Nonchordate classic cadherins have a structurally and functionally unique domain that is absent from chordate classic cadherins

Classic cadherins, which are adhesion molecules in cell-cell adherens junctions, have a large contribution to the construction of the animal body. Their molecular structures show clear differences between chordate and nonchordate metazoans. Although nonchordate classic cadherins have cadherin superfamily-specific extracellular repeats (CRs) and a highly conserved cytoplasmic domain (CP), these cadherins have a unique extracellular domain that is absent from vertebrate and ascidian classic cadherins. We called this the primitive classic cadherin domain (PCCD). To understand the roles of the PCCD, we constructed and characterized a series of mutant forms of the Drosophila classic cadherin DE-cadherin. Biochemical analyses indicated that the last two CRs and PCCD form a special structure with proteolytic cleavage. Mutations in the PCCD did not eliminate the cell-cell-binding function of DE-cadherin in cultured cells, but prevented the cadherin from efficiently translocating to the plasma membrane in epithelial cells of the developing embryo. In addition, genetic rescue assays suggested that although CP-mediated control plays a central role in tracheal fusion, the role of the PCCD in efficient recruitment of DE-cadherin to apical areas of the plasma membranes is also important for dynamic epithelial morphogenesis. We propose that there is a fundamental difference in the mode of classic cadherin-mediated cell-cell adhesion between chordate and nonchordate metazoans.     

Diversification of epithelial adherens junctions with independent reductive changes in cadherin form: identification of potential molecular synapomorphies among bilaterians

The adherens junction (AJ) is the most universal junction found in bilaterian epithelia and may represent one of the earliest types of cell-cell junctions. The adhesion molecules responsible for forming AJs are the classic cadherins (referred to simply as cadherins), whose extracellular domain organization displays marked variety among species examined so far. In this study, we attempted to reconstruct the evolution of cadherin by analyzing new data from several arthropods (two insects, one non-insect hexapod, three crustaceans, and one chelicerate) and previously published sequences for Drosophila melanogaster and other animals. The results of comparative analyses using the BLAST tool and immunohistochemical analyses revealed that the extracellular domain organizations of a decapod, an isopod, a spider, and a starfish cadherin, which are present at AJs in the embryonic epithelia are homologous. Independent reductive changes from the ancestral state were evident in the epithelia of hexapods+branchiopod, vertebrates+urochordates, and a cephalochordate. The form of cadherins in hexapods is more closely related to that of a branchiopod than to that of malacostracan crustaceans, and one of those of vertebrates is more closely related to that of urochordates than to that of a cephalochordate. Although the sampling of taxa is limited at this stage of research, we hypothesize that the reductive events in cadherin structure related to AJ formation in the epithelia may possess information about bilaterian relationships as molecular synapomorphies.     

Homoassociation of VE-cadherin follows a mechanism common to "classical" cadherins

Vascular endothelial cadherin (VE-cadherin/cadherin5) is specifically expressed in adherens junctions of endothelial cells and exerts important functions in cell-cell adhesion as well as signal transduction. To analyze the mechanism of VE-cadherin homoassociation, the ectodomains CAD1-5 were connected by linker sequences to the N terminus of the coiled-coil domain of cartilage matrix protein (CMP). The chimera VECADCMP were expressed in mammalian cells. The trimeric coiled-coil domain leads to high intrinsic domain concentrations and multivalency promoting self-association. Ca(2+)-dependent homophilic association of VECADCMP was detected in solid phase assays and cross-linking experiments. A striking analogy to homoassociation of type I ("classical") cadherins like E, N or P-cadherin was observed when interactions in VECADCMP and between these trimeric proteins were analyzed by electron microscopy. Ca(2+)-dependent ring-like and double ring-like arrangements suggest interactions between domains 1 and 2 of the ectodomains, which may be correlated with lateral and adhesive contacts in the adhesion process. Association to complexes composed of two VECADCMP molecules was also demonstrated by chemical cross-linking. No indication for an antiparallel association of VECAD ectodomains to hexameric complexes as proposed by Legrand et al. was found. Instead the data suggest that homoassociation of VE-cadherin follows the conserved mechanism of type I cadherins.     

Structure and interactions of desmosomal and other cadherins.

