Considerations on the folding topology and evolutionary origin of cadherin domains.

Cell-cell adhesion in zonula adherens and desmosomal junctions is mediated by cadherins, and recent crystal structures of the first domain from murine N-cadherin provide a plausible molecular basis for this adhesive action. A structure-based sequence analysis of this adhesive domain indicates that its fold is common to all extracellular cadherin domains. The cadherin folding topology is also shown to be similar to immunoglobulin-like domains and to other Greek-key beta-sandwich structures, as diverse as domains from plant cytochromes, bacterial cellulases, and eukaryotic transcription factors. Sequence similarities between cadherins and these other molecules are very low, however, and intron patterns are also different. On balance, independent origins for a favorable folding topology seem more likely than evolutionary divergence from an ancestor common to cadherins and immunoglobulins.

Contactus adherens, a special type of plaque-bearing adhering junction containing M-cadherin, in the granule cell layer of the cerebellar glomerulus.

In the glomeruli of the granule cell layer of mammalian cerebellum, neuronal extensions are interconnected by numerous small, nearly isodiametric (diameters up to 0.1 micron), junctions previously classified as puncta adherentia related to the vinculin-containing, actin microfilament-anchoring junctions of the zonula adherens of epithelial and certain other cells. Using immunofluorescence and immunoelectron microscopy, we have found, however, that these junctions are negative for E- and VE-cadherin, for desmosomal cadherins, and also for vinculin, alpha-actinin, and desmoplakin, but they do contain, in addition to the protein plakoglobin common to all forms of adhering junctions, the plaque proteins alpha- and beta-catenin and the transmembrane glycoprotein M-cadherin previously found as a spread--i.e., not junction bound--plasma membrane protein in certain fetal and regenerating muscle cells and in satellite cells of adult skeletal muscle. We conclude that these M-cadherin-containing junctions of the granule cell layer represent a special type of adhering junction, for which we propose the term contactus adherens (from the Latin contactus, for touch, site of bordering upon, also influence), and we discuss the differences between the various adhering junctions on the basis of their molecular constituents.

Anterior axis duplication in Xenopus induced by the over-expression of the cadherin-binding protein plakoglobin.

Plakoglobin interacts with both classical and desmosomal cadherins. It is closely related to Drosophila aramadillo (arm) gene product; arm acts in the wingless (wg)-signaling pathway to establish segment polarity. In Xenopus, homologs of wg--i.e., wnts, can produce anterior axis duplications by inducing dorsal mesoderm. Studies in Drosophila suggest that wnt acts by increasing the level of cytoplasmic armadillo protein (arm). To test whether simply increasing the level of plakoglobin mimics the effects of exogenous wnts in Xenopus, we injected fertilized eggs with RNA encoding an epitope-tagged form of plakoglobin; this induced both early radial gastrulation and anterior axis duplication. Exogenous plakoglobin accumulates in the nuclei of embryonic cells. Plakoglobin binds to the tail domain of the desmosomal cadherin desmoglein 1. When RNA encoding the tail domain of desmoglein was coinjected with plakoglobin RNA, both the dorsalizing effect and nuclear accumulation of plakoglobin were suppressed. Mutational analysis indicates that the central arm repeat region of plakoglobin is sufficient to induce axis duplication and that this polypeptide accumulates in the nuclei of embryonic cells. These data show that increased plakoglobin levels can, by themselves, generate the intracellular signals involved in the specification of dorsal mesoderm.

