α-Catenin-dependent cytoskeletal tension controls Yap activity in the heart.
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ABSTRACT:
SUBMITTER: Vite A
PROVIDER: S-EPMC5868989 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Adherens junctions (AJs) are essential for cell-cell contacts, morphogenesis, and the development of all higher eukaryotes. AJs are formed by calcium-dependent homotypic interactions of the ectodomains of single membrane-pass cadherin family receptors. These homotypic interactions in turn promote binding of the intracellular cytoplasmic tail domains of cadherin receptors with β-catenin, a multifunctional protein that plays roles in both transcription and AJs. The cadherin receptor-β-catenin complex binds to the cytoskeletal protein α-catenin, which is essential for both the formation and the stabilization of these junctions. Precisely how α-catenin contributes to the formation and stabilization of AJs is hotly debated, although the latter is thought to involve its interactions with the cytoskeletal protein vinculin. Here we report the crystal structure of the vinculin binding domain (VBD) of α-catenin in complex with the vinculin head domain (Vh1). This structure reveals that α-catenin is in a unique unfurled mode allowing dimer formation when bound to vinculin. Finally, binding studies suggest that vinculin must be in an activated state to bind to α-catenin and that this interaction is stabilized by the formation of a ternary α-catenin-vinculin-F-actin complex, which can be formed via the F-actin binding domain of either protein. We propose a feed-forward model whereby α-catenin-vinculin interactions promote their binding to the actin cytoskeleton to stabilize AJs.
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Project description:Mechanical forces are integrated at cadherin-based adhesion complexes to regulate morphology and strength of cell-cell junctions and organization of associated F-actin. A central mechanosensor at the cadherin complex is α-catenin, whose stretching recruits vinculin to regulate adhesion strength. The identity of the F-actin regulating signals that are also activated by mechanical forces at cadherin-based junctions has remained elusive. Here we identify the actin-regulators VASP, zyxin and TES as members of punctate, tensile cadherin-based junctions called Focal Adherens Junctions (FAJ) and show that they display mechanosensitive recruitment similar to that of vinculin. However, this recruitment is not altered by destroying or over-activating the α-catenin/vinculin module. Structured Illumination Microscopy (SIM) indicates that these tension sensitive proteins concentrate at locations within FAJs that are distinct from the core cadherin complex proteins. Furthermore, localization studies using mutated versions of VASP and zyxin indicate that these two proteins require binding to each other in order to localize to the FAJs. We conclude that there are multiple force sensitive modules present at the FAJ that are activated at distinct locations along the cadherin-F-actin axis and regulate specific aspects of junction dynamics.
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Project description:The cytosolic protein α-catenin is a postulated force transducer at cadherin complexes. The demonstration of force activation, identification of consequent downstream events in live cells, and development of tools to study these dynamic processes in living cells are central to elucidating the role of α-catenin in cellular mechanics and tissue function. Here we demonstrate that α-catenin is a force-activatable mechanotransducer at cell-cell junctions by using an engineered α-catenin conformation sensor based on fluorescence resonance energy transfer (FRET). This sensor reconstitutes α-catenin-dependent functions in α-catenin-depleted cells and recapitulates the behavior of the endogenous protein. Dynamic imaging of cells expressing the sensor demonstrated that α-catenin undergoes immediate, reversible conformation switching in direct response to different mechanical perturbations of cadherin adhesions. Combined magnetic twisting cytometry with dynamic FRET imaging revealed rapid, local conformation switching upon the mechanical stimulation of specific cadherin bonds. At acutely stretched cell-cell junctions, the immediate, reversible conformation change further reveals that α-catenin behaves like an elastic spring in series with cadherin and actin. The force-dependent recruitment of vinculin—a principal α-catenin effector—to junctions requires the vinculin binding site of the α-catenin sensor. In cells, the relative rates of force-dependent α-catenin conformation switching and vinculin recruitment reveal that α-catenin activation and vinculin recruitment occur sequentially, rather than in a concerted process, with vinculin accumulation being significantly slower. This engineered α-catenin sensor revealed that α-catenin is a reversible, stretch-activatable sensor that mechanically links cadherin complexes and actin and is an indispensable player in cadherin-specific mechanotransduction at intercellular junctions.
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Project description:N-glycosylation of E-cadherin has been shown to inhibit cell-cell adhesion. Specifically, our recent studies have provided evidence that the reduction of E-cadherin N-glycosylation promoted the recruitment of stabilizing components, vinculin and serine/threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs) and enhanced the association of AJs with the actin cytoskeleton. Here, we examined the details of how N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions. Using the hypoglycosylated E-cadherin variant, V13, we show that V13/?-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein. This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that increased association of PP2A with V13-containing AJs promoted their tethering to microtubules. On the other hand V13/?-catenin complexes associated more with vinculin, suggesting that they mediated the interaction of AJs with the actin cytoskeleton. N-glycosylation driven changes in the molecular organization of AJs were physiologically significant because transfection of V13 into A253 cancer cells, lacking both mature AJs and tight junctions (TJs), promoted the formation of stable AJs and enhanced the function of TJs to a greater extent than wild-type E-cadherin. These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through the assembly of distinct ?-catenin- and ?-catenin-containing scaffolds that impact the interaction with different cytoskeletal components.
