Cellular Mechanosensing Research Articles

Nuclease‐Resistant L‐DNA Tension Probes Enable Long‐Term Force Mapping of Single Cells and Cell Consortia

AbstractDNA‐based tension probes with precisely programmable force responses provide important insights into cellular mechanosensing. However, their degradability in cell culture limits their use for long‐term imaging, for instance, when cells migrate, divide, and differentiate. This is a critical limitation for providing insights into mechanobiology for these longer‐term processes. Here, we present DNA‐based tension probes that are entirely designed based on the stereoisomer of biological D‐DNA, i.e., L‐DNA. We demonstrate that L‐DNA tension probes are essentially indestructible by nucleases and provide days‐long imaging without significant loss in image quality. We also show their superiority already for short imaging times commonly used for classical D‐DNA tension probes. We showcase the potential of these resilient probes to image minute movements, and for generating long term force maps of single cells and of collectively migrating cell populations.

Cellular Nanointerface of Vertical Nanostructures: Impact of Size‐Modulated Nanopillar Arrays on Neuronal Morphology, Maturation, and Synapse Formation

Investigating the cellular mechanisms facilitated by highly ordered nanostructures in vitro neuronal networks is crucial for advancing biomedical research. However, the intricate effects of these nanostructures on cellular behavior remain unclear. Herein, we explore how variations in nanopillar (NP) array dimensions, defined by the spacing‐to‐diameter (S/D) ratio, influence cortical neuronal responses—including cellular morphology, mechanosensing, neuronal maturation, and synapse formation. NP arrays with a low S/D ratio accelerate neurite protrusion and enhance neuronal maturation. These topography‐driven changes are attributed to the degree of cell confinement on NPs, which can be visualized using focused ion beam scanning microscopy. Furthermore, the synaptic density in cortical neurons declines as the S/D ratio decreases, with neurons preferring to form synapses along the NPs. By utilizing correlative light and electron microscopy, the spatial organization of these synapses is mapped relative to the NP geometries, revealing specialized synaptic regions with remarkable clarity. This approach to designing high‐aspect‐ratio nanostructures with various S/D ratios holds broad applications in materials engineering, offering insights into nervous system engineering and neural‐interfacing bioelectronics.

Extracellular Matrix Components and Mechanosensing Pathways in Health and Disease.

Glycosaminoglycans (GAGs) and proteoglycans (PGs) are essential components of the extracellular matrix (ECM) with pivotal roles in cellular mechanosensing pathways. GAGs, such as heparan sulfate (HS) and chondroitin sulfate (CS), interact with various cell surface receptors, including integrins and receptor tyrosine kinases, to modulate cellular responses to mechanical stimuli. PGs, comprising a core protein with covalently attached GAG chains, serve as dynamic regulators of tissue mechanics and cell behavior, thereby playing a crucial role in maintaining tissue homeostasis. Dysregulation of GAG/PG-mediated mechanosensing pathways is implicated in numerous pathological conditions, including cancer and inflammation. Understanding the intricate mechanisms by which GAGs and PGs modulate cellular responses to mechanical forces holds promise for developing novel therapeutic strategies targeting mechanotransduction pathways in disease. This comprehensive overview underscores the importance of GAGs and PGs as key mediators of mechanosensing in maintaining tissue homeostasis and their potential as therapeutic targets for mitigating mechano-driven pathologies, focusing on cancer and inflammation.

Microscale geometrical modulation of PIEZO1 mediated mechanosensing through cytoskeletal redistribution

The microgeometry of the cellular microenvironment profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains unclear. Herein, we describe a fluorescent micropipette aspiration assay that allows for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time, under varied micropipette geometries. By integrating elastic shell finite element analysis with fluorescent lifetime imaging microscopy and employing PIEZO1-specific transgenic red blood cells and HEK cell lines, we demonstrate a direct correlation between the microscale geometry of aspiration and PIEZO1-mediated calcium signaling. We reveal that increased micropipette tip angles and physical constrictions lead to a significant reorganization of F-actin, accumulation at the aspirated cell neck, and subsequently amplify the tension stress at the dome of the cell to induce more PIEZO1’s activity. Disruption of the F-actin network or inhibition of its mobility leads to a notable decline in PIEZO1 mediated calcium influx, underscoring its critical role in cellular mechanosensing amidst geometrical constraints.

