Cell Mechanotransduction Research Articles

Extrafibrillarly Demineralized Dentin Matrix for Bone Regeneration (Adv. Healthcare Mater. 12/2023)

Extrafibrillar Demineralization In article 2202611 by Cui Huang, Hongye Yang, and co-workers, the extrafibrillar demineralization concept is introduced into the construction of tissue engineering scaffolds. The extrafibrillarly demineralized dentin facilitated mechanotransduction and osteogenic differentiation of stem cells, eventually brings about new ideas to develop hierarchical collagen-based biomaterials for bone regeneration.

Exposure to dietary fatty acids oleic and palmitic acid alters structure and mechanotransduction of intestinal cells in vitro

Intestinal cells are continuously exposed to food constituents while adapting to peristaltic movement and fluid shear stress. Oleic acid (OA) and palmitic acid (PA) are among the most prevalent fatty acids with respect to dietary lipids. Despite the central importance of dietary lipids for a balanced diet, awareness about potential detrimental effects related to excessive consumption is increasing; this includes toxicity, metabolic deregulation, and, particularly for cancer cells, a benefit from the uptake of fatty acids related to promotion of metastasis. Expanding on this, we started elucidating the effects of OA and PA (25–500 µM) on non-transformed human intestinal epithelial cells (HCEC-1CT) in comparison to colon carcinoma cells (HCT116), with regard to the mechanosensory apparatus. Hence, intestinal cells’ motility is on the one side essential to ensure adaption to peristaltic movement and barrier function, but also to enable metastatic progression. Incubation with both OA and PA (≥ 25 µM) significantly decreased membrane fluidity of HCT116 cells, whereas the effect on HCEC-1CT was more limited. Application of rhodamine-labelled PA demonstrated that the fatty acid is incorporated into the plasma membrane of HCT116, which could not be observed in the non-tumorigenic cell line. Down-streaming into the intracellular compartment, a pronounced rearrangement of actin cytoskeleton was evident in both cell lines (OA and PA; 25 and 100 µM). This was accompanied by a variation of translocation efficiency of the mechanosensitive co-transcription factor YAP1, albeit with a stronger effect seen for PA and the cancer cells. Untargeted proteomic analysis confirmed that exposure to OA and PA could alter the response capacity of HCT116 cells to fluid shear stress. Taken together, OA and PA were able to functionally modulate the mechanosensory apparatus of intestinal cells, implying a novel role for dietary fatty acids in the regulation of intestinal pathophysiology.

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining.

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level. Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

Stiff substrate induces nucleus pulposus cell ferroptosis via YAP and N-cadherin mediated mechanotransduction.

Increased tissues stiffness is associated with various pathological processes, such as fibrosis, inflammation, and aging. The matrix stiffness of the nucleus pulposus (NP) tissues increases gradually during intervertebral disc degeneration (IDD), while the mechanism through which NP cells sense and react to matrix stiffness remains unclear. In this study, our results indicated that ferroptosis was involved in stiff substrate-induced NP cell death. Further investigations indicated that the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4) increased in NP cells of the stiff group, which mediated lipid peroxidation and ferroptosis in NP cells. In addition, stiff substrate activated the Hippo signaling cascade and induced the nuclear translocation of yes-associated protein (YAP). Interestingly, inhibition of YAP was efficient to reverse the increase of ACSL4 expression caused by matrix stiffness. Furthermore, stiff substrate suppressed the expression of N-cadherin in NP cells. N-cadherin overexpression could inhibit YAP nuclear translocation via the formation of the N-cadherin/β-catenin/YAP complex, and reverse matrix stiffness-induced lipid peroxidation and ferroptosis in NP cells. Finally, the effects of YAP inhibition and N-cadherin overexpression on IDD progression was further illustrated in animal models. Our findings reveal a new mechanism of mechanotransduction in NP cells, providing novel insights into the development of therapies for the treatment of IDD. This article is protected by copyright. All rights reserved.

