Mechanosensitive Signaling Research Articles

Dormancy-inducing 3D engineered matrix uncovers mechanosensitive and drug-protective FHL2-p21 signaling axis.

Solid cancers frequently relapse with distant metastasis, despite local and systemic treatment. Cellular dormancy has been identified as an important mechanism underlying drug resistance enabling late relapse. Therefore, relapse from invisible, minimal residual cancer of seemingly disease-free patients call for in vitro models of dormant cells suited for drug discovery. Here, we explore dormancy-inducing 3D engineered matrices, which generate mechanical confinement and induce growth arrest and survival against chemotherapy in cancer cells. We characterized the dormant phenotype of solitary cells by P-ERKlow:P-p38high dormancy signaling ratio, along with Ki67- expression. As underlying mechanism, we identified stiffness-dependent nuclear localization of the four-and-a-half LIM domain 2 (FHL2) protein, leading to p53-independent high p21Cip1/Waf1 nuclear expression, validated in murine and human tissue. Suggestive of a resistance-causing role, cells in the dormancy-inducing matrix became sensitive against chemotherapy upon FHL2 down-regulation. Thus, our biomaterial-based approach will enable systematic screens for previously unidentified compounds suited to eradicate potentially relapsing dormant cancer cells.

The desmosome-intermediate filament system facilitates mechanotransduction at adherens junctions for epithelial homeostasis

Epithelial homeostasis can be critically influenced by how cells respond to mechanical forces, both local changes in force balance between cells and altered tissue-level forces.1 Coupling of specialized cell-cell adhesions to their cytoskeletons provides epithelia with diverse strategies to respond to mechanical stresses.2,3,4 Desmosomes confer tissue resilience when their associated intermediate filaments (IFs)2,3 stiffen in response to strain,5,6,7,8,9,10,11 while mechanotransduction associated with the E-cadherin apparatus12,13 at adherens junctions (AJs) actively modulates actomyosin by RhoA signaling. Although desmosomes and AJs make complementary contributions to mechanical homeostasis in epithelia,6,8 there is increasing evidence to suggest that these cytoskeletal-adhesion systems can interact functionally and biochemically.8,14,15,16,17,18,19,20 We now report that the desmosome-IF system integrated by desmoplakin (DP) facilitates active tension sensing at AJs for epithelial homeostasis. DP function is necessary for mechanosensitive RhoA signaling at AJs to be activated when tension was applied to epithelial monolayers. This effect required DP to anchor IFs to desmosomes and recruit the dystonin (DST) cytolinker to apical junctions. DP RNAi reduced the mechanical load that was applied to the cadherin complex by increased monolayer tension. Consistent with reduced mechanical signal strength, DP RNAi compromised assembly of the Myosin VI-E-cadherin mechanosensor that activates RhoA. The integrated DP-IF system therefore supports AJ mechanotransduction by enhancing the mechanical load of tissue tension that is transmitted to E-cadherin. This crosstalk was necessary for efficient elimination of apoptotic epithelial cells by apical extrusion, demonstrating its contribution to epithelial homeostasis.

Nano-mechanical Immunoengineering: Nanoparticle Elasticity Reprograms Tumor-Associated Macrophages via Piezo1.

The mechanical properties of nanoparticles play a crucial role in regulating nanobiointeractions, influencing processes such as blood circulation, tumor accumulation/penetration, and internalization into cancer cells. Consequently, they have a significant impact on drug delivery and therapeutic efficacy. However, it remains unclear whether and how macrophages alter their biological function in response to nanoparticle elasticity. Here, we report on the nano-mechanical biological effects resulting from the interactions between elastic silica nanoparticles (SNs) and macrophages. The SNs with variational elasticity Young's moduli ranging from 81 to 837 MPa were synthesized, and it was demonstrated that M2 [tumor-associated macrophages (TAMs)] could be repolarized to M1 by the soft SNs. Additionally, our findings revealed that cell endocytosis, membrane tension, the curvature protein Baiap2, and the cytoskeleton were all influenced by the elasticity of SNs. Moreover, the mechanically sensitive protein Piezo1 on the cell membrane was activated, leading to calcium ion influx, activation of the NF-κB pathway, and the initiation of an inflammatory response. In vivo experiments demonstrated that the softest 81 MPa SNs enhanced tumor penetration and accumulation and repolarized TAMs in intratumoral hypoxic regions, ultimately resulting in a significant inhibition of tumor growth. Taken together, this study has established a cellular feedback mechanism in response to nanoparticle elasticity, which induces plasma membrane deformation and subsequent activation of mechanosensitive signals. This provides a distinctive "nano-mechanical immunoengineering" strategy for reprogramming TAMs to enhance cancer immunotherapy.

