quiescence, distinguished by a unique gene expression signature that is different from starvation or dauer stages. When ribosome biogenesis is inhibited in volumetrically similar tissues, including body wall muscle, epidermis, pharynx, intestine, or germ line, it results in proportionally stunted growth across the organism to different degrees. We show that specifically inhibiting ribosome biogenesis in the epidermis is sufficient to trigger an organism-wide growth quiescence. Epidermis-specific ribosome depletion leads to larval growth quiescence at the L3 stage, reduces organism-wide protein synthesis, and induced cell nonautonomous gene expression alterations. Further molecular analysis reveals overexpression of secreted proteins, suggesting an organism-wide regulatory mechanism. We find that UNC-31, a dense-core vesicle (DCV) pathway component, plays a significant role in epidermal ribosome biogenesis-mediated growth quiescence. Our tissue-specific knockdown experiments reveal that the organism-wide growth quiescence induced by epidermal-specific ribosome biogenesis inhibition is suppressed by reducing unc-31 expression in the epidermis, but not in neurons or body wall muscles. Similarly, IDA-1, a membrane-associated protein of the DCV, is overexpressed, and its knockdown in epidermis suppresses the organism-wide growth quiescence in response to epidermal ribosome biogenesis inhibition. Finally, we observe an overall increase in DCV puncta labeled by IDA-1 when epidermal ribosome biogenesis is inhibited, and these puncta are present in or near epidermal cells. In conclusion, these findings suggest a novel mechanism of nutrition-independent multicellular growth coordination initiated from the epidermis tissue upon ribosome biogenesis inhibition.

ARVCF catenin controls force production during vertebrate convergent extension.

The design of an animal's body plan is encoded in the genome, and the execution of this program is a mechanical progression involving coordinated movement of proteins, cells, and whole tissues. Thus, a challenge to understanding morphogenesis is connecting events that occur across various length scales. Here, we describe how a poorly characterized adhesion effector, Arvcf catenin, controls Xenopus head-to-tail axis extension. We find that Arvcf is required for axis extension within the intact organism but not within isolated tissues. We show that the organism-scale phenotype results from a defect in tissue-scale force production. Finally, we determine that the force defect results from the dampening of the pulsatile recruitment of cell adhesion and cytoskeletal proteins to membranes. These results provide a comprehensive understanding of Arvcf function during axis extension and produce an insight into how a cellular-scale defect in adhesion results in an organism-scale failure of development.

Convergent extension requires adhesion-dependent biomechanical integration of cell crawling and junction contraction.

Convergent extension (CE) is an evolutionarily conserved collective cell movement that elongates several organ systems during development. Studies have revealed two distinct cellular mechanisms, one based on cell crawling and the other on junction contraction. Whether these two behaviors collaborate is unclear. Here, using live-cell imaging, we show that crawling and contraction act both independently and jointly but that CE is more effective when they are integrated via mechano-reciprocity. We thus developed a computational model considering both crawling and contraction. This model recapitulates the biomechanical efficacy of integrating the two modes and further clarifies how the two modes and their integration are influenced by cell adhesion. Finally, we use these insights to understand the function of an understudied catenin, Arvcf, during CE. These data are significant for providing interesting biomechanical and cell biological insights into a fundamental morphogenetic process that is implicated in human neural tube defects and skeletal dysplasias.

Mechanical heterogeneity along single cell-cell junctions is driven by lateral clustering of cadherins during vertebrate axis elongation.

Morphogenesis is governed by the interplay of molecular signals and mechanical forces across multiple length scales. The last decade has seen tremendous advances in our understanding of the dynamics of protein localization and turnover at subcellular length scales, and at the other end of the spectrum, of mechanics at tissue-level length scales. Integrating the two remains a challenge, however, because we lack a detailed understanding of the subcellular patterns of mechanical properties of cells within tissues. Here, in the context of the elongating body axis of Xenopus embryos, we combine tools from cell biology and physics to demonstrate that individual cell-cell junctions display finely-patterned local mechanical heterogeneity along their length. We show that such local mechanical patterning is essential for the cell movements of convergent extension and is imparted by locally patterned clustering of a classical cadherin. Finally, the patterning of cadherins and thus local mechanics along cell-cell junctions are controlled by Planar Cell Polarity signaling, a key genetic module for CE that is mutated in diverse human birth defects.

Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells.

Classic embryological studies have successfully applied genetics and cell biology principles to understand embryonic development. However, it remains unresolved how mechanics, as an integral driver of development, is involved in controlling tissue-scale cell fate patterning. Here we report a micropatterned human pluripotent stem (hPS)-cell-based neuroectoderm developmental model, in which pre-patterned geometrical confinement induces emergent patterning of neuroepithelial and neural plate border cells, mimicking neuroectoderm regionalization during early neurulation in vivo. In this hPS-cell-based neuroectoderm patterning model, two tissue-scale morphogenetic signals-cell shape and cytoskeletal contractile force-instruct neuroepithelial/neural plate border patterning via BMP-SMAD signalling. We further show that ectopic mechanical activation and exogenous BMP signalling modulation are sufficient to perturb neuroepithelial/neural plate border patterning. This study provides a useful microengineered, hPS-cell-based model with which to understand the biomechanical principles that guide neuroectoderm patterning and hence to study neural development and disease.

May the force be with you.

Understanding the coordination of the forces generated in embryos by two processes, convergent extension and convergent thickening, is key to understanding how a hollow sphere of cells develops into an elongated embryo.

Nanotopography regulates motor neuron differentiation of human pluripotent stem cells.

The regulation of human pluripotent stem cell (hPSC) behaviors has been mainly studied through exploration of biochemical factors. However, the current directed differentiation protocols for hPSCs that completely rely on biochemical factors remain suboptimal. It has recently become evident that coexisting biophysical signals in the stem cell microenvironment, including nanotopographic cues, can provide potent regulatory signals to mediate adult stem cell behaviors, including self-renewal and differentiation. Herein, we utilized a recently developed, large-scale nanofabrication technique based on reactive-ion etching (RIE) to generate random nanoscale structures on glass surfaces with high precision and reproducibility. We report here that hPSCs are sensitive to nanotopographic cues and such nanotopographic sensitivity can be leveraged for improving directed neuronal differentiation of hPSCs. We demonstrate early neuroepithelial conversion and motor neuron (MN) progenitor differentiation of hPSCs can be promoted using nanoengineered topographic substrates. We further explore how hPSCs sense the substrate nanotopography and relay this biophysical signal through a regulatory signaling network involving cell adhesion, the actomyosin cytoskeleton, and Hippo/YAP signaling to mediate the neuroepithelial induction of hPSCs. Our study provides an efficient method for large-scale production of MNs from hPSCs, useful for regenerative medicine and cell-based therapies.

Effects of substrate stiffness and actomyosin contractility on coupling between force transmission and vinculin-paxillin recruitment at single focal adhesions.

Focal adhesions (FAs) regulate force transfer between the cytoskeleton and ECM-integrin complexes. We previously showed that vinculin regulates force transmission at FAs. Vinculin residence time in FAs correlated with applied force, supporting a mechanosensitive model in which forces stabilize vinculin's active conformation to promote force transfer. In the present study, we examined the relationship between traction force and vinculin-paxillin localization to single FAs in the context of substrate stiffness and actomyosin contractility. We found that vinculin and paxillin FA area did not correlate with traction force magnitudes at single FAs, and this was consistent across different ECM stiffness and cytoskeletal tension states. However, vinculin residence time at FAs varied linearly with applied force for stiff substrates, and this was disrupted on soft substrates and after contractility inhibition. In contrast, paxillin residence time at FAs was independent of local applied force and substrate stiffness. Paxillin recruitment and residence time at FAs, however, were dependent on cytoskeletal contractility on lower substrate stiffness values. Finally, substrate stiffness and cytoskeletal contractility regulated whether vinculin and paxillin turnover dynamics are correlated to each other at single FAs. This analysis sheds new insights on the coupling among force, substrate stiffness, and FA dynamics.

Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis.

Mechanical homeostasis-a fundamental process by which cells maintain stable states under environmental perturbations-is regulated by two subcellular mechanotransducers: cytoskeleton tension and integrin-mediated focal adhesions (FAs). Here, we show that single-cell mechanical homeostasis is collectively driven by the distinct, graduated dynamics (rheostasis) of subcellular cytoskeleton tension and FAs. Such rheostasis involves a mechanosensitive pattern wherein ground states of cytoskeleton tension and FA determine their distinct reactive paths through either relaxation or reinforcement. Pharmacological perturbations of the cytoskeleton and molecularly modulated integrin catch-slip bonds biased the rheostasis and induced non-homeostasis of FAs, but not of cytoskeleton tension, suggesting a unique sensitivity of FAs in regulating homeostasis. Theoretical modelling revealed myosin-mediated cytoskeleton contractility and catch-slip-bond-like behaviours in FAs and the cytoskeleton as sufficient and necessary mechanisms for quantitatively recapitulating mechanosensitive rheostasis. Our findings highlight the previously underappreciated physical nature of the mechanical homeostasis of cells.

Desktop aligner for fabrication of multilayer microfluidic devices.

Multilayer assembly is a commonly used technique to construct multilayer polydimethylsiloxane (PDMS)-based microfluidic devices with complex 3D architecture and connectivity for large-scale microfluidic integration. Accurate alignment of structure features on different PDMS layers before their permanent bonding is critical in determining the yield and quality of assembled multilayer microfluidic devices. Herein, we report a custom-built desktop aligner capable of both local and global alignments of PDMS layers covering a broad size range. Two digital microscopes were incorporated into the aligner design to allow accurate global alignment of PDMS structures up to 4 in. in diameter. Both local and global alignment accuracies of the desktop aligner were determined to be about 20 µm cm(-1). To demonstrate its utility for fabrication of integrated multilayer PDMS microfluidic devices, we applied the desktop aligner to achieve accurate alignment of different functional PDMS layers in multilayer microfluidics including an organs-on-chips device as well as a microfluidic device integrated with vertical passages connecting channels located in different PDMS layers. Owing to its convenient operation, high accuracy, low cost, light weight, and portability, the desktop aligner is useful for microfluidic researchers to achieve rapid and accurate alignment for generating multilayer PDMS microfluidic devices.

Age-Associated Increase in Skin Fibroblast-Derived Prostaglandin E2 Contributes to Reduced Collagen Levels in Elderly Human Skin.

Production of type I collagen declines during aging, leading to skin thinning and impaired function. Prostaglandin E2 (PGE2) is a pleiotropic lipid mediator that is synthesized from arachidonic acid by the sequential actions of cyclooxygenases (COX) and PGE synthases (PTGES). PGE2 inhibits collagen production by fibroblasts in vitro. We report that PTGES1 and COX2 progressively increase with aging in sun-protected human skin. PTGES1 and COX2 mRNA were increased 3.4-fold and 2.7-fold, respectively, in the dermis of elderly (>80 years) versus young (21-30 years) individuals. Fibroblasts were the major cell source of both enzymes. PGE2 levels were increased 70% in elderly skin. Fibroblasts in aged skin display reduced spreading due to collagen fibril fragmentation. To investigate the relationship between spreading and PGE2 synthesis, fibroblasts were cultured on micropost arrays or hydrogels of varying mechanical compliance. Reduced spreading/mechanical force resulted in increased expression of both PTGES1 and COX2 and elevated levels of PGE2. Inhibition of PGE2 synthesis by diclofenac enhanced collagen production in skin organ cultures. These data suggest that reduced spreading/mechanical force of fibroblasts in aged skin elevates PGE2 production, contributing to reduced collagen production. Inhibition of PGE2 production may be therapeutically beneficial for combating age-associated collagen deficit in human skin.

Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells.

