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1.
Nat Phys ; 18(9): 1112-1121, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37220497

RESUMO

Cell behaviour is affected by the physical forces and mechanical properties of the cells and of their microenvironment. The viscosity of extracellular fluid - a component of the cellular microenvironment - can vary by orders of magnitude, but its effect on cell behaviour remains largely unexplored. Using bio-compatible polymers to increase the viscosity of the culture medium, we characterize how viscosity affects cell behaviour. We find that multiple types of adherent cells respond in an unexpected but similar manner to elevated viscosity. In a highly viscous medium, cells double their spread area, exhibit increased focal adhesion formation and turnover, generate significantly greater traction forces, and migrate nearly two times faster. We observe that when cells are immersed in regular medium, these viscosity-dependent responses require an actively ruffling lamellipodium - a dynamic membrane structure at the front of the cell. We present evidence that cells utilize membrane ruffling to sense changes in extracellular fluid viscosity and to trigger adaptive responses.

2.
Dev Cell ; 56(23): 3288-3302.e5, 2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34822787

RESUMO

Plasticity of cell mechanics underlies a wide range of cell and tissue behaviors allowing cells to migrate through narrow spaces, resist shear forces, and safeguard against mechanical damage. Such plasticity depends on spatiotemporal regulation of the actomyosin cytoskeleton, but mechanisms of adaptive change in cell mechanics remain elusive. Here, we report a mechanism of mechanically activated actin polymerization at focal adhesions (FAs), specifically requiring the actin elongation factor mDia1. By combining live-cell imaging with mathematical modeling, we show that actin polymerization at FAs exhibits pulsatile dynamics where spikes of mDia1 activity are triggered by contractile forces. The suppression of mDia1-mediated actin polymerization increases tension on stress fibers (SFs) leading to an increased frequency of spontaneous SF damage and decreased efficiency of zyxin-mediated SF repair. We conclude that tension-controlled actin polymerization acts as a safety valve dampening excessive tension on the actin cytoskeleton and safeguarding SFs against mechanical damage.


Assuntos
Citoesqueleto de Actina/fisiologia , Fibroblastos/fisiologia , Forminas/metabolismo , Fenômenos Mecânicos , Microtúbulos/fisiologia , Fibras de Estresse/fisiologia , Actinas/química , Actinas/metabolismo , Actomiosina , Animais , Fibroblastos/citologia , Adesões Focais , Forminas/genética , Humanos , Mecanotransdução Celular , Polimerização
3.
J Vis Exp ; (173)2021 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-34309600

RESUMO

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.


Assuntos
Lasers , Álcool de Polivinil
4.
J Vis Exp ; (171)2021 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-34096911

RESUMO

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out shape. Following the cell-substrate attachment, the cell forms a thin sheet of lamellipodia emanating from the cell body. In the lamellipodia, globular actin (G-actin) monomers polymerize into a dense filamentous actin (F-actin) meshwork that pushes against the plasma membrane, thereby providing the mechanical forces required for the cell to spread. Notably, the molecular players that control the actin polymerization in lamellipodia are essential for many other cellular processes, such as cell migration and endocytosis. Since spreading cells form continuous lamellipodia that span the entire cell periphery and persistently expand outward, cell spreading assays have become an efficient tool to assess the kinetics of lamellipodial protrusions. Although several technical implementations of the cell spreading assay have been developed, a detailed description of the workflow, which would include both a step-by-step protocol and computational tools for data analysis, is currently lacking. Here, we describe the experimental procedures of the cell spreading assay and present an open-source tool for quantitative and unbiased analysis of cell edge dynamics during spreading. When combined with pharmacological manipulations and/or gene-silencing techniques, this protocol is amenable to a large-scale screen of molecular players regulating lamellipodial protrusions.


