Mechanotransduction Mechanisms Research Articles

Structural Analysis of Piezo1 Ion Channel Reveals the Relationship between Amino Acid Sequence Mutations and Human Diseases

Since first identified in 2010, Piezo proteins have been found to perform as poreforming mechanosensitive ion channels across a wide range of animals. As a Piezo ortholog primarily expressed in mammalian systems, Piezo1 has been observed to distribute mainly in nonsensory tissues, regulating osmotic homeostasis, proprioception, and light touch. With previous studies on the putative structure of Piezo1, the gating system and several mechanotransduction mechanisms have been proposed. Besides, mutations of specific amino acid sequences in Piezo1 have been linked to several human diseases such as dehydrated hereditary xerocytosis (DHS) and congenital lymphatic dysplasia (CLD). However, most of these mutations have not been well characterized. To further elucidate the relations between these mutations and diseases, UCSF Chimera is used as the tool to visualize the structural importance of each of these mutated amino acids. With the aid from UCSF Chimera, this study has recorded and interpreted clashes and contacts originated from each of the mutations. Accordingly, specific mechanisms between mutations and human diseases are proposed, which pave the way for healing.

Mechanical and Topographical Guidance of Stem Cell Differentiation <i>in Vitro</i> Via YAP-Dependent Mechanotransduction: A Biomimetic Approach Toward Annulus Fibrosus Regeneration

The current engineered bionic disc tissue is considered as a potential way for treating intervertebral disc degeneration disease, which yet still remains challenging due to the complex radial gradient of natural annulus fibrosus (AF) tissue in cell phenotype, biochemical composition, topographical features, and mechanical properties. Previously, we have found that the differentiation of annulus fibrosus-derived stem cells (AFSCs) could be regulated by the elasticity of scaffold. In this study, we attempted to examine the combined effect of both mechanical and topographical features on the gene expression of AFSCs. Cells were cultured on four types of poly(ether carbonate urethane) urea (PECUU) scaffolds with controlled elasticity and fiber size (soft, small size; stiff, small size; soft, large size and stiff, large size). Results showed that when the fiber size of scaffold was kept constant, the expression of collagen-I (Col-I) in AFSCs increased with scaffold elasticity, while the expression of collagen-II (Col-II) and aggrecan genes showed an opposite trend. Moreover, when scaffold elasticity was controlled, the gene expression of Col-I in AFSCs increased with fiber size. In contrast, the expression of Col-II and aggrecan decreased. Such substrate elasticity and topography dependent changes of AFSC were similar to the gradient characteristics of native AF tissue. In addition, increasing elasticity and fiber size of scaffolds promoted YAP activation and its nuclear translocation. The results illustrate unambiguously that the mechanical property is a potent regulator of AFSC differentiation. Moreover, we revealed that fiber size of scaffold significantly affects spreading area, focal adhesion and differentiation of AFSCs. Therefore, both mechanical property and topography features of scaffolds regulate AFSC differentiation, possibly through a YAP-dependent mechanotransduction mechanism.

Investigation of Cellular Mechanotransduction Mechanism:

Investigation of Cellular Mechanotransduction Mechanism:

Mechanical forces in cell monolayers.

In various physiological processes, the cell collective is organized in a monolayer, such as seen in a simple epithelium. The advances in the understanding of mechanical behavior of the monolayer and its underlying cellular and molecular mechanisms will help to elucidate the properties of cell collectives. In this Review, we discuss recent in vitro studies on monolayer mechanics and their implications on collective dynamics, regulation of monolayer mechanics by physical confinement and geometrical cues and the effect of tissue mechanics on biological processes, such as cell division and extrusion. In particular, we focus on the active nematic property of cell monolayers and the emerging approach to view biological systems in the light of liquid crystal theory. We also highlight the mechanosensing and mechanotransduction mechanisms at the sub-cellular and molecular level that are mediated by the contractile actomyosin cytoskeleton and cell-cell adhesion proteins, such as E-cadherin and α-catenin. To conclude, we argue that, in order to have a holistic understanding of the cellular response to biophysical environments, interdisciplinary approaches and multiple techniques - from large-scale traction force measurements to molecular force protein sensors - must be employed.

