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1.
Nat Commun ; 11(1): 3282, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32612176

RESUMO

Osteocytes, cells ensconced within mineralized bone matrix, are the primary skeletal mechanosensors. Osteocytes sense mechanical cues by changes in fluid flow shear stress (FFSS) across their dendritic projections. Loading-induced reductions of osteocytic Sclerostin (encoded by Sost) expression stimulates new bone formation. However, the molecular steps linking mechanotransduction and Sost suppression remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced Sost suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces Sost mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/genética , Histona Desacetilases/genética , Mecanotransdução Celular/genética , Osteócitos/metabolismo , Transdução de Sinais/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Perfilação da Expressão Gênica/métodos , Histona Desacetilases/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteogênese/genética , Fosforilação
2.
Nature ; 584(7820): 268-273, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32728211

RESUMO

The ability of the skin to grow in response to stretching has been exploited in reconstructive surgery1. Although the response of epidermal cells to stretching has been studied in vitro2,3, it remains unclear how mechanical forces affect their behaviour in vivo. Here we develop a mouse model in which the consequences of stretching on skin epidermis can be studied at single-cell resolution. Using a multidisciplinary approach that combines clonal analysis with quantitative modelling and single-cell RNA sequencing, we show that stretching induces skin expansion by creating a transient bias in the renewal activity of epidermal stem cells, while a second subpopulation of basal progenitors remains committed to differentiation. Transcriptional and chromatin profiling identifies how cell states and gene-regulatory networks are modulated by stretching. Using pharmacological inhibitors and mouse mutants, we define the step-by-step mechanisms that control stretch-mediated tissue expansion at single-cell resolution in vivo.


Assuntos
Mecanotransdução Celular/fisiologia , Análise de Célula Única , Pele/citologia , Pele/crescimento & desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Redes Reguladoras de Genes/efeitos dos fármacos , Hidrogéis/administração & dosagem , Hidrogéis/farmacologia , Mecanotransdução Celular/efeitos dos fármacos , Mecanotransdução Celular/genética , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , RNA Mensageiro/genética , RNA-Seq , Pele/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Transativadores/antagonistas & inibidores , Transativadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcrição Genética/efeitos dos fármacos
3.
Nat Commun ; 11(1): 2997, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561714

RESUMO

PIEZO2 is the essential transduction channel for touch discrimination, vibration, and proprioception. Mice and humans lacking Piezo2 experience severe mechanosensory and proprioceptive deficits and fail to develop tactile allodynia. Bradykinin, a proalgesic agent released during inflammation, potentiates PIEZO2 activity. Molecules that decrease PIEZO2 function could reduce heightened touch responses during inflammation. Here, we find that the dietary fatty acid margaric acid (MA) decreases PIEZO2 function in a dose-dependent manner. Chimera analyses demonstrate that the PIEZO2 beam is a key region tuning MA-mediated channel inhibition. MA reduces neuronal action potential firing elicited by mechanical stimuli in mice and rat neurons and counteracts PIEZO2 sensitization by bradykinin. Finally, we demonstrate that this saturated fatty acid decreases PIEZO2 currents in touch neurons derived from human induced pluripotent stem cells. Our findings report on a natural product that inhibits PIEZO2 function and counteracts neuronal mechanical sensitization and reveal a key region for channel inhibition.


Assuntos
Ácidos Graxos/administração & dosagem , Canais Iônicos/antagonistas & inibidores , Mecanotransdução Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Propriocepção/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Algoritmos , Animais , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Canais Iônicos/genética , Canais Iônicos/metabolismo , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Neurônios/fisiologia , Propriocepção/genética , Propriocepção/fisiologia , Ratos , Tato/efeitos dos fármacos , Tato/fisiologia
5.
Artigo em Inglês | MEDLINE | ID: mdl-32159360