Bovine extracellular matrix proteins and potential role in meat quality: First in silico Bos taurus compendium

The extracellular matrix (ECM) is a three-dimensional set of proteins that binds tissues and defines their biomechanical properties. Among the ECM components known to be involved in beef sensory qualities, authors have often studied fibrillar collagens but also, although less often, proteoglycans and some glycoproteins. The ECM contains many other proteins. Our hypothesis is that to deepen the role of ECM proteins in beef qualities and to identify new ones among the vast amount of data generated by high throughput methods, it is necessary to have a list of proteins of this matrix to refer to for the bovine species. We have therefore defined the Bos taurus matrisome known as the set of genes encoding ECM (core matrisome proteins and matrisome-associated proteins). We have used orthology, as a reference method, and a bioinformatic approach based on a computational pipeline previously published for Homo sapiens, Mus musculus and Danio rerio for the definition of their respective matrisome. We have reported here that the Bos taurus matrisome is composed of 1022 genes that we have classified according to the different matrisome categories. This list is the only matrisome of a livestock species to be defined to date. SignificanceIn this study, we provide the first definition of matrisome of a livestock species, the Bos taurus. We believe that the Bos taurus matrisome will be of great interest for several reasons. It comes as a complement to the matrisomes of several other species such as Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster and Caenorabditis elegans previously defined by other authors. It could be used to identify matrisome molecules among the vast amount of data generated by the high throughput methods. It thus can be used in addition to the other matrisomes as a model by the scientific community to study cell behavior and mechanotransduction and could lead to the identification of novel biomarkers for several diseases and cancers in which ECM is involved. Moreover, in the domain of studies on livestock, the dataset that we have provided here can be used in the context of product quality studies, especially meat quality, but also, for example, for lactation studies.

Reduced Fibroblast Activation on Electrospun Polycaprolactone Scaffolds.

In vivo, quiescent fibroblasts reside in three-dimensional connective tissues and are activated in response to tissue injury before proliferating rapidly and becoming migratory and contractile myofibroblasts. When deregulated, chronic activation drives fibrotic disease. Fibroblasts cultured on stiff 2D surfaces display a partially activated phenotype, whilst many 3D environments limit fibroblast activation. Cell mechanotransduction, spreading, polarity, and integrin expression are controlled by material mechanical properties and micro-architecture. Between 3D culture systems, these features are highly variable, and the challenge of controlling individual properties without altering others has led to an inconsistent picture of fibroblast behaviour. Electrospinning offers greater control of mechanical properties and microarchitecture making it a valuable model to study fibroblast activation behaviour in vitro. Here, we present a comprehensive characterisation of the activation traits of human oral fibroblasts grown on a microfibrous scaffold composed of electrospun polycaprolactone. After over 7 days in the culture, we observed a reduction in proliferation rates compared to cells cultured in 2D, with low KI67 expression and no evidence of cellular senescence. A-SMA mRNA levels fell, and the expression of ECM protein-coding genes also decreased. Electrospun fibrous scaffolds, therefore, represent a tuneable platform to investigate the mechanisms of fibroblast activation and their roles in fibrotic disease.

Roles of focal adhesion proteins in skeleton and diseases.

The skeletal system, which contains bones, joints, tendons, ligaments and other elements, plays a wide variety of roles in body shaping, support and movement, protection of internal organs, production of blood cells and regulation of calcium and phosphate metabolism. The prevalence of skeletal diseases and disorders, such as osteoporosis and bone fracture, osteoarthritis, rheumatoid arthritis, and intervertebral disc degeneration, increases with age, causing pain and loss of mobility and creating a huge social and economic burden globally. Focal adhesions (FAs) are macromolecular assemblies that are composed of the extracellular matrix (ECM), integrins, intracellular cytoskeleton and other proteins, including kindlin, talin, vinculin, paxillin, pinch, Src, focal adhesion kinase (FAK) and integrin-linked protein kinase (ILK) and other proteins. FA acts as a mechanical linkage connecting the ECM and cytoskeleton and plays a key role in mediating cell-environment communications and modulates important processes, such as cell attachment, spreading, migration, differentiation and mechanotransduction, in different cells in skeletal system by impacting distinct outside-in and inside-out signaling pathways. This review aims to integrate the up-to-date knowledge of the roles of FA proteins in the health and disease of skeletal system and focuses on the specific molecular mechanisms and underlying therapeutic targets for skeletal diseases.

The mechanobiology of NK cells- 'Forcing NK to Sense' target cells.

Natural killer (NK) cells are innate immune lymphocytes that recognize and kill cancer and infected cells, which makes them unique 'off-the-shelf' candidates for a new generation of immunotherapies. Biomechanical forces in homeostasis and pathophysiology accrue additional immune regulation for NK immune responses. Indeed, cellular and tissue biomechanics impact NK receptor clustering, cytoskeleton remodeling, NK transmigration through endothelial cells, nuclear mechanics, and even NK-dendritic cell interaction, offering a plethora of unexplored yet important dynamic regulation for NK immunotherapy. Such events are made more complex by the heterogeneity of human NK cells. A significant question remains on whether and how biochemical and biomechanical cues collaborate for NK cell mechanotransduction, a process whereby mechanical force is sensed, transduced, and translated to downstream mechanical and biochemical signalling. Herein, we review recent advances in understanding how NK cells perceive and mechanotransduce biophysical cues. We focus on how the cellular cytoskeleton crosstalk regulates NK cell function while bearing in mind the heterogeneity of NK cells, the direct and indirect mechanical cues for NK anti-tumor activity, and finally, engineering advances that are of translational relevance to NK cell biology at the systems level.