Pressure-induced mesenchymal stem cell osteogenesis is dependent on intermediate filament remodeling.

Macroscale loading of bone generates a complex local mechanical microenvironment that drives osteogenesis and bone mechanoadaptation. One such mechanical stimulus generated is hydrostatic pressure (HP); however, the effect of HP on mesenchymal stem cells (MSCs) and the mechanotransduction mechanisms utilized by these cells to sense this stimulus are yet to be fully elucidated. In this study, we demonstrate that cyclic HP is a potent mediator of cytoskeletal reorganization and increases in osteogenic responses in MSCs. In particular, we demonstrate that the intermediate filament (IF) network undergoes breakdown and reorganization with centripetal translocation of IF bundles toward the perinuclear region. Furthermore, we show for the first time that this IF remodeling is required for loading-induced MSC osteogenesis, revealing a novel mechanism of MSC mechanotransduction. In addition, we demonstrate that chemical disruption of IFs with withaferin A induces a similar mechanism of IF breakdown and remodeling as well as a subsequent increase in osteogenic gene expression in MSCs, exhibiting a potential mechanotherapeutic effect to enhance MSC osteogenesis. This study therefore highlights a novel mechanotransduction mechanism of pressure-induced MSC osteogenesis involving the understudied cytoskeletal structure, the IF, and demonstrates a potential new therapy to enhance bone formation in bone-loss diseases such as osteoporosis.-Stavenschi, E., Hoey, D. A. Pressure-induced mesenchymal stem cell osteogenesis is dependent on intermediate filament remodeling.

An in vitro correlation of metastatic capacity and dual mechanostimulation.

Cells are under the influence of multiple forms of mechanical stimulation in vivo. For example, a cell is subjected to mechanical forces from tissue stiffness, shear and tensile stress and transient applied strain. Significant progress has been made in understanding the cellular mechanotransduction mechanisms in response to a single mechanical parameter. However, our knowledge of how a cell responds to multiple mechanical inputs is currently limited. In this study, we have tested the cellular response to the simultaneous application of two mechanical inputs: substrate compliance and transient tugging. Our results suggest that cells within a multicellular spheroid will restrict their response to a single mechanical input at a time and when provided with two mechanical inputs simultaneously, one will dominate. In normal and non-metastatic mammary epithelial cells, we found that they respond to applied stimulation and will override substrate compliance cues in favor of the applied mechanical stimulus. Surprisingly, however, metastatic mammary epithelial cells remain non-responsive to both mechanical cues. Our results suggest that, within our assay system, metastatic progression may involve the down-regulation of multiple mechanotransduction pathways.

Genetic variant at coronary artery disease and ischemic stroke locus 1p32.2 regulates endothelial responses to hemodynamics

Biomechanical cues dynamically control major cellular processes, but whether genetic variants actively participate in mechanosensing mechanisms remains unexplored. Vascular homeostasis is tightly regulated by hemodynamics. Exposure to disturbed blood flow at arterial sites of branching and bifurcation causes constitutive activation of vascular endothelium contributing to atherosclerosis, the major cause of coronary artery disease (CAD) and ischemic stroke (IS). Conversely, unidirectional flow promotes quiescent endothelium. Genome-wide association studies (GWAS) have identified chromosome 1p32.2 as strongly associated with CAD/IS; however, the causal mechanism related to this locus remains unknown. Using statistical analyses, assay of transposase accessible chromatin with whole-genome sequencing (ATAC-seq), H3K27ac/H3K4me2 ChIP with whole-genome sequencing (ChIP-seq), and CRISPR interference in human aortic endothelial cells (HAECs), our results demonstrate that rs17114036, a common noncoding polymorphism at 1p32.2, is located in an endothelial enhancer dynamically regulated by hemodynamics. CRISPR-Cas9-based genome editing shows that rs17114036-containing region promotes endothelial quiescence under unidirectional shear stress by regulating phospholipid phosphatase 3 (PLPP3). Chromatin accessibility quantitative trait locus (caQTL) mapping using HAECs from 56 donors, allelic imbalance assay from 7 donors, and luciferase assays demonstrate that CAD/IS-protective allele at rs17114036 in PLPP3 intron 5 confers increased endothelial enhancer activity. ChIP-PCR and luciferase assays show that CAD/IS-protective allele at rs17114036 creates a binding site for transcription factor Krüppel-like factor 2 (KLF2), which increases the enhancer activity under unidirectional flow. These results demonstrate that a human SNP contributes to critical endothelial mechanotransduction mechanisms and suggest that human haplotypes and related cis-regulatory elements provide a previously unappreciated layer of regulatory control in cellular mechanosensing mechanisms.