Assuntos
Actinas , Movimento Celular , Pseudópodes , Citoesqueleto de Actina , Membrana Celular , Humanos
5.
Methods Mol Biol ; 2299: 181-195, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028744

RESUMO

Myofibroblasts play important roles in physiological processes such as wound healing and tissue repair. While high contractile forces generated by the actomyosin network enable myofibroblasts to physically contract the wound and bring together injured tissue, prolonged and elevated levels of contraction also drive the progression of fibrosis and cancer. However, quantitative mapping of these forces has been difficult due to their extremely low magnitude ranging from 100 pN/µm2 to 2 nN/µm2. Here, we provide a protocol to measure cellular forces exerted on two-dimensional compliant elastic hydrogels. We describe the fabrication of polyacrylamide hydrogels labeled with fluorescent fiducial markers, functionalization of substrates with ECM proteins, setting up the experiment, and imaging procedures. We demonstrate the application of this technique for quantitative analysis of traction forces exerted by myofibroblasts.


Assuntos
Actinas/metabolismo , Fibroblastos/citologia , Miofibroblastos/fisiologia , Resinas Acrílicas , Animais , Adesão Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Humanos , Camundongos , Microscopia de Força Atômica , Contração Muscular , Miofibroblastos/citologia , Células NIH 3T3 , Estresse Mecânico
6.
Cells ; 10(5)2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33922651

RESUMO

Cells in the human body experience and integrate a wide variety of environmental cues. A growing interest in tissue mechanics in the past four decades has shown that the mechanical properties of tissue drive key biological processes and facilitate disease development. However, tissue stiffness is not only a potent behavioral cue, but also a product of cellular signaling activity. This review explores both roles of tissue stiffness in the context of inflammation and fibrosis, and the important molecular players driving such processes. During inflammation, proinflammatory cytokines upregulate tissue stiffness by increasing hydrostatic pressure, ECM deposition, and ECM remodeling. As the ECM stiffens, cells involved in the immune response employ intricate molecular sensors to probe and alter their mechanical environment, thereby facilitating immune cell recruitment and potentiating the fibrotic phenotype. This powerful feedforward loop raises numerous possibilities for drug development and warrants further investigation into the mechanisms specific to different fibrotic diseases.


Assuntos
Matriz Extracelular/imunologia , Fibrose/patologia , Inflamação/fisiopatologia , Mecanotransdução Celular , Animais , Fibrose/imunologia , Fibrose/metabolismo , Humanos
8.
Nat Mater ; 18(6): 638-649, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31114072

RESUMO

The interrelationship between microtubules and the actin cytoskeleton in mechanoregulation of integrin-mediated adhesions is poorly understood. Here, we show that the effects of microtubules on two major types of cell-matrix adhesion, focal adhesions and podosomes, are mediated by KANK family proteins connecting the adhesion protein talin with microtubule tips. Both total microtubule disruption and microtubule uncoupling from adhesions by manipulations with KANKs trigger a massive assembly of myosin IIA filaments, augmenting focal adhesions and disrupting podosomes. Myosin IIA filaments are indispensable effectors in the microtubule-driven regulation of integrin-mediated adhesions. Myosin IIA filament assembly depends on Rho activation by the RhoGEF GEF-H1, which is trapped by microtubules when they are connected with integrin-mediated adhesions via KANK proteins but released after their disconnection. Thus, microtubule capture by integrin-mediated adhesions modulates the GEF-H1-dependent effect of microtubules on the assembly of myosin IIA filaments. Subsequent actomyosin reorganization then remodels the focal adhesions and podosomes, closing the regulatory loop.


Assuntos
Adesões Focais/metabolismo , Integrinas/metabolismo , Microtúbulos/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto , Humanos , Mecanotransdução Celular , Podossomos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo
9.
Genetics ; 211(2): 597-615, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30514708

