The guidance receptor plexin D1 is a mechanosensor in endothelial cells.

Shear stress on arteries produced by blood flow is important for vascular development and homeostasis but can also initiate atherosclerosis1. Endothelial cells that line the vasculature use molecular mechanosensors to directly detect shear stress profiles that will ultimately lead to atheroprotective or atherogenic responses2. Plexins are key cell-surface receptors of the semaphorin family of cell-guidance signalling proteins and can regulate cellular patterning by modulating the cytoskeleton and focal adhesion structures3-5. However, a role for plexin proteins in mechanotransduction has not been examined. Here we show that plexin D1 (PLXND1) has a role in mechanosensation and mechanically induced disease pathogenesis. PLXND1 is required for the response of endothelial cells to shear stress in vitro and in vivo and regulates the site-specific distribution of atherosclerotic lesions. In endothelial cells, PLXND1 is a direct force sensor and forms a mechanocomplex with neuropilin-1 and VEGFR2 that is necessary and sufficient for conferring mechanosensitivity upstream of the junctional complex and integrins. PLXND1 achieves its binary functions as either a ligand or a force receptor by adopting two distinct molecular conformations. Our results establish a previously undescribed mechanosensor in endothelial cells that regulates cardiovascular pathophysiology, and provide a mechanism by which a single receptor can exhibit a binary biochemical nature.

Peptide-Protein Coassemblies into Hierarchical and Bioactive Tubular Membranes.

Multicomponent self-assembly offers opportunities for the design of complex and functional biomaterials with tunable properties. Here, we demonstrate how minor modifications in the molecular structures of peptide amphiphiles (PAs) and elastin-like recombinamers (ELs) can be used to generate coassembling tubular membranes with distinct structures, properties, and bioactivity. First, by introducing minor modifications in the charge density of PA molecules (PAK2, PAK3, PAK4), different diffusion-reaction processes can be triggered, resulting in distinct membrane microstructures. Second, by combining different types of these PAs prior to their coassembly with ELs, further modifications can be achieved, tuning the structures and properties of the tubular membranes. Finally, by introducing the cell adhesive peptide RGDS in either the PA or EL molecules, it is possible to harness the different diffusion-reaction processes to generate tubular membranes with distinct bioactivities. The study demonstrates the possibility to trigger and achieve minor but crucial differences in coassembling processes and tune material structure and bioactivity. The study demonstrates the possibility to use minor, yet crucial, differences in coassembling processes to tune material structure and bioactivity.

Engineering the cellular mechanical microenvironment - from bulk mechanics to the nanoscale.

The field of mechanobiology studies how mechanical properties of the extracellular matrix (ECM), such as stiffness, and other mechanical stimuli regulate cell behaviour. Recent advancements in the field and the development of novel biomaterials and nanofabrication techniques have enabled researchers to recapitulate the mechanical properties of the microenvironment with an increasing degree of complexity on more biologically relevant dimensions and time scales. In this Review, we discuss different strategies to engineer substrates that mimic the mechanical properties of the ECM and outline how these substrates have been applied to gain further insight into the biomechanical interaction between the cell and its microenvironment.

Syndecan-4 tunes cell mechanics by activating the kindlin-integrin-RhoA pathway.

Extensive research over the past decades has identified integrins to be the primary transmembrane receptors that enable cells to respond to external mechanical cues. We reveal here a mechanism whereby syndecan-4 tunes cell mechanics in response to localized tension via a coordinated mechanochemical signalling response that involves activation of two other receptors: epidermal growth factor receptor and ß1 integrin. Tension on syndecan-4 induces cell-wide activation of the kindlin-2/ß1 integrin/RhoA axis in a PI3K-dependent manner. Furthermore, syndecan-4-mediated tension at the cell-extracellular matrix interface is required for yes-associated protein activation. Extracellular tension on syndecan-4 triggers a conformational change in the cytoplasmic domain, the variable region of which is indispensable for the mechanical adaptation to force, facilitating the assembly of a syndecan-4/α-actinin/F-actin molecular scaffold at the bead adhesion. This mechanotransduction pathway for syndecan-4 should have immediate implications for the broader field of mechanobiology.

