Project description:The role of the RhoGTPase Rac1 in stabilizing mature endothelial adherens junctions (AJs) is not well understood. In this paper, using a photoactivatable probe to control Rac1 activity at AJs, we addressed the relationship between Rac1 and the dynamics of vascular endothelial cadherin (VE-cadherin). We demonstrated that Rac1 activation reduced the rate of VE-cadherin dissociation, leading to increased density of VE-cadherin at AJs. This response was coupled to a reduction in actomyosin-dependent tension across VE-cadherin adhesion sites. We observed that inhibiting myosin II directly or through photo-release of the caged Rho kinase inhibitor also reduced the rate of VE-cadherin dissociation. Thus, Rac1 functions by stabilizing VE-cadherin trans-dimers in mature AJs by counteracting the actomyosin tension. The results suggest a new model of VE-cadherin adhesive interaction mediated by Rac1-induced reduction of mechanical tension at AJs, resulting in the stabilization of VE-cadherin adhesions.
Project description:Primary hepatocytes from several different species rapidly lose viability and phenotypic functions on isolation from their native microenvironment of the liver. Stromal cells derived from both within and outside the liver can induce phenotypic functions in primary hepatocytes in vitro; however, the molecular mediators underlying this "coculture effect" have not been fully elucidated. We have previously developed a functional genomic screen utilizing cocultures of hepatocytes and 3T3 fibroblasts to identify such candidate hepatocyte-function-inducing molecules. In particular, truncated-cadherin (T-cadherin) was identified as a potential molecule of interest in induction of hepatic functions. Here we demonstrate that liver-specific functions of primary rat hepatocytes are induced on cocultivation with Chinese hamster ovary cells engineered to express T-cadherin on their surface as compared with wild-type controls. Additionally, culture of cells on substrata presenting recombinant T-cadherin protein (acellular presentation) enhanced hepatic functions in both pure hepatocyte cultures and in hepatocyte-stromal cocultures lacking endogenous T-cadherin expression. Collectively, these data indicate that both cellular and acellular presentation of T-cadherin can be used to modulate the hepatocyte phenotype in vitro for tissue engineering applications. Our work suggests potential avenues for investigating the role of T-cadherin on hepatocellular function in vivo in settings such as embryogenesis and liver pathology.
Project description:Mechanotransduction at cell-cell adhesions is crucial for the structural integrity, organization, and morphogenesis of epithelia. At cell-cell junctions, ternary E-cadherin/β-catenin/αE-catenin complexes sense and transmit mechanical load by binding to F-actin. The interaction with F-actin, described as a two-state catch bond, is weak in solution but is strengthened by applied force due to force-dependent transitions between weak and strong actin-binding states. Here, we provide direct evidence from optical trapping experiments that the catch bond property principally resides in the αE-catenin actin-binding domain (ABD). Consistent with our previously proposed model, the deletion of the first helix of the five-helix ABD bundle enables stable interactions with F-actin under minimal load that are well described by a single-state slip bond, even when αE-catenin is complexed with β-catenin and E-cadherin. Our data argue for a conserved catch bond mechanism for adhesion proteins with structurally similar ABDs. We also demonstrate that a stably bound ABD strengthens load-dependent binding interactions between a neighboring complex and F-actin, but the presence of the other αE-catenin domains weakens this effect. These results provide mechanistic insight to the cooperative binding of the cadherin-catenin complex to F-actin, which regulate dynamic cytoskeletal linkages in epithelial tissues.