In Vivo Osteocyte Mechanotransduction: Recent Developments and Future Directions.

Mechanical loading is an essential stimulus for skeletal tissues. Osteocytes are primarily responsible for sensing mechanical stimuli in bone and for orchestrating subsequent responses. This is critical for maintaining homeostasis, and responding to injury/disease. The osteocyte mechanotransduction pathway, and the downstream effects it mediates, is highly complex. In vivo models have proved invaluable in understanding this process. This review summarizes the commonly used models, as well as more recently developed ones, and describes how they are used to address emerging questions in the field. Minimally invasive animal models can be used to determine mechanisms of osteocyte mechanotransduction, at the cell and molecular level, while simultaneously reducing potentially confounding responses such as inflammation/wound-healing. The details of osteocyte mechanotransduction in bone are gradually becoming clearer. In vivo model systems are a key tool in pursing this question. Advances in this field are explored and discussed in this review.

Mesenchymal stem cell mechanotransduction is cAMP dependent and regulated by adenylyl cyclase 6 and the primary cilium.

Mechanical loading is a potent stimulus of bone adaptation, requiring the replenishment of the osteoblast from a progenitor population. One such progenitor is the mesenchymal stem cell (MSC), which undergoes osteogenic differentiation in response to oscillatory fluid shear. Yet, the mechanism mediating stem cell mechanotransduction, and thus the potential to target this therapeutically, is poorly understood. In this study, we demonstrate that MSCs utilise cAMP as a second messenger in mechanotransduction, which is required for flow-mediated increases in osteogenic gene expression. Furthermore, we demonstrate that this mechanosignalling is dependent on the primary cilium and the ciliary localised adenylyl cyclase 6. Finally, we also demonstrate that this mechanotransduction mechanism can be targeted therapeutically to enhance cAMP signalling and early osteogenic signalling, mimicking the beneficial effect of physical loading. Our findings therefore demonstrate a novel mechanism of MSC mechanotransduction that can be targeted therapeutically, demonstrating a potential mechanotherapeutic for bone-loss diseases such as osteoporosis.This article has an associated First Person interview with the first author of the paper.

First person – Gillian Johnson

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Gillian Johnson is the first author on ‘Mesenchymal stem cell mechanotransduction is cAMP dependent and regulated by adenylyl cyclase 6 and the primary cilium’, published in Journal of Cell Science. Gillian is a PhD student in the lab of David Hoey at Trinity College Dublin, Ireland, and works on deciphering the molecular mechanism of cilia-mediated stem cell mechanotransduction both in vitro and in vivo.

Double-String Model for Auditory Transduction of Drosophila

The Drosophila auditory system consists of four large basal segments: the arista, the funiculus, the pedicel, and the scape. When an acoustic stimulus is applied to the arista and the funiculus their mechanical vibrations are transmitted to chordotonal neurons in Johnston’s organ where mechanoelectric transduction arises. We study the mechanotransduction mechanism in the Drosophila auditory system by using a laser Doppler vibrometer (LDV) and extracellular electrophysiology. We find that large and small peaks appear alternatively and that the antenna vibration is asymmetric depending on whether the pedicel and the scape are fixed. Interestingly, we find that this asymmetric vibration accompanies the alternating neural peak structure. Here, we propose a mathematical model to explain the alternating peak structure by using a model consisting of two opposing neurons that are modeled as strings. Generally, strings have tension only when they are elongated. This property allows the alternating neural peaks for asymmetric antenna motion.