Shp2 contributes to the regulation of nuclear shape and cellular viscoelasticity in response to substrate spatial cues

Cell polarization can be guided by substrate topology through space constraints and adhesion induction, which are part of cellular mechanosensing pathways. Here, we demonstrated that protein tyrosine phosphatase Shp2 plays a crucial role in mediating the response of cells to substrate spatial cues. When compared to cells spreading on surfaces coated uniformly with fibronectin (FN), cells attached to 10 μm-width FN-strip micropattern (MP), which provides spatial cues for uniaxial spreading, exhibited elongated focal adhesions (FAs) and aligned stress fibers in the direction of the MP. As a result of uniaxial cell spreading, nuclei became elongated, dependent on ROCK-mediated actomyosin contractility. Additionally, intracellular viscoelasticity also increased. Shp2-deficient cells did not display elongated FAs mediated by MP, well-aligned stress fibers, or changes in nuclear shape and intracellular viscoelasticity. Overall, our data suggest that Shp2 is involved in regulating FAs and the actin cytoskeleton to modulate nuclear shape and intracellular physical properties in response to substrate spatial cues.

Synergistic Influence of Fibrous Pattern Orientation and Modulus on Cellular Mechanoresponse.

The fibrous extracellular matrix (ECM) is vital for tissue regeneration and impacts implanted device treatments. Previous research on fibrous biomaterials shows varying cellular reactions to surface orientation, often due to unclear interactions between surface topography and substrate elasticity. Our study addresses this gap by achieving the rapid creation of hydrogels with diverse fibrous topographies and varying substrate moduli through a surface printing strategy. Cells exhibit heightened traction force on nanopatterned soft hydrogels, particularly with randomly distributed patterns compared with regular soft hydrogels. Meanwhile, on stiff hydrogels featuring an aligned topography, optimal cellular mechanosensing is observed compared to random topography. Mechanistic investigations highlight that cellular force-sensing and adhesion are influenced by the interplay of pattern deformability and focal adhesion orientation, subsequently mediating stem cell differentiation. Our findings highlight the importance of combining substrate modulus and topography to guide cellular behavior in designing advanced tissue engineering biomaterials.

Tumor Biomechanics Alters Metastatic Dissemination of Triple Negative Breast Cancer via Rewiring Fatty Acid Metabolism.

In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin β1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages.

A TICT-AIE activated dual-channel fluorescence-on probe to reveal the dynamics mechanosensing of lipid droplets during ferroptosis

Mechanical forces play a crucial role in cellular processes, including ferroptosis, a form of regulated cell death associated with various diseases. However, the mechanical aspects of organelle lipid droplets (LDs) during ferroptosis are poorly understood. In this study, we designed and synthesized a fluorescent probe, TPE-V1, to enable real-time monitoring of LDs' viscosity using a dual-channel fluorescence-on model (red channel at 617 nm and NIR channel at 710 nm). The fluorescent imaging of using TPE-V1 was achieved due to the integrated mechanisms of the twisted intramolecular charge transfer (TICT) and aggregation-induced emission (AIE). Through dual-emission channel fluorescence imaging, we observed the enhanced mechanical energy of LDs triggering cellular mechanosensing, including ferroptosis and cell deformation. Theoretical calculations confirmed the probe's behavior, showing that high-viscosity media prevented the rotation processes and restored fluorescence quenching in low viscosity. These findings suggest that our TICT-TPE design strategy provides a practical approach to study LDs' mechanical properties during ferroptosis. This development enhances our understanding of the interplay between mechanical forces and LDs, contributing to the knowledge of ferroptotic cell death and potential therapeutic interventions targeting dysregulated cell death processes.

CHRFAM7A diversifies human immune adaption through Ca2+ signalling and actin cytoskeleton reorganization

Human restricted genes contribute to human specific traits in the immune system. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR), the highest Ca2+ conductor of the ACh receptors implicated in innate immunity. Understanding the mechanism of how CHRFAM7A affects the immune system remains unexplored. Two model systems are used, human induced pluripotent stem cells (iPSC) and human primary monocytes, to characterize α7 nAChR function, Ca2+ dynamics and decoders to elucidate the pathway from receptor to phenotype. CHRFAM7A/α7 nAChR is identified as a hypomorphic receptor with mitigated Ca2+ influx and prolonged channel closed state. This shifts the Ca2+ reservoir from the extracellular space to the endoplasmic reticulum (ER) leading to Ca2+ dynamic changes. Ca2+ decoder small GTPase Rac1 is then activated, reorganizing the actin cytoskeleton. Observed actin mediated phenotypes include cellular adhesion, motility, phagocytosis and tissue mechanosensation. CHRFAM7A introduces an additional, human specific, layer to Ca2+ regulation leading to an innate immune gain of function. Through the actin cytoskeleton it drives adaptation to the mechanical properties of the tissue environment leading to an ability to invade previously immune restricted niches. Human genetic diversity predicts profound translational significance as its understanding builds the foundation for successful treatments for infectious diseases, sepsis, and cancer metastasis. This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti) and in part by NIH grant R01HL163168 (Yongho Bae).