Mechanotransduction in talin through the interaction of the R8 domain with DLC1.

The mechanical unfolding of proteins is a cellular mechanism for force transduction with potentially broad implications in cell fate. Despite this, the mechanism by which protein unfolding elicits differential downstream signalling pathways remains poorly understood. Here, we used protein engineering, atomic force microscopy, and biophysical tools to delineate how protein unfolding controls cell mechanics. Deleted in liver cancer 1 (DLC1) is a negative regulator of Ras homolog family member A (RhoA) and cell contractility that regulates cell behaviour when localised to focal adhesions bound to folded talin. Using a talin mutant resistant to force-induced unfolding of R8 domain, we show that talin unfolding determines DLC1 downstream signalling and, consequently, cell mechanics. We propose that this new mechanism of mechanotransduction may have implications for a wide variety of associated cellular processes.

GPER is a mechanoregulator of pancreatic stellate cells and the tumor microenvironment.

The mechanical properties of the tumor microenvironment are emerging as attractive targets for the development of therapies. Tamoxifen, an agonist of the G protein-coupled estrogen receptor (GPER), is widely used to treat estrogen-positive breast cancer. Here, we show that tamoxifen mechanically reprograms the tumor microenvironment through a newly identified GPER-mediated mechanism. Tamoxifen inhibits the myofibroblastic differentiation of pancreatic stellate cells (PSCs) in the tumor microenvironment of pancreatic cancer in an acto-myosin-dependent manner via RhoA-mediated contractility, YAP deactivation, and GPER signaling. This hampers the ability of PSCs to remodel the extracellular matrix and to promote cancer cell invasion. Tamoxifen also reduces the recruitment and polarization to the M2 phenotype of tumor-associated macrophages. Our results highlight GPER as a mechanical regulator of the tumor microenvironment that targets the three hallmarks of pancreatic cancer: desmoplasia, inflammation, and immune suppression. The well-established safety of tamoxifen in clinics may offer the possibility to redirect the singular focus of tamoxifen on the cancer cells to the greater tumor microenvironment and lead a new strategy of drug repurposing.

Tamoxifen mechanically reprograms the tumor microenvironment via HIF-1A and reduces cancer cell survival.

The tumor microenvironment is fundamental to cancer progression, and the influence of its mechanical properties is increasingly being appreciated. Tamoxifen has been used for many years to treat estrogen-positive breast cancer. Here we report that tamoxifen regulates the level and activity of collagen cross-linking and degradative enzymes, and hence the organization of the extracellular matrix, via a mechanism involving both the G protein-coupled estrogen receptor (GPER) and hypoxia-inducible factor-1 alpha (HIF-1A). We show that tamoxifen reduces HIF-1A levels by suppressing myosin-dependent contractility and matrix stiffness mechanosensing. Tamoxifen also downregulates hypoxia-regulated genes and increases vascularization in PDAC tissues. Our findings implicate the GPER/HIF-1A axis as a master regulator of peri-tumoral stromal remodeling and the fibrovascular tumor microenvironment and offer a paradigm shift for tamoxifen from a well-established drug in breast cancer hormonal therapy to an alternative candidate for stromal targeting strategies in PDAC and possibly other cancers.

Retinoic Acid Receptor-ß Is Downregulated in Hepatocellular Carcinoma and Cirrhosis and Its Expression Inhibits Myosin-Driven Activation and Durotaxis in Hepatic Stellate Cells.

Hepatic stellate cells (HSCs) are essential perisinusoidal cells in both healthy and diseased liver. HSCs modulate extracellular matrix (ECM) homeostasis when quiescent, but in liver fibrosis, HSCs become activated and promote excess deposition of ECM molecules and tissue stiffening via force generation and mechanosensing. In hepatocellular carcinoma (HCC), activated HSCs infiltrate the stroma and migrate to the tumor core to facilitate paracrine signaling with cancer cells. Because the function of HSCs is known to be modulated by retinoids, we investigated the expression profile of retinoic acid receptor beta (RAR-ß) in patients with cirrhosis and HCC, as well as the effects of RAR-ß activation in HSCs. We found that RAR-ß expression is significantly reduced in cirrhotic and HCC tissues. Using a comprehensive set of biophysical methods combined with cellular and molecular biology, we have elucidated the biomechanical mechanism by which all trans-retinoic acid promotes HSC deactivation via RAR-ß-dependent transcriptional downregulation of myosin light chain 2 expression. Furthermore, this also abrogated mechanically driven migration toward stiffer substrates. Conclusion: Targeting mechanotransduction in HSCs at the transcriptional level may offer therapeutic options for a range of liver diseases.