RESUMO

Approximately 75% of xenobiotics are primarily eliminated through metabolism; thus the accurate scaling of metabolic clearance is vital to successful drug development. Yet, when data is scaled from in vitro to in vivo, hepatic metabolic clearance, the primary source of metabolism, is still commonly underpredicted. Over the past decades, with biophysics used as a key component to restore aspects of the in vivo environment, several new cell culture settings have been investigated to improve hepatocyte functionalities. Most of these studies have focused on shear stress, i.e., flow mediated by a pressure gradient. One potential conclusion of these studies is that hepatocytes are naturally "mechanosensitive," i.e., they respond to a change in their biophysical environment. We demonstrate that hepatocytes also respond to an increase in hydrostatic pressure that, we suggest, is directly linked to the lobule geometry and vessel density. Furthermore, we demonstrate that hydrostatic pressure improves albumin production and increases cytochrome P-450 (CYP) 1A2 expression levels in an aryl hydrocarbon-dependent manner in human hepatocytes. Increased albumin production and CYP function are commonly attributed to the impacts of shear stress in microfluidic experiments. Therefore, our results highlight evidence of a novel link between hydrostatic pressure and CYP metabolism and demonstrate that the spectrum of hepatocyte mechanosensitivity might be larger than previously thought.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Citocromo P-450 CYP1A2/genética , Fígado/metabolismo , Mecanotransdução Celular/genética , Receptores de Hidrocarboneto Arílico/genética , Técnicas de Cultura de Células , Regulação da Expressão Gênica/genética , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Pressão Hidrostática , Inativação Metabólica/genética , Fígado/efeitos dos fármacos , Transdução de Sinais/genética
6.
Nat Cell Biol ; 22(4): 498-511, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203420

RESUMO

Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.


Assuntos
Citoesqueleto/genética , Proteínas Ativadoras de GTPase/genética , Integrinas/genética , Mecanotransdução Celular/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Células COS , Adesão Celular , Linhagem Celular , Movimento Celular , Chlorocebus aethiops , Biologia Computacional , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Cães , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Proteínas Ativadoras de GTPase/classificação , Proteínas Ativadoras de GTPase/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Integrinas/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Pan troglodytes , Domínios Proteicos , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho/classificação , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
7.
Cell ; 180(5): 956-967.e17, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32084332

RESUMO

Mechanotransduction, the conversion of mechanical stimuli into electrical signals, is a fundamental process underlying essential physiological functions such as touch and pain sensing, hearing, and proprioception. Although the mechanisms for some of these functions have been identified, the molecules essential to the sense of pain have remained elusive. Here we report identification of TACAN (Tmem120A), an ion channel involved in sensing mechanical pain. TACAN is expressed in a subset of nociceptors, and its heterologous expression increases mechanically evoked currents in cell lines. Purification and reconstitution of TACAN in synthetic lipids generates a functional ion channel. Finally, a nociceptor-specific inducible knockout of TACAN decreases the mechanosensitivity of nociceptors and reduces behavioral responses to painful mechanical stimuli but not to thermal or touch stimuli. We propose that TACAN is an ion channel that contributes to sensing mechanical pain.


Assuntos
Mecanotransdução Celular/genética , Nociceptores/metabolismo , Dor/genética , Tato/genética , Animais , Regulação da Expressão Gênica/genética , Humanos , Lipídeos/genética , Camundongos , Camundongos Knockout , Dor/fisiopatologia , Técnicas de Patch-Clamp , Estresse Mecânico , Tato/fisiologia
8.
J Cell Biol ; 219(2)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31914171

RESUMO

Hemidesmosomes are specialized cell-matrix adhesion structures that are associated with the keratin cytoskeleton. Although the adhesion function of hemidesmosomes has been extensively studied, their role in mechanosignaling and transduction remains largely unexplored. Here, we show that keratinocytes lacking hemidesmosomal integrin α6ß4 exhibit increased focal adhesion formation, cell spreading, and traction-force generation. Moreover, disruption of the interaction between α6ß4 and intermediate filaments or laminin-332 results in similar phenotypical changes. We further demonstrate that integrin α6ß4 regulates the activity of the mechanosensitive transcriptional regulator YAP through inhibition of Rho-ROCK-MLC- and FAK-PI3K-dependent signaling pathways. Additionally, increased tension caused by impaired hemidesmosome assembly leads to a redistribution of integrin αVß5 from clathrin lattices to focal adhesions. Our results reveal a novel role for hemidesmosomes as regulators of cellular mechanical forces and establish the existence of a mechanical coupling between adhesion complexes.


