RESUMO
Vinculin binds to specific sites of mechanically unfolded talin rod domains to reinforce the coupling of the cell's exterior to its force generation machinery. Force-dependent vinculin-talin complexation and dissociation was previously observed as contraction or extension of the unfolded talin domains respectively using magnetic tweezers. However, the structural mechanism underlying vinculin recognition of unfolded vinculin binding sites (VBSs) in talin remains unknown. Using molecular dynamics simulations, we demonstrate that a VBS dynamically refolds under force, and that vinculin can recognize and bind to partially unfolded VBS states. Vinculin binding enables refolding of the mechanically strained VBS and stabilizes its folded α-helical conformation, providing resistance against mechanical stress. Together, these results provide an understanding of a recognition mechanism of proteins unfolded by force and insight into the initial moments of how vinculin binds unfolded talin rod domains during the assembly of this mechanosensing meshwork.
Assuntos
Simulação de Dinâmica Molecular , Ligação Proteica , Talina , Vinculina , Vinculina/metabolismo , Vinculina/química , Talina/metabolismo , Talina/química , Sítios de Ligação , Desdobramento de Proteína , Dobramento de Proteína , Estresse Mecânico , HumanosRESUMO
Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix. Quantitating its contribution to the reinforcement of newly forming adhesions, however, requires ultrasensitive cell force assays covering short time and low force ranges. Here, we have combined atomic force microscopy-based single-cell force spectroscopy (SCFS) and optical tweezers force spectroscopy to investigate the role of vinculin in reinforcement of individual nascent adhesions during the first 5 min of cell contact with fibronectin or vitronectin. At minimal adhesion times (5-10 s), mouse embryonic fibroblast (MEF) wildtype (wt) and vinculin knock-out (vin(-/-) ) cells develop comparable adhesion forces on the scale of several individual integrin-ligand bonds, confirming that vinculin is dispensable for adhesion initiation. In contrast, after 60 to 120 s, adhesion strength and traction reinforce quickly in wt cells, while remaining low in vin(-/-) cells. Re-expression of full-length vinculin or a constitutively active vinculin mutant (vinT12) in MEF vin(-/-) cells restored adhesion and traction with the same efficiency, while vinculin with a mutated talin-binding head region (vinA50I) or missing the actin-binding tail-domain (vin880) was ineffective. Integrating total internal reflection fluorescence imaging into the SCFS setup furthermore enabled us to correlate vinculin-green fluorescent protein (GFP) recruitment to nascent adhesion sites with the built-up of vinculin-dependent adhesion forces directly. Vinculin recruitment and cell adhesion reinforcement followed synchronous biphasic patterns, suggesting vinculin recruitment, but not activation, as the rate-limiting step for adhesion reinforcement. Combining sensitive SCFS with fluorescence microscopy thus provides insight into the temporal sequence of vinculin-dependent mechanical reinforcement in nascent integrin adhesions.
Assuntos
Fibroblastos , Adesões Focais , Animais , Camundongos , Adesão Celular/fisiologia , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Integrinas/metabolismo , Talina/genética , Talina/química , Talina/metabolismo , Vinculina/genética , Vinculina/química , Vinculina/metabolismoRESUMO
Extracellular matrix (ECM) stiffness regulates various cell behaviors, including cell differentiation, proliferation and migration. Vinculin and vinexin α (an isoform encoded by the SORBS3 gene), both of which localize to focal adhesions, cooperatively function as mechanosensors of ECM stiffness. On a rigid ECM, vinexin α interacts with vinculin and induces a conformational change in vinculin to give an 'open' form, which promotes nuclear localization of Yes-associated protein (YAP, also known as YAP1) and transcriptional coactivator with a PDZ-binding motif (TAZ, also known as WWTR1) (hereafter YAP/TAZ). However, the detailed mechanism by which vinexin α induces the conformational change in vinculin has not been revealed. Here, we identify an amphipathic helix named H2 as a novel vinculin-binding site in vinexin α. The H2 helix interacts with the vinculin D1b subdomain and promotes the formation of a talin-vinculin-vinexin α ternary complex. Mutations in the H2 region not only impair the ability of vinexin α to induce the ECM stiffness-dependent conformational change in vinculin but also to promote nuclear localization of YAP/TAZ on rigid ECM. Taken together, these results demonstrate that the H2 helix in vinexin α plays a critical role in ECM stiffness-dependent regulation of vinculin and cell behaviors.