Cell-geometry-dependent changes in plasma membrane order direct stem cell signalling and fate.

Cell size and shape affect cellular processes such as cell survival, growth and differentiation1-4, thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications.

FAK controls the mechanical activation of YAP, a transcriptional regulator required for durotaxis.

Focal adhesion kinase (FAK) is a key molecule in focal adhesions and regulates fundamental processes in cells such as growth, survival, and migration. FAK is one of the first molecules recruited to focal adhesions in response to external mechanical stimuli and therefore is a pivotal mediator of cell mechanosignaling, and relays these stimuli to other mechanotransducers within the cytoplasm. Yes-associated protein (YAP) has been identified recently as one of these core mechanotransducers. YAP translocates to the nucleus following changes in cell mechanics to promote the expression of genes implicated in motility, apoptosis, proliferation, and organ growth. Here, we show that FAK controls the nuclear translocation and activation of YAP in response to mechanical activation and submit that the YAP-dependent process of durotaxis requires a cell with an asymmetric distribution of active and inactive FAK molecules.-Lachowski, D., Cortes, E., Robinson, B., Rice, A., Rombouts, K., Del Río Hernández, A. E. FAK controls the mechanical activation of YAP, a transcriptional regulator required for durotaxis.

Mechanical unfolding reveals stable 3-helix intermediates in talin and α-catenin.

Mechanical stability is a key feature in the regulation of structural scaffolding proteins and their functions. Despite the abundance of α-helical structures among the human proteome and their undisputed importance in health and disease, the fundamental principles of their behavior under mechanical load are poorly understood. Talin and α-catenin are two key molecules in focal adhesions and adherens junctions, respectively. In this study, we used a combination of atomistic steered molecular dynamics (SMD) simulations, polyprotein engineering, and single-molecule atomic force microscopy (smAFM) to investigate unfolding of these proteins. SMD simulations revealed that talin rod α-helix bundles as well as α-catenin α-helix domains unfold through stable 3-helix intermediates. While the 5-helix bundles were found to be mechanically stable, a second stable conformation corresponding to the 3-helix state was revealed. Mechanically weaker 4-helix bundles easily unfolded into a stable 3-helix conformation. The results of smAFM experiments were in agreement with the findings of the computational simulations. The disulfide clamp mutants, designed to protect the stable state, support the 3-helix intermediate model in both experimental and computational setups. As a result, multiple discrete unfolding intermediate states in the talin and α-catenin unfolding pathway were discovered. Better understanding of the mechanical unfolding mechanism of α-helix proteins is a key step towards comprehensive models describing the mechanoregulation of proteins.

Role of Extracellular Matrix in Development and Cancer Progression.

The immense diversity of extracellular matrix (ECM) proteins confers distinct biochemical and biophysical properties that influence cell phenotype. The ECM is highly dynamic as it is constantly deposited, remodelled, and degraded during development until maturity to maintain tissue homeostasis. The ECM's composition and organization are spatiotemporally regulated to control cell behaviour and differentiation, but dysregulation of ECM dynamics leads to the development of diseases such as cancer. The chemical cues presented by the ECM have been appreciated as key drivers for both development and cancer progression. However, the mechanical forces present due to the ECM have been largely ignored but recently recognized to play critical roles in disease progression and malignant cell behaviour. Here, we review the ways in which biophysical forces of the microenvironment influence biochemical regulation and cell phenotype during key stages of human development and cancer progression.

Talin: a mechanosensitive molecule in health and disease.