Assuntos
Hemidesmossomos/genética , Integrina alfa6beta4/genética , Queratinas/genética , Mecanotransdução Celular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Moléculas de Adesão Celular/genética , Movimento Celular/genética , Junções Célula-Matriz/genética , Junções Célula-Matriz/metabolismo , Células Cultivadas , Citoesqueleto/genética , Adesões Focais/genética , Adesões Focais/metabolismo , Humanos , Filamentos Intermediários/genética , Filamentos Intermediários/metabolismo , Queratinócitos/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Quinases Associadas a rho/genética
9.
Biochim Biophys Acta Biomembr ; 1862(4): 183176, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31923411

RESUMO

Bacterial mechanosensitive channels gate in response to membrane tension, driven by shifts in environmental osmolarity. The mechanosensitive channels of small conductance (MscS) and large conductance (MscL) from Escherichia coli (Ec) gate in response to mechanical force applied to the membrane. Ec-MscS is the foundational member of the MscS superfamily of ion channels, a diverse family with at least fifteen subfamilies identified by homology to the pore lining helix of Ec-MscS, as well as significant diversity on the N- and C-termini. The MscL family of channels are homologous to Ec-MscL. In a rhizosphere associated bacterium, Paraburkholderia graminis C4D1M, mechanosensitive channels are essential for cell survival during changing osmotic environments such as a rainstorm. Utilizing bioinformatics, we predicted six MscS superfamily members and a single MscL homologue. The MscS superfamily members fall into at least three subfamilies: bacterial cyclic nucleotide gated, multi-TM, and extended N-terminus. Osmotic downshock experiments show that wildtype P. graminis cells contain a survival mechanism that prevents cell lysis in response to hypoosmotic shock. To determine if this rescue is due to mechanosensitive channels, we developed a method to create giant spheroplasts of P. graminis to explore the single channel response to applied mechanical tension. Patch clamp electrophysiology on these spheroplasts shows two unique conductances: MscL-like and MscS-like. These conductances are due to likely three unique proteins. This indicates that channels that gate in response to mechanical tension are present in the membrane. Here, we report the first single channel evidence of mechanosensitive ion channels from P. graminis membranes.


Assuntos
Burkholderiaceae/genética , Mecanotransdução Celular/genética , Concentração Osmolar , Esferoplastos/genética , Burkholderiaceae/metabolismo , Sobrevivência Celular/genética , Microambiente Celular/genética , Biologia Computacional , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Canais Iônicos/genética , Canais Iônicos de Abertura Ativada por Ligante/genética , Pressão Osmótica , Rizosfera , Homologia de Sequência de Aminoácidos
10.
Biomolecules ; 10(2)2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31991559

RESUMO

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase with key roles in the regulation of cell adhesion migration, proliferation and survival. In cancer FAK is a major driver of invasion and metastasis and its upregulation is associated with poor patient prognosis. FAK is autoinhibited in the cytosol, but activated upon localisation into a protein complex, known as focal adhesion complex. This complex forms upon cell adhesion to the extracellular matrix (ECM) at the cytoplasmic side of the plasma membrane at sites of ECM attachment. FAK is anchored to the complex via multiple sites, including direct interactions with specific membrane lipids and connector proteins that attach focal adhesions to the actin cytoskeleton. In migrating cells, the contraction of actomyosin stress fibres attached to the focal adhesion complex apply a force to the complex, which is likely transmitted to the FAK protein, causing stretching of the FAK molecule. In this review we discuss the current knowledge of the FAK structure and how specific structural features are involved in the regulation of FAK signalling. We focus on two major regulatory mechanisms known to contribute to FAK activation, namely interactions with membrane lipids and stretching forces applied to FAK, and discuss how they might induce structural changes that facilitate FAK activation.