RESUMO

G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in eukaryotes, with multiple GPCR mutations linked to human diseases. With the growing number of sequenced human genomes, determining the pathogenicity of a mutation is challenging, but can be aided by a direct measurement of GPCR-mediated signaling. This is particularly difficult for the visual pigment rhodopsin-a GPCR activated by light-for which hundreds of mutations have been linked to inherited degenerative retinal diseases such as retinitis pigmentosa. In this study, we successfully engineered, for the first time, activation by human rhodopsin of the yeast mating pathway, resulting in signaling via a fluorescent reporter. We combine this novel assay for rhodopsin light-dependent activation with studies of subcellular localization, and the upregulation of the unfolded protein response in response to misfolded rhodopsin protein. We use these assays to characterize a panel of rhodopsin mutations with known molecular phenotypes, finding that rhodopsin maintains a similar molecular phenotype in yeast, with some interesting differences. Furthermore, we compare our assays in yeast with clinical phenotypes from patients with novel disease-linked mutations. We demonstrate that our engineered yeast strain can be useful in rhodopsin mutant classification, and in helping to determine the molecular mechanisms underlying their pathogenicity. This approach may also be applied to better understand the clinical relevance of other human GPCR mutations, furthering the use of yeast as a tool for investigating molecular mechanisms relevant to human disease.


Assuntos
Mutação de Sentido Incorreto , Retinose Pigmentar/genética , Rodopsina/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Genes Fúngicos Tipo Acasalamento/genética , Humanos , Retinose Pigmentar/patologia , Rodopsina/química , Rodopsina/genética , Saccharomyces cerevisiae
10.
G3 (Bethesda) ; 9(2): 561-570, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30573469

RESUMO

Several examples of transcription factors that show stochastic, unsynchronized pulses of nuclear localization have been described. Here we show that under constant calcium stress, nuclear localization pulses of the transcription factor Crz1 follow stochastic variations in cytosolic calcium concentration. We find that the size of the stochastic calcium bursts is positively correlated with the number of subsequent Crz1 pulses. Based on our observations, we propose a simple stochastic model of how the signaling pathway converts a constant external calcium concentration into a digital number of Crz1 pulses in the nucleus, due to the time delay from nuclear transport and the stochastic decoherence of individual Crz1 molecule dynamics. We find support for several additional predictions of the model and suggest that stochastic input to nuclear transport may produce noisy digital responses to analog signals in other signaling systems.


Assuntos
Sinalização do Cálcio , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Teóricos , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Saccharomyces cerevisiae/metabolismo , Processos Estocásticos
11.
Cell Rep ; 25(9): 2401-2416.e5, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30485809

RESUMO

Cells in multicellular organisms are arranged in complex three-dimensional patterns. This requires both transient and stable adhesions with the extracellular matrix (ECM). Integrin adhesion receptors bind ECM ligands outside the cell and then, by binding the protein talin inside the cell, assemble an adhesion complex connecting to the cytoskeleton. The activity of talin is controlled by several mechanisms, but these have not been well studied in vivo. By generating mice containing the activating point mutation E1770A in talin (Tln1), which disrupts autoinhibition, we show that talin autoinhibition controls cell-ECM adhesion, cell migration, and wound healing in vivo. In particular, blocking autoinhibition gives rise to more mature, stable focal adhesions that exhibit increased integrin activation. Mutant cells also show stronger attachment to ECM and decreased traction force. Overall, these results demonstrate that modulating talin function via autoinhibition is an important mechanism for regulating multiple aspects of integrin-mediated cell-ECM adhesion in vivo.


Assuntos
Matriz Extracelular/metabolismo , Talina/metabolismo , Cicatrização , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Adesão Celular , Movimento Celular , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Integrinas/metabolismo , Camundongos , Mutação/genética , Fenótipo , Transdução de Sinais , Talina/genética
12.
Biophys J ; 112(4): 780-794, 2017 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-28256237