Correlating Material Properties to Osteoprotegerin Expression on Nanoparticulate Mineralized Collagen Glycosaminoglycan Scaffolds.

Precision material design directed by cell biological processes represents a frontier in developing clinically translatable regenerative technologies. While understanding cell-material interactions on multipotent progenitor cells yields insights on target tissue differentiation, equally if not more important is the quantification of indirect multicellular interactions. In this work, the relationship of two material properties, phosphate content and stiffness, of a nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG) in the expression of an endogenous anti-osteoclastogenic secreted protein, osteoprotegerin (OPG) by primary human mesenchymal stem cells (hMSCs) is evaluated. The phosphate content of MC-GAG requires the type III sodium phosphate symporter PiT-1/SLC20A1 for OPG expression, correlating with β-catenin downregulation, but is independent of the effects of phosphate ion on osteogenic differentiation. Using three stiffness MC-GAG variants that do not differ significantly by osteogenic differentiation, it is observed that the softest material elicited ≈1.6-2 times higher OPG expression than the stiffer materials. Knockdown of the mechanosensitive signaling axis of YAP, TAZ, β-catenin and combinations thereof in hMSCs on MC-GAG demonstrates that β-catenin downregulation increases OPG expression by 1.5-fold. Taken together, these data constitute a roadmap for material properties that can used to suppress osteoclast activation via osteoprotegerin expression separately from the anabolic processes of osteogenesis.

Mechanosensitive membrane domains regulate calcium entry in arterial endothelial cells to protect against inflammation.

Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, and this supports an anti-inflammatory phenotype. High laminar shear stress also induces flow-aligned cell elongation and front-rear polarity, but whether these are required for the anti-inflammatory phenotype is unclear. Here, we showed that Caveolin-1-rich microdomains polarize to the downstream end of ECs that are exposed to continuous high laminar flow. These microdomains were characterized by high membrane rigidity, filamentous actin (F-actin), and raft-associated lipids. Transient receptor potential vanilloid-type 4 (TRPV4) ion channels were ubiquitously expressed on the plasma membrane but mediated localized Ca2+ entry only at these microdomains where they physically interacted with clustered Caveolin-1. These focal Ca2+ bursts activated endothelial nitric oxide synthase (eNOS) within the confines of these domains. Importantly, we found that signaling at these domains required both cell body elongation and sustained flow. Finally, TRPV4 signaling at these domains was necessary and sufficient to suppress inflammatory gene expression, and exogenous activation of TRPV4 channels ameliorated the inflammatory response to stimuli both in vitro and in vivo. Our work revealed a polarized mechanosensitive signaling hub in arterial ECs that dampens inflammatory gene expression and promotes cell resilience.

Ion channel Piezo1 activation aggravates the endothelial dysfunction under a high glucose environment

BackgroundVasculopathy is the most common complication of diabetes. Endothelial cells located in the innermost layer of blood vessels are constantly affected by blood flow or vascular components; thus, their mechanosensitivity plays an important role in mediating vascular regulation. Endothelial damage, one of the main causes of hyperglycemic vascular complications, has been extensively studied. However, the role of mechanosensitive signaling in hyperglycemic endothelial damage remains unclear.MethodsVascular endothelial-specific Piezo1 knockout mice were generated to investigate the effects of Piezo1 on Streptozotocin-induced hyperglycemia and vascular endothelial injury. In vitro activation or knockdown of Piezo1 was performed to evaluate the effects on the proliferation, migration, and tubular function of human umbilical vein endothelial cells in high glucose. Reactive oxygen species production, mitochondrial membrane potential alternations, and oxidative stress-related products were used to assess the extent of oxidative stress damage caused by Piezo1 activation.ResultsOur study found that in VECreERT2;Piezo1flox/flox mice with Piezo1 conditional knockout in vascular endothelial cells, Piezo1 deficiency alleviated streptozotocin-induced hyperglycemia with reduced apoptosis and abscission of thoracic aortic endothelial cells, and decreased the inflammatory response of aortic tissue caused by high glucose. Moreover, the knockout of Piezo1 showed a thinner thoracic aortic wall, reduced tunica media damage, and increased endothelial nitric oxide synthase expression in transgenic mice, indicating the relief of endothelial damage caused by hyperglycemia. We also showed that Piezo1 activation aggravated oxidative stress injury and resulted in severe dysfunction through the Ca2+-induced CaMKII-Nrf2 axis in human umbilical vein endothelial cells. In Piezo1 conditional knockout mice, Piezo1 deficiency partially restored superoxide dismutase activity and reduced malondialdehyde content in the thoracic aorta. Mechanistically, Piezo1 deficiency decreased CaMKII phosphorylation and restored the expression of Nrf2 and its downstream molecules HO-1 and NQO1.ConclusionIn summary, our study revealed that Piezo1 is involved in high glucose-induced oxidative stress injury and aggravated endothelial dysfunction, which have great significance for alleviating endothelial damage caused by hyperglycemia.