The cadherin superfamily of cell-cell adhesion molecules is now known to include proteins of the desmosome as well as of the adherens type of junction. The desmosomal cadherins consist of two families of proteins, the desmocollins and the desmogleins, both of which are represented by different isoforms which are differentially expressed in epidermis. The desmocollins are quite similar to the classic cadherins in overall structure, but with alternatively spliced variants; the desmogleins have extra cytoplasmic sequences added onto the basic cadherin structure. The cytoplasmic domains are specialized for binding to 'mediator' proteins, such as plakoglobin, which interconnect to the intermediate filament system rather than the actin filaments as do the classic cadherins.     

The cadherin cytoplasmic domain is unstructured in the absence of beta-catenin. A possible mechanism for regulating cadherin turnover

Cadherins are single pass transmembrane proteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion by linking the cytoskeletons of adjacent cells. In adherens junctions, the cytoplasmic domain of cadherins bind to beta-catenin, which in turn binds to the actin-associated protein alpha-catenin. The physical properties of the E-cadherin cytoplasmic domain and its interactions with beta-catenin have been investigated. Proteolytic sensitivity, tryptophan fluorescence, circular dichroism, and (1)H NMR measurements indicate that murine E-cadherin cytoplasmic domain is unstructured. Upon binding to beta-catenin, the domain becomes resistant to proteolysis, suggesting that it structures upon binding. Cadherin-beta-catenin complex stability is modestly dependent on ionic strength, indicating that, contrary to previous proposals, the interaction is not dominated by electrostatics. Comparison of 18 cadherin sequences indicates that their cytoplasmic domains are unlikely to be structured in isolation. This analysis also reveals the presence of PEST sequences, motifs associated with ubiquitin/proteosome degradation, that overlap the previously identified beta-catenin-binding site. It is proposed that binding of cadherins to beta-catenin prevents recognition of degradation signals that are exposed in the unstructured cadherin cytoplasmic domain, favoring a cell surface population of catenin-bound cadherins capable of participating in cell adhesion.     

Modulation of Cell–Cell Adherens Junctions by Surface Clustering of the N-Cadherin Cytoplasmic Tail

Cadherins mediate the formation of cell–cell adherens junctions (AJ) by homophilic interactions through their extracellular domains as well as by interacting with the actin cytoskeleton via their cytoplasmic portions. Cadherin clustering initiates cytoplasmic signaling that results in the assembly of structural components into cell–cell AJ. To elucidate the function of the cytoplasmic tail of cadherins in initiating the assembly signal, we generated and characterized a chimeric cadherin tail fused to an inert transmembrane anchor. The chimera enabled us to cluster the cadherin cytoplasmic tail in the absence of extracellular portions of the molecule. The transfected cadherin tail chimera localized to cell–cell AJ of epithelial cells, indicating that the submembrane junctional plaque has the capacity to recruit additional cadherins, with no involvement of their extracellular domains. Expression of the chimera in cells of mesenchymal origin resulted in dominant negative effects on the formation of cell–cell AJ. Surface clustering of cadherin cytoplasmic tails induced the recruitment of components and structural assembly of cell–cell AJ, thereby reversing the initial dominant–negative effects. We conclude that the cadherin cytoplasmic tail contains information required to direct the molecule to cell–cell AJ. Its function as modulator of cell–cell AJ depends on cell type and on whether the tail is clustered.     

Characterization of microRNAs in cephalochordates reveals a correlation between microRNA repertoire homology and morphological similarity in chordate evolution

SUMMARY Cephalochordates, urochordates, and vertebrates comprise the three extant groups of chordates. Although higher morphological and developmental similarity exists between cephalochordates and vertebrates, molecular phylogeny studies have instead suggested that the morphologically simplified urochordates are the closest relatives to vertebrates. MicroRNAs (miRNAs) are regarded as the major factors driving the increase of morphological complexity in early vertebrate evolution, and are extensively characterized in vertebrates and in a few species of urochordates. However, the comprehensive set of miRNAs in the basal chordates, namely the cephalochordates, remains undetermined. Through extensive sequencing of a small RNA library and genomic homology searches, we characterized 100 miRNAs from the cephalochordate amphioxus, Branchiostoma japonicum , and B. floridae . Analysis of the evolutionary history of the cephalochordate miRNAs showed that cephalochordates possess 54 miRNA families homologous to those of vertebrates, which is threefold higher than those shared between urochordates and vertebrates. The miRNA contents demonstrated a clear correlation between the extent of miRNA overlapping and morphological similarity among the three chordate groups, providing a strong evidence of miRNAs being the major genetic factors driving morphological complexity in early chordate evolution.     

