RESUMO
Many mechanobiological processes that govern development and tissue homeostasis are regulated on the level of individual molecular linkages, and a number of proteins experiencing piconewton-scale forces in cells have been identified. However, under which conditions these force-bearing linkages become critical for a given mechanobiological process is often still unclear. Here, we established an approach to revealing the mechanical function of intracellular molecules using molecular optomechanics. When applied to the integrin activator talin, the technique provides direct evidence that its role as a mechanical linker is indispensable for the maintenance of cell-matrix adhesions and overall cell integrity. Applying the technique to desmoplakin shows that mechanical engagement of desmosomes to intermediate filaments is expendable under homeostatic conditions yet strictly required for preserving cell-cell adhesion under stress. These results reveal a central role of talin and desmoplakin as mechanical linkers in cell adhesion structures and demonstrate that molecular optomechanics is a powerful tool to investigate the molecular details of mechanobiological processes.
Assuntos
Integrinas , Talina , Talina/metabolismo , Desmoplaquinas/genética , Desmoplaquinas/metabolismo , Adesão Celular/fisiologia , Integrinas/metabolismo , Filamentos IntermediáriosRESUMO
Hypoxia is an essential regulator of cell metabolism, affects cell migration and angiogenesis during development and contributes to a wide range of pathological conditions. Multiple techniques to assess hypoxia through oxygen-imaging have been developed. However, significant limitations include low spatiotemporal resolution, limited tissue penetration of exogenous probes and non-dynamic signals due to irreversible probe-chemistry. First genetically-encoded reporters only partly overcame these limitations as the green and red fluorescent proteins (GFP/RFP) families require molecular oxygen for fluorescence. For the herein presented ratiometric and FRET-FLIM reporters dUnORS and dUnOFLS, we exploited oxygen-dependent maturation in combination with the hypoxia-tolerant fluorescent-protein UnaG. For ratiometric measurements, UnaG was fused to the orange large Stokes Shift protein CyOFP1, allowing excitation with a single light-source, while fusion of UnaG with mOrange2 allowed FRET-FLIM analysis. Imaging live or fixed cultured cells for calibration, we applied both reporters in spheroid and tumor transplantation-models and obtained graded information on oxygen-availability at cellular resolution, establishing these sensors as promising tools for visualizing oxygen-gradients in-vivo.
Assuntos
Técnicas Biossensoriais , Microscopia , Humanos , Oxigênio , Ionóforos , Corantes Fluorescentes , HipóxiaRESUMO
Genetically encoded Förster Resonance Energy Transfer (FRET)-based tension sensors were developed to enable the quantification of piconewton (pN)-scale forces that act across distinct proteins in living cells and organisms. An important extension of this technology is the multiplexing of tension sensors to monitor several independent FRET probes in parallel. Here we describe how pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) can be implemented to enable the analysis of two co-expressed tension sensor constructs. Our protocol covers all essential steps from biosensor expression and live cell PIE image acquisition to lifetime calculations.
Assuntos
Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas , Microscopia de Fluorescência/métodosRESUMO
Mechanoelectrical transduction is mediated by the opening of different types of force-sensitive ion channels, including Piezo1/2 and the TREK/TRAAK K2P channels. Piezo1 curves the membrane locally into an inverted dome that reversibly flattens in response to force application. Moreover, Piezo1 forms numerous preferential interactions with various membrane lipids, including cholesterol. Whether this structural architecture influences the functionality of neighboring membrane proteins is unknown. Here, we show that Piezo1/2 increase TREK/TRAAK current amplitude, slow down activation/deactivation, and remove inactivation upon mechanical stimulation. These findings are consistent with a mechanism whereby Piezo1/2 cause a local depletion of membrane cholesterol associated with a prestress of TREK/TRAAK channels. This regulation occurs in mouse fibroblasts between endogenous Piezo1 and TREK-1/2, both channel types acting in concert to delay wound healing. In conclusion, we demonstrate a community effect between different structural and functional classes of mechanosensitive ion channels.