Multiscale computational model of aortic remodeling following postnatal disruption of TGFβ signaling

The healthy adult aorta is a remarkably resilient structure, able to resist relentless cardiac-induced and hemodynamic loads under normal conditions. Fundamental to such mechanical homeostasis is the mechano-sensitive cell signaling that controls gene products and thus the structural integrity of the wall. Mouse models have shown that smooth muscle cell-specific disruption of transforming growth factor-beta (TGFβ) signaling during postnatal development compromises this resiliency, rendering the aortic wall susceptible to aneurysm and dissection under normal mechanical loading. By contrast, disruption of such signaling in the adult aorta appears to introduce a vulnerability that remains hidden under normal loading, but manifests under increased loading as experienced during hypertension. We present a multiscale (transcript to tissue) computational model to examine possible reasons for compromised mechanical homeostasis in the adult aorta following reduced TGFβ signaling in smooth muscle cells.

ZO-1 Regulates Hippo-Independent YAP Activity and Cell Proliferation via a GEF-H1- and TBK1-Regulated Signalling Network.

Tight junctions are a barrier-forming cell-cell adhesion complex and have been proposed to regulate cell proliferation. However, the underlying mechanisms are not well understood. Here, we used cells deficient in the junction scaffold ZO-1 alone or together with its paralog ZO-2, which disrupts the junctional barrier. We found that ZO-1 knockout increased cell proliferation, induced loss of cell density-dependent proliferation control, and promoted apoptosis and necrosis. These phenotypes were enhanced by double ZO-1/ZO-2 knockout. Increased proliferation was dependent on two transcriptional regulators: YAP and ZONAB. ZO-1 knockout stimulated YAP nuclear translocation and activity without changes in Hippo-dependent phosphorylation. Knockout promoted TANK-binding kinase 1 (TBK1) activation and increased expression of the RhoA activator GEF-H1. Knockdown of ZO-3, another paralog interacting with ZO1, was sufficient to induce GEF-H1 expression and YAP activity. GEF-H1, TBK1, and mechanotransduction at focal adhesions were found to cooperate to activate YAP/TEAD in ZO-1-deficient cells. Thus, ZO-1 controled cell proliferation and Hippo-independent YAP activity by activating a GEF-H1- and TBK1-regulated mechanosensitive signalling network.

Abstract 5569: Mechano-chemical control of secretory cues regulating immune tolerance, survival and invasion in oral squamous cell carcinomas

Abstract Tissue fibrosis and extracellular matrix (ECM) stiffening have been shown to promote tumor cell proliferation, metastasis, and chemoresistance in a range of cancers. Stiffness of the ECM can lead to changes in cancer cell behavior, including enhanced proliferation, activation of mechano-sensitive signaling pathways, and potential genomic damage, with implications for cancer progression and immune responses. Recent research suggests stiff matrix may also induce the release of tumor-derived small extracellular vesicles (sEVs) that promote tumor growth. Tumor-derived sEVs carry bioactive cargo, such as micro-RNAs (miRNAs) and DNA fragments, and function as a unique form of intracellular communication promoting cell survival, growth, and metastasis. Understanding these mechano-sensing mechanisms is essential for identifying potential targets in precision medicine for solid tumor treatment. However, the influence of tumor stiffness on secretory cues, and their subsequent role in immunomodulation and/or chemo-resistance has not been extensively studied. This project aims to explore how tumor stiffness can alter cell responses to genotoxic stress and how these responses can perpetuate feedback to the immune system through the release of secretory cues such as sEVs. Here, we outline the isolation and characterization of tumor-derived sEVs obtained from culture media of oral squamous cell carcinoma (OSCC) spheroids, cultured at varying stiffness (0.2kPa-1.5kPa). In this project, we use OSCC lines that are either sensitive or resistant to cisplatin (HSC-3 and HN-5, respectively). To explore the immunomodulatory effects of these tumor-derived sEVs, we first established a consistent supply of CD206+ve macrophages differentiated from human induced pluripotent stem cells (iMacs), following the established protocol of Douthwaite et al (2021). iMacs were then treated with sEVs with their resulting immunophenotype, and activation assessed. These preliminary experiments provide a step towards understanding how the biophysical environment influences tumor cell resistance to therapy and how sEVs can modulate the relationship between cancer cells and macrophages. Citation Format: Bethany R. Bareham, Matthew Dibble, Leila Mouhib, Aitor Bermejo Arteagabeitia, Subhankar Mukhopadhyay, Maddy Parsons. Mechano-chemical control of secretory cues regulating immune tolerance, survival and invasion in oral squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5569.