Assuntos
Adesão Celular/genética , Quinase 1 de Adesão Focal/genética , Adesões Focais/genética , Mecanotransdução Celular/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Movimento Celular , Quinase 1 de Adesão Focal/ultraestrutura , Adesões Focais/ultraestrutura , Humanos , Membranas/ultraestrutura , Fosforilação , Transdução de Sinais/genética
11.
Nat Rev Cardiol ; 17(1): 52-63, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31366922

RESUMO

Flowing blood generates a frictional force called shear stress that has major effects on vascular function. Branches and bends of arteries are exposed to complex blood flow patterns that exert low or low oscillatory shear stress, a mechanical environment that promotes vascular dysfunction and atherosclerosis. Conversely, physiologically high shear stress is protective. Endothelial cells are critical sensors of shear stress but the mechanisms by which they decode complex shear stress environments to regulate physiological and pathophysiological responses remain incompletely understood. Several laboratories have advanced this field by integrating specialized shear-stress models with systems biology approaches, including transcriptome, methylome and proteome profiling and functional screening platforms, for unbiased identification of novel mechanosensitive signalling pathways in arteries. In this Review, we describe these studies, which reveal that shear stress regulates diverse processes and demonstrate that multiple pathways classically known to be involved in embryonic development, such as BMP-TGFß, WNT, Notch, HIF1α, TWIST1 and HOX family genes, are regulated by shear stress in arteries in adults. We propose that mechanical activation of these pathways evolved to orchestrate vascular development but also drives atherosclerosis in low shear stress regions of adult arteries.


Assuntos
Aterosclerose/genética , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mecanotransdução Celular/genética , Animais , Aterosclerose/metabolismo , Aterosclerose/fisiopatologia , Endotélio Vascular/fisiopatologia , Predisposição Genética para Doença , Humanos , Neovascularização Fisiológica/genética , Fenótipo , Fluxo Sanguíneo Regional , Fatores de Risco , Estresse Mecânico , Remodelação Vascular/genética
12.
Biochim Biophys Acta Gen Subj ; 1864(1): 129433, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31520681

RESUMO

BACKGROUND: Kidney disease modeling and assessment of drug-induced kidney injury can be advanced using three-dimensional (3D) microfluidic models that recapitulate in vivo characteristics. Fluid shear stress (FSS) has been depicted as main modulator improving in vitro physiology in proximal tubule epithelial cells (PTECs). We aimed to elucidate the role of FSS and primary cilia on transport activity and morphology in PTECs. METHODS: Human conditionally immortalized PTEC (ciPTEC-parent) was cultured in a microfluidic 3D device, the OrganoPlate, under a physiological peak FSS of 2.0 dyne/cm2 or low peak FSS of 0.5 dyne/cm2. Upon a 9-day exposure to FSS, albumin-FITC uptake, activity of P-glycoprotein (P-gp) and multidrug resistance-associated proteins 2/4 (MRP2/4), cytotoxicity and cell morphology were determined. RESULTS: A primary cilium knock-out cell model, ciPTEC-KIF3α-/-, was successfully established via CRISPR-Cas9 genome editing. Under physiological peak FSS, albumin-FITC uptake (p = .04) and P-gp efflux (p = .002) were increased as compared to low FSS. Remarkably, a higher albumin-FITC uptake (p = .03) and similar trends in activity of P-gp and MRP2/4 were observed in ciPTEC-KIF3α-/-. FSS induced cell elongation corresponding with the direction of flow in both cell models, but had no effect on cyclosporine A-induced cytotoxicity. CONCLUSIONS: FSS increased albumin uptake, P-gp efflux and cell elongation, but this was not attributed to a mechanosensitive mechanism related to primary cilia in PTECs, but likely to microvilli present at the apical membrane. GENERAL SIGNIFICANCE: FSS-induced improvements in biological characteristics and activity in PTECs was not mediated through a primary cilium-related mechanism.