Collective mechanical responses of cadherin-based adhesive junctions as predicted by simulations

Cadherin-based adherens junctions and desmosomes help stabilize cell-cell contacts with additional function in mechano-signaling, while clustered protocadherin junctions are responsible for directing neuronal circuits assembly. Structural models for adherens junctions formed by epithelial cadherin (CDH1) proteins indicate that their long, curved ectodomains arrange to form a periodic, two-dimensional lattice stabilized by tip-to-tip trans interactions (across junction) and lateral cis contacts. Less is known about the exact architecture of desmosomes, but desmoglein (DSG) and desmocollin (DSC) cadherin proteins are also thought to form ordered junctions. In contrast, clustered protocadherin (PCDH)-based cell-cell contacts in neuronal tissues are thought to be responsible for self-recognition and avoidance, and structural models for clustered PCDH junctions show a linear arrangement in which their long and straight ectodomains form antiparallel overlapped trans complexes. Here, we report all-atom molecular dynamics simulations testing the mechanics of minimalistic adhesive junctions formed by CDH1, DSG2 coupled to DSC1, and PCDHγB4, with systems encompassing up to 3.7 million atoms. Simulations generally predict a favored shearing pathway for the adherens junction model and a two-phased elastic response to tensile forces for the adhesive adherens junction and the desmosome models. Complexes within these junctions first unbend at low tensile force and then become stiff to unbind without unfolding. However, cis interactions in both the CDH1 and DSG2-DSC1 systems dictate varied mechanical responses of individual dimers within the junctions. Conversely, the clustered protocadherin PCDHγB4 junction lacks a distinct two-phased elastic response. Instead, applied tensile force strains trans interactions directly, as there is little unbending of monomers within the junction. Transient intermediates, influenced by new cis interactions, are observed after the main rupture event. We suggest that these collective, complex mechanical responses mediated by cis contacts facilitate distinct functions in robust cell-cell adhesion for classical cadherins and in self-avoidance signaling for clustered PCDHs.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. X. Genes for cell junctions and extracellular matrix

Cell junctions and the extracellular matrix (ECM) are crucial components in intercellular communication. These systems are thought to have become highly diversified during the course of vertebrate evolution. In the present study, we have examined whether the ancestral chordate already had such vertebrate systems for intercellular communication, for which we have searched the genome of the ascidian Ciona intestinalis. From this molecular perspective, the Ciona genome contains genes that encode protein components of tight junctions, hemidesmosomes and connexin-based gap junctions, as well as of adherens junctions and focal adhesions, but it does not have those for desmosomes. The latter omission is curious, and the ascidian type-I cadherins may represent an ancestral form of the vertebrate type-I cadherins and desmosomal cadherins, while Ci-Plakin may represent an ancestral protein of the vertebrate desmoplakins and plectins. If this is the case, then ascidians may have retained ancestral desmosome-like structures, as suggested by previous electron-microscopic observations. In addition, ECM genes that have been regarded as vertebrate-specific were also found in the Ciona genome. These results suggest that the last common ancestor shared by ascidians and vertebrates, the ancestor of the entire chordate clade, had essentially the same systems of cell junctions as those in extant vertebrates. However, the number of such genes for each family in the Ciona genome is far smaller than that in vertebrate genomes. In vertebrates these ancestral cell junctions appear to have evolved into more diverse, and possibly more complex, forms, compared with those in their urochordate siblings.     