Capturing physiological hemodynamic flow and mechanosensitive cell signaling in vessel-on-a-chip platforms.

Recent advances in organ chip (or, "organ-on-a-chip") technologies and microphysiological systems (MPS) have enabled in vitro investigation of endothelial cell function in biomimetic three-dimensional environments under controlled fluid flow conditions. Many current organ chip models include a vascular compartment; however, the design and implementation of these vessel-on-a-chip components varies, with consequently varied impact on their ability to capture and reproduce hemodynamic flow and associated mechanosensitive signaling that regulates key characteristics of healthy, intact vasculature. In this review, we introduce organ chip and vessel-on-a-chip technology in the context of existing in vitro and in vivo vascular models. We then briefly discuss the importance of mechanosensitive signaling for vascular development and function, with focus on the major mechanosensitive signaling pathways involved. Next, we summarize recent advances in MPS and organ chips with an integrated vascular component, with an emphasis on comparing both the biomimicry and adaptability of the diverse approaches used for supporting and integrating intravascular flow. We review current data showing how intravascular flow and fluid shear stress impacts vessel development and function in MPS platforms and relate this to existing work in cell culture and animal models. Lastly, we highlight new insights obtained from MPS and organ chip models of mechanosensitive signaling in endothelial cells, and how this contributes to a deeper understanding of vessel growth and function in vivo. We expect this review will be of broad interest to vascular biologists, physiologists, and cardiovascular physicians as an introduction to organ chip platforms that can serve as viable model systems for investigating mechanosensitive signaling and other aspects of vascular physiology.

Magnetic nano-tweezer for interrogating mechanosensitive signaling proteins in space and time.

Spatiotemporal interrogation of signal transduction at the single-cell level is necessary to understand how extracellular cues are converted into biochemical signals and differentially regulate cellular responses. Using single-cell perturbation tools such as optogenetics, specific biochemical cues can be delivered to selective molecules or cells at any desired location and time. By measuring cellular responses to provided perturbations, investigators have decoded and deconstructed the working mechanisms of a variety of neuroelectric and biochemical signaling processes. However, analogous methods for deciphering the working mechanisms of mechanosensitive signaling by regulating mechanical inputs to cell receptors have remained elusive. To address this unmet need, we have recently developed a nanotechnology-based single-cell and single-molecule perturbation tool, termed mechanogenetics, that enables precise spatial and mechanical control over genetically encoded cell-surface receptors in live cells. This tool combines a magnetofluorescent nanoparticle (MFN) actuator, which provides precise spatial and mechanical signals to receptors via target-specific one-to-one interaction, with a micromagnetic tweezersthat remotely controls the force exerted on a single nanoparticle. This chapter provides comprehensive experimental protocols of mechanogenetics consisting of four stages: (i) chemical synthesis of MFNs, (ii) bio-conjugation and purification of monovalent MFNs, (iii) establishment of cells with genetically encoded mechanosensitive proteins, and (iv) modular targeting and control of cell-surface receptors in live cells. The entire procedure takes up to 1 week. This mechanogenetic tool can be generalized to study many outstanding questions related to the dynamics of cell signaling and transcriptional control, including the mechanism of mechanically activated receptor.