Assuntos
Lesão Renal Aguda/metabolismo , Cílios/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Lesão Renal Aguda/induzido quimicamente , Lesão Renal Aguda/genética , Transporte Biológico/efeitos dos fármacos , Cílios/efeitos dos fármacos , Ciclosporina/toxicidade , Células Epiteliais/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/metabolismo , Mecanotransdução Celular/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Resistência ao Cisalhamento , Estresse Mecânico
13.
Acta Biochim Pol ; 66(4): 491-498, 2019 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-31883439

RESUMO

Mesenchymal Stem/Stromal Cells (MSCs) have been widely considered as a promising source of cells for tissue regeneration. Among other stem cells, they are characterized by a high osteogenic potential. Intensive studies in this field had shown that even if basic osteogenic differentiation is relatively simple, its clinical application requires more sophisticated approaches to prepare effective and safe cell therapy products. The aim of this review is to underline biological, physical and chemical factors which play a crucial role in osteogenic differentiation of MSCs. Existence of two distinct mechanisms of ossification (intramembranous and endochondral) indicate that choosing a proper source of MSCs may be critical for successful regeneration of a particular bone type. In this context, Dental Pulp Stem Cells representing a group of MSCs and originating from neural crest ( a structure responsible for development of cranial bones) are considered as the most promising for skull bone defect repair. Factors which facilitate osteogenic differentiation of MSCs include changes in forces exerted on cells during development. Thus, culturing of cells in hydrogels or on biocompatible three-dimensional scaffolds improves osteogenic differentiation of MSCs by both, the mechanotransductive and chemical impact on cells. Moreover, atmospheric oxygen concentration routinely used for cell cultures in vitro does not correspond to lower oxygen concentration present in stem cell niches. A decrease in oxygen concentration allows to create more physiological cell culture conditions, mimicking the ones in stem cell niches, which promote the MSCs stemness. Altogether, factors discussed in this review provide exciting opportunities to boost MSCs propagation and osteogenic differentiation which is crucial for successful clinical applications.


Assuntos
Regeneração Óssea/genética , Técnicas de Cultura de Células , Células-Tronco Mesenquimais , Osteogênese/genética , Tecido Adiposo/crescimento & desenvolvimento , Tecido Adiposo/metabolismo , Diferenciação Celular/genética , Humanos , Hidrogéis/farmacologia , Mecanotransdução Celular/genética , Oxigênio/metabolismo , Nicho de Células-Tronco/genética , Tecidos Suporte
14.
Elife ; 82019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31880537

RESUMO

Prokaryotic mechanosensitive (MS) channels open by sensing the physical state of the membrane. As such, lipid-protein interactions represent the defining molecular process underlying mechanotransduction. Here, we describe cryo-electron microscopy (cryo-EM) structures of the E. coli small-conductance mechanosensitive channel (MscS) in nanodiscs (ND). They reveal a novel membrane-anchoring fold that plays a significant role in channel activation and establish a new location for the lipid bilayer, shifted ~14 Å from previous consensus placements. Two types of lipid densities are explicitly observed. A phospholipid that 'hooks' the top of each TM2-TM3 hairpin and likely plays a role in force sensing, and a bundle of acyl chains occluding the permeation path above the L105 cuff. These observations reshape our understanding of force-from-lipids gating in MscS and highlight the key role of allosteric interactions between TM segments and phospholipids bound to key dynamic components of the channel.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/ultraestrutura , Lipídeos/química , Mecanotransdução Celular/genética , Microscopia Crioeletrônica , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Canais Iônicos/química , Canais Iônicos/genética , Bicamadas Lipídicas/química , Lipídeos/genética , Nanoestruturas/química
15.
ACS Appl Mater Interfaces ; 11(49): 45520-45530, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31714734