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Project description:Shortly after birth, muscle cells of the mammalian heart lose their ability to divide. Thus, they are unable to effectively replace dying cells in the injured heart. The recent discovery that the transcriptional coactivator Yes-associated protein (Yap) is necessary and sufficient for cardiomyocyte proliferation has gained considerable attention. However, the upstream regulators and signaling pathways that control Yap activity in the heart are poorly understood.To investigate the role of ?-catenins in the heart using cardiac-specific ?E- and ?T-catenin double knockout mice.We used 2 cardiac-specific Cre transgenes to delete both ?E-catenin (Ctnna1) and ?T-catenin (Ctnna3) genes either in the perinatal or in the adult heart. Perinatal depletion of ?-catenins increased cardiomyocyte number in the postnatal heart. Increased nuclear Yap and the cell cycle regulator cyclin D1 accompanied cardiomyocyte proliferation in the ?-catenin double knockout hearts. Fetal genes were increased in the ?-catenin double knockout hearts indicating a less mature cardiac gene expression profile. Knockdown of ?-catenins in neonatal rat cardiomyocytes also resulted in increased proliferation, which could be blocked by knockdown of Yap. Finally, inactivation of ?-catenins in the adult heart using an inducible Cre led to increased nuclear Yap and cardiomyocyte proliferation and improved contractility after myocardial infarction.These studies demonstrate that ?-catenins are critical regulators of Yap, a transcriptional coactivator essential for cardiomyocyte proliferation. Furthermore, we provide proof of concept that inhibiting ?-catenins might be a useful strategy to promote myocardial regeneration after injury.
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Project description:Gastrointestinal stromal tumours (GISTs), the most common mesenchymal neoplasm of the gastrointestinal tract, result from deregulated proliferation of transformed KIT-positive interstitial cells of Cajal that share mesenchymal progenitors with smooth muscle cells. Despite the identification of selective KIT inhibitors, primary resistance and relapse remain a major concern. Moreover, most patients develop resistance partly through reactivation of KIT and its downstream signalling pathways. We previously identified the Limb Expression 1 (LIX1) gene as a unique marker of digestive mesenchyme immaturity. We also demonstrated that LIX1 regulates mesenchymal progenitor proliferation and differentiation by controlling the Hippo effector YAP1, which is constitutively activated in many sarcomas. Therefore, we wanted to determine LIX1 role in GIST development. We found that LIX1 is strongly up-regulated in GIST samples and this is associated with unfavourable prognosis. Moreover, LIX1 controls GIST cell proliferation in vitro and in vivo. Upon LIX1 inactivation in GIST cells, YAP1/TAZ activity is reduced, KIT (the GIST signature) is down-regulated, and cells acquire smooth muscle lineage features. Our data highlight LIX1 role in digestive mesenchyme-derived cell-fate decisions and identify this novel regulator as a target for drug design for GIST treatment by influencing its differentiation status.
| S-EPMC7417687 | biostudies-literature
Project description:BackgroundAdherens junctions (AJs) facilitate cell-cell contact and contribute to cellular communication as well as signaling under physiological and pathological conditions. Aberrant expression of AJ proteins is frequently observed in human cancers; however, how these factors contribute to tumorigenesis is poorly understood. In addition, for some factors such as α-catenin contradicting data has been described. In this study we aim to decipher how the AJ constituent α-catenin contributes to liver cancer formation.MethodsTCGA data was used to detect transcript changes in 23 human tumor types. For the detection of proteins, liver cancer tissue microarrays were analyzed by immunohistochemistry. Liver cancer cell lines (HLF, Hep3B, HepG2) were used for viability, proliferation, and migration analyses after RNAinterference-mediated gene silencing. To investigate the tumor initiating potential, vectors coding for α-catenin and myristoylated AKT were injected in mice by hydrodynamic gene delivery. A BioID assay combined with mass spectrometry was performed to identify α-catenin binding partners. Results were confirmed by proximity ligation and co-immunoprecipitation assays. Binding of transcriptional regulators at gene promoters was investigated using chromatin-immunoprecipitation.Resultsα-catenin mRNA was significantly reduced in many human malignancies (e.g., colon adenocarcinoma). In contrast, elevated α-catenin expression in other cancer entities was associated with poor clinical outcome (e.g., for hepatocellular carcinoma; HCC). In HCC cells, α-catenin was detectable at the membrane as well as cytoplasm where it supported tumor cell proliferation and migration. In vivo, α-catenin facilitated moderate oncogenic properties in conjunction with AKT overexpression. Cytokinesis regulator centrosomal protein 55 (CEP55) was identified as a novel α-catenin-binding protein in the cytoplasm of HCC cells. The physical interaction between α-catenin and CEP55 was associated with CEP55 stabilization. CEP55 was highly expressed in human HCC tissues and its overexpression correlated with poor overall survival and cancer recurrence. Next to the α-catenin-dependent protein stabilization, CEP55 was transcriptionally induced by a complex consisting of TEA domain transcription factors (TEADs), forkhead box M1 (FoxM1), and yes-associated protein (YAP). Surprisingly, CEP55 did not affect HCC cell proliferation but significantly supported migration in conjunction with α-catenin.ConclusionMigration-supporting CEP55 is induced by two independent mechanisms in HCC cells: stabilization through interaction with the AJ protein α-catenin and transcriptional activation via the FoxM1/TEAD/YAP complex.
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