Architecture of the VE-cadherin hexamer

Vascular endothelial-cadherin (VE-cadherin) is the major constituent of the adherens junctions of endothelial cells and plays a key role in angiogenesis and vascular permeability. The ectodomains EC1-4 of VE-cadherin are known to form hexamers in solution. To examine the mechanism of homotypic association of VE-cadherin, we have made a 3D reconstruction of the EC1-4 hexamer using electron microscopy and produced a homology model based on the known structure of C-cadherin EC1-5. The hexamer consists of a trimer of dimers with each N-terminal EC1 module making an antiparallel dimeric contact, and the EC4 modules forming extensive trimeric interactions. Each EC1-4 molecule makes a helical curve allowing some torsional flexibility to the edifice. While there is no direct evidence for the existence of hexamers of cadherin at adherens junctions, the model that we have produced provides indirect evidence since it can be used to explain some of the disparate results for adherens junctions. It is in accord with the X-ray and electron microscopy results, which demonstrate that the EC1 dimer is central to homotypic cadherin interaction. It provides an explanation for the force measurements of the interaction between opposing cadherin layers, which have previously been interpreted as resulting from three different interdigitating interactions. It is in accord with observations of native junctions by cryo-electron microscopy. The fact that this hexameric model of VE-cadherin can be used to explain more of the existing data on adherens junctions than any other model alone argues in favour of the existence of the hexamer at the adherens junction. In the context of the cell-cell junction these cis-trimers close to the membrane, and trans-dimers from opposing membranes, would increase the avidity of the bond.     

Allosteric Cross Talk between Cadherin Extracellular Domains

Atomic force microscopy and surface force apparatus measurements determined the functional impact of the cadherin point mutation W2A and domain deletion mutations on C-cadherin binding signatures. Direct comparison of results obtained using both experimental approaches demonstrates that C-cadherin ectodomains form multiple independent bonds that require different structural regions. The results presented reveal significant interdomain cross talk. They further demonstrate that the mutation W2A not only abolishes adhesion between N-terminal domains, but allosterically modulates other binding states that require functional domains distal to the N-terminal binding site. Such allosteric effects may play a prominent role in modulating adhesion by Type I classic cadherins, cadherin oligomerization at junctional contacts, and propagation of binding information to the cytoplasmic region.     

Enhancer evolution in chordates: Lessons from functional analyses of cephalochordate cis-regulatory modules

Chordates comprise three major groups, cephalochordates (amphioxus), tunicates (urochordates), and vertebrates. Since cephalochordates were the early branching group, comparisons between amphioxus and other chordates help us to speculate about ancestral chordates. Here, I summarize accumulating data from functional studies analyzing amphioxus cis-regulatory modules (CRMs) in model systems of other chordate groups, such as mice, chickens, clawed frogs, fish, and ascidians. Conservatism and variability of CRM functions illustrate how gene regulatory networks have evolved in chordates. Amphioxus CRMs, which correspond to CRMs deeply conserved among animal phyla, govern reporter gene expression in conserved expression domains of the putative target gene in host animals. In addition, some CRMs located in similar genomic regions (intron, upstream, or downstream) also possess conserved activity, even though their sequences are divergent. These conservative CRM functions imply ancestral genomic structures and gene regulatory networks in chordates. However, interestingly, if expression patterns of amphioxus genes do not correspond to those of orthologs of experimental models, some amphioxus CRMs recapitulate expression patterns of amphioxus genes, but not those of endogenous genes, suggesting that these amphioxus CRMs are close to the ancestral states of chordate CRMs, while vertebrates/tunicates innovated new CRMs to reconstruct gene regulatory networks subsequent to the divergence of the cephalochordates. Alternatively, amphioxus CRMs may have secondarily lost ancestral CRM activity and evolved independently. These data help to solve fundamental questions of chordate evolution, such as neural crest cells, placodes, a forebrain/midbrain, and genome duplication. Experimental validation is crucial to verify CRM functions and evolution.     