Assuntos
Ativação do Canal Iônico , Canais Iônicos/fisiologia , Mecanotransdução Celular , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Colesterol/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Gengiva/citologia , Gengiva/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Potássio de Domínios Poros em Tandem/genéticaRESUMO
Talin is a cell adhesion molecule that is indispensable for the development and function of multicellular organisms. Despite its central role for many cell biological processes, suitable methods to investigate the nanoscale organization of talin in its native environment are missing. Here, we overcome this limitation by combining single-molecule resolved PAINT (points accumulation in nanoscale topography) imaging with the IRIS (image reconstruction by integrating exchangeable single-molecule localization) approach, enabling the quantitative analysis of genetically unmodified talin molecules in cells. We demonstrate that a previously reported peptide can be utilized to specifically label the two major talin isoforms expressed in mammalian tissues with a localization precision of <10â nm. Our experiments show that the methodology performs equally well as state-of-the-art single-molecule localization techniques, and the first applications reveal a thus far undescribed cell adhesion structure in differentiating stem cells. Furthermore, we demonstrate the applicability of this peptide-PAINT technique to mouse tissues paving the way to single-protein imaging of endogenous talin proteins under physiologically relevant conditions.
Assuntos
Peptídeos/metabolismo , Células-Tronco/metabolismo , Talina/metabolismo , Animais , Adesão Celular , Camundongos , Microscopia de Fluorescência , Peptídeos/química , Células-Tronco/química , Talina/químicaRESUMO
The ability of cells to generate mechanical forces, but also to sense, adapt to, and respond to mechanical signals, is crucial for many developmental, postnatal homeostatic, and pathophysiological processes. However, the molecular mechanisms underlying cellular mechanotransduction have remained elusive for many decades, as techniques to visualize and quantify molecular forces across individual proteins in cells were missing. The development of genetically encoded molecular tension sensors now allows the quantification of piconewton-scale forces that act upon distinct molecules in living cells and even whole organisms. In this review, we discuss the physical principles, advantages, and limitations of this increasingly popular method. By highlighting current examples from the literature, we demonstrate how molecular tension sensors can be utilized to obtain access to previously unappreciated biophysical parameters that define the propagation of mechanical forces on molecular scales. We discuss how the methodology can be further developed and provide a perspective on how the technique could be applied to uncover entirely novel aspects of mechanobiology in the future.
Assuntos
Mecanotransdução Celular , Animais , Transferência Ressonante de Energia de Fluorescência , Humanos , ProteínasRESUMO
Leukocyte extravasation is an essential step during the immune response and requires the destabilization of endothelial junctions. We have shown previously that this process depends in vivo on the dephosphorylation of VE-cadherin-Y731. Here, we reveal the underlying mechanism. Leukocyte-induced stimulation of PECAM-1 triggers dissociation of the phosphatase SHP2 which then directly targets VE-cadherin-Y731. The binding site of PECAM-1 for SHP2 is needed for VE-cadherin dephosphorylation and subsequent endocytosis. Importantly, the contribution of PECAM-1 to leukocyte diapedesis in vitro and in vivo was strictly dependent on the presence of Y731 of VE-cadherin. In addition to SHP2, dephosphorylation of Y731 required Ca2+ -signaling, non-muscle myosin II activation, and endothelial cell tension. Since we found that ß-catenin/plakoglobin mask VE-cadherin-Y731 and leukocyte docking to endothelial cells exert force on the VE-cadherin-catenin complex, we propose that leukocytes destabilize junctions by PECAM-1-SHP2-triggered dephosphorylation of VE-cadherin-Y731 which becomes accessible by actomyosin-mediated mechanical force exerted on the VE-cadherin-catenin complex.
Assuntos
Antígenos CD/química , Antígenos CD/genética , Caderinas/química , Caderinas/genética , Leucócitos/citologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Actomiosina/metabolismo , Animais , Sinalização do Cálcio , Técnicas de Introdução de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucócitos/metabolismo , Camundongos , Fosforilação , Migração Transendotelial e Transepitelial , Tirosina/químicaRESUMO
Single-molecule localization microscopy (SMLM) enabling the investigation of individual proteins on molecular scales has revolutionized how biological processes are analysed in cells. However, a major limitation of imaging techniques reaching single-protein resolution is the incomplete and often unknown labeling and detection efficiency of the utilized molecular probes. As a result, fundamental processes such as complex formation of distinct molecular species cannot be reliably quantified. Here, we establish a super-resolution microscopy framework, called quantitative single-molecule colocalization analysis (qSMCL), which permits the identification of absolute molecular quantities and thus the investigation of molecular-scale processes inside cells. The method combines multiplexed single-protein resolution imaging, automated cluster detection, in silico data simulation procedures, and widely applicable experimental controls to determine absolute fractions and spatial coordinates of interacting species on a true molecular level, even in highly crowded subcellular structures. The first application of this framework allowed the identification of a long-sought ternary adhesion complex-consisting of talin, kindlin and active ß1-integrin-that specifically forms in cell-matrix adhesion sites. Together, the experiments demonstrate that qSMCL allows an absolute quantification of multiplexed SMLM data and thus should be useful for investigating molecular mechanisms underlying numerous processes in cells.