Minimal mutation of the cytoplasmic tail inhibits the ability of E-cadherin to activate Rac but not phosphatidylinositol 3-kinase - Direct evidence of a role for cadherin-activated Rac signaling in adhesion and contact formation

Classic cadherins are adhesion-activated cell signaling receptors. In particular, homophilic cadherin ligation can directly activate Rho family GTPases and phosphatidylinositol 3-kinase (PI3-kinase), signaling molecules with the capacity to support the morphogenetic effects of these adhesion molecules during development and disease. However, the molecular basis for cadherin signaling has not been elucidated, nor is its precise contribution to cadherin function yet understood. One attractive hypothesis is that cadherin-activated signaling participates in stabilizing adhesive contacts ( Yap, A. S., and Kovacs, E. M. ( 2003) J. Cell Biol. 160, 11-16). We now report that minimal mutation of the cadherin cytoplasmic tail to uncouple binding of p120-ctn ablated the ability of E-cadherin to activate Rac. This was accompanied by profound defects in the capacity of cells to establish stable adhesive contacts, defects that were rescued by sustained Rac signaling. These data provide direct evidence for a role of cadherin-activated Rac signaling in contact formation and adhesive stabilization. In contrast, cadherin-activated PI3-kinase signaling was not affected by loss of p120-ctn binding. The molecular requirements for E-cadherin to activate Rac signaling thus appear distinct from those that stimulate PI3-kinase, and we postulate that p120-ctn may play a central role in the E-cadherin-Rac signaling pathway.

Direct cadherin-activated cell signaling: a view from the plasma membrane

Classical cadherin adhesion molecules are key determinants of cell recognition and tissue morphogenesis, with diverse effects on cell behavior. Recent developments indicate that classical cadherins are adhesion-activated signaling receptors. In particular, early-immediate Rac signaling is emerging as a mechanism to coordinate cadherin-actin integration at the plasma membrane.

Identifying the molecular phenotype of renal progenitor cells

Although many of the molecular interactions in kidney development are now well understood, the molecules involved in the specification of the metanephric mesenchyme from surrounding intermediate mesoderm and, hence, the formation of the renal progenitor population are poorly characterized. In this study, cDNA microarrays were used to identify genes enriched in the murine embryonic day 10.5 (E10.5) uninduced metanephric mesenchyme, the renal progenitor population, in comparison with more rostral derivatives of the intermediate mesoderm. Microarray data were analyzed using R statistical software to determine accurately genes differentially expressed between these populations. Microarray outliers were biologically verified, and the spatial expression pattern of these genes at E10.5 and subsequent stages of early kidney development was determined by RNA in situ hybridization. This approach identified 21 genes preferentially expressed by the E10.5 metanephric mesenchyme, including Ewing sarcoma homolog, 14-3-3 theta, retinoic acid receptor-alpha, stearoyl-CoA desaturase 2, CD24, and cadherin-11, that may be important in formation of renal progenitor cells. Cell surface proteins such as CD24 and cadherin-11 that were strongly and specifically expressed in the uninduced metanephric mesenchyme and mark the renal progenitor population may prove useful in the purification of renal progenitor cells by FACS. These findings may assist in the isolation and characterization of potential renal stem cells for use in cellular therapies for kidney disease.

The challenges of abundance: epithelial junctions and small GTPase signalling

Small GTPases of the Ras superfamily play critical roles in epithelial biogenesis. Many key morphogenetic functions occur when small GTPases act at epithelial junctions, where they mediate an increasingly complex interplay between cell-cell adhesion molecules and fundamental cellular processes, such as cytoskeletal activity, polarity and trafficking. Important recent advances in this field include the role of additional members of the Ras superfamily in cell-cell contact stability and the capacity for polarity determinants to regulate small GTPase signalling. Interestingly, small GTPases may participate in the cross-talk between different adhesive receptors: in tissues classical cadherins can selectively regulate other junctions through cell signalling rather than through a global influence on cell-cell cohesion.

Dynamic microtubules regulate the local concentration of E-cadherin at cell-cell contacts

In contrast to the well-established relationship between cadherins and the actin cytoskeleton, the potential link between cadherins and microtubules (MTs) has been less extensively investigated. We now identify a pool of MTs that extend radially into cell-cell contacts and are inhibited by manoeuvres that block the dynamic activity of MT plus-ends (e.g. in the presence of low concentrations of nocodazole and following expression of a CLIP-170 mutant). Blocking dynamic MTs perturbed the ability of cells to concentrate and accumulate E-cadherin at cell-cell contacts, as assessed both by quantitative immunofluorescence microscopy and fluorescence recovery after photobleaching (FRAP) analysis, but did not affect either transport of E-cadherin to the plasma membrane or the amount of E-cadherin expressed at the cell surface. This indicated that dynamic MTs allow cells to concentrate E-cadherin at cell-cell contacts by regulating the regional distribution of E-cadherin once it reaches the cell surface. Importantly, dynamic MTs were necessary for myosin II to accumulate and be activated at cadherin adhesive contacts, a mechanism that supports the focal accumulation of E-cadherin. We propose that this population of MTs represents a novel form of cadherin-MT cooperation, where cadherin adhesions recruit dynamic MTs that, in turn, support the local concentration of cadherin molecules by regulating myosin II activity at cell-cell contacts.

Ena/VASP proteins can regulate distinct modes of actin organization at cadherin-adhesive contacts

Functional interactions between classical cadherins and the actin cytoskeleton involve diverse actin activities, including filament nucleation, cross-linking, and bundling. In this report, we explored the capacity of Ena/VASP proteins to regulate the actin cytoskeleton at cadherin-adhesive contacts. We extended the observation that Ena/vasodilator-stimulated phosphoprotein (VASP) proteins localize at cell-cell contacts to demonstrate that E-cadherin homophilic ligation is sufficient to recruit Mena to adhesion sites. Ena/VASP activity was necessary both for F-actin accumulation and assembly at cell-cell contacts. Moreover, we identified two distinct pools of Mena within individual homophilic adhesions that cells made when they adhered to cadherin-coated substrata. These Mena pools localized with Arp2/3-driven cellular protrusions as well as at the tips of cadherin-based actin bundles. Importantly, Ena/VASP activity was necessary for both modes of actin activity to be expressed. Moreover, selective depletion of Ena/VASP proteins from the tips of cadherin-based bundles perturbed the bundles without affecting the protrusive F-actin pool. We propose that Ena/VASP proteins may serve as higher order regulators of the cytoskeleton at cadherin contacts through their ability to modulate distinct modes of actin organization at those contacts.

Cinderella no longer: alpha-catenin steps out of cadherin's shadow

To date, alpha-catenin has been best understood as an important cytoplasmic component of the classical cadherin complex responsible for cell-cell adhesion. By virtue of its capacity to bind F-actin, alpha-catenin was commonly envisaged to support cadherin function by coupling the adhesion receptor to the actin cytoskeleton. But is alpha-catenin solely the cadherin's handmaiden? A range of recent developments suggest, instead, that its biological activity is much more complex than previously appreciated. Evidence from cellular systems and model organisms demonstrates a clear, often dramatic, role for alpha-catenin in tissue organization and morphogenesis. The morphogenetic impact of alpha-catenin reflects its capacity to mediate functional cooperation between cadherins and the actin cytoskeleton, but is not confined to this. alpha-Catenin has a role in regulating cell proliferation and cadherin-independent pools of alpha-catenin may contribute to its functional impact.

Differential downregulation of e-cadherin and desmoglein by epidermal growth factor.

Modulation of cell : cell junctions is a key event in cutaneous wound repair. In this study we report that activation of the epidermal growth factor (EGF) receptor disrupts cell : cell adhesion, but with different kinetics and fates for the desmosomal cadherin desmoglein and for E-cadherin. Downregulation of desmoglein preceded that of E-cadherin in vivo and in an EGF-stimulated in vitro wound reepithelialization model. Dual immunofluorescence staining revealed that neither E-cadherin nor desmoglein-2 internalized with the EGF receptor, or with one another. In response to EGF, desmoglein-2 entered a recycling compartment based on predominant colocalization with the recycling marker Rab11. In contrast, E-cadherin downregulation was accompanied by cleavage of the extracellular domain. A broad-spectrum matrix metalloproteinase inhibitor protected E-cadherin but not the desmosomal cadherin, desmoglein-2, from EGF-stimulated disruption. These findings demonstrate that although activation of the EGF receptor regulates adherens junction and desmosomal components, this stimulus downregulates associated cadherins through different mechanisms.