Diffusion mediated rigidity-gradient hydrogel substrate for cellular mechanosensing

Diffusion mediated rigidity-gradient hydrogel substrate for cellular mechanosensing

Ionic Liquid Interface as a Cell Scaffold.

In sharp contrast to conventional solid/hydrogel platforms, water-immiscible liquids, such as perfluorocarbons and silicones, allow the adhesion of mammalian cells via protein nanolayers (PNLs) formed at the interface. However, fluorocarbons and silicones, which are typically used for liquid cell culture, possess only narrow ranges of physicochemical parameters and have not allowed for a wide variety of cell culturing environments. In this paper, it is proposed that water-immiscible ionic liquids (ILs) are a new family of liquid substrates with tunable physicochemical properties and high solvation capabilities. Tetraalkylphosphonium-based ILs are identified as non-cytotoxic ILs, whereon human mesenchymal stem cells are successfully cultured. By reducing the cation charge distribution, or ionicity, via alkyl chain elongation, the interface allows cell spreading with matured focal contacts. High-speed atomic force microscopy observations of the PNL formation process suggest that the cation charge distribution significantly altered the protein adsorption dynamics, which are associated with the degree of protein denaturation and the PNL mechanics. Moreover, by exploiting dissolution capability of ILs, an ion-gel cell scaffold is fabricated. This enables to further identify the significant contribution of bulk subphase mechanics to cellular mechanosensing in liquid-based culture scaffolds.

Flow induces common and specific transcriptional changes in renal tubular epithelial cells involving the PI3K pathway.

Flow-induced shear stress affects renal epithelial cells in the nephron tubule with potential implications for differential functionalities of the individual segments. Disruptions of cellular mechanosensation or flow conditions are associated with the development and progression of various renal diseases. This study investigates the effects of flow on the transcriptome of various renal tubular epithelial cell types. We analyzed the transcriptome of induced renal epithelial cells (iREC) cultured under physiological flow (0.57 ± 0.05 dyn/cm2 ) or in static conditions for 72 h. RNA sequencing showed 861 differentially expressed genes (DEGs), with 503 up- and 358 downregulated under flow. DEGs were linked to extracellular matrix (ECM) components (e.g. Col1a1, Col4a3, Col4a4, Fn1, Smoc2), junctions (Gja1, Tubb5), channel activities (Abcc4, Aqp1), and transcription factors (Foxq1, Lgr6). Next, we performed a meta-analysis comparing our data with three published datasets that subjected epithelial cell lines from distinct segments to flow, including proximal tubule and collecting duct cells. We found that TGF-ß, p53, MAPK, and PI3K are common flow-regulated pathways. Tfrc expression and thus the capability of iron uptake is commonly upregulated under flow. Many DEGs were related to kidney diseases, such as fibrosis (e.g. Tgfb1-3 and Serpine1). To obtain further mechanistic insights we investigated the role of the PI3K pathway in flow sensing. Applying flow and inhibition of PI3K showed significantly altered expression of transcripts related to ECM remodeling, angiogenesis, and ion transport. This suggests that the PI3K pathway is a critical mediator in flow-dependent cellular processes and gene expression, potentially influencing renal development and tissue remodeling. Finally, we derived a cross-cell-line summary of common as well as segment-specific transcriptomic effects, thus providing insights into the molecular mechanisms underlying flow sensing in the nephron tubule.

Membrane-bound myosin IC drives the chiral rotation of the gliding actin filament around its longitudinal axis

Myosin IC, a single-headed member of the myosin I family, specifically interacts with anionic phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) in the cell membrane via the pleckstrin homology domain located in the myosin IC tail. Myosin IC is widely expressed and physically links the cell membrane to the actin cytoskeleton; it plays various roles in membrane-associated physiological processes, including establishing cellular chirality, lipid transportation, and mechanosensing. In this study, we evaluated the motility of full-length myosin IC of Drosophila melanogaster via the three-dimensional tracking of quantum dots bound to actin filaments that glided over a membrane-bound myosin IC-coated surface. The results revealed that myosin IC drove a left-handed rotational motion in the gliding actin filament around its longitudinal axis, indicating that myosin IC generated a torque perpendicular to the gliding direction of the actin filament. The quantification of the rotational motion of actin filaments on fluid membranes containing different PI(4,5)P2 concentrations revealed that the rotational pitch was longer at lower PI(4,5)P2 concentrations. These results suggest that the torque generated by membrane-bound myosin IC molecules can be modulated based on the phospholipid composition of the cell membrane.