Assuntos
Matriz Extracelular/metabolismo , Proteínas Musculares/metabolismo , Vinculina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Matriz Extracelular/química , Matriz Extracelular/genética , Camundongos , Proteínas Musculares/química , Proteínas Musculares/genética , Estrutura Secundária de Proteína , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , Vinculina/química , Vinculina/genética , Proteínas de Sinalização YAPRESUMO
Vinculin and its heart-specific splice variant metavinculin are key regulators of cell adhesion processes. These membrane-bound cytoskeletal proteins regulate the cell shape by binding to several other proteins at cell-cell and cell-matrix junctions. Vinculin and metavinculin link integrin adhesion molecules to the filamentous actin network. Loss of both proteins prevents cell adhesion and cell spreading and reduces the formation of stress fibers, focal adhesions, or lamellipodia extensions. The binding of talin at cell-matrix junctions or of α-catenin at cell-cell junctions activates vinculin and metavinculin by releasing their autoinhibitory head-tail interaction. Once activated, vinculin and metavinculin bind F-actin via their five-helix bundle tail domains. Unlike vinculin, metavinculin has a 68-amino-acid insertion before the second α-helix of this five-helix F-actin-binding domain. Here, we present the full-length cryogenic electron microscopy structure of metavinculin that captures the dynamics of its individual domains and unveiled a hallmark structural feature, namely a kinked isoform-specific α-helix in its F-actin-binding domain. Our identified conformational landscape of metavinculin suggests a structural priming mechanism that is consistent with the cell adhesion functions of metavinculin in response to mechanical and cellular cues. Our findings expand our understanding of metavinculin function in the heart with implications for the etiologies of cardiomyopathies.
Assuntos
Microscopia Crioeletrônica , Citoesqueleto/química , Domínios e Motivos de Interação entre Proteínas , Vinculina/química , Actinas/química , Actinas/metabolismo , Adesão Celular , Membrana Celular/química , Membrana Celular/metabolismo , Cristalografia por Raios X , Citoesqueleto/metabolismo , Humanos , Modelos Moleculares , Peptídeos , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Vinculina/metabolismoRESUMO
Talin and vinculin, both actin-cytoskeleton-related proteins, have been documented to participate in establishing bacterial infections, respectively, as the adapter protein to mediate cytoskeleton-driven dynamics of the plasma membrane. However, little is known regarding the potential role of the talin-vinculin complex during spotted fever group rickettsial and Ebola virus infections, two dreadful infectious diseases in humans. Many functional properties of proteins are determined by their participation in protein-protein complexes, in a temporal and/or spatial manner. To resolve the limitation of application in using mouse primary antibodies on archival, multiple formalin-fixed mouse tissue samples, which were collected from experiments requiring high biocontainment, we developed a practical strategic proximity ligation assay (PLA) capable of employing one primary antibody raised in mouse to probe talin-vinculin spatial proximal complex in mouse tissue. We observed an increase of talin-vinculin spatial proximities in the livers of spotted fever Rickettsia australis or Ebola virus-infected mice when compared with mock mice. Furthermore, using EPAC1-knockout mice, we found that deletion of EPAC1 could suppress the formation of spatial proximal complex of talin-vinculin in rickettsial infections. In addition, we observed increased colocalization between spatial proximity of talin-vinculin and filamentous actin-specific phalloidin staining in single survival mouse from an ordinarily lethal dose of rickettsial or Ebola virus infection. These findings may help to delineate a fresh insight into the mechanisms underlying liver specific pathogenesis during infection with spotted fever rickettsia or Ebola virus in the mouse model.
Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Doença pelo Vírus Ebola/metabolismo , Fígado/metabolismo , Talina/metabolismo , Vinculina/metabolismo , Animais , Células Cultivadas , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Fígado/microbiologia , Fígado/virologia , Camundongos Knockout , Ligação Proteica , Rickettsia/fisiologia , Rickettsiose do Grupo da Febre Maculosa/metabolismo , Rickettsiose do Grupo da Febre Maculosa/microbiologia , Talina/química , Vinculina/químicaRESUMO
This study reports novel findings that link E-cadherin (also known as CDH1)-mediated force-transduction signaling to vinculin targeting to intercellular junctions via epidermal growth factor receptor (EGFR) and integrins. These results build on previous findings that demonstrated that mechanically perturbed E-cadherin receptors activate phosphoinositide 3-kinase and downstream integrins in an EGFR-dependent manner. Results of this study show that this EGFR-mediated kinase cascade controls the force-dependent recruitment of vinculin to stressed E-cadherin complexes - a key early signature of cadherin-based mechanotransduction. Vinculin targeting requires its phosphorylation at tyrosine 822 by Abl family kinases (hereafter Abl), but the origin of force-dependent Abl activation had not been identified. We now present evidence that integrin activation, which is downstream of EGFR signaling, controls Abl activation, thus linking E-cadherin to Abl through a mechanosensitive signaling network. These findings place EGFR and integrins at the center of a positive-feedback loop, through which force-activated E-cadherin signals regulate vinculin recruitment to cadherin complexes in response to increased intercellular tension.This article has an associated First Person interview with the first author of the paper.
Assuntos
Caderinas/metabolismo , Receptores ErbB/metabolismo , Integrinas/metabolismo , Junções Intercelulares/metabolismo , Vinculina/química , Vinculina/metabolismo , Caderinas/genética , Linhagem Celular Tumoral , Receptores ErbB/genética , Humanos , Integrinas/genética , Junções Intercelulares/genética , Mecanotransdução Celular , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Vinculina/genéticaRESUMO
Mechanotransduction by α-catenin facilitates the force-dependent development of adherens junctions (AJs) by recruiting vinculin to reinforce actin anchoring of AJs. The α-catenin mechanotransducing action is facilitated by its force-sensing device region that autoinhibits the vinculin-binding site 1 (VBS1). Here, we report the high-resolution structure of the force-sensing device region of α-catenin, which shows the autoinhibited form comprised of helix bundles E, F and G. The cryptic VBS1 is embedded into helix bundle E stabilized by direct interactions with the autoinhibitory region forming helix bundles F and G. Our molecular dissection study showed that helix bundles F and G are stable in solution in each isolated form, whereas helix bundle E that contains VBS1 is unstable and intrinsically disordered in solution in the isolated form. We successfully identified key residues mediating the autoinhibition and produced mutated α-catenins that display variable force sensitivity and autoinhibition. Using these mutants, we demonstrate both in vitro and in vivo that, in the absence of this stabilization, the helix bundle containing VBS1 would adopt an unfolded form, thus exposing VBS for vinculin binding. We provide evidence for importance of mechanotransduction with the intrinsic force sensitivity for vinculin recruitment to adherens junctions of epithelial cell sheets with mutated α-catenins.
