Cellular levels of p120 catenin function as a set point for cadherin expression levels in microvascular endothelial cells.
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The mechanisms by which catenins regulate cadherin function are not fully understood, and the precise function of p120 catenin (p120ctn) has remained particularly elusive. In microvascular endothelial cells, p120ctn colocalized extensively with cell surface VE-cadherin, but failed to colocalize with VE-cadherin that had entered intracellular degradative compartments. To test the possibility that p120ctn binding to VE-cadherin regulates VE-cadherin internalization, a series of approaches were undertaken to manipulate p120ctn availability to endogenous VE-cadherin. Expression of VE-cadherin mutants that competed for p120ctn binding triggered the degradation of endogenous VE-cadherin. Similarly, reducing levels of p120ctn using siRNA caused a dramatic and dose-related reduction in cellular levels of VE-cadherin. In contrast, overexpression of p120ctn increased VE-cadherin cell surface levels and inhibited entry of cell surface VE-cadherin into degradative compartments. These results demonstrate that cellular levels of p120ctn function as a set point mechanism that regulates cadherin expression levels, and that a major function of p120ctn is to control cadherin internalization and degradation. (+info)
p120 catenin associates with kinesin and facilitates the transport of cadherin-catenin complexes to intercellular junctions.
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p120 catenin (p120) is a component of adherens junctions and has been implicated in regulating cadherin-based cell adhesion as well as the activity of Rho small GTPases, but its exact roles in cell-cell adhesion are unclear. Using time-lapse imaging, we show that p120-GFP associates with vesicles and exhibits unidirectional movements along microtubules. Furthermore, p120 forms a complex with kinesin heavy chain through the p120 NH2-terminal head domain. Overexpression of p120, but not an NH2-terminal deletion mutant deficient in kinesin binding, recruits endogenous kinesin to N-cadherin. Disruption of the interaction between N-cadherin and p120, or the interaction between p120 and kinesin, leads to a delayed accumulation of N-cadherin at cell-cell contacts during calcium-initiated junction reassembly. Our analyses identify a novel role of p120 in promoting cell surface trafficking of cadherins via association and recruitment of kinesin. (+info)
A novel cell-cell junction system: the cortex adhaerens mosaic of lens fiber cells.
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The anucleate prismoid fiber cells of the eye lens are densely packed to form a tissue in which the plasma membranes and their associated cytoplasmic coat form a single giant cell-cell adhesive complex, the cortex adhaerens. Using biochemical and immunoprecipitation methods in various species (cow, pig, rat), in combination with immunolocalization microscopy, we have identified two different major kinds of cortical complex. In one, the transmembrane glycoproteins N-cadherin and cadherin-11 [which also occur in heterotypic ('mixed') complexes] are associated with alpha- and beta-catenin, plakoglobin (proportions variable among species), p120ctn and vinculin. The other complex contains ezrin, periplakin, periaxin and desmoyokin (and so is called the EPPD complex), usually together with moesin, spectrin(s) and plectin. In sections through lens fiber tissue, the short sides of the lens fiber hexagons appear to be enriched in the cadherin-based complexes, whereas the EPPD complexes also occur on the long sides. Moreover, high resolution double-label fluorescence microscopy has revealed, on the short sides, a finer, almost regular mosaicism of blocks comprising the cadherin-based, catenin-containing complexes, alternating with patches formed by the EPPD complexes. The latter, a new type of junctional plaque ensemble of proteins hitherto known only from certain other cell types, must be added to the list of major lens cortex proteins. We here discuss its possible functional importance for the maintenance of lens structure and functions, notably clear and sharp vision. (+info)
Lamellipodium extension and cadherin adhesion: two cell responses to cadherin activation relying on distinct signalling pathways.
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Cell adhesion molecules of the cadherin family contribute to the regulation of cell shape and fate by mediating strong intercellular adhesion through Ca2+-dependent interaction of their ectodomain and association of their cytoplasmic tail to actin. However, the mechanisms co-ordinating cadherinmediated adhesion with the reorganization of the actin cytoskeleton remain elusive. Here, the formation of de novo contacts was dissected by spreading cells on a highly active N-cadherin homophilic ligand. Cells responded to N-cadherin activation by extending lamellipodium and organizing cadherin-catenin complexes and actin filaments in cadherin adhesions. Lamellipodium protrusion, associated with actin polymerization at the leading edge sustained the extension of cadherin contacts through a phosphoinositide 3-kinase (PI 3-kinase)-Rac1 pathway. Cadherin adhesions were formed by PI 3-kinase-independent, Rac1-dependent co-recruitment of adhesion complexes and actin filaments. The expression and localization of p120 at the plasma membrane, associated with an increase in membrane-associated Rac1 was required for both cell responses, consistent with a major role of p120 in signalling pathways initiated by cadherin activation and contributing to Rac1-dependent contact extension and maturation. These results provide additional information on the mechanisms by which cadherin coordinates adhesion with dynamic changes in the cytoskeleton to control cell shape and intercellular junction organization. (+info)
p120 catenin associates with microtubules: inverse relationship between microtubule binding and Rho GTPase regulation.
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p120 catenin (p120ctn), an armadillo protein and component of the cadherin adhesion complex, has been found recently to induce a dendritic morphology by regulating Rho family GTPases. We have identified specific serines within the Arm repeat domain that, when mutated to alanine, promote p120ctn association with interphase microtubules, leading to microtubule reorganization and stabilization. The mutant p120ctn also localized to the mitotic spindle and centrosomes. In contrast to wild-type p120ctn, the microtubule-associated p120ctn mutant did not activate Rac1 and did not induce a dendritic morphology. In addition, we show that a basic motif within the p120ctn Arm repeat domain known to be required for the inhibition of RhoA is also required for binding to microtubules. We therefore propose that binding of p120ctn to microtubules is inversely related to its ability to regulate Rho GTPases. (+info)
VE-cadherin-p120 interaction is required for maintenance of endothelial barrier function.
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Interaction of p120 with juxtamembrane domain (JMD) of VE-cadherin has been implicated in regulation of endothelial cell-cell adhesion. We used a number of approaches to alter the level of p120 available for binding to VE-cadherin as a means to investigate the role of p120-VE-cadherin interaction in regulation of barrier function in confluent endothelial monolayers. Expression of an epitope-tagged fragment corresponding to JMD of VE-cadherin resulted in a decrease in endothelial barrier function as assessed by changes in albumin clearance and electrical resistance. Binding of JMD-Flag to p120 resulted in a decreased level of p120. In addition to decreasing p120 level, expression of JMD also decreased level of VE-cadherin. Expression of JMD also caused an increase in MLC phosphorylation and rearrangement of actin cytoskeleton, which, coupled with decreased cadherin, can contribute to loss of barrier function. Reducing p120 by siRNA resulted in a decrease in VE-cadherin, whereas increasing the level of p120 increased the level of VE-cadherin, demonstrating that p120 regulates the level of VE-cadherin. Overexpression of p120 was, however, associated with decreased barrier function and rearrangement of the actin cytoskeleton. Interestingly, expression of p120 was able to inhibit thrombin-induced increases in MLC phosphorylation, suggesting that p120 inhibits activation of Rho/Rho kinase pathway in endothelial cells. Excess p120 also prevented JMD-induced increases in MLC phosphorylation, correlating this phosphorylation with Rho/Rho kinase pathway. These findings show p120 plays a major role in regulating endothelial barrier function, as either a decrease or increase of p120 resulted in disruption of permeability across cell monolayers. (+info)
A novel interaction between kinesin and p120 modulates p120 localization and function.
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p120-catenin exists in a membrane-associated cadherin-bound pool, a cytosolic pool that affects Rho GTPases, and a nuclear pool that is thought to associate with the methylation-relevant transcriptional repressor Kaiso. We show here that cytoplasmic p120 can also associate both directly and indirectly with the microtubule network, and that p120 traffics along microtubules toward their plus ends. The direct binding required most of the armadillo repeats and was mutually exclusive for interaction with E-cadherin. Perturbing the p120-microtubule interaction with nocodazole or taxol markedly affected both the tubulin interaction and the balance between cytoplasmic and nuclear p120. The indirect binding occurred via a novel interaction between a segment of the p120 N-terminal domain and conventional kinesin heavy chains. Selective uncoupling of the p120-kinesin interaction by overexpression of the respective p120 and kinesin-binding fragments promoted nuclear p120 accumulation. In addition, expression of full-length kinesin reduced the nuclear accumulation of p120 and blocked the branching phenotype associated with p120 overexpression. Taken together, the data suggest that kinesin affects both the targeting and activity of p120 at several cellular locations. (+info)
Laminar shear stress differentially modulates gene expression of p120 catenin, Kaiso transcription factor, and vascular endothelial cadherin in human coronary artery endothelial cells.
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We demonstrated previously that laminar shear stress (LSS) enhances human coronary artery endothelial cell (HCAEC) wound closure via a vascular endothelial cadherin (VE-cadherin)-dependent mechanism. VE-cadherin can interact with p120 catenin (p120(ctn)) to mediate cell locomotion and proliferation. In this study, we hypothesized that p120(ctn) and an interacting protein, Kaiso, a transcriptional factor with which p120(ctn) may interact, would be expressed differentially at the wound border and away from the wound border in HCAEC exposed to LSS. One of the major goals in this study was to assess the differential gene expression of p120(ctn), Kaiso, and VE-cadherin in HCAEC at specific locations along the wound border to further our understanding of the molecular mechanisms involved in wound closure. We combined the technique of laser capture microdissection with quantitative real time PCR to compare p120(ctn), Kaiso, and VE-cadherin mRNA expression in HCAEC at and away from the wound border under LSS. Total RNA was isolated from 200-1,000 laser-captured HCAEC and reverse transcribed into cDNA. Detection of p120(ctn), Kaiso, and VE-cadherin mRNA was carried out using quantitative real time PCR. Normalization of cDNA templates was achieved by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) quantification. Quantitative real time PCR analysis revealed p120(ctn):GAPDH ratios, Kaiso: GAPDH ratios, and VE-cadherin:GAPDH ratios, relative to static control for each set, of 0.99-4.18 (mean +/- S.E., 1.94 +/- 0.404), 1.0-5.24 (2.11 +/- 0.51), and 0.99-1.42 (1.09 +/- 0.09) after 3 h of LSS, respectively. With these techniques, we found that p120(ctn) and Kaiso transcripts were increased in laser-captured HCAEC at the wound border compared with HCAEC away from the wound border. In addition, differential expression of p120(ctn) and Kaiso mRNA was observed in HCAEC depending on how LSS was applied in relation to the wounding process. These techniques may have wide applicability for studying wound healing because gene expression of key adhesion molecules in HCAEC may now be determined from select regions of the endothelial wound border. (+info)