Talin is a ubiquitous, large focal adhesion protein that links intracellular networks with the extracellular matrix (ECM) via its connection with the actin cytoskeleton and membrane integrins. It is one of a handful molecules that can expose new recognition sites when undergoing force-induced mechanical unfolding, and it can bind and recruit cytoskeletal proteins that are involved in mechanotransduction. Talin has attracted great interest in the field of mechanobiology because of its plasticity in undergoing conformational changes under force stimulation as well as its cellular localization that bridges the cytoskeleton with the ECM. In addition to these roles in healthy cells, the dysregulation of talin activators can lead to disease states in which aberrant integrin activation and mechanotransduction precipitate changes in cell spreading, migration, and survival. New data have implicated a role for talin in diseases that are highly regulated by mechanical cues. In this review, we present the current understanding of talin structure, its relationship to binding partners, and its role in disease states.-Haining, A. W. M., Lieberthal, T. J., del Río Hernández, A. Talin: a mechanosensitive molecule in health and disease.

Mechanical and Functional Responses in Astrocytes under Alternating Deformation Modes Using Magneto-Active Substrates.

This work introduces NeoMag, a system designed to enhance cell mechanics assays in substrate deformation studies. NeoMag uses multidomain magneto-active materials to mechanically actuate the substrate, transmitting reversible mechanical cues to cells. The system boasts full flexibility in alternating loading substrate deformation modes, seamlessly adapting to both upright and inverted microscopes. The multidomain substrates facilitate mechanobiology assays on 2D and 3D cultures. The integration of the system with nanoindenters allows for precise evaluation of cellular mechanical properties under varying substrate deformation modes. The system is used to study the impact of substrate deformation on astrocytes, simulating mechanical conditions akin to traumatic brain injury and ischemic stroke. The results reveal local heterogeneous changes in astrocyte stiffness, influenced by the orientation of subcellular regions relative to substrate strain. These stiffness variations, exceeding 50% in stiffening and softening, and local deformations significantly alter calcium dynamics. Furthermore, sustained deformations induce actin network reorganization and activate Piezo1 channels, leading to an initial increase followed by a long-term inhibition of calcium events. Conversely, fast and dynamic deformations transiently activate Piezo1 channels and disrupt the actin network, causing long-term cell softening. These findings unveil mechanical and functional alterations in astrocytes during substrate deformation, illustrating the multiple opportunities this technology offers.

Retinoic acid receptor ß modulates mechanosensing and invasion in pancreatic cancer cells via myosin light chain 2.

Pancreatic ductal adenocarcinoma (PDAC) is the most common and lethal form of pancreatic cancer, characterised by stromal remodelling, elevated matrix stiffness and high metastatic rate. Retinoids, compounds derived from vitamin A, have a history of clinical use in cancer for their anti-proliferative and differentiation effects, and more recently have been explored as anti-stromal therapies in PDAC for their ability to induce mechanical quiescence in cancer associated fibroblasts. Here, we demonstrate that retinoic acid receptor ß (RAR-ß) transcriptionally represses myosin light chain 2 (MLC-2) expression in pancreatic cancer cells. As a key regulatory component of the contractile actomyosin machinery, MLC-2 downregulation results in decreased cytoskeletal stiffness and traction force generation, impaired response to mechanical stimuli via mechanosensing and reduced ability to invade through the basement membrane. This work highlights the potential of retinoids to target the mechanical drivers of pancreatic cancer.

Mesenchymal Stem Cells Sense the Toughness of Nanomaterials and Interfaces.

Stem cells are known to sense and respond to the mechanical properties of biomaterials. In turn, cells exert forces on their environment that can lead to striking changes in shape, size and contraction of associated tissues, and may result in mechanical disruption and functional failure. However, no study has so far correlated stem cell phenotype and biomaterials toughness. Indeed, disentangling toughness-mediated cell response from other mechanosensing processes has remained elusive as it is particularly challenging to uncouple Youngs' or shear moduli from toughness, within a range relevant to cell-generated forces. In this report, it is shown how the design of the macromolecular architecture of polymer nanosheets regulates interfacial toughness, independently of interfacial shear storage modulus, and how this controls the expansion of mesenchymal stem cells at liquid interfaces. The viscoelasticity and toughness of poly(l-lysine) nanosheets assembled at liquid-liquid interfaces is characterised via interfacial shear rheology. The local (microscale) mechanics of nanosheets are characterised via magnetic tweezer-assisted interfacial microrheology and the thickness of these assemblies is determined from in situ ellipsometry. Finally, the response of mesenchymal stem cells to adhesion and culture at corresponding interfaces is investigated via immunostaining and confocal microscopy.