RESUMO

Recent studies have found discordant mechanosensitive outcomes when comparing 2D and 3D, highlighting the need for tools to study mechanotransduction in 3D across a wide spectrum of stiffness. A gelatin methacryloyl (GelMA) hydrogel with a continuous stiffness gradient ranging from 5 to 38 kPa was developed to recapitulate physiological stiffness conditions. Adipose-derived stem cells (ASCs) were encapsulated in this hydrogel, and their morphological characteristics and expression of both mechanosensitive proteins (Lamin A, YAP, and MRTFa) and differentiation markers (PPARγ and RUNX2) were analyzed. Low-stiffness regions (∼8 kPa) permitted increased cellular and nuclear volume and enhanced mechanosensitive protein localization in the nucleus. This trend was reversed in high stiffness regions (∼30 kPa), where decreased cellular and nuclear volumes and reduced mechanosensitive protein nuclear localization were observed. Interestingly, cells in soft regions exhibited enhanced osteogenic RUNX2 expression, while those in stiff regions upregulated the adipogenic regulator PPARγ, suggesting that volume, not substrate stiffness, is sufficient to drive 3D stem cell differentiation. Inhibition of myosin II (Blebbistatin) and ROCK (Y-27632), both key drivers of actomyosin contractility, resulted in reduced cell volume, especially in low-stiffness regions, causing a decorrelation between volume expansion and mechanosensitive protein localization. Constitutively active and inactive forms of the canonical downstream mechanotransduction effector TAZ were stably transfected into ASCs. Activated TAZ resulted in higher cellular volume despite increasing stiffness and a consistent, stiffness-independent translocation of YAP and MRTFa into the nucleus. Thus, volume adaptation as a function of 3D matrix stiffness can control stem cell mechanotransduction and differentiation.


Assuntos
Adipogenia/genética , Diferenciação Celular/efeitos dos fármacos , Mecanotransdução Celular/genética , Osteogênese/genética , Citoesqueleto de Actina/genética , Actomiosina/genética , Adipogenia/efeitos dos fármacos , Amidas/farmacologia , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Encapsulamento de Células/métodos , Núcleo Celular/química , Tamanho Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Gelatina/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Lamina Tipo A/genética , Células-Tronco Mesenquimais/citologia , Miosina Tipo II/genética , PPAR gama/genética , Piridinas/farmacologia , Transativadores/genética , Fatores de Transcrição/genética , Quinases Associadas a rho/genética
16.
PLoS Genet ; 15(11): e1008454, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31697683

RESUMO

α-catenin is a key protein of adherens junctions (AJs) with mechanosensory properties. It also acts as a tumor suppressor that limits tissue growth. Here we analyzed the function of Drosophila α-Catenin (α-Cat) in growth regulation of the wing epithelium. We found that different α-Cat levels led to a differential activation of Hippo/Yorkie or JNK signaling causing tissue overgrowth or degeneration, respectively. α-Cat can modulate Yorkie-dependent tissue growth through recruitment of Ajuba, a negative regulator of Hippo signaling to AJs but also through a mechanism independent of Ajuba recruitment to AJs. Both mechanosensory regions of α-Cat, the M region and the actin-binding domain (ABD), contribute to growth regulation. Whereas M is dispensable for α-Cat function in the wing, individual M domains (M1, M2, M3) have opposing effects on growth regulation. In particular, M1 limits Ajuba recruitment. Loss of M1 causes Ajuba hyper-recruitment to AJs, promoting tissue-tension independent overgrowth. Although M1 binds Vinculin, Vinculin is not responsible for this effect. Moreover, disruption of mechanosensing of the α-Cat ABD affects tissue growth, with enhanced actin interactions stabilizing junctions and leading to tissue overgrowth. Together, our findings indicate that α-Cat acts through multiple mechanisms to control tissue growth, including regulation of AJ stability, mechanosensitive Ajuba recruitment, and dynamic direct F-actin interactions.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas com Domínio LIM/genética , Asas de Animais/crescimento & desenvolvimento , alfa Catenina/genética , Citoesqueleto de Actina/genética , Actinas/genética , Junções Aderentes/genética , Animais , Morte Celular/genética , Citoesqueleto/genética , Drosophila melanogaster/crescimento & desenvolvimento , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases/genética , Mecanotransdução Celular/genética , Proteínas Nucleares/genética , Domínios Proteicos/genética , Proteínas Serina-Treonina Quinases/genética , Transativadores/genética , Vinculina/genética , Asas de Animais/metabolismo
17.
Proc Natl Acad Sci U S A ; 116(34): 16949-16954, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31391300