Assuntos
Proteínas do Citoesqueleto/química , Integrina beta1/química , Proteínas Musculares/química , Imagem Individual de Molécula/métodos , Talina/química , Animais , Adesão Celular , Linhagem Celular , Humanos , Camundongos , Imagem Individual de Molécula/instrumentaçãoRESUMO
Vinculin is a ubiquitously expressed protein, crucial for the regulation of force transduction in cells. Muscle cells express a vinculin splice-isoform called metavinculin, which has been associated with cardiomyopathies. However, the molecular function of metavinculin has remained unclear and its role for heart muscle disorders undefined. Here, we have employed a set of piconewton-sensitive tension sensors to probe metavinculin mechanics in cells. Our experiments reveal that metavinculin bears higher molecular forces but is less frequently engaged as compared to vinculin, leading to altered force propagation in cell adhesions. In addition, we have generated knockout mice to investigate the consequences of metavinculin loss in vivo. Unexpectedly, these animals display an unaltered tissue response in a cardiac hypertrophy model. Together, the data reveal that the transduction of cell adhesion forces is modulated by expression of metavinculin, yet its role for heart muscle function seems more subtle than previously thought.
Assuntos
Adesão Celular/fisiologia , Miocárdio/citologia , Vinculina/metabolismo , Animais , Fibroblastos , Recuperação de Fluorescência Após Fotodegradação , Adesões Focais/fisiologia , Expressão Gênica , Células HEK293 , Humanos , Integrinas/metabolismo , Junções Intercelulares/fisiologia , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Talina/metabolismo , Vinculina/genéticaRESUMO
Genetically encoded Förster resonance energy transfer (FRET)-based tension sensors measure piconewton-scale forces across individual molecules in living cells or whole organisms. These biosensors show comparably high FRET efficiencies in the absence of tension, but FRET quickly decreases when forces are applied. In this article, we describe how such biosensors can be generated for a specific protein of interest, and we discuss controls to confirm that the observed differences in FRET efficiency reflect changes in molecular tension. These FRET efficiency changes can be related to mechanical forces as the FRET-force relationship of the employed tension sensor modules are calibrated. We provide information on construct generation, expression in cells, and image acquisition using live-cell fluorescence lifetime imaging microscopy (FLIM). Moreover, we describe how to analyze, statistically evaluate, and interpret the resulting data sets. Together, these protocols should enable the reader to plan, execute, and interpret FRET-based tension sensor experiments. © 2019 by John Wiley & Sons, Inc.
Assuntos
Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência/métodos , DNA/química , Escherichia coli/genética , Técnicas de Amplificação de Ácido Nucleico , Transformação GenéticaRESUMO
Cells in developing organisms are subjected to particular mechanical forces that shape tissues and instruct cell fate decisions. How these forces are sensed and transmitted at the molecular level is therefore an important question, one that has mainly been investigated in cultured cells in vitro. Here, we elucidate how mechanical forces are transmitted in an intact organism. We studied Drosophila muscle attachment sites, which experience high mechanical forces during development and require integrin-mediated adhesion for stable attachment to tendons. Therefore, we quantified molecular forces across the essential integrin-binding protein Talin, which links integrin to the actin cytoskeleton. Generating flies expressing 3 Förster resonance energy transfer (FRET)-based Talin tension sensors reporting different force levels between 1 and 11 piconewton (pN) enabled us to quantify physiologically relevant molecular forces. By measuring primary Drosophila muscle cells, we demonstrate that Drosophila Talin experiences mechanical forces in cell culture that are similar to those previously reported for Talin in mammalian cell lines. However, in vivo force measurements at developing flight muscle attachment sites revealed that average forces across Talin are comparatively low and decrease even further while attachments mature and tissue-level tension remains high. Concomitantly, the Talin concentration at attachment sites increases 5-fold as quantified by fluorescence correlation spectroscopy (FCS), suggesting that only a small proportion of Talin molecules are mechanically engaged at any given time. Reducing Talin levels at late stages of muscle development results in muscle-tendon rupture in the adult fly, likely as a result of active muscle contractions. We therefore propose that a large pool of adhesion molecules is required to share high tissue forces. As a result, less than 15% of the molecules experience detectable forces at developing muscle attachment sites at the same time. Our findings define an important new concept of how cells can adapt to changes in tissue mechanics to prevent mechanical failure in vivo.