Mechanobiological Strategies to Augment Cancer Treatment.

Cancer cells exhibit aberrant extracellular matrix mechanosensing due to the altered expression of mechanosensory cytoskeletal proteins. Such aberrant mechanosensing of the tumor microenvironment (TME) by cancer cells is associated with disease development and progression. In addition, recent studies show that such mechanosensing changes the mechanobiological properties of cells, and in turn cells become susceptible to mechanical perturbations. Due to an increasing understanding of cell biomechanics and cellular machinery, several approaches have emerged to target the mechanobiological properties of cancer cells and cancer-associated cells to inhibit cancer growth and progression. In this Perspective, we summarize the progress in developing mechano-based approaches to target cancer by interfering with the cellular mechanosensing machinery and overall TME.

Examination of the mechanism of Piezo ion channel in 5-HT synthesis in the enterochromaffin cell and its association with gut motility.

In the gastrointestinal tract, serotonin (5-hydroxytryptamine, 5-HT) is an important monoamine that regulates intestinal dynamics. QGP-1 cells are human-derived enterochromaffin cells that secrete 5-HT and functionally express Piezo ion channels associated with cellular mechanosensation. Piezo ion channels can be blocked by Grammostola spatulata mechanotoxin 4 (GsMTx4), a spider venom peptide that inhibits cationic mechanosensitive channels. The primary aim of this study was to explore the effects of GsMTx4 on 5-HT secretion in QGP-1 cells in vitro. We investigated the transcript and protein levels of the Piezo1/2 ion channel, tryptophan hydroxylase 1 (TPH1), and mitogen-activated protein kinase signaling pathways. In addition, we observed that GsMTx4 affected mouse intestinal motility in vivo. Furthermore, GsMTx4 blocked the response of QGP-1 cells to ultrasound, a mechanical stimulus.The prolonged presence of GsMTx4 increased the 5-HT levels in the QGP-1 cell culture system, whereas Piezo1/2 expression decreased, and TPH1 expression increased. This effect was accompanied by the increased phosphorylation of the p38 protein. GsMTx4 increased the entire intestinal passage time of carmine without altering intestinal inflammation. Taken together, inhibition of Piezo1/2 can mediate an increase in 5-HT, which is associated with TPH1, a key enzyme for 5-HT synthesis. It is also accompanied by the activation of the p38 signaling pathway. Inhibitors of Piezo1/2 can modulate 5-HT secretion and influence intestinal motility.

Programmable and Reversible Integrin-Mediated Cell Adhesion Reveals Hysteresis in Actin Kinetics that Alters Subsequent Mechanotransduction.

Dynamically evolving adhesions between cells and extracellular matrix (ECM) transmit time-varying signals that control cytoskeletal dynamics and cell fate. Dynamic cell adhesion and ECM stiffness regulate cellular mechanosensing cooperatively, but it has not previously been possible to characterize their individual effects because of challenges with controlling these factors independently. Therefore, a DNA-driven molecular system is developed wherein the integrin-binding ligand RGD can be reversibly presented and removed to achieve cyclic cell attachment/detachment on substrates of defined stiffness. Using this culture system, it is discovered that cyclic adhesion accelerates F-actin kinetics and nuclear mechanosensing in human mesenchymal stem cells (hMSCs), with the result that hysteresis can completely change how hMSCs transduce ECM stiffness. Results are dramatically different from well-known results for mechanotransduction on static substrates, but are consistent with a mathematical model of F-actin fragments retaining structure following loss of integrin ligation and participating in subsequent repolymerization. These findings suggest that cyclic integrin-mediated adhesion alters the mechanosensing of ECM stiffness by hMSCs through transient, hysteretic memory that is stored in F-actin.

Joining forces: crosstalk between mechanosensitive PIEZO1 ion channels and integrin-mediated focal adhesions.