RESUMO

Focal adhesions (FAs) are integrin-based transmembrane assemblies that connect a cell to its extracellular matrix (ECM). They are mechanosensors through which cells exert actin cytoskeleton-mediated traction forces to sense the ECM stiffness. Interestingly, FAs themselves are dynamic structures that adapt their growth in response to mechanical force. It is unclear how the cell manages the plasticity of the FA structure and the associated traction force to accurately sense ECM stiffness. Strikingly, FA traction forces oscillate in time and space, and govern the cell mechanosensing of ECM stiffness. However, precisely how and why the FA traction oscillates is unknown. We developed a model of FA growth that integrates the contributions of the branched actin network and stress fibers (SFs). Using the model in combination with experimental tests, we show that the retrograde flux of the branched actin network promotes the proximal growth of the FA and contributes to a traction peak near the FA's distal tip. The resulting traction gradient within the growing FA favors SF formation near the FA's proximal end. The SF-mediated actomyosin contractility further stabilizes the FA and generates a second traction peak near the center of the FA. Formin-mediated SF elongation negatively feeds back with actomyosin contractility, resulting in central traction peak oscillation. This underpins the observed FA traction oscillation and, importantly, broadens the ECM stiffness range over which FAs can accurately adapt to traction force generation. Actin cytoskeleton-mediated FA growth and maturation thus culminate with FA traction oscillation to drive efficient FA mechanosensing.


Assuntos
Actinas/metabolismo , Adesões Focais/metabolismo , Fenômenos Mecânicos , Animais , Fenômenos Biomecânicos , Matriz Extracelular/metabolismo , Retroalimentação Fisiológica , Camundongos
13.
Elife ; 52016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27552250

RESUMO

A new study reveals that a protein called talin forms a vital link between microtubules and focal adhesions at the surface of cells.


Assuntos
Adesões Focais , Talina , Adesão Celular , Microtúbulos
14.
Nat Commun ; 7: 11714, 2016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-27226243

RESUMO

Cell migration is fundamental for both physiological and pathological processes. Migrating cells usually display high dynamics in morphology, which is orchestrated by an integrative array of signalling pathways. Here we identify a novel pathway, we term lateral signalling, comprised of the planar cell polarity (PCP) protein Pk1 and the RhoGAPs, Arhgap21/23. We show that the Pk1-Arhgap21/23 complex inhibits RhoA, is localized on the non-protrusive lateral membrane cortex and its disruption leads to the disorganization of the actomyosin network and altered focal adhesion dynamics. Pk1-mediated lateral signalling confines protrusive activity and is regulated by Smurf2, an E3 ubiquitin ligase in the PCP pathway. Furthermore, we demonstrate that dynamic interplay between lateral and protrusive signalling generates cyclical fluctuations in cell shape that we quantify here as shape volatility, which strongly correlates with migration speed. These studies uncover a previously unrecognized lateral signalling pathway that coordinates shape volatility during productive cell migration.


Assuntos
Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Forma Celular/fisiologia , Transdução de Sinais/fisiologia , Actomiosina/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Interferência de RNA , Imagem com Lapso de Tempo/métodos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
15.
Biochem J ; 473(4): 397-410, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26611753

RESUMO

Ca(2+) release is tightly sequestered in eukaryotic cells to enable fine spatio-temporal control of signalling but how Ca(2+) release from the endoplasmic reticulum (ER) is linked to cell adhesions is not defined. We examined the spatial restriction of Ca(2+) release through the inositol 1,4,5-triphosphate receptor 1 (IP3R1) in response to interleukin-1 (IL-1) and the functions of the adhesion-associated proteins, focal adhesion kinase (FAK) and protein tyrosine phosphatase-α (PTPα). In cultured fibroblasts IL-1 treatment promoted co-localization of PTPα and FAK with the ER and increased association of IP3R1 with PTPα and FAK at focal adhesions (FAs). GST pull-down assays of purified proteins demonstrated that PTPα and FAK directly interacted with IP3R1. These interactions depended on the focal adhesion-targeting (FAT) and band4.1-ezrin-radixin-moesin (FERM) domains of FAK. PTPα was required for the association of IP3R1 with Src, which mediated IP3R1 phosphorylation and consequently ER Ca(2+) release. Collectively, these data indicate that PTPα and FAK, which are enriched in FAs, interact with IP3R1 at adjacent ER sites to spatially sequester IL-1-induced Ca(2+) signalling.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Interleucina-1/farmacologia , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Animais , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Camundongos , Camundongos Knockout , Ligação Proteica
16.
Proc Natl Acad Sci U S A ; 112(19): E2447-56, 2015 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-25918420

