RESUMEN
Although extracellular matrix (ECM) stiffness is an important aspect of the extracellular microenvironment and is known to direct the lineage specification of stem cells and affect cancer progression, the molecular mechanisms that sense ECM stiffness have not yet been elucidated. In this study, we show that the proline-rich linker (PRL) region of vinculin and the PRL-region-binding protein vinexin are involved in sensing the stiffness of ECM substrates. A rigid substrate increases the level of cytoskeleton-associated vinculin, and the fraction of vinculin stably localizing at focal adhesions (FAs) is larger on rigid ECM than on soft ECM. Mutations in the PRL region or the depletion of vinexin expression impair these responses to ECM stiffness. Furthermore, vinexin depletion impairs the stiffness-dependent regulation of cell migration. These results suggest that the interaction of the PRL region of vinculin with vinexin α plays a crucial role in sensing ECM stiffness and in mechanotransduction.
Asunto(s)
Matriz Extracelular/metabolismo , Proteínas Musculares/metabolismo , Animales , Células Cultivadas , Dicroismo Circular , Recuperación de Fluorescencia tras Fotoblanqueo , Inmunoprecipitación , Ratones , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiologíaRESUMEN
Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct measurements have been made, and there is little mechanistic insight. Using vinculin-null cells expressing vinculin mutants, we demonstrate that vinculin is not required for transmission of adhesive and traction forces but is necessary for myosin contractility-dependent adhesion strength and traction force and for the coupling of cell area and traction force. Adhesion strength and traction forces depend differentially on vinculin head (V(H)) and tail domains. V(H) enhances adhesion strength by increasing ECM-bound integrin-talin complexes, independently from interactions with vinculin tail ligands and contractility. A full-length, autoinhibition-deficient mutant (T12) increases adhesion strength compared with VH, implying roles for both vinculin activation and the actin-binding tail. In contrast to adhesion strength, vinculin-dependent traction forces absolutely require a full-length and activated molecule; V(H) has no effect. Physical linkage of the head and tail domains is required for maximal force responses. Residence times of vinculin in focal adhesions, but not T12 or V(H), correlate with applied force, supporting a mechanosensitive model for vinculin activation in which forces stabilize vinculin's active conformation to promote force transfer.
Asunto(s)
Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Adhesiones Focales/metabolismo , Vinculina/metabolismo , Actinas/metabolismo , Animales , Western Blotting , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Integrinas/metabolismo , Ratones , Ratones Noqueados , Microscopía Fluorescente , Modelos Biológicos , Unión Proteica , Estrés Mecánico , Talina/metabolismo , Vinculina/genéticaRESUMEN
Integrin-based focal adhesions (FA) transmit anchorage and traction forces between the cell and the extracellular matrix (ECM). To gain further insight into the physical parameters of the ECM that control FA assembly and force transduction in non-migrating cells, we used fibronectin (FN) nanopatterning within a cell adhesion-resistant background to establish the threshold area of ECM ligand required for stable FA assembly and force transduction. Integrin-FN clustering and adhesive force were strongly modulated by the geometry of the nanoscale adhesive area. Individual nanoisland area, not the number of nanoislands or total adhesive area, controlled integrin-FN clustering and adhesion strength. Importantly, below an area threshold (0.11 µm(2)), very few integrin-FN clusters and negligible adhesive forces were generated. We then asked whether this adhesive area threshold could be modulated by intracellular pathways known to influence either adhesive force, cytoskeletal tension, or the structural link between the two. Expression of talin- or vinculin-head domains that increase integrin activation or clustering overcame this nanolimit for stable integrin-FN clustering and increased adhesive force. Inhibition of myosin contractility in cells expressing a vinculin mutant that enhances cytoskeleton-integrin coupling also restored integrin-FN clustering below the nanolimit. We conclude that the minimum area of integrin-FN clusters required for stable assembly of nanoscale FA and adhesive force transduction is not a constant; rather it has a dynamic threshold that results from an equilibrium between pathways controlling adhesive force, cytoskeletal tension, and the structural linkage that transmits these forces, allowing the balance to be tipped by factors that regulate these mechanical parameters.
Asunto(s)
Citoesqueleto de Actina/fisiología , Matriz Extracelular/fisiología , Adhesiones Focales/metabolismo , Integrinas/metabolismo , Amidas/farmacología , Animales , Fenómenos Biomecánicos , Adhesión Celular , Fibronectinas/metabolismo , Fibronectinas/ultraestructura , Adhesiones Focales/fisiología , Adhesiones Focales/ultraestructura , Ratones , Células 3T3 NIH , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Piridinas/farmacología , Talina/química , Talina/metabolismo , Vinculina/química , Vinculina/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
Vinculin, an actin-binding protein, is emerging as an important regulator of adherens junctions. In focal-adhesions, vinculin is activated by simultaneous binding of talin to its head domain and actin filaments to its tail domain. Talin is not present in adherens junctions. Consequently, the identity of the ligand that activates vinculin in cell-cell junctions is not known. Here we show that in the presence of F-actin, α-catenin, a cytoplasmic component of the cadherin adhesion complex, activates vinculin. Direct binding of α-catenin to vinculin is critical for this event because a point mutant (α-catenin L344P) lacking high affinity binding does not activate vinculin. Furthermore, unlike all known vinculin activators, α-catenin binds to and activates vinculin independently of an A50I substitution in the vinculin head, a mutation that inhibits vinculin binding to talin and IpaA. Collectively, these data suggest that α-catenin employs a novel mechanism to activate vinculin and may explain how vinculin is differentially recruited and/or activated in cell-cell and cell-matrix adhesions.
Asunto(s)
Adhesiones Focales/metabolismo , Vinculina/metabolismo , alfa Catenina/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Sustitución de Aminoácidos , Animales , Adhesión Celular/fisiología , Adhesiones Focales/genética , Células HEK293 , Humanos , Ratones , Mutación Puntual , Unión Proteica , Vinculina/genética , alfa Catenina/genéticaRESUMEN
Conformational change is believed to be important to vinculin's function at sites of cell adhesion. However, nothing is known about vinculin's conformation in living cells. Using a Forster resonance energy transfer probe that reports on changes in vinculin's conformation, we find that vinculin is in the actin-binding conformation in a peripheral band of adhesive puncta in spreading cells. However, in fully spread cells with established polarity, vinculin's conformation is variable at focal adhesions. Time-lapse imaging reveals a gradient of conformational change that precedes loss of vinculin from focal adhesions in retracting regions. At stable or protruding regions, recruitment of vinculin is not necessarily coupled to the actin-binding conformation. However, a different measure of vinculin conformation, the recruitment of vinexin beta by activated vinculin, shows that autoinhibition of endogenous vinculin is relaxed at focal adhesions. Beyond providing direct evidence that vinculin is activated at focal adhesions, this study shows that the specific functional conformation correlates with regional cellular dynamics.
Asunto(s)
Membrana Celular/metabolismo , Adhesiones Focales/metabolismo , Vinculina/química , Vinculina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos Bacterianos/farmacología , Proteínas Bacterianas/farmacología , Adhesión Celular/fisiología , Línea Celular , Membrana Celular/ultraestructura , Forma de la Célula/fisiología , Extensiones de la Superficie Celular/metabolismo , Extensiones de la Superficie Celular/ultraestructura , Adhesiones Focales/ultraestructura , Humanos , Ratones , Microscopía por Video , Proteínas Musculares/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Conformación ProteicaRESUMEN
Vinculin is a highly conserved intracellular protein with a crucial role in the maintenance and regulation of cell adhesion and migration. In the cytosol, vinculin adopts a default autoinhibited conformation. On recruitment to cell-cell and cell-matrix adherens-type junctions, vinculin becomes activated and mediates various protein-protein interactions that regulate the links between F-actin and the cadherin and integrin families of cell-adhesion molecules. Here we describe the crystal structure of the full-length vinculin molecule (1,066 amino acids), which shows a five-domain autoinhibited conformation in which the carboxy-terminal tail domain is held pincer-like by the vinculin head, and ligand binding is regulated both sterically and allosterically. We show that conformational changes in the head, tail and proline-rich domains are linked structurally and thermodynamically, and propose a combinatorial pathway to activation that ensures that vinculin is activated only at sites of cell adhesion when two or more of its binding partners are brought into apposition.
Asunto(s)
Vinculina/química , Vinculina/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Rastreo Diferencial de Calorimetría , Adhesión Celular , Pollos , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
A direct, transient interaction between vinculin and Arp2/3 is required to promote receptor-stimulated lamellipodial extension and cell spreading. Vinculin selectively recruits Arp2/3 to the leading edge of the lamellipodium, where it may couple the actin polymerization machinery to adhesion complexes to promote membrane protrusion over ruffling.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Seudópodos/fisiología , Vinculina/metabolismo , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , MovimientoRESUMEN
Vinculin is a highly conserved and abundant cytoskeletal protein involved in linking the actin cytoskeleton to the cell membrane at sites of cellular adhesion. At these sites of adhesion, vinculin plays a role in physiological processes such as cell motility, migration, development, and wound healing. Loss of normal vinculin function has been associated with cancer phenotypes, cardiovascular disease, and lethal errors in embryogenesis. The tail domain of vinculin (Vt) binds to acidic phospholipids and has been proposed to play a role in vinculin activation and focal adhesion turnover. To better characterize Vt-lipid specificity, we conducted a series of lipid co-sedimentation experiments and find that Vt shows specific association with phosphatidylinositol 4,5-bisphosphate (PIP2), compared with phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), or phosphatidylinositol (PI) in the context of mixed lipid vesicles. The C terminus of Vt has been proposed to be important for PIP2 association, as various mutations and deletions within the C-terminal reduce PIP2 association. Lipid co-sedimentation and NMR analyses indicate that removal of the hydrophobic hairpin does not alter Vt structure or PIP2 association. However, more extensive deletions within the C-terminal introduce Vt structural perturbations and reduce PIP2 binding. Intriguingly, a significant increase in PIP2 binding was observed for multiple Vt variants that perturb interactions between the N-terminal strap and helix bundle, suggesting that a rearrangement of this N-terminal strap may be required for PIP2 binding.
Asunto(s)
Proteínas Aviares/química , Fosfatidilinositol 4,5-Difosfato/química , Vinculina/química , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Adhesión Celular/fisiología , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Movimiento Celular/fisiología , Pollos , Citoesqueleto/química , Citoesqueleto/genética , Citoesqueleto/metabolismo , Desarrollo Embrionario/fisiología , Interacciones Hidrofóbicas e Hidrofílicas , Resonancia Magnética Nuclear Biomolecular , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Vinculina/genética , Vinculina/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
Vinculin regulates cell adhesion by strengthening contacts between extracellular matrix and the cytoskeleton. Binding of the integrin ligand, talin, to the head domain of vinculin and F-actin to its tail domain is a potential mechanism for this function, but vinculin is autoinhibited by intramolecular interactions between its head and tail domain and must be activated to bind talin and actin. Because autoinhibition of vinculin occurs by synergism between two head and tail interfaces, one hypothesis is that activation could occur by two ligands that coordinately disrupt both interfaces. To test this idea we use a fluorescence resonance energy transfer probe that reports directly on activation of vinculin. Neither talin rod, VBS3 (a talin peptide that mimics a postulated activated state of talin), nor F-actin alone can activate vinculin. But in the presence of F-actin either talin rod or VBS3 induces dose-dependent activation of vinculin. The activation data are supported by solution phase binding studies, which show that talin rod or VBS3 fails to bind vinculin, whereas the same two ligands bind tightly to vinculin head domain (K(d) approximately 100 nM). These data strongly support a combinatorial mechanism of vinculin activation; moreover, they are inconsistent with a model in which talin or activated talin is sufficient to activate vinculin. Combinatorial activation implies that at cell adhesion sites vinculin is a coincidence detector awaiting simultaneous signals from talin and actin polymerization to unleash its scaffolding activity.
Asunto(s)
Actinas/fisiología , Proteínas Aviares/fisiología , Talina/fisiología , Vinculina/metabolismo , Actinas/química , Secuencia de Aminoácidos , Animales , Proteínas Aviares/química , Proteínas Aviares/metabolismo , Línea Celular , Pollos , Técnicas Químicas Combinatorias , Transferencia Resonante de Energía de Fluorescencia , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Talina/química , Vinculina/antagonistas & inhibidores , Vinculina/químicaRESUMEN
Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrin-cytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or alpha-actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to Kd approximately 10(-7) is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells.
Asunto(s)
Vinculina/química , Fluorescencia , Humanos , Integrina beta1/metabolismo , Mutación , Unión Proteica , Conformación Proteica , Talina/metabolismo , Vinculina/genética , Vinculina/metabolismoRESUMEN
Vinculin is autoinhibited by an intramolecular interaction that masks binding sites for talin and F-actin. Although a recent structural model explains autoinhibition solely in terms of the interaction between vinculin tail (Vt) and residues 1-258 (D1), we find an absolute requirement for an interface involving the D4 domain of head (Vh residues 710-836) and Vt. Charge-to-alanine mutations in Vt revealed a class of mutants, T12 and T19, distal to the V-(1-258) binding site, which showed increases in their Kd values for head binding of 100- and 42-fold, respectively. Reciprocal mutation of residues in the D4 domain that contact Vt yielded a head-tail interaction mutant of comparable magnitude to T19. These findings account for the approximately 120-fold difference in Kd values between Vt binding to V-(1-258), as opposed to full-length Vh-(1-851). The significance of a bipartite autoinhibitory site is evidenced by its effects on talin binding to Vh. Whereas Vt fails to compete with the talin rod domain for binding to V-(1-258), competition occurs readily with full-length Vh, and this requires the D4 interface. Moreover in intact vinculin, mutations in the D4-Vt interface stabilize association of vinculin and talin rod. In cells, these head-tail interaction mutants induce hypertrophy and elongation of focal adhesions. Definition of a second autoinhibitory site, the D4-Vt interface, supports the competing model of vinculin activation that invokes cooperative action of ligands at two sites. Together the D1-Vt and D4-Vt interfaces provide the high affinity (approximately 10(-9)) autoinhibition observed in full-length vinculin.
Asunto(s)
Talina/metabolismo , Vinculina/metabolismo , Actinas/química , Alanina/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Unión Competitiva , Línea Celular , Relación Dosis-Respuesta a Droga , Fibronectinas/química , Adhesiones Focales/metabolismo , Vectores Genéticos , Humanos , Immunoblotting , Cinética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Talina/química , Talina/genética , Transfección , Vinculina/química , Vinculina/genéticaRESUMEN
Colchicine, a known microtubule disrupting agent, produces a human myopathy, characterized by accumulation of lysosomes. We have created a reliable animal model of colchicine myopathy that replicates the subacute myopathy seen in humans, reproducing the chronic proximal weakness and vacuolar changes in nonnecrotic myofibers. If a microtubule network plays a role in lysosomal function in muscle, disturbance of it could alter degradation of intrinsic membrane receptors, presumably at some intracellular processing site or at exocytosis. Thus, we examined, as a possible cellular pathogenesis of colchicine myopathy, how the muscle cytoskeleton affects the degradation of membrane proteins, which are processed through the endosomal/lysosomal pathway. We used the acetylcholine receptor as a model membrane component in cultured myotubes allowed to preincubate with colchicine. We tested at which step colchicine interferes with receptor trafficking by accounting for internalization, delivery to lysosomes, hydrolysis, or exocytotic release of debris. We report that colchicine significantly decreases the exocytosis of AChRs but does not affect receptor internalization, lysosomal hydrolysis, or the number of surface membrane receptors. Further, our immunofluorescence observations revealed a morphologic tubulin network in rat skeletal muscle that is more densely distributed in white (mitochondria-poor) muscle fibers than in red (mitochondria-rich) fibers but is present in both. Ultrastructurally, immunogold labeling localized tubulin in the intermyofibrillar region in a long and linear fashion, unassociated with myofibers or mitochondria. Taken together, our findings suggest the following: (1) Microtubules likely play a functional role in the pathway of lysosomal degradation in normal adult skeletal muscle; (2) The observed decrease in overall apparent degradation of membrane receptors by colchicine must be due primarily to inhibition of exocytosis. These data indicate that lysosomal "constipation" underlies colchicine myopathy. (3) An animal model faithful to the human disorder will allow further pathogenetic studies.