Both integrin-mediated focal adhesions (FAs) and mechanosensitive ion channels such as PIEZO1 are critical in mechanotransduction processes that influence cell differentiation, development, and cancer. Ample evidence now exists for regulatory crosstalk between FAs and PIEZO1 channels with the molecular mechanisms underlying this process remaining unclear. However, an emerging picture is developing based on spatial crosstalk between FAs and PIEZO1 revealing a synergistic model involving the cytoskeleton, extracellular matrix (ECM) and calcium-dependent signaling. Already cell type, cell contractility, integrin subtypes and ECM composition have been shown to regulate this crosstalk, implying a highly fine-tuned relationship between these two major mechanosensing systems. In this review, we summarize the latest advances in this area, highlight the physiological implications of this crosstalk and identify gaps in our knowledge that will improve our understanding of cellular mechanosensing.

Enhancement effects of extracellular fluid viscosity and matrix stiffness on cancer cell mechanosensing

Cancer cells are in vivo situated in a complex and heterogeneous tumor microenvironment (TME) that includes various biochemical and biophysical cues, such as the elasticity of extracellular matrix (ECMwo) and the viscosity of extracellular fluid (ECF). ECF viscosity in TME is much higher than in normal tissue, but it is unclear how this increased viscosity works simultaneously with other biophysical cues (e.g., ECM stiffness) to influence cancer cell behavior. We experimentally observed that ECF viscosity can significantly enhance cellular mechanosensing behaviors (e.g., cell spreading, cell adhesion, and YAP/TAZ nuclear translocation) only on a stiff substrate, representing a novel enhancement of cell behaviors by distinct mechanical signals. To explore the mechanical mechanisms behind such enhancement phenomenon, we developed a viscosity-based motor-clutch model, with which we found that cells sense and respond to ECF viscosity and ECM stiffness by regulating integrin-ECM bonds in cell adhesion dynamics. These findings help us understand how different mechanical signals in the complex tumor microenvironment collaborate to influence cancer cell behaviors during the development of cancers.

Vaginal Fibroblast Behavior as a Function of Stiffness Changes in a Polyisocyanide Hydrogel for Prolapse Repair.

There is an urgent need for improved outcomes in the treatment of pelvic organ prolapse (POP). Success of primary surgery relies on the load bearing capacity of plicated connective tissue underneath the vaginal wall, which is compromised due to an altered vaginal fibroblast function and collagen composition. There is an important factor in connective tissue repair that relates to changes in stiffness of the vaginal fibroblast microenvironment, which influences cell activity through cellular mechanosensing. The aim of this study is to investigate the effect of stiffness changes on vaginal fibroblast functions that relate to connective tissue healing in prolapse repair. The substrate stiffness was controlled by changing the polymer concentration in the fibrous and strongly biomimetic polyisocyanide (PIC) hydrogel. We analyzed stiffness during cell culture and assessed the consequential fibroblast proliferation, morphology, collagen deposition, and contraction. Our results show that increasing stiffness coincides with vaginal fibroblast alignment, promotes collagen deposition, and inhibits PIC gel contraction. These findings suggest that the matrix stiffness directly influences vaginal fibroblast functionality. Moreover, we observed a buildup in stiffness and collagen, with an enhanced fibroblast and collagen organization on the PIC-substrate, which indicate an enhanced structural integrity of the hydrogel-cell construct. An improved tissue structure during healing is relevant in the functional repair of POP. Therefore, this study encourages future research in the use of PIC gels as a supplement in prolapse surgery, whereby the hydrogel stiffness should be considered.

Importance of mechanical cues in regulating musculoskeletal circadian clock rhythmicity: Implications for articular cartilage.

The circadian clock, a collection of endogenous cellular oscillators with an approximate 24-h cycle, involves autoregulatory transcriptional/translational feedback loops to enable synchronization within the body. Circadian rhythmicity is controlled by a master clock situated in the hypothalamus; however, peripheral tissues are also under the control of autonomous clocks which are coordinated by the master clock to regulate physiological processes. Although light is the primary signal required to entrain the body to the external day, non-photic zeitgeber including exercise also entrains circadian rhythmicity. Cellular mechano-sensing is imperative for functionality of physiological systems including musculoskeletal tissues. Over the last decade, mechano-regulation of circadian rhythmicity in skeletal muscle, intervertebral disc, and bone has been demonstrated to impact tissue homeostasis. In contrast, few publications exist characterizing the influence of mechanical loading on the circadian rhythm in articular cartilage, a musculoskeletal tissue in which loading is imperative for function; importantly, a dysregulated cartilage clock contributes to development of osteoarthritis. Hence, this review summarizes the literature on mechano-regulation of circadian clocks in musculoskeletal tissues and infers on their collective importance in understanding the circadian clock and its synchronicity for articular cartilage mechanobiology.