Cross-Talk between Adherens Junctions and Desmosomes Depends on Plakoglobin

Squamous epithelial cells have both adherens junctions and desmosomes. The ability of these cells to organize the desmosomal proteins into a functional structure depends upon their ability first to organize an adherens junction. Since the adherens junction and the desmosome are separate structures with different molecular make up, it is not immediately obvious why formation of an adherens junction is a prerequisite for the formation of a desmosome. The adherens junction is composed of a transmembrane classical cadherin (E-cadherin and/or P-cadherin in squamous epithelial cells) linked to either β-catenin or plakoglobin, which is linked to α-catenin, which is linked to the actin cytoskeleton. The desmosome is composed of transmembrane proteins of the broad cadherin family (desmogleins and desmocollins) that are linked to the intermediate filament cytoskeleton, presumably through plakoglobin and desmoplakin. To begin to study the role of adherens junctions in the assembly of desmosomes, we produced an epithelial cell line that does not express classical cadherins and hence is unable to organize desmosomes, even though it retains the requisite desmosomal components. Transfection of E-cadherin and/or P-cadherin into this cell line did not restore the ability to organize desmosomes; however, overexpression of plakoglobin, along with E-cadherin, did permit desmosome organization. These data suggest that plakoglobin, which is the only known common component to both adherens junctions and desmosomes, must be linked to E-cadherin in the adherens junction before the cell can begin to assemble desmosomal components at regions of cell–cell contact. Although adherens junctions can form in the absence of plakoglobin, making use only of β-catenin, such junctions cannot support the formation of desmosomes. Thus, we speculate that plakoglobin plays a signaling role in desmosome organization.Squamous epithelial cells typically contain two prominent types of cell–cell junctions: the adherens junction and the desmosome. The adherens junction is an intercellular adhesion complex that is composed of a transmembrane protein (a classical cadherin) and numerous cytoplasmic proteins (α-catenin, β-catenin and plakoglobin, vinculin and α-actinin; for reviews see Takeichi, 1990; Geiger and Ayalon, 1992). The cadherins are directly responsible for adhesive interactions via a Ca²⁺-dependent, homotypic mechanism, i.e., in the presence of sufficient Ca²⁺, cadherin on one cell binds to an identical molecule on an adjacent cell. The desmosome, also an intercellular adhesion complex, is composed of at least two different transmembrane proteins (desmoglein and desmocollin) as well as several cytoplasmic proteins, including desmoplakins and plakoglobin (Koch and Franke, 1994). The transmembrane components of the desmosome are members of the broadly defined cadherin family and also require Ca²⁺ for adhesive activity. However, decisive experimental evidence for homophilic or heterophilic interactions between desmosomal cadherins via their extracellular domains has not yet been presented (Koch and Franke, 1994; Kowalczyk et al., 1996). While members of the cadherin family constitute the transmembrane portion of both adherens junctions and desmosomes, the different classes of cadherins are linked to different cytoskeletal elements by the cytoplasmic components of each junction. Specifically, the classical cadherins are linked to actin filaments and the desmosomal cadherins to intermediate filaments.The organization of the proteins within the adherens junction is well understood (for reviews see Kemler, 1993; Cowin, 1994; Wheelock et al., 1996). Specifically, the intracellular domain of cadherin interacts directly with either plakoglobin or β-catenin, which in turns binds to α-catenin (Jou et al., 1995; Sacco et al., 1995). α-Catenin interacts with α-actinin and actin filaments, thereby linking the cadherin/ catenin complex to the cytoskeleton (Knudsen et al., 1995; Rimm et al., 1995). Cadherin/catenin complexes include either plakoglobin or β-catenin but not both (Näthke et al., 1994). The importance of the classical cadherins to the formation of adherens junctions and desmosomes has been demonstrated. Keratinocytes maintained in medium with low Ca²⁺ (i.e., 30 μM) grow as a monolayer and do not exhibit adherens junctions or desmosomes; however, elevation of Ca²⁺ concentration induces the rapid formation of adherens junctions followed by the formation of desmosomes (Hennings et al., 1980; Tsao et al., 1982; Boyce and Ham, 1983; Hennings and Holbrook, 1983; O''Keefe et al., 1987; Wheelock and Jensen, 1992; Hodivala and Watt, 1994; Lewis et al., 1994). Simultaneous blocking with functionperturbing antibodies against the two classical cadherins (E- and P-cadherin) found in keratinocytes inhibits not only Ca²⁺-induced adherens junction formation but also severely limits desmosome formation (Lewis et al., 1994; Jensen et al., 1996). Consistent with these findings, expression of a dominant-negative cadherin by keratinocytes results in decreased E-cadherin expression and delayed assembly of desmosomes (Fujimori and Takeicki, 1993; Amagai, et al., 1995). These data suggest some form of cross-talk between the proteins of the adherens junction and those of the desmosome. One candidate protein that might mediate such cross-talk is plakoglobin, since it is the only known common component of both junctions.Plakoglobin is found to be associated with the cytoplasmic domains of both the classical cadherins and the desmosomal cadherins. Despite the high degree of identity between plakoglobin and β-catenin (65% at the amino acid level; Fouquet et al., 1992), β-catenin only associates with the classical cadherins and not with the desmosomal cadherins. In the adherens junction, plakoglobin and β-catenin have at least one common function, i.e., the linking of cadherin to α-catenin and thus to actin. However, there is emerging evidence that other functions of these two proteins are not identical. For example, in a study by Navarro et al. (1993), E-cadherin transfected into a spindle cell carcinoma was shown to associate with α- and β-catenin, but not with the low levels of endogenous plakoglobin. The transfected cells did not revert to a more epithelial morphology in spite of the presence of functional E-cadherin, and the authors suggested that the lack of plakoglobin may have prevented such morphological reversion.In the present study, we have tested the hypothesis that plakoglobin, through its interaction with E- or P-cadherin, serves as a regulatory molecule for desmosome organization. Even though plakoglobin is not an essential structural component of the adherens junction (Sacco et al., 1995), our data indicate that plakoglobin can function as a regulator of desmosome formation only when it is associated with a classical cadherin. Thus, we propose that plakoglobin has at least two functions: (a) as a structural component of the adherens junction and the desmosome and (b) as a signaling molecule that regulates communication between the adherens junction and the desmosome.     