Our understanding of the intrinsic mechanosensitive properties of human pluripotent stem cells (hPSCs), in particular the effects that the physical microenvironment has on their differentiation, remains elusive. Here, we show that neural induction and caudalization of hPSCs can be accelerated by using a synthetic microengineered substrate system consisting of poly(dimethylsiloxane) micropost arrays (PMAs) with tunable mechanical rigidities. The purity and yield of functional motor neurons derived from hPSCs within 23 days of culture using soft PMAs were improved more than fourfold and tenfold, respectively, compared with coverslips or rigid PMAs. Mechanistic studies revealed a multi-targeted mechanotransductive process involving Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosin cytoskeleton integrity and contractility. Our findings suggest that substrate rigidity is an important biophysical cue influencing neural induction and subtype specification, and that microengineered substrates can thus serve as a promising platform for large-scale culture of hPSCs.

Nanoroughened surfaces for efficient capture of circulating tumor cells without using capture antibodies.

Circulating tumor cells (CTCs) detached from both primary and metastatic lesions represent a potential alternative to invasive biopsies as a source of tumor tissue for the detection, characterization and monitoring of cancers. Here we report a simple yet effective strategy for capturing CTCs without using capture antibodies. Our method uniquely utilized the differential adhesion preference of cancer cells to nanorough surfaces when compared to normal blood cells and thus did not depend on their physical size or surface protein expression, a significant advantage as compared to other existing CTC capture techniques.

Live-cell subcellular measurement of cell stiffness using a microengineered stretchable micropost array membrane.

Forces are increasingly recognized as major regulators of cell structure and function, and the mechanical properties of cells, such as cell stiffness, are essential to the mechanisms by which cells sense forces, transmit them to the cell interior or to other cells, and transduce them into chemical signals that impact a spectrum of cellular responses. Here we reported a new whole-cell cell stiffness measurement technique with a subcellular spatial resolution. This technique was based on a novel cell stretching device that allowed for quantitative control and real-time measurements of mechanical stimuli and cellular biomechanical responses. Our strategy involved a microfabricated array of silicone elastomeric microposts integrated onto a stretchable elastomeric membrane. Using a computer-controlled vacuum, this micropost array membrane (mPAM) was activated to apply equibiaxial cell stretching forces to adherent cells attached on the tops of the microposts. The micropost top positions before and after mPAM stretches were recorded using fluorescence microscopy and further utilized to quantify local cell stretching forces and cell area increments. A robust computation scheme was developed and implemented for subcellular quantifications of cell stiffness using the data of local cell stretching forces and cell area increments generated from mPAM cell stretch assays. Our cell stiffness studies using the mPAM revealed strong positive correlations among cell stiffness, cellular traction force, and cell spread area, and illustrated the important functional roles of actin polymerization and myosin II-mediated cytoskeleton contractility in regulating cell stiffness. Collectively, our work reported a new approach for whole-cell stiffness measurements with a subcellular spatial resolution, which would help likely explain the complex biomechanical functions and force-sensing mechanisms of cells and design better materials for cell and tissue engineering and other applications in vivo.

Microengineered synthetic cellular microenvironment for stem cells.

Stem cells possess the ability of self-renewal and differentiation into specific cell types. Therefore, stem cells have great potentials in fundamental biology studies and clinical applications. The most urgent desire for stem cell research is to generate appropriate artificial stem cell culture system, which can mimic the dynamic complexity and precise regulation of the in vivo biochemical and biomechanical signals, to regulate and direct stem cell behaviors. Precise control and regulation of the biochemical and biomechanical stimuli to stem cells have been successfully achieved using emerging micro/nanoengineering techniques. This review provides insights into how these micro/nanoengineering approaches, particularly microcontact printing and elastomeric micropost array, are applied to create dynamic and complex environment for stem cells culture.

Nanotopography influences adhesion, spreading, and self-renewal of human embryonic stem cells.