Hyaluronic acid selective anchoring to the cytoskeleton: An atomic force microscopy study.

The hyaluronic acid component of the glycocalyx plays a role in cell mechanotransduction by selectively transmitting mechanical signals to the cell cytoskeleton or to the cell membrane. The aim of this study was to evaluate the mechanical link between the hyaluronic acid molecule and the cell cytoskeleton by means of atomic force microscopy single molecule force spectroscopy. Hyaluronic acid molecules on live cells were targeted with probes coated with hyaluronic acid binding protein. Two different types of events were observed when the detachment of the target molecule from the probe occurred, suggesting the presence of cytoskeleton- and membrane-anchored molecules. Membrane-anchored molecules facilitated the formation of tethers when pulled. About 15% of the tested hyaluronic acid molecules were shown to be anchored to the cytoskeleton. When multiple molecules bonded to the probe, specific detachment patterns were observed, suggesting that a cytoskeletal bond needed to be broken to improve the ability to pull tethers from the cell membrane. This likely resulted in the formation of tethering structures maintaining a cytoskeletal core similar to the ones observed for cells over-expressing HA synthases. The different observed rupture events were associated with separate mechanotransductive mechanisms in an analogous manner to that previously proposed for the endothelial glycocalyx. Single cytoskeleton anchored rupture events represent HA molecules linked to the cytoskeleton and therefore transmitting mechanical stimuli into the inner cell compartments. Single membrane tethers would conversely represent the glycocalyx molecules connected to areas of the membrane where an abundance of signalling molecules reside.

A novel micro-grooved collagen substrate for inducing vascular smooth muscle differentiation through cell tissue arrangement and nucleus remodeling

Vascular smooth muscle cells (SMCs) actively remodel arterial walls through biomechanical signals and dedifferentiate from the contractile to the synthetic state under pathological conditions. It is important to determine the differentiation mechanism of SMCs to understand their pathophysiology in disease. Previously, we found that the F-actin cytoskeleton in dedifferentiated SMCs on dishes was firmly connected to the nucleus, and that internal mechanical signals in SMCs are transmitted directly to the nucleus, indicating that nuclear-cytoskeletal interactions could be associated with SMC differentiation. However, mechanical environments in vivo are quite different from those of cultured cells: SMCs in vivo show an elongated shape and form a tissue that aligns with the circumferential direction of the walls. Thus, in the present study, we established a simple technique to fabricate a novel micro-grooved native collagen substrate that mimics the elongated cell shapes and alignment observed in vivo. The substrates had “wavy wrinkle” grooves with a width of ~5 µm and a Young's modulus of ~500 kPa, which were quite similar to those of the elastic lamina in vascular tissues. Using confocal microscopy image-based analysis, and nano-indentation imaging with atomic force microscopy, we found that SMCs on the micro-grooved collagen formed significant cell tissue arrangement, and changed their nuclear morphology to a “slim ellipsoid” in response to the force-reduction caused by F-actin remodeling, which consequently improved SMC differentiation. These findings indicated that this type of intracellular force-reduction around the nucleus has a crucial effect on SMC differentiation. Our micro-grooved collagen substrate is a powerful tool to investigate the mechanism of vascular SMC mechanotransduction.