RESUMO

The advent of molecular tension probes for real-time mapping of piconewton forces in living systems has had a major impact on mechanobiology. For example, DNA-based tension probes have revealed roles for mechanics in platelet, B cell, T cell, and fibroblast function. Nonetheless, imaging short-lived forces transmitted by low-abundance receptors remains a challenge. This is a particular problem for mechanoimmunology where ligand-receptor bindings are short lived, and a few antigens are sufficient for cell triggering. Herein, we present a mechanoselection strategy that uses locking oligonucleotides to preferentially and irreversibly bind DNA probes that are mechanically strained over probes at rest. Thus, infrequent and short-lived mechanical events are tagged. This strategy allows for integration and storage of mechanical information into a map of molecular tension history. Upon addition of unlocking oligonucleotides that drive toehold-mediated strand displacement, the probes reset to the real-time state, thereby erasing stored mechanical information. As a proof of concept, we applied this strategy to study OT-1 T cells, revealing that the T cell receptor (TCR) mechanically samples antigens carrying single amino acid mutations. Such events are not detectable using conventional tension probes. Each mutant peptide ligand displayed a different level of mechanical sampling and spatial scanning by the TCR that strongly correlated with its functional potency. Finally, we show evidence that T cells transmit pN forces through the programmed cell death receptor-1 (PD1), a major target in cancer immunotherapy. We anticipate that mechanical information storage will be broadly useful in studying the mechanobiology of the immune system.


Assuntos
Antígenos , Sondas de DNA , Mecanotransdução Celular , Peptídeos , Receptores de Antígenos de Linfócitos T , Linfócitos T , Antígenos/química , Antígenos/genética , Antígenos/imunologia , Linhagem Celular , Sondas de DNA/química , Sondas de DNA/genética , Sondas de DNA/imunologia , Humanos , Mecanotransdução Celular/genética , Mecanotransdução Celular/imunologia , Mutação , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/química , Linfócitos T/citologia , Linfócitos T/imunologia
18.
J Biomed Sci ; 26(1): 56, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31387590

RESUMO

BACKGROUND: Endothelial cell (EC) dysfunctions, including turnover enrichment, gap junction disruption, inflammation, and oxidation, play vital roles in the initiation of vascular disorders and atherosclerosis. Hemodynamic forces, i.e., atherprotective pulsatile (PS) and pro-atherogenic oscillatory shear stress (OS), can activate mechanotransduction to modulate EC function and dysfunction. This review summarizes current studies aiming to elucidate the roles of epigenetic factors, i.e., histone deacetylases (HDACs), non-coding RNAs, and DNA methyltransferases (DNMTs), in mechanotransduction to modulate hemodynamics-regulated EC function and dysfunction. OS enhances the expression and nuclear accumulation of class I and class II HDACs to induce EC dysfunction, i.e., proliferation, oxidation, and inflammation, whereas PS induces phosphorylation-dependent nuclear export of class II HDACs to inhibit EC dysfunction. PS induces overexpression of the class III HDAC Sirt1 to enhance nitric oxide (NO) production and prevent EC dysfunction. In addition, hemodynamic forces modulate the expression and acetylation of transcription factors, i.e., retinoic acid receptor α and krüppel-like factor-2, to transcriptionally regulate the expression of microRNAs (miRs). OS-modulated miRs, which stimulate proliferative, pro-inflammatory, and oxidative signaling, promote EC dysfunction, whereas PS-regulated miRs, which induce anti-proliferative, anti-inflammatory, and anti-oxidative signaling, inhibit EC dysfunction. PS also modulates the expression of long non-coding RNAs to influence EC function. i.e., turnover, aligmant, and migration. On the other hand, OS enhances the expression of DNMT-1 and -3a to induce EC dysfunction, i.e., proliferation, inflammation, and NO repression. CONCLUSION: Overall, epigenetic factors play vital roles in modulating hemodynamic-directed EC dysfunction and vascular disorders, i.e., atherosclerosis. Understanding the detailed mechanisms through which epigenetic factors regulate hemodynamics-directed EC dysfunction and vascular disorders can help us to elucidate the pathogenic mechanisms of atherosclerosis and develop potential therapeutic strategies for atherosclerosis treatment.