Assuntos
Desenvolvimento Muscular/fisiologia , Sarcômeros/metabolismo , Talina/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Western Blotting , Células Cultivadas , Drosophila , Matriz Extracelular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Adesões Focais/metabolismo , Adesões Focais/fisiologia , Integrinas/genética , Integrinas/metabolismo , Masculino , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Ligação Proteica , Talina/genética , Tendões/metabolismoRESUMO
The ability of cells to sense and respond to mechanical stimuli is crucial for many developmental and homeostatic processes, while mechanical dysfunction of cells has been associated with numerous pathologies including muscular dystrophies, cardiovascular defects and epithelial disorders. Yet, how cells detect and process mechanical information is still largely unclear. In this review, we outline major mechanisms underlying cellular mechanotransduction and we summarize the current understanding of how cells integrate information from distinct mechanosensitive structures to mediate complex mechanoresponses. We also discuss the concept of mechanical memory and describe how cells store information on previous mechanical events for different periods of time.
Assuntos
Mecanotransdução Celular , Animais , Fenômenos Biofísicos , Humanos , Canais Iônicos/metabolismo , Transdução de SinaisRESUMO
Desmosomes are intercellular adhesion complexes that connect the intermediate filament cytoskeletons of neighboring cells, and are essential for the mechanical integrity of mammalian tissues. Mutations in desmosomal proteins cause severe human pathologies including epithelial blistering and heart muscle dysfunction. However, direct evidence for their load-bearing nature is lacking. Here we develop Förster resonance energy transfer (FRET)-based tension sensors to measure the forces experienced by desmoplakin, an obligate desmosomal protein that links the desmosomal plaque to intermediate filaments. Our experiments reveal that desmoplakin does not experience significant tension under most conditions, but instead becomes mechanically loaded when cells are exposed to external mechanical stresses. Stress-induced loading of desmoplakin is transient and sensitive to the magnitude and orientation of the applied tissue deformation, consistent with a stress absorbing function for desmosomes that is distinct from previously analyzed cell adhesion complexes.
Assuntos
Desmossomos/química , Animais , Adesão Celular , Citoesqueleto/química , Citoesqueleto/metabolismo , Desmoplaquinas/química , Desmoplaquinas/metabolismo , Desmossomos/metabolismo , Cães , Filamentos Intermediários/química , Filamentos Intermediários/metabolismo , Células Madin Darby de Rim Canino , Estresse MecânicoRESUMO
Förster resonance energy transfer (FRET)-based tension sensor modules (TSMs) are available for investigating how distinct proteins bear mechanical forces in cells. Yet, forces in the single piconewton (pN) regime remain difficult to resolve, and tools for multiplexed tension sensing are lacking. Here, we report the generation and calibration of a genetically encoded, FRET-based biosensor called FL-TSM, which is characterized by a near-digital force response and increased sensitivity at 3-5 pN. In addition, we present a method allowing the simultaneous evaluation of coexpressed tension sensor constructs using two-color fluorescence lifetime microscopy. Finally, we introduce a procedure to calculate the fraction of mechanically engaged molecules within cells. Application of these techniques to new talin biosensors reveals an intramolecular tension gradient across talin-1 that is established upon integrin-mediated cell adhesion. The tension gradient is actomyosin- and vinculin-dependent and sensitive to the rigidity of the extracellular environment.
Assuntos
Talina/química , Calibragem , Transferência Ressonante de Energia de Fluorescência , Adesões Focais/química , Microscopia de Fluorescência , Miosinas/químicaRESUMO
The ability of cells to adhere and sense their mechano-chemical environment is key to many developmental, postnatal homeostatic and pathological processes; however, the underlying molecular mechanisms are still poorly understood. Here, we summarize recent progress that indicates how cell adhesion, mechanotransduction and chemical signaling are coordinated in cells, and we discuss how the combination of novel experimental approaches with theoretical studies is currently utilized to unravel the molecular mechanisms governing mechano-chemical coupling during cell adhesion.