Finite element modeling of living cells for AFM indentation-based biomechanical characterization

Mechanotransduction—the process living cells sense and respond to forces—is essential for maintenance of normal cell, tissue, and organ functioning. To promote the knowledge of mechanotransduction, atomic force microscope (AFM) force-indentation has been broadly used to quantify the mechanical properties of living cells. However, most studies treated the cells as a homogeneous elastic or viscoelastic material, which is far from the real structure of cells, and the quantified mechanical properties cannot be used to investigate the inner working mechanism of mechanotransduction, such as internal force distribution/transduction. Therefore, a new viscoelastic finite element method (FEM) model is proposed in this study to simulate the force response of living cells during AFM force-indentation measurement by accounting for both the cell elasticity and viscoelasticity. The cell is modeled as a multi-layered structure with different mechanical characteristics of each layer to account for the depth-dependent mechanical behavior of living cells. This FEM model was validated by comparing the simulated force-indentation curves with the AFM experimental data on living NIH/3T3 cells, and the simulation error was less than 10% with respect to the experiment results. Therefore, the proposed FEM model can accurately simulate the force response of living cells and has a potential to be utilized to study and predict the intracellular force transduction and distribution.

A variational homogenization approach applied to the multiscale analysis of the viscoelastic behavior of tendon fascicles

This work presents a variational homogenization approach based on representative volume elements (RVE) in order to investigate the macro- and microviscoelastic behavior of tendon fascicles. A three-dimensional hexagonal–helicoidal finite element RVE is proposed to properly account for the morphology of tendon fascicles observed in serial block-face scanning electron microscopy. Two material phases (collagen fibers and cells) comprising three finite strain variational viscoelastic models (fibrils, matrix of fibers and cells) compose the proposed multiscale model. The material parameters of the micromechanical models were identified with the aid of atomic force microscopy experiments extracted from the literature. A set of multiscale simulations of tensile tests under physiological strain amplitudes were performed, providing the following results. Firstly, numerical predictions corroborate experimental findings: collagen fibrils are the main load-bearing structures of tendons; the cellular matrix contributes neither to the stiffness nor to the energy dissipation of tendons. Secondly, the model brings insights about microscale mechanics of tendon fascicles not completely understood: prediction of uncoiling of fibers during axial loads which may explain the large apparent Poisson ratios and fluid loss, and significant strain localization in cells, which may lead to important mechanotransduction mechanisms. Moreover, the distribution of dissipated power became available, pointing out the fibrils as the main source of dissipation of fascicles under high macroscopic strain rates and during the unloading phase in cyclic regimes.

PO-064 A potential role of stretch-activated channels in anabolic mechanotransduction in the atrophied rat soleus muscle

Objective Prolonged immobilization or unloading of skeletal muscle causes muscle disuse atrophy, which is characterized by a reduction in muscle cross-sectional area and compromised contractile function. To date, the mechanisms of anabolic mechanotransduction in the atrophied mammalian skeletal muscle remain poorly understood. The aim of the present study was to assess a possible role of stretch-activated ion channels (SAC) in the propagation of a mechanical signal to anabolic signaling and protein synthesis (PS) in an isolated rat soleus muscle following mechanical unloading.
 Methods The mechanical unloading was performed via hindlimb suspension (HS). Twenty-eight male Wistar rats weighing were randomly assigned to the following 4 groups (n=7/group): 1) vivarium control (C), 2) control + SAC inhibitor (gadolinium) (C+Gd3+), 3) 7-day HS (HS), 4) 7-day HS + SAC inhibitor (HS+Gd3+). Following unloading, an isolated rat soleus was placed in an organ culture medium and subjected to a bout of eccentric contractions (EC). Upon completion of the EC, muscles were collected for Western blot analyses to determine the content of the key anabolic markers. The rate of PS was measured by SUnSET technique.
 Results EC-induced increase in PS was significantly less in the HS and HS+ Gd3+ groups vs. the C group. There was no statistically significant difference between the HS and HS+ Gd3+ groups in terms of EC-induced increase in muscle PS. A decrease in EC-induced phosphorylation of p70S6K, 4E-BP1, RPS6 and GSK-3beta in the 7-day unloaded soleus treated with SAC inhibitor did not differ from that of the 7-day unloaded soleus without SAC blockade. Thus, the inhibition of SAC with gadolinium did not lead to further decline in EC-induced phosphorylation of the key anabolic markers and muscle PS.
 Conclusions The results of the study suggest that attenuation of mTORC1-signaling and PS in response to EC in unloaded soleus muscle may be associated with inactivation of SAC. The study was supported by the RFBR grant # 16-34-60055.