Assuntos
Actinas/metabolismo , Junções Aderentes/fisiologia , Mecanotransdução Celular , Vinculina/metabolismo , alfa Catenina/química , alfa Catenina/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Vinculina/química , Vinculina/genética , alfa Catenina/genéticaRESUMO
The main cause of death globally remains debilitating heart conditions, such as dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), which are often due to mutations of specific components of adhesion complexes. Vinculin regulates these complexes and plays essential roles in intercalated discs that are necessary for muscle cell function and coordinated movement and in the development and function of the heart. Humans bearing familial or sporadic mutations in vinculin suffer from chronic, progressively debilitating DCM that ultimately leads to cardiac failure and death, whereas autosomal dominant mutations in vinculin can also provoke HCM, causing acute cardiac failure. The DCM/HCM-associated mutants of vinculin occur in the 68-residue insert unique to the muscle-specific, alternatively spliced isoform of vinculin, termed metavinculin (MV). Contrary to studies that suggested that phosphoinositol-4,5-bisphosphate (PIP2) only induces vinculin homodimers, which are asymmetric, we show that phospholipid binding results in a domain-swapped symmetric MV dimer via a quasi-equivalent interface compared with vinculin involving R975. Although one of the two PIP2 binding sites is preserved, the symmetric MV dimer that bridges two PIP2 molecules differs from the asymmetric vinculin dimer that bridges only one PIP2 Unlike vinculin, wild-type MV and the DCM/HCM-associated R975W mutant bind PIP2 in their inactive conformations, and R975W MV fails to dimerize. Mutating selective vinculin residues to their corresponding MV residues, or vice versa, switches the isoform's dimeric constellation and lipid binding site. Collectively, our data suggest that MV homodimerization modulates microfilament attachment at muscular adhesion sites and furthers our understanding of MV-mediated cardiac remodeling.
Assuntos
Fosfatidilinositol 4,5-Difosfato/química , Vinculina/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Modelos Moleculares , Mutação , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica , Vinculina/genética , Vinculina/metabolismoRESUMO
Intercellular epithelial junctions formed by classical cadherins, ß-catenin, and the actin-binding protein α-catenin link the actin cytoskeletons of adjacent cells into a structural continuum. These assemblies transmit forces through the tissue and respond to intracellular and extracellular signals. However, the mechanisms of junctional assembly and regulation are poorly understood. Studies of cadherin-catenin assembly in a number of metazoans have revealed both similarities and unexpected differences in the biochemical properties of the cadherin·catenin complex that likely reflect the developmental and environmental requirements of different tissues and organisms. Here, we report the structural and biochemical characterization of HMP-1, the Caenorhabditis elegans α-catenin homolog, and compare it with mammalian α-catenin. HMP-1 shares overall similarity in structure and actin-binding properties, but displayed differences in conformational flexibility and allosteric regulation from mammalian α-catenin. HMP-1 bound filamentous actin with an affinity in the single micromolar range, even when complexed with the ß-catenin homolog HMP-2 or when present in a complex of HMP-2 and the cadherin homolog HMR-1, indicating that HMP-1 binding to F-actin is not allosterically regulated by the HMP-2·HMR-1 complex. The middle (i.e. M) domain of HMP-1 appeared to be less conformationally flexible than mammalian α-catenin, which may underlie the dampened effect of HMP-2 binding on HMP-1 actin-binding activity compared with that of the mammalian homolog. In conclusion, our data indicate that HMP-1 constitutively binds ß-catenin and F-actin, and although the overall structure and function of HMP-1 and related α-catenins are similar, the vertebrate proteins appear to be under more complex conformational regulation.
Assuntos
Actinas/química , Caderinas/química , Proteínas de Caenorhabditis elegans/química , Proteínas do Citoesqueleto/química , alfa Catenina/química , beta Catenina/química , Sítio Alostérico , Animais , Caenorhabditis elegans , Adesão Celular , Cristalografia por Raios X , Glutationa Transferase/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Coelhos , Relação Estrutura-Atividade , Vinculina/químicaRESUMO
Vinculin is a highly conserved protein involved in cell adhesion and mechanotransduction, and both gain and loss of its activity causes defective cell behaviour. Here, we examine how altering vinculin activity perturbs integrin function within the context of Drosophila development. Whereas loss of vinculin produced relatively minor phenotypes, gain of vinculin activity, through a loss of head-tail autoinhibition, caused lethality. The minimal domain capable of inducing lethality is the talin-binding D1 domain, and this appears to require talin-binding activity, as lethality was suppressed by competition with single vinculin-binding sites from talin. Activated Drosophila vinculin triggered the formation of cytoplasmic adhesion complexes through the rod of talin, but independently of integrin. These complexes contain a subset of adhesion proteins but no longer link the membrane to actin. The negative effects of hyperactive vinculin were segregated into morphogenetic defects caused by its whole head domain and lethality caused by its D1 domain. These findings demonstrate the crucial importance of the tight control of the activity of vinculin.