Mechanosensitive hormone signaling promotes mammary progenitor expansion and breast cancer risk

Tissue stem-progenitor cell frequency has been implicated in tumor risk and progression, but tissue-specific factors linking these associations remain ill-defined. We observed that stiff breast tissue from women with high mammographic density, who exhibit increased lifetime risk for breast cancer, associates with abundant stem-progenitor epithelial cells. Using genetically engineered mouse models of elevated integrin mechanosignaling and collagen density, syngeneic manipulations, and spheroid models, we determined that a stiff matrix and high mechanosignaling increase mammary epithelial stem-progenitor cell frequency and enhance tumor initiation invivo. Augmented tissue mechanics expand stemness by potentiating extracellular signal-related kinase (ERK) activity to foster progesterone receptor-dependent RANK signaling. Consistently, we detected elevated phosphorylated ERK and progesterone receptors and increased levels of RANK signaling in stiff breast tissue from women with high mammographic density. The findings link fibrosis and mechanosignaling to stem-progenitor cell frequency and breast cancer risk and causally implicate epidermal growth factor receptor-ERK-dependent hormone signaling in this phenotype.

Mind the Viscous Modulus: The Mechanotransductive Response to the Viscous Nature of Isoelastic Matrices Regulates Stem Cell Chondrogenesis.

The design of hydrogels as mimetics of tissues' matrices typically disregards the viscous nature of native tissues and focuses only on their elastic properties. In the case of stem cell chondrogenesis, this has led to contradictory results, likely due to unreported changes in the matrices' viscous modulus. Here, by employing isoelastic matrices with Young's modulus of ≈12kPa, variations in viscous properties alone (i.e., loss tangent between 0.1 and 0.25) are demonstrated to be sufficient to drive efficient growth factor-free chondrogenesis of human mesenchymal stem cells, both in 2D and 3D cultures. The increase of the viscous component of RGD-functionalized polyacrylamide or polyethylene glycol maleimide hydrogels promotes a phenotype with reduced adhesion, alters mechanosensitive signaling, and boosts cell-cell contacts. In turn, this upregulates the chondrogenic transcription factor SOX9 and supports neocartilage formation, demonstrating that the mechanotransductive response to the viscous nature of the matrix can be harnessed to direct cell fate.

Differential sensitivity to longitudinal and transverse stretch mediates transcriptional responses in mouse neonatal ventricular myocytes.

To identify how cardiomyocyte mechanosensitive signaling pathways are regulated by anisotropic stretch, micropatterned mouse neonatal cardiomyocytes were stretched primarily longitudinally or transversely to the myofiber axis. Four hours of static, longitudinal stretch induced differential expression of 557 genes, compared with 30 induced by transverse stretch, measured using RNA-seq. A logic-based ordinary differential equation model of the cardiac myocyte mechanosignaling network, extended to include the transcriptional regulation and expression of 784 genes, correctly predicted measured expression changes due to anisotropic stretch with 69% accuracy. The model also predicted published transcriptional responses to mechanical load in vitro or in vivo with 63-91% accuracy. The observed differences between transverse and longitudinal stretch responses were not explained by differential activation of specific pathways but rather by an approximately twofold greater sensitivity to longitudinal stretch than transverse stretch. In vitro experiments confirmed model predictions that stretch-induced gene expression is more sensitive to angiotensin II and endothelin-1, via RhoA and MAP kinases, than to the three membrane ion channels upstream of calcium signaling in the network. Quantitative cardiomyocyte gene expression differs substantially with the axis of maximum principal stretch relative to the myofilament axis, but this difference is due primarily to differences in stretch sensitivity rather than to selective activation of mechanosignaling pathways.NEW & NOTEWORTHY Anisotropic stretch applied to micropatterned neonatal mouse ventricular myocytes induced markedly greater acute transcriptional responses when the major axis of stretch was parallel to the myofilament axis than when it was transverse. Analysis with a novel quantitative network model of mechanoregulated cardiomyocyte gene expression suggests that this difference is explained by higher cell sensitivity to longitudinal loading than transverse loading than by the activation of differential signaling pathways.

Extracellular matrix stiffness activates mechanosensitive signals but limits breast cancer cell spheroid proliferation and invasion.