Project description:Cadherins are homophilic cell-cell adhesion molecules whose aberrant expression has often been shown to correlate with different stages of tumor progression. In this work, we investigate the interaction of two peptidomimetic ligands with the extracellular portion of human E-cadherin using a combination of NMR and computational techniques. Both ligands have been previously developed as mimics of the tetrapeptide sequence Asp1-Trp2-Val3-Ile4 of the cadherin adhesion arm, and have been shown to inhibit E-cadherin-mediated adhesion in epithelial ovarian cancer cells with millimolar potency. To sample a set of possible interactions of these ligands with the E-cadherin extracellular portion, STD-NMR experiments in the presence of two slightly different constructs, the wild type E-cadherin-EC1-EC2 fragment and the truncated E-cadherin-(Val3)-EC1-EC2 fragment, were carried out at three temperatures. Depending on the protein construct, a different binding epitope of the ligand and also a different temperature effect on STD signals were observed, both suggesting an involvement of the Asp1-Trp2 protein sequence among all the possible binding events. To interpret the experimental results at the atomic level and to probe the role of the cadherin adhesion arm in the dynamic interaction with the peptidomimetic ligand, a computational protocol based on docking calculations and molecular dynamics simulations was applied. In agreement with NMR data, the simulations at different temperatures unveil high variability/dynamism in ligand-cadherin binding, thus explaining the differences in ligand binding epitopes. In particular, the modulation of the signals seems to be dependent on the protein flexibility, especially at the level of the adhesive arm, which appears to participate in the interaction with the ligand. Overall, these results will help the design of novel cadherin inhibitors that might prevent the swap dimer formation by targeting both the Trp2 binding pocket and the adhesive arm residues.
Project description:Adiponectin is a highly abundant protein hormone secreted by adipose tissue. It elicits diverse biological responses, including anti-diabetic, anti-inflammatory, anti-tumor, and anti-atherosclerotic effects. Adiponectin consists of a globular domain and a collagen-like domain, and it occurs in three major oligomeric forms that self-assemble: trimers, hexamers, and high-molecular-weight oligomers. Adiponectin has been reported to bind to two seven-transmembrane domain receptors, AdipoR1 and AdipoR2, as well as to the protein T-cadherin, which is highly expressed in the cardiovascular system and binds only the high-molecular-weight form of adiponectin. The molecular mechanisms underlying this specificity remain unclear. Here we used a combination of X-ray crystallography and protein engineering to define the details of adiponectin's interaction with T-cadherin. We found that T-cadherin binds to the globular domain of adiponectin, relying on structural stabilization of this domain by bound metal ions. Moreover, we show that the adiponectin globular domain can be engineered to enhance its binding affinity for T-cadherin. These results help to define the molecular basis for the interaction between adiponectin and T-cadherin, and our engineered globular domain variants may be useful tools for further investigating adiponectin's functions.
Project description:Endothelial p120-catenin (p120) maintains the level of vascular endothelial cadherin (VE-Cad) by inhibiting VE-Cad endocytosis. Loss of p120 results in a decrease in VE-Cad levels, leading to the formation of monolayers with decreased barrier function (as assessed by transendothelial electrical resistance [TEER]), whereas overexpression of p120 increases VE-Cad levels and promotes a more restrictive monolayer. To test whether reduced endocytosis mediated by p120 is required for VE-Cad formation of a restrictive barrier, we restored VE-Cad levels using an endocytic-defective VE-Cad mutant. This endocytic-defective mutant was unable to rescue the loss of TEER associated with p120 or VE-Cad depletion. In contrast, the endocytic-defective mutant was able to prevent sprout formation in a fibrin bead assay, suggesting that p120•VE-Cad interaction regulates barrier function and angiogenic sprouting through different mechanisms. Further investigation found that depletion of p120 increases Src activity and that loss of p120 binding results in increased VE-Cad phosphorylation. In addition, expression of a Y658F-VE-Cad mutant or an endocytic-defective Y658F-VE-Cad double mutant were both able to rescue TEER independently of p120 binding. Our results show that in addition to regulating endocytosis, p120 also allows the phosphorylated form of VE-Cad to participate in the formation of a restrictive monolayer.
Project description:EPB41L5 belongs to the band 4.1 superfamily. We investigate here the involvement of EPB41L5 in epithelial-mesenchymal transition (EMT) during mouse gastrulation. EPB41L5 expression is induced during TGFbeta-stimulated EMT, whereas silencing of EPB41L5 by siRNA inhibits this transition. In EPB41L5 mutants, cell-cell adhesion is enhanced, and EMT is greatly impaired during gastrulation. Moreover, cell attachment, spreading, and mobility are greatly reduced by EPB41L5 deficiency. Gene transcription regulation during EMT occurs normally at the mRNA level; EPB41L5 siRNA does not affect either the decrease in E-cadherin or the increase in integrin expression. However, at the protein level, the decrease in E-cadherin and increase in integrin are inhibited in both EPB41L5 siRNA-treated NMuMG cells and mutant mesoderm. We find that EPB41L5 binds p120ctn through its N-terminal FERM domain, inhibiting p120ctn-E-cadherin binding. EPB41L5 overexpression causes E-cadherin relocalization into Rab5-positive vesicles in epithelial cells. At the same time, EPB41L5 binds to paxillin through its C terminus, enhancing integrin/paxillin association, thereby stimulating focal adhesion formation.
Project description:A fundamental question in stem cell biology concerns the regulatory strategies that control the choice between multipotency and differentiation. Drosophila blood progenitors or prohemocytes exhibit key stem cell characteristics, including multipotency, quiescence, and niche dependence. As a result, studies of Drosophila hematopoiesis have provided important insights into the molecular mechanisms that control these processes. Here, we show that E-cadherin is an important regulator of prohemocyte fate choice, maintaining prohemocyte multipotency and blocking differentiation. These functions are reminiscent of the role of E-cadherin in mammalian embryonic stem cells. We also show that mis-expression of E-cadherin in differentiating hemocytes disrupts the boundary between these cells and undifferentiated prohemocytes. Additionally, upregulation of E-cadherin in differentiating hemocytes increases the number of intermediate cell types expressing the prohemocyte marker, Patched. Furthermore, our studies indicate that the Drosophila GATA transcriptional co-factor, U-shaped, is required for E-cadherin expression. Consequently, E-cadherin is a downstream target of U-shaped in the maintenance of prohemocyte multipotency. In contrast, we showed that forced expression of the U-shaped GATA-binding partner, Serpent, repressed E-cadherin expression and promoted lamellocyte differentiation. Thus, U-shaped may maintain E-cadherin expression by blocking the inhibitory activity of Serpent. Collectively, these observations suggest that GATA:FOG complex formation regulates E-cadherin levels and, thereby, the choice between multipotency and differentiation. The work presented in this report further defines the molecular basis of prohemocyte cell fate choice, which will provide important insights into the mechanisms that govern stem cell biology.
Project description:This study aims to define the role of E-cadherin (Ecad) engagement in cell-cell contact during membrane-cortex interaction. As a tool, we used a hydrodynamic membrane tube extrusion technique to characterize the mechanical interaction between the plasma membrane and the underlying cortical cytoskeleton. Cells were anchored on 4.5 microm beads coated with polylysine (PL) to obtain nonspecific cell adhesion or with an antibody against Ecad to mimic specific Ecad-mediated cell adhesion. We investigated tube length dynamics L(t) over time and through successive extrusions applied to the cell at regular time intervals. A constant slow velocity was observed for the first extrusion, for PL-attached cells. Subsequent extrusions had two phases: an initial high-velocity regime followed by a low-velocity regime. Successive extrusions gradually weakened the binding of the membrane around the tube neck to the underlying cortical cytoskeleton. Cells specifically attached via Ecad first exhibited a very low extrusion velocity regime followed by a faster extrusion regime similar to nonspecific extrusion. This indicates that Ecad strengthens the membrane-cortical cytoskeleton interaction, but only in a restricted area corresponding to the site of contact between the cell and the bead. Occasional giant "cortex" tubes were extruded with specifically anchored cells, demonstrating that the cortex remained tightly bound to the membrane through Ecad-mediated adhesion at the contact site.