Assuntos
Drosophila melanogaster/metabolismo , Integrinas/metabolismo , Vinculina/metabolismo , Animais , Adesão Celular , Citoplasma/metabolismo , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Modelos Biológicos , Músculos/embriologia , Músculos/metabolismo , Agregados Proteicos , Ligação Proteica , Domínios Proteicos , Vinculina/químicaRESUMO
The focal adhesion protein vinculin has been implicated in associating with soluble and membranous phospholipids. Detailed investigations over the past ten years describe the intermolecular interactions of the vinculin tail domain with soluble and membrane phospholipids. Previous studies have implied that the tail's unstructured C-terminal region affects the mechanical behavior of cells and that the same region, at the molecular level, has bi-stable behavior sensitive to different protonation states. The aim of this short communication is to discuss whether the C-terminal vinculin tail (Vt) domain interacts favorably with membrane-embedded phospholipids such as PIP2 and that the region is also an anchor for lipid membranes.
Assuntos
Fosfolipídeos/metabolismo , Vinculina/metabolismo , Animais , Dicroísmo Circular , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipídeos/química , Ligação Proteica , Domínios Proteicos , Vinculina/químicaRESUMO
Fluorescence live imaging has become an essential methodology in modern cell biology. However, fluorescence requires excitation light, which can sometimes cause potential problems, such as autofluorescence, phototoxicity, and photobleaching. Furthermore, combined with recent optogenetic tools, the light illumination can trigger their unintended activation. Because luminescence imaging does not require excitation light, it is a good candidate as an alternative imaging modality to circumvent these problems. The application of luminescence imaging, however, has been limited by the two drawbacks of existing luminescent protein probes, such as luciferases: namely, low brightness and poor color variants. Here, we report the development of bright cyan and orange luminescent proteins by extending our previous development of the bright yellowish-green luminescent protein Nano-lantern. The color change and the enhancement of brightness were both achieved by bioluminescence resonance energy transfer (BRET) from enhanced Renilla luciferase to a fluorescent protein. The brightness of these cyan and orange Nano-lanterns was â¼20 times brighter than wild-type Renilla luciferase, which allowed us to perform multicolor live imaging of intracellular submicron structures. The rapid dynamics of endosomes and peroxisomes were visualized at around 1-s temporal resolution, and the slow dynamics of focal adhesions were continuously imaged for longer than a few hours without photobleaching or photodamage. In addition, we extended the application of these multicolor Nano-lanterns to simultaneous monitoring of multiple gene expression or Ca(2+) dynamics in different cellular compartments in a single cell.