Breast cancer is characterized by physical changes that occur in the tumor microenvironment throughout growth and metastasis of tumors. Extracellular matrix stiffness increases as tumors develop and spread, with stiffer environments thought to correlate with poorer disease prognosis. Changes in extracellular stiffness and other physical characteristics are sensed by integrins which integrate these extracellular cues to intracellular signaling, resulting in modulation of proliferation and invasion. However, the co-ordination of mechano-sensitive signaling with functional changes to groups of tumor cells within 3-dimensional environments remains poorly understood. Here we provide evidence that increasing the stiffness of collagen scaffolds results in increased activation of ERK1/2 and YAP in human breast cancer cell spheroids. We also show that ERK1/2 acts upstream of YAP activation in this context. We further demonstrate that YAP, matrix metalloproteinases and actomyosin contractility are required for collagen remodeling, proliferation and invasion in lower stiffness scaffolds. However, the increased activation of these proteins in higher stiffness 3-dimensional collagen gels is correlated with reduced proliferation and reduced invasion of cancer cell spheroids. Our data collectively provide evidence that higher stiffness 3-dimensional environments induce mechano-signaling but contrary to evidence from 2-dimensional studies, this is not sufficient to promote pro-tumorigenic effects in breast cancer cell spheroids.

The actin-bundling protein, PLS3, is part of the mechanoresponsive machinery that regulates osteoblast mineralization.

Plastin-3 (PLS3) is a calcium-sensitive actin-bundling protein that has recently been linked to the development of childhood-onset osteoporosis. Clinical data suggest that PLS3 mutations lead to a defect in osteoblast function, however the underlying mechanism remains elusive. To investigate the role of PLS3 in bone mineralization, we generated MC3T3-E1 preosteoblast cells that are stably depleted of PLS3. Analysis of osteogenic differentiation of control and PLS3 knockdown (PLS3 KD) cells showed that depletion of PLS3 does not alter the first stage of osteoblast mineralization in which a collagen matrix is deposited, but severely affects the subsequent mineralization of that matrix. During this phase, osteoblasts heavily rely on mechanosensitive signaling pathways to sustain mineral deposition in response to increasing stiffness of the extracellular matrix (ECM). PLS3 prominently localizes to focal adhesions (FAs), which are intricately linked to mechanosensation. In line with this, we observed that depletion of PLS3 rendered osteoblasts unresponsive to changes in ECM stiffness and showed the same cell size, FA lengths and number of FAs when plated on soft (6kPa) versus stiff (100kPa) substrates in contrast to control cells, which showed an increased in each of these parameters when plated on 100kPa substrates. Defective cell spreading of PLS3 KD cells on stiff substrates could be rescued by expression of wildtype PLS3, but not by expression of three PLS3 mutations that were identified in patients with early onset osteoporosis and that have aberrant actin-bundling activity. Altogether, our results show that actin-bundling by PLS3 is part of the mechanosensitive mechanism that promotes osteoblast mineralization and thus begins to elucidate how PLS3 contributes to the development of bone defects such as osteoporosis.

NG2/CSPG4 attenuates motility in mandibular fibrochondrocytes under serum starvation conditions.

The migration of mandibular fibrochondrocytes is important for the development of the mandible, the homeostasis of the mandibular cartilage, and for the capacity of the tissue to respond to injury. Mandibular fibrochondrocytes have to overcome formidable obstacles during migration including a dense and heterogeneous three-dimensional matrix. Guiding the direction of cell migration and commitment to a migratory phenotype in this microenvironment necessitates a multivalent response to chemotactic and extracellular matrix-mediated stimuli. One of the key matrix components in the cartilage of the temporomandibular joint is type VI collagen. Neuron/glial antigen 2 (NG2/CSPG4) is a transmembrane proteoglycan that binds with collagen VI and has been implicated in a wide range of cell behaviors including cell migration, motility, adhesion, and proliferation. While NG2/CSPG4 has been shown to be a key regulator of mandibular cartilage homeostasis, its role in the migration of mandibular fibrochondrocytes during normal and cell stress conditions has yet to be resolved. Here, we address this gap in knowledge by characterizing NG2/CSPG4-dependent migration in mandibular fibrochondrocytes using primary mandibular fibrochondrocytes isolated from control and full length NG2/CSPG4 knockout mice, in primary mandibular fibrochondrocytes isolated from NG2|DsRed reporter mice and in an immortalized mandibular fibrochondrocyte cell line with a mutated NG2/CSPG4 ectodomain. All threecells demonstrate similar results, with loss of the full length or truncated NG2/CSPG4 increasing the rate of cell migration in serum starvation/cell stress conditions. These findings clearly implicate NG2/CSPG4 as a key molecule in the regulation of cell migration in mandibular fibrochondrocytes in normal and cell stress conditions, underscoring the role of NG2/CSPG4 as a mechanosensitive signaling hub in the mandibular cartilage.