Highly specific and non-invasive imaging of Piezo1-dependent activity across scales using GenEPi.

Mechanosensing is a ubiquitous process to translate external mechanical stimuli into biological responses. Piezo1 ion channels are directly gated by mechanical forces and play an essential role in cellular mechanotransduction. However, readouts of Piezo1 activity are mainly examined by invasive or indirect techniques, such as electrophysiological analyses and cytosolic calcium imaging. Here, we introduce GenEPi, a genetically-encoded fluorescent reporter for non-invasive optical monitoring of Piezo1-dependent activity. We demonstrate that GenEPi has high spatiotemporal resolution for Piezo1-dependent stimuli from the single-cell level to that of the entire organism. GenEPi reveals transient, local mechanical stimuli in the plasma membrane of single cells, resolves repetitive contraction-triggered stimulation of beating cardiomyocytes within microtissues, and allows for robust and reliable monitoring of Piezo1-dependent activity in vivo. GenEPi will enable non-invasive optical monitoring of Piezo1 activity in mechanochemical feedback loops during development, homeostatic regulation, and disease.

RGD density along with substrate stiffness regulate hPSC hepatocyte functionality through YAP signalling.

Human pluripotent stem cell-derived hepatocytes (hPSC-Heps) may be suitable for treating liver diseases, but differentiation protocols often fail to yield adult-like cells. We hypothesised that replicating healthy liver niche biochemical and biophysical cues would produce hepatocytes with desired metabolic functionality. Using 2D synthetic hydrogels which independently control mechanical properties and biochemical cues, we found that culturing hPSC-Heps on surfaces matching the stiffness of fibrotic liver tissue upregulated expression of genes for RGD-binding integrins, and increased expression of YAP/TAZ and their transcriptional targets. Alternatively, culture on soft, healthy liver-like substrates drove increases in cytochrome p450 activity and ureagenesis. Knockdown of ITGB1 or reducing RGD-motif-containing peptide concentration in stiff hydrogels reduced YAP activity and improved metabolic functionality; however, on soft substrates, reducing RGD concentration had the opposite effect. Furthermore, targeting YAP activity with verteporfin or forskolin increased cytochrome p450 activity, with forskolin dramatically enhancing urea synthesis. hPSC-Heps could also be successfully encapsulated within RGD peptide-containing hydrogels without negatively impacting hepatic functionality, and compared to 2D cultures, 3D cultured hPSC-Heps secreted significantly less fetal liver-associated alpha-fetoprotein, suggesting furthered differentiation. Our platform overcomes technical hurdles in replicating the liver niche, and allowed us to identify a role for YAP/TAZ-mediated mechanosensing in hPSC-Hep differentiation.

Eukaryotic initiation factor 6 regulates mechanical responses in endothelial cells.

The repertoire of extratranslational functions of components of the protein synthesis apparatus is expanding to include control of key cell signaling networks. However, very little is known about noncanonical functions of members of the protein synthesis machinery in regulating cellular mechanics. We demonstrate that the eukaryotic initiation factor 6 (eIF6) modulates cellular mechanobiology. eIF6-depleted endothelial cells, under basal conditions, exhibit unchanged nascent protein synthesis, polysome profiles, and cytoskeleton protein expression, with minimal effects on ribosomal biogenesis. In contrast, using traction force and atomic force microscopy, we show that loss of eIF6 leads to reduced stiffness and force generation accompanied by cytoskeletal and focal adhesion defects. Mechanistically, we show that eIF6 is required for the correct spatial mechanoactivation of ERK1/2 via stabilization of an eIF6-RACK1-ERK1/2-FAK mechanocomplex, which is necessary for force-induced remodeling. These results reveal an extratranslational function for eIF6 and a novel paradigm for how mechanotransduction, the cellular cytoskeleton, and protein translation constituents are linked.