Assuntos
Luciferases/química , Luminescência , Proteínas Luminescentes/química , Proteínas Recombinantes de Fusão/química , Animais , Cálcio/metabolismo , Linhagem Celular , DNA/química , Cães , Células-Tronco Embrionárias/citologia , Endossomos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Adesões Focais , Regulação da Expressão Gênica , Luciferases de Renilla/metabolismo , Camundongos , Dados de Sequência Molecular , Oligonucleotídeos/química , Peroxissomos/metabolismo , Regiões Promotoras Genéticas , Renilla , Vinculina/químicaRESUMO
Focal adhesions are dynamic constructs at the leading edge of migrating cells, linking them to the extracellular matrix and enabling force sensing and transmission. The lifecycle of a focal adhesion is a highly coordinated process involving spatial and temporal variations of protein composition, interaction, and cellular tension. The assembly of focal adhesions requires the recruitment and activation of vinculin. Vinculin is present in the cytoplasm in an autoinhibited conformation in which its tail is held pincerlike by its head domains, further stabilized by two high-affinity head-tail interfaces. Vinculin has binding sites for talin and F-actin, but effective binding requires vinculin activation to release its head-tail associations. In migrating cells, it has been shown that the locations of vinculin activation coincide with areas of high cellular tension, and that the highest recorded tensions across vinculin are associated with adhesion assembly. Here, we use a structure-based model to investigate vinculin activation by talin modulated by tensile force generated by transient associations with F-actin. We show that vinculin activation may proceed from an intermediate state stabilized by partial talin-vinculin association. There is a low-force regime and a high-force regime where vinculin activation is dominated by two different pathways with distinct responses to force. Specifically, at zero or low forces, vinculin activation requires substantial destabilization of the main head-tail interface, which is rigid and undergoes very limited fluctuations, despite the other being relatively flexible. This pathway is not significantly affected by force; instead, higher forces favor an alternative pathway, which seeks to release the vinculin tail from its pincerlike head domains before destabilizing the head-tail interfaces. This pathway has a force-sensitive activation barrier and is significantly accelerated by force. Experimental data of vinculin during various stages of the focal adhesion lifecycle are consistent with the proposed force-regulated activation pathway.
Assuntos
Adesões Focais/metabolismo , Vinculina/metabolismo , Actinas/química , Actinas/metabolismo , Fenômenos Biomecânicos , Simulação por Computador , Citoplasma/química , Citoplasma/metabolismo , Adesões Focais/química , Humanos , Cinética , Modelos Moleculares , Domínios Proteicos , Estabilidade Proteica , Estrutura Secundária de Proteína , Talina/química , Talina/metabolismo , Vinculina/químicaRESUMO
Pleiotropy is the phenomenon by which the same gene can result in multiple phenotypes. Pleiotropic proteins are emerging as important contributors to rare and common disorders. Nevertheless, little is known on the mechanisms underlying pleiotropy and the characteristic of pleiotropic proteins. We analyzed disease-causing proteins reported in UniProt and observed that 12% are pleiotropic (variants in the same protein cause more than one disease). Pleiotropic proteins were enriched in deleterious and rare variants, but not in common variants. Pleiotropic proteins were more likely to be involved in the pathogenesis of neoplasms, neurological, and circulatory diseases and congenital malformations, whereas non-pleiotropic proteins in endocrine and metabolic disorders. Pleiotropic proteins were more essential and had a higher number of interacting partners compared with non-pleiotropic proteins. Significantly more pleiotropic than non-pleiotropic proteins contained at least one intrinsically long disordered region (P < 0.001). Deleterious variants occurring in structurally disordered regions were more commonly found in pleiotropic, rather than non-pleiotropic proteins. In conclusion, pleiotropic proteins are an important contributor to human disease. They represent a biologically different class of proteins compared with non-pleiotropic proteins and a better understanding of their characteristics and genetic variants can greatly aid in the interpretation of genetic studies and drug design.
Assuntos
Estudos de Associação Genética , Pleiotropia Genética , Predisposição Genética para Doença , Biologia Computacional , Bases de Dados Genéticas , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Razão de Chances , Ligação Proteica , Conformação Proteica , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Biologia de Sistemas/métodos , Vinculina/química , Vinculina/genética , Vinculina/metabolismoRESUMO
Mechanosensing of the micro-environments has been shown to be essential for cell survival, growth, differentiation and migration. The mechanosensing pathways are mediated by a set of mechanosensitive proteins located at focal adhesion and cell-cell adherens junctions as well as in the cytoskeleton network. Here we review the applications of magnetic tweezers on elucidating the molecular mechanisms of the mechanosensing proteins. The scope of this review includes the principles of the magnetic tweezers technology, theoretical analysis of force-dependent stability and interaction of mechanosensing proteins, and recent findings obtained using magnetic tweezers.