Ultrasonic High-Resolution Imaging and Acoustic Tweezers Using Ultrahigh Frequency Transducer: Integrative Single-Cell Analysis.

Ultrasound imaging is a highly valuable tool in imaging human tissues due to its non-invasive and easily accessible nature. Despite advances in the field of ultrasound research, conventional transducers with frequencies lower than 20 MHz face limitations in resolution for cellular applications. To address this challenge, we employed ultrahigh frequency (UHF) transducers and demonstrated their potential applications in the field of biomedical engineering, specifically for cell imaging and acoustic tweezers. The lateral resolution achieved with a 110 MHz UHF transducer was 20 μm, and 6.5 μm with a 410 MHz transducer, which is capable of imaging single cells. The results of our experiments demonstrated the successful imaging of a single PC-3 cell and a 15 μm bead using an acoustic scanning microscope equipped with UHF transducers. Additionally, the dual-mode multifunctional UHF transducer was used to trap and manipulate single cells and beads, highlighting its potential for single-cell studies in areas such as cell deformability and mechanotransduction.

Visualization of mechanical forces in 3D cell models with molecular DNA tension probe.

Mechanical force can act as an important stimulus to regulate cellular processes. Accurate measurement of these forces plays a fundamental role in our understanding of cell mechanosensing and mechanotransduction. Current methods have focused on investigating the cell-matrix and intercellular forces on 2D monolayer cell models. In contrast, cells in real biological systems exist in multilayers and expose to forces from 3D directions. Exploring cellular mechanical forces in 3D cell models is thus critical to reveal their native interaction patterns in organs or tissues. We have recently developed a type of lipid-DNA-based molecular tension probes that have emerged as a potent tool for imaging intercellular mechanical forces. In this work, we have further investigated the potential usage of these molecular DNA tension probes for measuring mechanical forces in 3D spheroids and organoids. Using tumor spheroids and stem cell organoids of different sizes and shapes, we first characterized and optimized the probe penetration and cell membrane modification kinetics and efficiencies of the lipid-DNA conjugates. Afterwards, as a proof of concept, E-cadherin-mediated intercellular tensile forces were imaged and quantified in these 3D cell models using designer ratiometric DNA tension probes. Such membrane tensile force measurement was enabled because these DNA hairpin-based probes can convert cellular mechanical forces into digital fluorescence signals. These optimized DNA tension probes can also be used to investigate the dynamic changes and spatial distributions of different cellular mechanotransduction processes. We believe this is an important study that can potentially fill the gap between current studies of 2D cell mechanics and that in real 3D biological models.

Cholesterol as a tool to probe the role of membrane in cochlear hair cell mechanotransduction.

Most mechanically gated channels are sensitive to force translated through the membrane. The lipid bilayer can modulate channel function directly through lipid/protein interactions or indirectly based on membrane mechanical properties. Cholesterol is an important component of the membrane and a major modulator of membrane mechanical properties. While other ion channels and mechanically gated channels can be activated or modulated by changes to the membrane cholesterol, the functional role of membrane cholesterol in regulation of mammalian cochlear mechanotransduction (MET) channels has not been closely investigated. Using whole-cell patch clamping and live-cell fluorescence lifetime imaging (FLIM) of a viscosity-sensitive molecular rotor BODIPY 1c for the first time in the inner ear, we examined the role of membrane cholesterol in modulating the MET response properties of rat cochlear hair cells. Molecular rotors are fluorophores for which the fluorescence lifetime (the average time a fluorophore remains in the excited state) increase with increasing viscosity of their immediate environment. We confirmed extraction of cholesterol, using methyl β cyclodextrin (MβCD), with reduced filipin staining in both inner and outer hair bundles. MβCD results in reversible reduction in fluorescence lifetime in hair bundles, suggesting initial reduction followed by gradual recovery in the stereocilia membrane viscosity. MβCD reversibly increases the channel resting open probability, suggesting that cholesterol depletion increases force transfer to the MET channel. Together this data suggests that the cell membrane is part of the force relay machinery to the MET channel and could possibly interact directly with components of the MET machinery. Further studies are needed to generate causal link between MET channel gating and membrane mechanics.