Direct application of mechanical stimulation to cell adhesion sites using a novel magnetic-driven micropillar substrate.

Cells change the traction forces generated at their adhesion sites, and these forces play essential roles in regulating various cellular functions. Here, we developed a novel magnetic-driven micropillar array PDMS substrate that can be used for the mechanical stimulation to cellular adhesion sites and for the measurement of associated cellular traction forces. The diameter, length, and center-to-center spacing of the micropillars were 3, 9, and 9μm, respectively. Sufficient quantities of iron particles were successfully embedded into the micropillars, enabling the pillars to bend in response to an external magnetic field. We established two methods to apply magnetic fields to the micropillars (Suresh 2007). Applying a uniform magnetic field of 0.3T bent all of the pillars by ~4μm (Satcher et al. 1997). Creating a magnetic field gradient in the vicinity of the substrate generated a well-defined local force on the pillars. Deflection of the micropillars allowed transfer of external forces to the actin cytoskeleton through adhesion sites formed on the pillar top. Using the magnetic field gradient method, we measured the traction force changes in cultured vascular smooth muscle cells (SMCs) after local cyclic stretch stimulation at one edge of the cells. We found that the responses of SMCs were quite different from cell to cell, and elongated cells with larger pre-tension exhibited significant retraction following stretch stimulation. Our magnetic-driven micropillar substrate should be useful in investigating cellular mechanotransduction mechanisms.

Correction: A method to quantify mechanobiologic forces during zebrafish cardiac development using 4-D light sheet imaging and computational modeling.

[This corrects the article DOI: 10.1371/journal.pcbi.1005828.].

Correction: Mechanosensitive caveolin-1 activation-induced PI3K/Akt/mTOR signaling pathway promotes breast cancer motility, invadopodia formation and metastasis in vivo.

[This corrects the article DOI: 10.18632/oncotarget.7583.].

Different mechanisms activated by mild versus strong loading in rat Achilles tendon healing.

BackgroundMechanical loading stimulates Achilles tendon healing. However, various degrees of loading appear to have different effects on the mechanical properties of the healing tendon, and strong loading might create microdamage in the tissue. This suggests that different mechanisms might be activated depending on the magnitude of loading. The aim of this study was to investigate these mechanisms further.MethodsFemale rats had their right Achilles tendon cut transversely and divided into three groups: 1) unloading (calf muscle paralysis by Botox injections, combined with joint fixation by a steel-orthosis), 2) mild loading (Botox only), 3) strong loading (free cage activity). Gene expression was analyzed by PCR, 5 days post-injury, and mechanical testing 8 days post-injury. The occurrence of microdamage was analyzed 3, 5, or 14 days post-injury, by measuring leakage of injected fluorescence-labelled albumin in the healing tendon tissue.ResultsPeak force, peak stress, and elastic modulus of the healing tendons gradually improved with increased loading as well as the expression of extracellular matrix genes. In contrast, only strong loading increased transverse area and affected inflammation genes. Strong loading led to higher fluorescence (as a sign of microdamage) compared to mild loading at 3 and 5 days post-injury, but not at 14 days.DiscussionOur results show that strong loading improves both the quality and quantity of the healing tendon, while mild loading only improves the quality. Strong loading also induces microdamage and alters the inflammatory response. This suggests that mild loading exert its effect via mechanotransduction mechanisms, while strong loading exert its effect both via mechanotransduction and the creation of microdamage.ConclusionIn conclusion, mild loading is enough to increase the quality of the healing tendon without inducing microdamage and alter the inflammation in the tissue. This supports the general conception that early mobilization of a ruptured tendon in patients is advantageous.