Assuntos
Aterosclerose/fisiopatologia , Células Endoteliais/fisiologia , Epigênese Genética , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Metilação de DNA/genética , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Hemodinâmica , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Mecanotransdução Celular/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
19.
Nano Lett ; 19(9): 5982-5990, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31389241

RESUMO

KANK proteins mediate cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix. KANKs interact with the integrin/actin-binding protein talin and with several components of microtubule-stabilizing cortical complexes. Because of actomyosin contractility, the talin-KANK complex is likely under mechanical force, and its mechanical stability is expected to be a critical determinant of KANK recruitment to focal adhesions. Here, we quantified the lifetime of the complex of the talin rod domain R7 and the KN domain of KANK1 under shear-force geometry and found that it can withstand forces for seconds to minutes over a physiological force range up to 10 pN. Complex stability measurements combined with cell biological experiments suggest that shear-force stretching promotes KANK1 localization to the periphery of focal adhesions. These results indicate that the talin-KANK1 complex is mechanically strong, enabling it to support the cross-talk between microtubule and actin cytoskeleton at focal adhesions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas do Citoesqueleto/química , Adesões Focais/química , Complexos Multiproteicos/química , Talina/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Actomiosina/química , Actomiosina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adesão Celular/genética , Proteínas do Citoesqueleto/genética , Matriz Extracelular/química , Matriz Extracelular/genética , Adesões Focais/genética , Células HeLa , Humanos , Integrinas/química , Integrinas/genética , Fenômenos Mecânicos , Mecanotransdução Celular/genética , Microtúbulos/química , Microtúbulos/genética , Complexos Multiproteicos/genética , Contração Muscular/genética , Resistência ao Cisalhamento/fisiologia , Talina/genética
20.
Oncogene ; 38(42): 6818-6834, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31406247

RESUMO

Tumor-repopulating cells (TRCs) are cancer stem cell (CSC)-like cells with highly tumorigenic and self-renewing abilities, which were selected from tumor cells in soft three-dimensional (3D) fibrin gels with unidentified mechanisms. Here we evaluated the transcriptome alteration during TRCs generation in 3D culture and revealed that a variety of molecules related with integrin/membrane and stemness were continuously altered by mechanical environment. Some key regulators such as MYC/STAT3/hsa-miR-199a-5p, were changed in the TRCs generation. They regulated membrane genes and the downstream mechanotransduction pathways such as Hippo/WNT/TGF-ß/PI3K-AKT pathways, thus further affecting the expression of downstream cancer-related genes. By integrating networks for membrane proteins, the WNT pathway and cancer-related genes, we identified key molecules in the selection of TRCs, such as ATF4, SLC3A2, CCT3, and hsa-miR-199a-5p. Silencing ATF4 or CCT3 inhibited the selection and growth of TRCs whereas reduction of SLC3A2 or hsa-miR-199a-5p promoted TRCs growth. Further studies showed that CCT3 promoted cell proliferation and stemness in vitro, while its suppression inhibited TRCs-induced tumor formation. We also contemplated CCT3 as a stemness-related gene. Our findings provide insights in the mechanism of TRCs selection through transcriptome analysis.


Assuntos
Proliferação de Células/genética , Redes Reguladoras de Genes , Mecanotransdução Celular/genética , Células-Tronco Neoplásicas/patologia , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos
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