RESUMO

Actin filaments and integrin-based focal adhesions (FAs) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator, inverted formin 2 (INF2), localizes specifically to FAs and dorsal stress fibers (SFs) in fibroblasts. High-resolution fluorescence microscopy and manipulation of INF2 levels in cells indicate that INF2 plays a critical role at the SF-FA junction by promoting actin polymerization via free barbed end generation and centripetal elongation of an FA-associated actin bundle to form dorsal SF. INF2 assembles into FAs during maturation rather than during their initial generation, and once there, acts to promote rapid FA elongation and maturation into tensin-containing fibrillar FAs in the cell center. We show that INF2 is required for fibroblasts to organize fibronectin into matrix fibers and ultimately 3D matrices. Collectively our results indicate an important role for the formin INF2 in specifying the function of fibrillar FAs through its ability to generate dorsal SFs. Thus, dorsal SFs and fibrillar FAs form a specific class of integrated adhesion-associated actin structure in fibroblasts that mediates generation and remodeling of ECM.


Assuntos
Matriz Extracelular/metabolismo , Adesões Focais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fibras de Estresse/metabolismo , Actinas/metabolismo , Animais , Adesão Celular , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Forminas , Proteínas de Fluorescência Verde/metabolismo , Humanos , Integrinas/metabolismo , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Isoformas de Proteínas , Pseudópodes/metabolismo , RNA Interferente Pequeno/metabolismo
17.
Curr Biol ; 25(2): 175-186, 2015 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-25544611

RESUMO

BACKGROUND: Cell migration requires coordinated formation of focal adhesions (FAs) and assembly and contraction of the actin cytoskeleton. Nonmuscle myosin II (MII) is a critical mediator of contractility and FA dynamics in cell migration. Signaling downstream of the small GTPase Rac1 also regulates FA and actin dynamics, but its role in regulation of MII during migration is less clear. RESULTS: We found that Rac1 promotes association of MIIA with FA. Live-cell imaging showed that, whereas most MIIA at the leading edge assembled into dorsal contractile arcs, a substantial subset assembled in or was captured within maturing FA, and this behavior was promoted by active Rac1. Protein kinase C (PKC) activation was necessary and sufficient for integrin- and Rac1-dependent phosphorylation of MIIA heavy chain (HC) on serine1916 (S1916) and recruitment to FA. S1916 phosphorylation of MIIA HC and localization in FA was enhanced during cell spreading and ECM stiffness mechanosensing, suggesting upregulation of this pathway during physiological Rac1 activation. Phosphomimic and nonphosphorylatable MIIA HC mutants demonstrated that S1916 phosphorylation was necessary and sufficient for the capture and assembly of MIIA minifilaments in FA. S1916 phosphorylation was also sufficient to promote the rapid assembly of FAs to enhance cell migration and for the modulation of traction force, spreading, and migration by ECM stiffness. CONCLUSIONS: Our study reveals for the first time that Rac1 and integrin activation regulates MIIA HC phosphorylation through a PKC-dependent mechanism that promotes MIIA association with FAs and acts as a critical modulator of cell migration and mechanosensing.


Assuntos
Movimento Celular , Adesões Focais/metabolismo , Proteínas Motores Moleculares/genética , Cadeias Pesadas de Miosina/genética , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/genética , Linhagem Celular , Humanos , Mecanotransdução Celular/fisiologia , Proteínas Motores Moleculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Fosforilação , Proteínas rac1 de Ligação ao GTP/metabolismo
18.
Methods Cell Biol ; 123: 367-94, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24974038