Assuntos
Matriz Extracelular/metabolismo , Mecanotransdução Celular , Animais , Adesão Celular , Humanos , Integrinas/metabolismo , Modelos Biológicos , Transdução de SinaisRESUMO
The ability of cells to sense and respond to mechanical forces is crucial for a wide range of developmental and pathophysiological processes. The molecular mechanisms underlying cellular mechanotransduction, however, are largely unknown because suitable techniques to measure mechanical forces across individual molecules in cells have been missing. In this article, we highlight advances in the development of molecular force sensing techniques and discuss our recently expanded set of FRET-based tension sensors that allows the analysis of mechanical forces with piconewton sensitivity in cells. In addition, we provide a theoretical framework for the design of additional tension sensor modules with adjusted force sensitivity.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Mecanotransdução Celular , Microscopia de Força Atômica/métodos , Fenômenos Mecânicos , Estresse MecânicoRESUMO
The development of calibrated Förster resonance energy transfer (FRET)-based tension sensors has allowed the first analyses of mechanical processes with piconewton (pN) sensitivity in cells. Here, we introduce the working principle of this emerging microscopy method and discuss how it has been utilized to obtain quantitative insights into the mechanisms of intracellular force transduction in cell-matrix adhesions, cell-cell junctions, and at the cell cortex. These examples demonstrate that genetically encoded tension sensors are powerful tools to unravel force transduction mechanisms, but also indicate current limitations. We propose that further technical improvements are needed to develop a truly molecular understanding of mechanobiological processes in cells and tissues.
Assuntos
Células/metabolismo , Fenômenos Biomecânicos , Calibragem , Adesão Celular , Transferência Ressonante de Energia de Fluorescência , Humanos , Modelos BiológicosRESUMO
The ability of cells to adhere and sense differences in tissue stiffness is crucial for organ development and function. The central mechanisms by which adherent cells detect extracellular matrix compliance, however, are still unknown. Using two single-molecule-calibrated biosensors that allow the analysis of a previously inaccessible but physiologically highly relevant force regime in cells, we demonstrate that the integrin activator talin establishes mechanical linkages following cell adhesion, which are indispensable for cells to probe tissue stiffness. Talin linkages are exposed to a range of piconewton forces and bear, on average, 7-10 pN during cell adhesion depending on their association with F-actin and vinculin. Disruption of talin's mechanical engagement does not impair integrin activation and initial cell adhesion but prevents focal adhesion reinforcement and thus extracellular rigidity sensing. Intriguingly, talin mechanics are isoform specific so that expression of either talin-1 or talin-2 modulates extracellular rigidity sensing.
Assuntos
Técnicas Biossensoriais/métodos , Matriz Extracelular/metabolismo , Adesões Focais/metabolismo , Talina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Western Blotting , Adesão Celular , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fenômenos Mecânicos , Camundongos Knockout , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Pinças Ópticas , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Talina/genética , Vinculina/genética , Vinculina/metabolismoRESUMO
The ability of cells to sense and respond to mechanical forces is central to a wide range of biological processes and plays an important role in numerous pathologies. The molecular mechanisms underlying cellular mechanotransduction, however, have remained largely elusive because suitable methods to investigate subcellular force propagation were missing. Here, we review recent advances in the development of biosensors that allow molecular force measurements. We describe the underlying principle of currently available techniques and propose a strategy to systematically evaluate new Förster resonance energy transfer (FRET)-based biosensors.
RESUMO
BACKGROUND: Actomyosin-based contractility acts on cadherin junctions to support tissue integrity and morphogenesis. The actomyosin apparatus of the epithelial zonula adherens (ZA) is built by coordinating junctional actin assembly with Myosin II activation. However, the physical interaction between Myosin and actin filaments that is necessary for contractility can induce actin filament turnover, potentially compromising the contractile apparatus itself. RESULTS: We now identify tension-sensitive actin assembly as one cellular solution to this design paradox. We show that junctional actin assembly is maintained by contractility in established junctions and increases when contractility is stimulated. The underlying mechanism entails the tension-sensitive recruitment of vinculin to the ZA. Vinculin, in turn, directly recruits Mena/VASP proteins to support junctional actin assembly. By combining strategies that uncouple Mena/VASP from vinculin or ectopically target Mena/VASP to junctions, we show that tension-sensitive actin assembly is necessary for junctional integrity and effective contractility at the ZA. CONCLUSIONS: We conclude that tension-sensitive regulation of actin assembly represents a mechanism for epithelial cells to resolve potential design contradictions that are inherent in the way that the junctional actomyosin system is assembled. This emphasizes that maintenance and regulation of the actin scaffolds themselves influence how cells generate contractile tension.