Rethinking replating.

JGP study (In this issue, Osten et al. https://doi.org/10.1085/jgp.202313377) suggests that, by altering mechanosensitive signaling pathways, replating stem cell-derived cardiomyocytes changes myosin expression and contractile function.

Defined extracellular matrix compositions support stiffness-insensitive cell spreading and adhesion signaling

Integrin-dependent adhesion to the extracellular matrix (ECM) mediates mechanosensing and signaling in response to altered microenvironmental conditions. In order to provide tissue- and organ-specific cues, the ECM is composed of many different proteins that temper the mechanical properties and provide the necessary structural diversity. Despite most human tissues being soft, the prevailing view from predominantly in vitro studies is that increased stiffness triggers effective cell spreading and activation of mechanosensitive signaling pathways. To address the functional coupling of ECM composition and matrix rigidity on compliant substrates, we developed a matrix spot array system to screen cell phenotypes against different ECM mixtures on defined substrate stiffnesses at high resolution. We applied this system to both cancer and normal cells and surprisingly identified ECM mixtures that support stiffness-insensitive cell spreading on soft substrates. Employing the motor-clutch model to simulate cell adhesion on biochemically distinct soft substrates, with varying numbers of available ECM-integrin-cytoskeleton (clutch) connections, we identified conditions in which spreading would be supported on soft matrices. Combining simulations and experiments, we show that cell spreading on soft is supported by increased clutch engagement on specific ECM mixtures and even augmented by the partial inhibition of actomyosin contractility. Thus, "stiff-like" spreading on soft is determined by a balance of a cell's contractile and adhesive machinery. This provides a fundamental perspective for in vitro mechanobiology studies, identifying a mechanism through which cells spread, function, and signal effectively on soft substrates.

Abstract P1140: Acute Mechanosensitive Adaptation To Phenylephrine In Cardiomyocytes And Fibroblasts

Mechanosensitive pathways are fine tuned to the physiological environment of the cell. Adrenergic stimulation induces cell type specific responses in the heart, including cardiomyocyte hypertrophy and fibroblast proliferation. We hypothesized acute induction of hypertrophy by phenylephrine (PHE) would activate mechanosensitive pathways in fibroblasts and myocytes, revealing novel molecular features of early hypertrophic adaptation. Male mice were subcutaneously injected with 20 mg/kg of PHE or PBS every other day for one week. Echocardiography revealed significant cardiac hypertrophy with increased ejection fraction (72.45% + 6.15 PHE v 56.32 + 5.32 PBS, p<0.0001) and LV wall thickness (2.25 mm + 0.29 PHE v 1.62 + 0.13 PBS, p<0.0001), along with diastolic impairment, as measured by elevated E/e’ ratio (37.25 + 7.29 PHE v 23.67 + 3.62 PBS, p<0.0001). Interestingly, histological analysis showed increased fibrotic deposition and cardiomyocyte cross-sectional area was preferential increased in fibrotic areas (265.5 um 2 + 67.5 PHE fibrotic v 195.1 + 47.46 PHE non-fibrotic v 165.6 + 46.96 PBS). While cardiac fibroblasts from PHE-treated animals did not show alpha smooth muscle actin staining, a traditional marker for activated myofibroblasts, fibroblasts from PHE-treated mice showed increased f-actin stress fibers and nuclear localization of Mrtfa, a mechanosensitive transcriptional coactivator. We performed RNA-seq on isolated cardiomyocytes, demonstrating activation of myocyte-specific responses to increased pressure ( Des, Nppa ), as well as activation of fibrotic genes ( Tgfbr1, Tgfbr2, Tgfb2 ) in myocytes . We sought to investigate mechanosensitive pathways initiated by PHE treatment in cardiomyocytes and identified transcriptional changes in Ankrd1, a Titin binding protein which translocates to the nucleus upon stress. We demonstrate a PHE induced increase in Ankrd1 protein expression and nuclear localization (nuclear area occupancy, as measured by microscopy, 17.04% + 8.76 PHE v 11.91% + 9.134 PBS, p=0.002) in cardiomyocytes. Together these findings demonstrate distinct acute phenotyptic adaptation to adrenergic stress in myocytes and fibroblasts, revealing cell type-specific mechanosensitive signaling mechanisms.