RESUMO

Cellular forces generated by the actomyosin cytoskeleton and transmitted to the extracellular matrix (ECM) through discrete, integrin-based protein assemblies, that is, focal adhesions, are critical to developmental morphogenesis and tissue homeostasis, as well as disease progression in cancer. However, quantitative mapping of these forces has been difficult since there has been no experimental technique to visualize nanonewton forces at submicrometer spatial resolution. Here, we provide detailed protocols for measuring cellular forces exerted on two-dimensional elastic substrates with a high-resolution traction force microscopy (TFM) method. We describe fabrication of polyacrylamide substrates labeled with multiple colors of fiducial markers, functionalization of the substrates with ECM proteins, setting up the experiment, and imaging procedures. In addition, we provide the theoretical background of traction reconstruction and experimental considerations important to design a high-resolution TFM experiment. We describe the implementation of a new algorithm for processing of images of fiducial markers that are taken below the surface of the substrate, which significantly improves data quality. We demonstrate the application of the algorithm and explain how to choose a regularization parameter for suppression of the measurement error. A brief discussion of different ways to visualize and analyze the results serves to illustrate possible uses of high-resolution TFM in biomedical research.


Assuntos
Análise de Célula Única/métodos , Algoritmos , Animais , Fenômenos Biomecânicos , Linhagem Celular , Módulo de Elasticidade , Marcadores Fiduciais , Análise de Fourier , Humanos , Mecanotransdução Celular , Microscopia de Fluorescência
19.
Curr Opin Cell Biol ; 25(5): 619-26, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23830911

RESUMO

The ability of cells to move directionally toward areas of stiffer extracellular matrix (ECM) via a process known as 'durotaxis' is thought to be critical for development and wound healing, but durotaxis can also drive cancer metastasis. Migration is driven by integrin-mediated focal adhesions (FAs), protein assemblies that couple contractile actomyosin bundles to the plasma membrane, transmit force generated by the cytoskeleton to the ECM, and convert the mechanical properties of the microenvironment into biochemical signals. To probe the stiffness of the ECM, motile fibroblasts modulate FA mechanics on the nanoscale and exert forces that are reminiscent of repeated tugging on the ECM. Within a single cell, all FAs tug autonomously and thus act as local rigidity sensors, allowing discernment of differences in the extracellular matrix rigidity at high spatial resolution. In this article, we review current advances that may shed light on the mechanism of traction force fluctuations within FAs. We also examine plausible downstream effectors of tugging forces which may regulate cytoskeletal and FA dynamics to guide cell migration in response to ECM stiffness gradients.


Assuntos
Movimento Celular , Adesões Focais/metabolismo , Animais , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Humanos , Integrinas/metabolismo , Microtúbulos/metabolismo
20.
J Cell Biol ; 202(1): 163-77, 2013 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-23836933

RESUMO

In migrating cells, integrin-based focal adhesions (FAs) assemble in protruding lamellipodia in association with rapid filamentous actin (F-actin) assembly and retrograde flow. How dynamic F-actin is coupled to FA is not known. We analyzed the role of vinculin in integrating F-actin and FA dynamics by vinculin gene disruption in primary fibroblasts. Vinculin slowed F-actin flow in maturing FA to establish a lamellipodium-lamellum border and generate high extracellular matrix (ECM) traction forces. In addition, vinculin promoted nascent FA formation and turnover in lamellipodia and inhibited the frequency and rate of FA maturation. Characterization of a vinculin point mutant that specifically disrupts F-actin binding showed that vinculin-F-actin interaction is critical for these functions. However, FA growth rate correlated with F-actin flow speed independently of vinculin. Thus, vinculin functions as a molecular clutch, organizing leading edge F-actin, generating ECM traction, and promoting FA formation and turnover, but vinculin is dispensible for FA growth.


Assuntos
Actinas/metabolismo , Adesões Focais/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteólise , Vinculina/metabolismo , Substituição de Aminoácidos , Animais , Movimento Celular , Clonagem Molecular , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Adesões Focais/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação Puntual , Ligação Proteica , Transporte Proteico , Pseudópodes/metabolismo , Vinculina/genética
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