Mechanotransduction for Muscle Protein Synthesis via Mechanically Activated Ion Channels

Cell mechanotransduction, the ability to detect physical forces and convert them into a series of biochemical events, is important for a wide range of physiological processes. Cells express an array of mechanosensors transducing physical forces into intracellular signaling cascades, including ion channels. Ion channels that can be directly activated by mechanical cues are known as mechanically activated (MA), or stretch-activated (SA), channels. In response to repeated exposures to mechanical stimulation in the form of resistance training, enhanced protein synthesis and fiber hypertrophy are elicited in skeletal muscle, whereas a lack of mechanical stimuli due to inactivity/mechanical unloading leads to reduced muscle protein synthesis and fiber atrophy. To date, the role of MA channels in the transduction of mechanical load to intracellular signaling pathways regulating muscle protein synthesis is poorly described. This review article will discuss MA channels in striated muscle, their regulation, and putative roles in the anabolic processes in muscle cells/fibers in response to mechanical stimuli.

Dissecting Physical and Biochemical Effects in Nanotopographical Regulation of Cell Behavior.

Regulating cell behavior using nanotopography has been widely implemented. To facilitate cell adhesion, physical nanotopography is usually coated with adhesive proteins such as fibronectin (FN). However, the confounding effects of physical and biochemical cues of nanotopography hinder the understanding of nanotopography in regulating cell behavior, which ultimately limits the biomedical applications of nanotopography. To delineate the roles of the physical and biochemical cues in cell regulation, we fabricate substrates that have either the same physical nanotopography but different biochemical (FN) nanopatterns or identical FN nanopatterns but different physical nanotopographies. We then examine the influences of physical and biochemical cues of nanotopography on spreading, nuclear deformation, mechanotransduction, and function of human mesenchymal stem cells (hMSCs). Our results reveal that physical topographies, especially nanogratings, dominantly control cell spreading, YAP localization, proliferation, and differentiation of hMSCs. However, biochemical FN nanopatterns affect hMSC elongation, YAP intracellular localization, and lamin a/c (LAMAC) expression. Furthermore, we find that physical nanogratings induce nanoscale curvature of nuclei at the basal side, which attenuates the osteogenic differentiation of hMSCs. Collectively, our study highlights the dominant effect of physical nanotopography in regulating stem cell functions, while suggesting that fine-tuning of cell behavior can be achieved through altering the presentation of biochemical cues on substrate surfaces.

Cover Picture

The cover picture depicts the stretchable electrochemical sensor for in‐situ inducing and real‐time monitoring of cell mechanotransduction. The stretchable sensor is mainly fabricated by different nanomaterials including nanoparticles, nanosheets and nanowires/nanotubes, which form continuous pathways for electron transport on elastic substrate. To further achieve the tracking of transient biochemical molecules released during cell mechanotransduction, the stretchable sensing interface is equipped with excellent sensitivity, selectivity and stability by combining functional materials such as natural enzyme, nanocatalyst and biomimetic molecule. More details are given in the review by Liu et al. on page 443—457.image

Meet the First Authors.

Meet the First Authors.

Vascular mechanotransduction.

This review aims to survey the current state of mechanotransduction in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), including their sensing of mechanical stimuli and transduction of mechanical signals that result in the acute functional modulation and longer-term transcriptomic and epigenetic regulation of blood vessels. The mechanosensors discussed include ion channels, plasma membrane-associated structures and receptors, and junction proteins. The mechanosignaling pathways presented include the cytoskeleton, integrins, extracellular matrix, and intracellular signaling molecules. These are followed by discussions on mechanical regulation of transcriptome and epigenetics, relevance of mechanotransduction to health and disease, and interactions between VSMCs and ECs. Throughout this review, we offer suggestions for specific topics that require further understanding. In the closing section on conclusions and perspectives, we summarize what is known and point out the need to treat the vasculature as a system, including not only VSMCs and ECs but also the extracellular matrix and other types of cells such as resident macrophages and pericytes, so that we can fully understand the physiology and pathophysiology of the blood vessel as a whole, thus enhancing the comprehension, diagnosis, treatment, and prevention of vascular diseases.

Microtubule Assists Actomyosin to Regulate Cell Nuclear Mechanics and Chromatin Accessibility.

Cellular behaviors and functions can be regulated by mechanical cues from microenvironments, which are transmitted to nucleus through the physical connections of cytoskeletons in the cells. How these physical connections determine transcriptional activity were not clearly known. The actomyosin, which generates intracellular traction force, has been recognized to control the nuclear morphology. Here, we have revealed that microtubule, the stiffest cytoskeleton, is also involved in the process of nuclear morphology alteration. The microtubule negatively regulates the actomyosin-induced nuclear invaginations but not the nuclear wrinkles. Moreover, these nuclear shape changes are proven to mediate the chromatin remodeling, which essentially mediates cell gene expression and phenotype determination. The actomyosin disruption leads to the loss of chromatin accessibility, which can be partly recovered by microtubule interference through nuclear shape control. This finding answers the question of how mechanical cues regulate chromatin accessibility and cell behaviors. It also provides new insights into cell mechanotransduction and nuclear mechanics.