Assuntos
Moléculas de Adesão Celular/fisiologia , Mecanotransdução Celular , Moléculas de Adesão Celular/química , Espectroscopia de Ressonância de Spin Eletrônica , Matriz Extracelular/química , Matriz Extracelular/fisiologia , Adesões Focais/química , Adesões Focais/fisiologia , Fenômenos Magnéticos , Estabilidade Proteica , Talina/química , Talina/fisiologia , Vinculina/química , Vinculina/fisiologia , alfa Catenina/química , alfa Catenina/fisiologiaRESUMO
Phosphoinositols are an important class of phospholipids that are involved in a myriad of cellular processes, from cell signaling to motility and adhesion. Vinculin (Vn) is a major adaptor protein that regulates focal adhesions in conjunction with PIP2 in lipid membranes and other cytoskeletal components. The binding and unbinding transitions of Vn at the membrane interface are an important link to understanding the coordination of cell signaling and motility. Using different biophysical tools, including atomic force microscopy combined with confocal fluorescence microscopy and Fourier transform infrared spectroscopy, we studied the nanoscopic interactions of activated and autoinhibited states of Vn with lipid membranes. We hypothesize that a weak interaction occurs between Vn and lipid membranes, which leads to binding of autoinhibited Vn to supported lipid bilayers, and to unbinding in freestanding lipid vesicles. Likely driving forces may include tethering of the C-terminus to the lipid membrane, as well as hydrophobic helix-membrane interactions. Conversely, activated Vn binds strongly to membranes through specific interactions with clusters of PIP2 embedded in lipid membranes. Activated Vn harbored on PIP2 clusters may form small oligomeric interaction platforms for further interaction partners, which is necessary for the proper function of focal adhesion points.
Assuntos
Fosfolipídeos/química , Lipossomas Unilamelares/química , Vinculina/antagonistas & inibidores , Vinculina/química , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Microscopia Confocal , Microscopia de Fluorescência , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Vinculina/metabolismoRESUMO
Mechanotransduction at E-cadherin junctions has been postulated to be mediated in part by a force-dependent conformational activation of α-catenin. Activation of α-catenin allows it to interact with vinculin in addition to F-actin, resulting in a strengthening of junctions. Here, using E-cadherin adhesions reconstituted on synthetic, nanopatterned membranes, we show that activation of α-catenin is dependent on E-cadherin clustering, and is sustained in the absence of mechanical force or association with F-actin or vinculin. Adhesions were formed by filopodia-mediated nucleation and micron-scale assembly of E-cadherin clusters, which could be distinguished as either peripheral or central assemblies depending on their relative location at the cell-bilayer adhesion. Whereas F-actin, vinculin, and phosphorylated myosin light chain associated only with the peripheral assemblies, activated α-catenin was present in both peripheral and central assemblies, and persisted in the central assemblies in the absence of actomyosin tension. Impeding filopodia-mediated nucleation and micron-scale assembly of E-cadherin adhesion complexes by confining the movement of bilayer-bound E-cadherin on nanopatterned substrates reduced the levels of activated α-catenin. Taken together, these results indicate that although the initial activation of α-catenin requires micron-scale clustering that may allow the development of mechanical forces, sustained force is not required for maintaining α-catenin in the active state.