Human embryonic stem cells (hESCs) have great potentials for future cell-based therapeutics. However, their mechanosensitivity to biophysical signals from the cellular microenvironment is not well characterized. Here we introduced an effective microfabrication strategy for accurate control and patterning of nanoroughness on glass surfaces. Our results demonstrated that nanotopography could provide a potent regulatory signal over different hESC behaviors, including cell morphology, adhesion, proliferation, clonal expansion, and self-renewal. Our results indicated that topological sensing of hESCs might include feedback regulation involving mechanosensory integrin-mediated cell-matrix adhesion, myosin II, and E-cadherin. Our results also demonstrated that cellular responses to nanotopography were cell-type specific, and as such, we could generate a spatially segregated coculture system for hESCs and NIH/3T3 fibroblasts using patterned nanorough glass surfaces.

A silicone-based stretchable micropost array membrane for monitoring live-cell subcellular cytoskeletal response.

External forces are increasingly recognized as major regulators of cellular structure and function, yet the underlying mechanism by which cells sense forces and transduce them into intracellular biochemical signals and behavioral responses ('mechanotransduction') is largely undetermined. To aid in the mechanistic study of mechanotransduction, herein we devised a cell stretching device that allowed for quantitative control and real-time measurement of mechanical stimuli and cellular biomechanical responses. Our strategy involved a microfabricated array of silicone elastomeric microposts integrated onto a stretchable elastomeric membrane. Using a computer-controlled vacuum, this micropost array membrane (mPAM) was activated to apply equibiaxial cell stretching forces to adherent cells attached to the microposts. Using the mPAM, we studied the live-cell subcellular dynamic responses of contractile forces in vascular smooth muscle cells (VSMCs) to a sustained static equibiaxial cell stretch. Our data showed that in response to a sustained cell stretch, VSMCs regulated their cytoskeletal (CSK) contractility in a biphasic manner: they first acutely enhanced their contraction to resist rapid cell deformation ('stiffening') before they allowed slow adaptive inelastic CSK reorganization to release their contractility ('softening'). The contractile response across entire single VSMCs was spatially inhomogeneous and force-dependent. Our mPAM device and live-cell subcellular contractile measurements will help elucidate the mechanotransductive system in VSMCs and thus contribute to our understanding of pressure-induced vascular disease processes.

Synergistic regulation of cell function by matrix rigidity and adhesive pattern.

Cell-extracellular matrix (ECM) interactions play a critical role in regulating cellular behaviors. Recent studies of cell-ECM interactions have mainly focused on the actomyosin based and adhesion mediated mechanosensing pathways to understand how individual mechanical signals in the cell microenvironment, such as matrix rigidity and adhesive ECM pattern, are sensed by the cell and further trigger downstream intracellular signaling cascades and cellular responses. However, synergistic and collective regulation of cellular behaviors by matrix rigidity and adhesive ECM pattern are still elusive and largely uncharacterized. Here, we generated a library of microfabricated polydimethylsiloxane (PDMS) micropost arrays to study the synergistic and independent effects of matrix rigidity and adhesive ECM pattern on mechanoresponsive behaviors of both NIH/3T3 fibroblasts and human umbilical vein endothelial cells (HUVECs). We showed that both cell types were mechanosensitive and their cell spreading, FA formation, cytoskeletal contractility, and proliferation were all strongly dependent on both substrate rigidity and adhesive ECM pattern. We further showed that under the same substrate rigidity condition, smaller and closer adhesive ECM islands would cause both cells to spread out more, form more adhesion structures, and have a higher proliferation rate. The influence of adhesive ECM pattern on rigidity-mediated cytoskeletal contractility was cell type specific and was only significant for NIH/3T3. Morphometric analysis of cell populations revealed a strong correlation between focal adhesion and cell spreading, regardless of substrate rigidity and adhesive ECM pattern. We also observed a strong correlation between cellular traction force and cell spreading, with a substantially smaller independent effect of substrate rigidity on traction force. Our study here had determined key aspects of the biomechanical responses of adherent cells to independent and collective changes of substrate rigidity and adhesive ECM pattern.