Mechanotransduction of mesenchymal stem cells and hemodynamic implications.

Mesenchymal stem cells (MSCs) possess the capacity for self-renewal and multipotency. The traditional approach to manipulating MSC's fate choice predominantly relies on biochemical stimulation. Accumulating evidence also suggests the role of physical input in MSCs differentiation. Therefore, investigating mechanotransduction at the molecular level and related to tissue-specific cell functions sheds light on the responses secondary to mechanical forces. In this review, a new frontier aiming to optimize the cultural parameters was illustrated, i.e. spatial boundary condition, which recapitulates in vivo physiology and facilitates the investigations of cellular behavior. The concept of mechanical memory was additionally addressed to appreciate how MSCs store imprints from previous culture niches. Besides, different types of forces as physical stimuli were of interest based on the association with the respective signaling pathways and the differentiation outcome. The downstream mechanoreceptors and their corresponding effects were further pinpointed. The cardiovascular system or immune system may share similar mechanisms of mechanosensing and mechanotransduction; for example, resident stem cells in a vascular wall and recruited MSCs in the bloodstream experience mechanical forces such as stretch and fluid shear stress. In addition, baroreceptors or mechanosensors of endothelial cells detect changes in blood flow, pass over signals induced by mechanical stimuli and eventually maintain arterial pressure at the physiological level. These mechanosensitive receptors transduce pressure variation and regulate endothelial barrier functions. The exact signal transduction is considered context dependent but still elusive. In this review, we summarized the current evidence of how mechanical stimuli impact MSCs commitment and the underlying mechanisms. Future perspectives are anticipated to focus on the application of cardiovascular bioengineering and regenerative medicine.

Mechanobiology in the development and progression of non-alcoholic fatty liver disease: an updated review

Mechanobiology is a rapidly emerging field focused on the biological impact of physical forces at the molecular, cellular, and tissue level. Living cells perceive mechanical cues and transform them into biochemical signals through mechanotransduction. Mechanotransduction is a complex process that involves mechanosensors (which are located in the plasma membrane or within the cell) and mechanotransmission to the nucleus (which occurs either by physical connection between the mechanosensor and the nucleus or by mechanosignaling through biochemical pathways). Essential biological functions, including development, growth, motility, and metabolism, depend on the mechanoresponses generated by these events. Multiple lines of evidence indicate that disruption of mechanical homeostasis may contribute to the pathogenesis of non-alcoholic fatty liver disease (NAFLD), a highly prevalent metabolic disorder characterized by abnormal accumulation of lipid droplets in hepatocytes (steatosis) and often associated with inflammation and liver cell injury (steatohepatitis). While predicting individual predisposition to adverse outcomes in NAFLD remains a challenge, there is increasing evidence that steatosis and steatohepatitis trigger mechanoresponses that contribute to the early stages of pathogenesis in NAFLD and critically impact disease progression. Lipid accumulation and lipotoxicity modify liver viscoelasticity, alter the biomechanics of liver sinusoids, and initiate aberrant pathways of mechanotransduction in hepatocytes and non-parenchymal liver cells, such as sinusoidal endothelial cells and hepatic stellate cells. Interactions of these cells at mechanical interfaces with each other, with extracellular matrix, and with sinusoidal blood flow are profoundly altered by steatosis and steatohepatitis; such changes may promote a pro-angiogenic and pro-fibrotic milieu. A better understanding of liver mechanobiology may facilitate the identification of novel molecular and cellular targets in the management of NAFLD.

Photoresponsive Hydrogels for Studying Mechanotransduction of Cells.

Hydrogels are important platform materials for in vitro cellular studies. Mechanistic studies on durotaxis, the directional movement of a cell affected by a spatial gradient of stiffness of the underlying substrate, requires materials such as polyacrylamide, polyethylene glycol, or PDMS, in which the stiffness can be controlled in a spatiotemporal manner. Here, we describe the synthesis of an o-nitrobenzyl-based photocleavable cross-linker and its incorporation into a polyacrylamide hydrogel to render it photoresponsive. Precise control over the physical properties of the gel allows observation of glioblastoma durotaxis under surface stiffness conditions relevant to the actual brain environment.