Elastic versus brittle mechanical responses predicted for dimeric cadherin complexes

Cadherins are a superfamily of adhesion proteins involved in a variety of biological processes that include the formation of intercellular contacts, the maintenance of tissue integrity, and the development of neuronal circuits. These transmembrane proteins are characterized by ectodomains composed of a variable number of extracellular cadherin (EC) repeats that are similar but not identical in sequence and fold. E-cadherin, along with desmoglein and desmocollin proteins, are three classical-type cadherins that have slightly curved ectodomains and engage in homophilic and heterophilic interactions through an exchange of conserved tryptophan residues in their N-terminal EC1 repeat. In contrast, clustered protocadherins are straighter than classical cadherins and interact through an antiparallel homophilic binding interface that involves overlapped EC1 to EC4 repeats. Here we present molecular dynamics simulations that model the adhesive domains of these cadherins using available crystal structures, with systems encompassing up to 2.8 million atoms. Simulations of complete classical cadherin ectodomain dimers predict a two-phased elastic response to force in which these complexes first softly unbend and then stiffen to unbind without unfolding. Simulated α, β, and γ clustered protocadherin homodimers lack a two-phased elastic response, are brittle and stiffer than classical cadherins and exhibit complex unbinding pathways that in some cases involve transient intermediates. We propose that these distinct mechanical responses are important for function, with classical cadherin ectodomains acting as molecular shock absorbers and with stiffer clustered protocadherin ectodomains facilitating overlap that favors binding specificity over mechanical resilience. Overall, our simulations provide insights into the molecular mechanics of single cadherin dimers relevant in the formation of cellular junctions essential for tissue function.     

Intestinal LI-cadherin acts as a Ca2+-dependent adhesion switch

Cadherins are Ca(2+)-dependent transmembrane glycoproteins that mediate cell-cell adhesion and are important for the structural integrity of epithelia. LI-cadherin and the classical E-cadherin are the predominant two cadherins in the intestinal epithelium. LI-cadherin consists of seven extracellular cadherin repeats and a short cytoplasmic part that does not interact with catenins. In contrast, E-cadherin is composed of five cadherin repeats and a large cytoplasmic domain that is linked via catenins to the actin cytoskeleton. Whereas E-cadherin is concentrated in adherens junctions, LI-cadherin is evenly distributed along the lateral contact area of intestinal epithelial cells. To investigate if the particular structural properties of LI-cadherin result in a divergent homotypic adhesion mechanism, we analyzed the binding parameters of LI-cadherin on the single molecule and the cellular level using atomic force microscopy, affinity chromatography and laser tweezer experiments. Homotypic trans-interaction of LI-cadherin exhibits low affinity binding with a short lifetime of only 1.4 s. Interestingly, LI-cadherin binding responds to small changes in extracellular Ca(2+) below the physiological plasma concentration with a high degree of cooperativity. Thus, LI-cadherin might serve as a Ca(2+)-regulated switch for the adhesive system on basolateral membranes of the intestinal epithelium.     