Assuntos
Caderinas/metabolismo , Adesão Celular/fisiologia , Mecanotransdução Celular/fisiologia , alfa Catenina/metabolismo , Actinas/química , Actinas/metabolismo , Antígenos CD , Caderinas/química , Caderinas/genética , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Bicamadas Lipídicas/química , Mecanotransdução Celular/efeitos dos fármacos , Microscopia Confocal , Microscopia de Fluorescência , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Estresse Mecânico , Vinculina/química , Vinculina/metabolismo , alfa Catenina/química , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/química , Quinases Associadas a rho/metabolismoRESUMO
Vasodilator-stimulated phosphoprotein (VASP) can catalyze actin polymerization by elongating actin filaments. The elongation mechanism involves VASP oligomerization and its binding to profilin, a G-actin chaperone. Actin polymerization is required for tension generation during the contraction of airway smooth muscle (ASM); however, the role of VASP in regulating actin dynamics in ASM is not known. We stimulated ASM cells and tissues with the contractile agonist acetylcholine (ACh) or the adenylyl cyclase activator, forskolin (FSK), a dilatory agent. ACh and FSK stimulated VASP Ser(157) phosphorylation by different kinases. Inhibition of VASP Ser(157) phosphorylation by expression of the mutant VASP S157A in ASM tissues suppressed VASP phosphorylation and membrane localization in response to ACh, and also inhibited contraction and actin polymerization. ACh but not FSK triggered the formation of VASP-VASP complexes as well as VASP-vinculin and VASP-profilin complexes at membrane sites. VASP-VASP complex formation and the interaction of VASP with vinculin and profilin were inhibited by expression of the inactive vinculin mutant, vinculin Y1065F, but VASP phosphorylation and membrane localization were unaffected. We conclude that VASP phosphorylation at Ser(157) mediates its localization at the membrane, but that VASP Ser(157) phosphorylation and membrane localization are not sufficient to activate its actin catalytic activity. The interaction of VASP with activated vinculin at membrane adhesion sites is a necessary prerequisite for VASP-mediated molecular processes necessary for actin polymerization. Our results show that VASP is a critical regulator of actin dynamics and tension generation during the contractile activation of ASM.
Assuntos
Actinas/química , Moléculas de Adesão Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Contração Muscular , Músculo Liso/fisiologia , Fosfoproteínas/metabolismo , Multimerização Proteica , Traqueia/fisiologia , Vinculina/metabolismo , Acetilcolina/farmacologia , Animais , Biocatálise , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Galinhas , Colforsina/farmacologia , Cães , Epinefrina/farmacologia , Humanos , Contração Muscular/efeitos dos fármacos , Músculo Liso/citologia , Fosforilação/efeitos dos fármacos , Profilinas/metabolismo , Estrutura Quaternária de Proteína , Transporte Proteico/efeitos dos fármacos , Tirosina/metabolismo , Vinculina/químicaRESUMO
Cell-matrix adhesion and cell-cell contacts are essential for the metabolism, protein synthesis, survival, and cancer metastasis of cells. Major transmembrane receptors are the integrins, which are responsible for cell-matrix adhesions, and the cadherins, which are important for cell-cell adhesions. Adherent cells anchor via focal adhesion proteins to the extracellular matrix, whereas cell-cell contacts connect via focal adherens junction proteins. The temporal formation of these connections is greatly strengthened either through externally applied stresses on the cell or by myosin-driven cell contractility. The mechanism by which protein(s) within these connections sense, transmit, and respond to mechanochemical signaling is currently strongly debated and various candidates have been named. Vinculin has been described as one of the key players in cell-matrix and cell-cell adhesions that build a strong physical connection for transmitting forces between the cytoskeleton, the extracellular matrix, and cell-cell connections.
Assuntos
Mecanotransdução Celular/fisiologia , Vinculina/metabolismo , Actinas/metabolismo , Junções Aderentes/metabolismo , Adesão Celular , Proteína Substrato Associada a Crk/metabolismo , Matriz Extracelular/metabolismo , Humanos , Integrinas/metabolismo , Ligação Proteica , Vinculina/químicaRESUMO
Mechanical forces are central to developmental, physiological and pathological processes. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with piconewton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent. We show that tension across vinculin in stable FAs is approximately 2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently.