RESUMEN
The cytoskeleton of the cell is constantly exposed to physical forces that regulate cellular functions. Selected members of the LIM (Lin-11, Isl-1, and Mec-3) domain-containing protein family accumulate along force-bearing actin fibers, with evidence supporting that the LIM domain is solely responsible for this force-induced interaction. However, LIM domain's force-induced interactions are not limited to actin. LIMK1 and LMO1, both containing only two tandem LIM domains, are recruited to force-bearing keratin fibers in epithelial cells. This unique recruitment is mediated by their LIM domains and regulated by the sequences outside the LIM domains. Based on in vitro reconstitution of this interaction, LIMK1 and LMO1 directly interact with stretched keratin 8/18 fibers. These results show that LIM domain's mechano-sensing abilities extend to the keratin cytoskeleton, highlighting the diverse role of LIM proteins in force-regulated signaling.
Asunto(s)
Filamentos Intermedios , Queratinas , Proteínas con Dominio LIM , Quinasas Lim , Proteínas con Dominio LIM/metabolismo , Humanos , Quinasas Lim/metabolismo , Queratinas/metabolismo , Filamentos Intermedios/metabolismo , Unión Proteica , Animales , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Most species of aconite contain highly toxic aconitines, the oral ingestion of which can be fatal, primarily because they cause ventricular arrhythmias. We describe a case of severe aconite poisoning that was successfully treated through veno-arterial extracorporeal membrane oxygenation (VA-ECMO) and in which detailed toxicological analyses of the aconite roots and biological samples were performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). CASE SUMMARY: A 23-year-old male presented to the emergency room with circulatory collapse and ventricular arrhythmia after ingesting approximately half of a root labeled, "Aconitum japonicum Thunb". Two hours after arrival, VA-ECMO was initiated as circulatory collapse became refractory to antiarrhythmics and vasopressors. Nine hours after arrival, an electrocardiogram revealed a return to sinus rhythm. The patient was weaned off VA-ECMO and the ventilator on hospital days 3 and 5, respectively. On hospital day 15, he was transferred to a psychiatric hospital. The other half of the root and his biological samples were toxicologically analyzed using LC-MS/MS, revealing 244.3 mg/kg of aconitine and 24.7 mg/kg of mesaconitine in the root. Serum on admission contained 1.50 ng/mL of aconitine. Beyond hospital day 2, neither were detected. Urine on admission showed 149.09 ng/mL of aconitine and 3.59 ng/mL of mesaconitine, but these rapidly decreased after hospital day 3. CONCLUSION: The key to saving the life of a patient with severe aconite poisoning is to introduce VA-ECMO as soon as possible.
RESUMEN
Biomolecular condensates are nonmembranous structures that are mainly formed through liquid-liquid phase separation. Tensins are focal adhesion (FA) proteins linking the actin cytoskeleton to integrin receptors. Here, we report that GFP-tagged tensin-1 (TNS1) proteins phase-separate to form biomolecular condensates in cells. Live-cell imaging showed that new TNS1 condensates are budding from the disassembling ends of FAs, and the presence of these condensates is cell cycle dependent. TNS1 condensates dissolve immediately prior to mitosis and rapidly reappear while postmitotic daughter cells establish new FAs. TNS1 condensates contain selected FA proteins and signaling molecules such as pT308Akt but not pS473Akt, suggesting previously unknown roles of TNS1 condensates in disassembling FAs, as the storage of core FA components and the signaling intermediates.
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Adhesiones Focales , Transducción de Señal , Tensinas , Adhesiones Focales/metabolismo , Proteínas , División Celular , Adhesión CelularRESUMEN
Protein homeostasis, including protein folding, refolding, and degradation, is thought to decline with aging. HSPB5 (also known as αB-crystallin) prevents target protein aggregation as a molecular chaperone and exhibits a cytoprotective function against various cell stresses. To elucidate the effect of HSPB5 on endoplasmic reticulum (ER) stress, we searched for novel binding proteins of HSPB5 using the proximity-dependent biotin labeling method. Proteins presumed to interact with HSPB5 in cells treated with the proteasome inhibitor MG132 were identified by a reversible biotin-binding capacity method combining tamavidin2-REV magnetic beads and mass spectrometry. We discovered a new binding protein for HSPB5, polo-like kinase 2 (PLK2), which is an apoptosis-related enzyme. The expression of PLK2 was upregulated by MG132 treatment, and it was co-localized with HSPB5 near the ER in L6 muscle cells. Inhibition of PLK2 decreased ER stress-induced phosphorylation of serine 19 in HSPB5 and increased apoptosis by activation of caspase 3 under ER stress. Overexpression of HSPB5 (WT) suppressed the ER stress-induced caspase 3 activity, but this was not observed with phospho-deficient HSPB5 (3A) mutants. These results clarify the role of HSPB5 phosphorylation during ER stress and suggest that the PLK2/HSPB5 pathway plays an essential role in cytoprotection against proteasome inhibition-induced ER stress.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Inhibidores de Proteasoma , Biotina/metabolismo , Caspasa 3/metabolismo , Citoprotección , Leupeptinas , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Agregado de Proteínas , Serina/metabolismoRESUMEN
We found that specific biomedical Ti and its alloys, such as CP Ti, Ti-29Nb-13Ta-4.6Zr, and Ti-36Nb-2Ta-3Zr-0.3O, form a bright white oxide layer after a particular oxidation heat treatment. In this paper, the interfacial microstructure of the oxide layer on Ti-29Nb-13Ta-4.6Zr and the exfoliation resistance of commercially pure (CP) Ti, Ti-29Nb-13Ta-4.6Zr, and Ti-36Nb-2Ta-3Zr-0.3O were investigated. The alloys investigated were oxidized at 1273 or 1323 K for 0.3-3.6 ks in an air furnace. The exfoliation stress of the oxide layer was high in Ti-29Nb-13Ta-4.6Zr and Ti-36Nb-2Ta-3Zr-0.3O, and the maximum exfoliation stress was as high as 70 MPa, which is almost the same as the stress exhibited by epoxy adhesives, whereas the exfoliation stress of the oxide layer on CP Ti was less than 7 MPa, regardless of duration time. The nanoindentation hardness and frictional coefficients of the oxide layer on Ti-29Nb-13Ta-4.6Zr suggested that the oxide layer was hard and robust enough for artificial tooth coating. The cross-sectional transmission electron microscopic observations of the microstructure of oxidized Ti-29Nb-13Ta-4.6Zr revealed that a continuous oxide layer formed on the surface of the alloys. The Au marker method revealed that both in- and out-diffusion occur during oxidation in Ti-29Nb-13Ta-4.6Zr and Ti-36Nb-2Ta-3Zr-0.3O, whereas only out-diffusion governs oxidation in CP Ti. The obtained results indicate that the high exfoliation resistance of the oxide layer on Ti-29Nb-13Ta-4.6Zr and Ti-36Nb-2Ta-3Zr-0.3O are attributed to their dense microstructures composing of fine particles, and a composition-graded interfacial microstructure. On the basis of the results of our microstructural observations, the oxide formation mechanism of the Ti-Nb-Ta-Zr alloy is discussed.
RESUMEN
At the onset of mitosis, centrosomes expand the pericentriolar material (PCM) to maximize their microtubule-organizing activity. This step, termed centrosome maturation, ensures proper spindle organization and faithful chromosome segregation. However, as the centrosome expands, how PCM proteins are recruited and held together without membrane enclosure remains elusive. We found that endogenously expressed pericentrin (PCNT), a conserved PCM scaffold protein, condenses into dynamic granules during late G2/early mitosis before incorporating into mitotic centrosomes. Furthermore, the N-terminal portion of PCNT, enriched with conserved coiled-coils (CCs) and low-complexity regions (LCRs), phase separates into dynamic condensates that selectively recruit PCM proteins and nucleate microtubules in cells. We propose that CCs and LCRs, two prevalent sequence features in the centrosomal proteome, are preserved under evolutionary pressure in part to mediate liquid-liquid phase separation, a process that bestows upon the centrosome distinct properties critical for its assembly and functions.
Asunto(s)
Antígenos , Centrosoma , Humanos , Microtúbulos , Mitosis , Huso AcromáticoRESUMEN
Cadherin cell-cell adhesion proteins play key roles in tissue morphogenesis and wound healing. Cadherin ectodomains bind in two conformations, X-dimers and strand-swap dimers, with different adhesive properties. However, the mechanisms by which cells regulate ectodomain conformation are unknown. Cadherin intracellular regions associate with several actin-binding proteins including vinculin, which are believed to tune cell-cell adhesion by remodeling the actin cytoskeleton. Here, we show at the single-molecule level, that vinculin association with the cadherin cytoplasmic region allosterically converts weak X-dimers into strong strand-swap dimers and that this process is mediated by myosin II-dependent changes in cytoskeletal tension. We also show that in epithelial cells, â¼70% of apical cadherins exist as strand-swap dimers while the remaining form X-dimers, providing two cadherin pools with different adhesive properties. Our results demonstrate the inside-out regulation of cadherin conformation and establish a mechanistic role for vinculin in this process.
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Cadherinas/química , Cadherinas/metabolismo , Actinas/metabolismo , Animales , Adhesión Celular , Citoesqueleto , Perros , Células de Riñón Canino Madin Darby , Miosina Tipo II/metabolismo , Unión Proteica , Vinculina/metabolismoRESUMEN
Sensing physical forces is a critical first step in mechano-transduction of cells. Zyxin, a LIM domain-containing protein, is recruited to force-bearing actin filaments and is thought to repair and strengthen them. Yet, the precise force-induced protein interactions surrounding zyxin remain unclear. Using BioID analysis, we identified proximal proteins surrounding zyxin under normal and force-bearing conditions by label-free mass spectrometry analysis. Under force-bearing conditions, increased biotinylation of α-actinin 1, α-actinin 4, and AFAP1 were detected, and these proteins accumulated along force-bearing actin fibers independently from zyxin, albeit at a lower intensity than zyxin. VASP also accumulated along force-bearing actin fibers in a zyxin-dependent manner, but the biotinylation of VASP remained constant regardless of force, supporting the model of a free zyxin-VASP complex in the cytoplasm being corecruited to tensed actin fibers. In addition, ARHGAP42, a RhoA GAP, was also identified as a proximal protein of zyxin and colocalized with zyxin along contractile actin bundles. The overexpression of ARHGAP42 reduced the rate of small wound closure, a zyxin-dependent process. These results demonstrate that the application of proximal biotinylation can resolve the proximity and composition of protein complexes as a function of force, which had not been possible with traditional biochemical analysis.
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Fenómenos Biomecánicos/fisiología , Zixina/metabolismo , Zixina/fisiología , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Perros , Adhesiones Focales/metabolismo , Células de Riñón Canino Madin Darby , Fenómenos Mecánicos , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Estrés Mecánico , Zixina/químicaRESUMEN
Epithelia are continuously self-renewed, but how epithelial integrity is maintained during the morphological changes that cells undergo in mitosis is not well understood. Here, we show that as epithelial cells round up when they enter mitosis, they exert tensile forces on neighboring cells. We find that mitotic cell-cell junctions withstand these tensile forces through the mechanosensitive recruitment of the actin-binding protein vinculin to cadherin-based adhesions. Surprisingly, vinculin that is recruited to mitotic junctions originates selectively from the neighbors of mitotic cells, resulting in an asymmetric composition of cadherin junctions. Inhibition of junctional vinculin recruitment in neighbors of mitotic cells results in junctional breakage and weakened epithelial barrier. Conversely, the absence of vinculin from the cadherin complex in mitotic cells is necessary to successfully undergo mitotic rounding. Our data thus identify an asymmetric mechanoresponse at cadherin adhesions during mitosis, which is essential to maintain epithelial integrity while at the same time enable the shape changes of mitotic cells.
Asunto(s)
Uniones Adherentes/fisiología , Células Epiteliales/fisiología , Epitelio/fisiología , Uniones Intercelulares/fisiología , Mitosis/fisiología , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Uniones Adherentes/metabolismo , Animales , Cadherinas/metabolismo , Línea Celular , Perros , Células Epiteliales/metabolismo , Epitelio/metabolismo , Uniones Intercelulares/metabolismo , Células de Riñón Canino Madin Darby , Proteínas de Microfilamentos/metabolismoRESUMEN
Aquaporin (AQP) water channels facilitate passive transport of water across cellular membranes following an osmotic gradient. AQPs are expressed in a multitude of epithelia, endothelia, and other cell types where they play important roles in physiology, especially in the regulation of body water homeostasis, skin hydration, and fat metabolism. AQP dysregulation is associated with many pathophysiological conditions, including nephrogenic diabetes insipidus, chronic kidney disease, and congestive heart failure. Moreover, AQPs have emerged as major players in a multitude of cancers where high expression correlates with metastasis and poor prognosis. Besides water transport, AQPs have been shown to be involved in cellular signaling, cell migration, cell proliferation, and regulation of junctional proteins involved in cell-cell adhesion; all cellular processes which are dysregulated in cancer. This review focuses on AQPs as regulators of junctional proteins involved in cell-cell adhesion.
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Acuaporinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Adhesión Celular , Neoplasias/metabolismo , Agua/metabolismo , Animales , Acuaporinas/química , Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Humanos , Neoplasias/patología , Estado de Hidratación del Organismo , Conformación Proteica , Transducción de Señal , Relación Estructura-Actividad , Equilibrio HidroelectrolíticoRESUMEN
Aquaporins (AQPs) are water channels that facilitate transport of water across cellular membranes. AQPs are overexpressed in several cancers. Especially in breast cancer, AQP5 overexpression correlates with spread to lymph nodes and poor prognosis. Previously, we showed that AQP5 expression reduced cell-cell adhesion by reducing levels of adherens and tight-junction proteins (e.g., ZO-1, plakoglobin, and ß-catenin) at the actual junctions. Here, we show that, when targeted to the plasma membrane, the AQP5 COOH-terminal tail domain regulated junctional proteins and, moreover, that AQP5 interacted with ZO-1, plakoglobin, ß-catenin, and desmoglein-2, which were all reduced at junctions upon AQP5 overexpression. Thus, our data suggest that AQP5 mediates the effect on cell-cell adhesion via interactions with junctional proteins independently of AQP5-mediated water transport. AQP5 overexpression in cancers may thus contribute to carcinogenesis and cancer spread by two independent mechanisms: reduced cell-cell adhesion, a characteristic of epithelial-mesenchymal transition, and increased cell migration capacity via water transport.
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Acuaporina 5/metabolismo , Adhesión Celular/fisiología , Animales , Línea Celular , Membrana Celular/metabolismo , Movimiento Celular/fisiología , Perros , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Proteínas de Uniones Estrechas/metabolismo , beta Catenina/metabolismo , gamma Catenina/metabolismoRESUMEN
We combine proximity labeling and single molecule binding assays to discover transmembrane protein interactions in cells. We first screen for candidate binding partners by tagging the extracellular and cytoplasmic regions of a "bait" protein with BioID biotin ligase and identify proximal proteins that are biotin tagged on both their extracellular and intracellular regions. We then test direct binding interactions between proximal proteins and the bait, using single molecule atomic force microscope binding assays. Using this approach, we identify binding partners for the extracellular region of E-cadherin, an essential cell-cell adhesion protein. We show that the desmosomal proteins desmoglein-2 and desmocollin-3, the focal adhesion protein integrin-α2ß1, the receptor tyrosine kinase ligand ephrin-B1, and the classical cadherin P-cadherin, all directly interact with E-cadherin ectodomains. Our data shows that combining extracellular and cytoplasmic proximal tagging with a biophysical binding assay increases the precision with which transmembrane ectodomain interactors can be identified.
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Cadherinas/genética , Efrina-B1/genética , Unión Proteica/genética , Mapas de Interacción de Proteínas/genética , Cadherinas/ultraestructura , Adhesión Celular/genética , Citoplasma/genética , Citoplasma/ultraestructura , Desmocolinas , Desmogleína 2/genética , Desmogleína 2/ultraestructura , Desmoplaquinas/genética , Desmoplaquinas/ultraestructura , Desmosomas/genética , Desmosomas/ultraestructura , Efrina-B1/ultraestructura , Humanos , Integrinas/genética , Integrinas/ultraestructura , Microscopía de Fuerza Atómica , Dominios Proteicos/genética , Imagen Individual de MoléculaRESUMEN
Cystic kidney disease is the progressive development of multiple fluid-filled cysts that may severely compromise kidney functions and lead to renal failure. TNS1 (tensin-1) knockout mice develop cystic kidneys and die from renal failure. Here, we have established TNS1-knockout MDCK cells and applied 3D culture system to investigate the mechanism leading to cyst formation. Unlike wild-type MDCK cells, which form cysts with a single lumen, TNS1-knockout cysts contain multiple lumens and upregulated Mek/Erk activities. The multiple lumen phenotype and Mek/Erk hyperactivities are rescued by re-expression of wild-type TNS1 but not the TNS1 mutant lacking a fragment essential for its cell-cell junction localization. Furthermore, Mek inhibitor treatments restore the multiple lumens back to single lumen cysts. Mek/Erk hyperactivities are also detected in TNS1-knockout mouse kidneys. Treatment with the Mek inhibitor trametinib significantly reduces the levels of interstitial infiltrates, fibrosis and dilated tubules in TNS1-knockout kidneys. These studies establish a critical role of subcellular localization of TNS1 in suppressing Mek/Erk signaling and maintaining lumenogenesis, and provide potential therapeutic strategies by targeting the Mek/Erk pathway for cystic kidney diseases.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Enfermedades Renales Poliquísticas/metabolismo , Tensinas/metabolismo , Animales , Proliferación Celular , Ratones , Ratones Noqueados , TransfecciónRESUMEN
The cytoskeleton provides structural integrity to cells and serves as a key component in mechanotransduction. Tensins are thought to provide a force-bearing linkage between integrins and the actin cytoskeleton; yet, direct evidence of tensin's role in mechanotransduction is lacking. We here report that local force application to epithelial cells using a micrometer-sized needle leads to rapid accumulation of cten (tensin 4), but not tensin 1, along a fibrous intracellular network. Surprisingly, cten-positive fibers are not actin fibers; instead, these fibers are keratin intermediate filaments. The dissociation of cten from tension-free keratin fibers depends on the duration of cell stretch, demonstrating that the external force favors maturation of cten-keratin network interactions over time and that keratin fibers retain remarkable structural memory of a cell's force-bearing state. These results establish the keratin network as an integral part of force-sensing elements recruiting distinct proteins like cten and suggest the existence of a mechanotransduction pathway via keratin network.
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Citoesqueleto/química , Células Epiteliales/química , Mecanotransducción Celular , Estrés Mecánico , Tensinas/química , Animales , Movimiento Celular , Perros , Humanos , Procesamiento de Imagen Asistido por Computador , Queratinas/química , Células de Riñón Canino Madin Darby , Proteínas de Microfilamentos/químicaRESUMEN
Protein-protein interactions are the molecular basis of cell signaling. Recently, proximity based biotin identification (BioID) has emerged as an alternative approach to traditional co-immunoprecipitation. In this protocol, a mutant biotin ligase promiscuously labels proximal binding partners with biotin, and resulting biotinylated proteins are purified using streptavidin conjugated beads. This approach does not require preservation of protein complexes in vitro, making it an ideal approach to identify transient or weak protein complexes. However, due to the high affinity bond between streptavidin and biotin, elution of biotinylated proteins from streptavidin conjugated beads requires harsh denaturing conditions, which are often incompatible with downstream processing. To effectively release biotinylated proteins bound to streptavidin conjugated beads, we designed a series of experiments to determine optimal binding and elution conditions. Interestingly, the concentrations of SDS and IGEPAL-CA630 during the incubation with streptavidin conjugated beads were the key to effective elution of biotinylated proteins using excess biotin and heating. This protocol provides an alternative method to isolate biotinylated proteins from streptavidin conjugated beads that is suitable for further downstream analysis.
Asunto(s)
Biotina/química , Proteínas/química , Proteínas/aislamiento & purificación , Animales , Biotinilación , Western Blotting , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Perros , Electroforesis en Gel de Poliacrilamida , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Calor , Inmunoprecipitación , Células de Riñón Canino Madin Darby , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Solubilidad , EstreptavidinaRESUMEN
Wnt5a-Ror signaling constitutes a developmental pathway crucial for embryonic tissue morphogenesis, reproduction and adult tissue regeneration, yet the molecular mechanisms by which the Wnt5a-Ror pathway mediates these processes are largely unknown. Using a proteomic screen, we identify the kinesin superfamily protein Kif26b as a downstream target of the Wnt5a-Ror pathway. Wnt5a-Ror, through a process independent of the canonical Wnt/ß-catenin-dependent pathway, regulates the cellular stability of Kif26b by inducing its degradation via the ubiquitin-proteasome system. Through this mechanism, Kif26b modulates the migratory behavior of cultured mesenchymal cells in a Wnt5a-dependent manner. Genetic perturbation of Kif26b function in vivo caused embryonic axis malformations and depletion of primordial germ cells in the developing gonad, two phenotypes characteristic of disrupted Wnt5a-Ror signaling. These findings indicate that Kif26b links Wnt5a-Ror signaling to the control of morphogenetic cell and tissue behaviors in vertebrates and reveal a new role for regulated proteolysis in noncanonical Wnt5a-Ror signal transduction.
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Cinesinas/metabolismo , Transducción de Señal , Proteína Wnt-5a/metabolismo , Animales , Línea Celular , Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Cinesinas/genética , Ratones , Ratones Endogámicos C57BL , Morfogénesis/efectos de los fármacos , Proteómica , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Vía de Señalización Wnt , Proteína Wnt-5a/farmacología , beta Catenina/metabolismoRESUMEN
Physical force has emerged as a key regulator of tissue homeostasis, and plays an important role in embryogenesis, tissue regeneration, and disease progression. Currently, the details of protein interactions under elevated physical stress are largely missing, therefore, preventing the fundamental, molecular understanding of mechano-transduction. This is in part due to the difficulty isolating large quantities of cell lysates exposed to force-bearing conditions for biochemical analysis. We designed a simple, easy-to-fabricate, large-scale cell stretch device for the analysis of force-sensitive cell responses. Using proximal biotinylation (BioID) analysis or phospho-specific antibodies, we detected force-sensitive biochemical changes in cells exposed to prolonged cyclic substrate stretch. For example, using promiscuous biotin ligase BirA* tagged α-catenin, the biotinylation of myosin IIA increased with stretch, suggesting the close proximity of myosin IIA to α-catenin under a force bearing condition. Furthermore, using phospho-specific antibodies, Akt phosphorylation was reduced upon stretch while Src phosphorylation was unchanged. Interestingly, phosphorylation of GSK3ß, a downstream effector of Akt pathway, was also reduced with stretch, while the phosphorylation of other Akt effectors was unchanged. These data suggest that the Akt-GSK3ß pathway is force-sensitive. This simple cell stretch device enables biochemical analysis of force-sensitive responses and has potential to uncover molecules underlying mechano-transduction.
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Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Western Blotting , Perros , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células de Riñón Canino Madin Darby , Mecanotransducción Celular/genética , Mecanotransducción Celular/fisiología , Fosforilación/genética , Fosforilación/fisiología , Unión Proteica/genética , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Estrés MecánicoRESUMEN
Collective migration of epithelial cells is an integral part of embryonic development, wound healing, tissue renewal and carcinoma invasion. While previous studies have focused on cell-extracellular matrix adhesion as a site of migration-driving, traction force-transmission, cadherin mediated cell-cell adhesion is also capable of force-transmission. Using a soft elastomer coated with purified N-cadherin as a substrate and a Hepatocyte Growth Factor-treated, transformed MDCK epithelial cell line as a model system, we quantified traction transmitted by N-cadherin-mediated contacts. On a substrate coated with purified extracellular domain of N-cadherin, cell surface N-cadherin proteins arranged into puncta. N-cadherin mutants (either the cytoplasmic deletion or actin-binding domain chimera), however, failed to assemble into puncta, suggesting the assembly of focal adhesion like puncta requires the cytoplasmic domain of N-cadherin. Furthermore, the cytoplasmic domain deleted N-cadherin expressing cells exerted lower traction stress than the full-length or the actin binding domain chimeric N-cadherin. Our data demonstrate that N-cadherin junctions exert significant traction stress that requires the cytoplasmic domain of N-cadherin, but the loss of the cytoplasmic domain does not completely eliminate traction force transmission.
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Cadherinas/genética , Células Epiteliales/metabolismo , Mecanotransducción Celular/genética , Mutación , Citoesqueleto de Actina/metabolismo , Animales , Fenómenos Biomecánicos , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Adhesión Celular/fisiología , Perros , Elastómeros/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Fibronectinas/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Células de Riñón Canino Madin Darby , Mecanotransducción Celular/efectos de los fármacos , Mecanotransducción Celular/fisiología , Microscopía Confocal , Estrés Mecánico , Propiedades de Superficie , Imagen de Lapso de Tiempo/métodosRESUMEN
Self-assembly and self-organization have recently emerged as robust scaffold-free tissue engineering methodologies that can be used to generate various tissues, including cartilage, vessel, and liver. Self-assembly, in particular, is a scaffold-free platform for tissue engineering that does not require the input of exogenous energy to the system. Although self-assembly can generate functional tissues, most notably neocartilage, the mechanisms of self-assembly remain unclear. To study the self-assembling process, we used articular chondrocytes as a model to identify parameters that can affect this process. Specifically, the roles of cell-cell and cell-matrix adhesion molecules, surface-bound collagen, and the actin cytoskeletal network were investigated. Using time-lapse imaging, we analyzed the early stages of chondrocyte self-assembly. Within hours, chondrocytes rapidly coalesced into cell clusters before compacting to form tight cellular structures. Chondrocyte self-assembly was found to depend primarily on integrin function and secondarily on cadherin function. In addition, actin or myosin II inhibitors prevented chondrocyte self-assembly, suggesting that cell adhesion alone is not sufficient, but rather the active contractile actin cytoskeleton is essential for proper chondrocyte self-assembly and the formation of neocartilage. Better understanding of the self-assembly mechanisms allows for the rational modulation of this process toward generating neocartilages with improved properties. These findings are germane to understanding self-assembly, an emerging platform for tissue engineering of a plethora of tissues, especially as these neotissues are poised for translation.
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Cartílago Articular/metabolismo , Condrocitos/metabolismo , Citoesqueleto/metabolismo , Modelos Biológicos , Animales , Cartílago Articular/citología , Bovinos , Condrocitos/citologíaRESUMEN
Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, ß-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only ß-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.