Loss of ancestral genes in the genomic evolution of Ciona intestinalis

Comparison of the predicted protein sets encoded by the complete genomes of two vertebrates (human and pufferfish), the urochordate Ciona intestinalis, three nonchordate animals, and two fungi were used to reconstruct a set of gene families present in the common ancestor of chordates. These ancestral families were much more likely to be lost in Ciona than in either vertebrate. In addition, of 256 duplicate gene pairs that arose by duplication prior to the most recent common ancestor of vertebrates and insects, one of the duplicate genes was four times as likely to be lost in Ciona as in the vertebrates. These results show that the genome of Ciona is not representative of the ancestral chordate genome with respect to gene content but rather shows derived features that may reflect adaptation of the specific ecological niche of urochordates.     

EGFR and ADAMs cooperate to regulate shedding and endocytic trafficking of the desmosomal cadherin desmoglein 2

Regulation of classic cadherins plays a critical role in tissue remodeling during development and cancer; however, less attention has been paid to the importance of desmosomal cadherins. We previously showed that EGFR inhibition results in accumulation of the desmosomal cadherin, desmoglein 2 (Dsg2), at cell-cell interfaces accompanied by inhibition of matrix metalloprotease (MMP)-dependent shedding of the Dsg2 ectodomain and tyrosine phosphorylation of its cytoplasmic domain. Here, we show that EGFR inhibition stabilizes Dsg2 at intercellular junctions by interfering with its accumulation in an internalized cytoplasmic pool. Furthermore, MMP inhibition and ADAM17 RNAi, blocked shedding and depleted internalized Dsg2, but less so E-cadherin, in highly invasive SCC68 cells. ADAM9 and 15 silencing also impaired Dsg2 processing, supporting the idea that this desmosomal cadherin can be regulated by multiple ADAM family members. In contrast, ADAM10 siRNA enhanced accumulation of a 100-kDa Dsg2 cleavage product and internalized pool of Dsg2. Although both MMP and EGFR inhibition increased intercellular adhesive strength in control cells, the response to MMP-inhibition was Dsg2-dependent. These data support a role for endocytic trafficking in regulating desmosomal cadherin turnover and function and raise the possibility that internalization and regulation of desmosomal and classic cadherin function can be uncoupled mechanistically.     

Cadherin-catenin complex: Protein interactions and their implications for cadherin function

Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms. In epithelial cells, E-cadherin represents a key molecule in the establishment and stabilization of cellular junctions. On the cellular level, E-cadherin is concentrated at the adherens junction and interacts homophilically with E-cadherin molecules of adjacent cells. Significant progress has been made in understanding the extra- and intracellular interactions of E-cadherin. Recent success in solving the three-dimensional structure of an extracellular cadherin domain provides a structural basis for understanding the homophilic interaction mechanism and the calcium requirement of cadherins. According to the crystal structure, individual cadherin molecules cooperate to form a linear cell adhesion zipper. The intracellular anchorage of cadherins is regulated by the dynamic association with cytoplasmic proteins, termed catenins. The cytoplasmic domain of E-cadherin is complexed with either β-catenin or plakoglobin (γ-catenin). β-catenin and plakoglobin bind directly to α-catenin, giving rise to two distinct cadherin-catenin complexes (CCC). α-catenin is thought to link both CCC's to actin filaments. The anchorage of cadherins to the cytoskeleton appears to be regulated by tyrosine phosphorylation. Phosphorylation-induced junctional disassembly targets the catenins, indicating that catenins are components of signal transduction pathways. The unexpected association of catenins with the product of the tumor suppressor gene APC has led to the discovery of a second, cadherin-independent catenin complex. Two separate catenin complexes are therefore involved in the cross-talk between cell adhesion and signal transduction. In this review we focus on protein interactions regulating the molecular architecture and function of the CCC. In the light of a fundamental role of the CCC during mammalian development and tissue morphogenesis, we also discuss the phenotypes of embryos lacking E-cadherin or β-catenin. © 1996 Wiley-Liss, Inc.