Mobility and cytoskeletal interactions of cell adhesion receptors.

Clustering of cell adhesion receptors and their interactions with the cytoskeleton are key events in the formation and function of cell adhesion structures. On the free cell surface, cadherin molecules interact with the cytoskeleton/membrane skeleton by being bound or corralled, and such interactions are greatly enhanced by the formation of cadherin oligomers. Corralled cadherin molecules undergo hop diffusion from one compartment to an adjacent one (membrane skeleton fence model), which prompts the initial formation of small adhesion clusters at cell-cell contact sites, but larger-scale assemblies of cadherin and actin filaments might require a further co-ordinated recruitment of these molecules.     

Cadherin dimers in cell-cell adhesion

While the critical function of classic cadherin in cell-cell junctions is well established, the molecular mechanism of cadherin-based adhesion remains unclear. The elusive but principal part of this adhesion process is the cadherin-cadherin interaction maintaining the intercellular contacts. This interaction is believed to be weak, suggesting that the adhesive contacts are strengthened by the cytoskeleton-dependent clustering of numerous cadherin molecules. An examination of cadherin homodimers in living cells has shown, however, that cadherin adhesive interaction is surprisingly strong. This observation implies that the strength of the adhesive contacts is regulated by the processes disintegrating cadherin dimers. The molecular structure of these dimers and mechanisms potentially responsible for their dynamics in living cells are discussed in this review.     

Computer simulations of cell sorting due to differential adhesion

The actions of cell adhesion molecules, in particular, cadherins during embryonic development and morphogenesis more generally, regulate many aspects of cellular interactions, regulation and signaling. Often, a gradient of cadherin expression levels drives collective and relative cell motions generating macroscopic cell sorting. Computer simulations of cell sorting have focused on the interactions of cells with only a few discrete adhesion levels between cells, ignoring biologically observed continuous variations in expression levels and possible nonlinearities in molecular binding. In this paper, we present three models relating the surface density of cadherins to the net intercellular adhesion and interfacial tension for both discrete and continuous levels of cadherin expression. We then use then the Glazier-Graner-Hogeweg (GGH) model to investigate how variations in the distribution of the number of cadherins per cell and in the choice of binding model affect cell sorting. We find that an aggregate with a continuous variation in the level of a single type of cadherin molecule sorts more slowly than one with two levels. The rate of sorting increases strongly with the interfacial tension, which depends both on the maximum difference in number of cadherins per cell and on the binding model. Our approach helps connect signaling at the molecular level to tissue-level morphogenesis.     

Regulation of cell adhesion by the cadherin cytoplasmic domain

Juxtacrine cell interactions associated to cadherin-mediated cell-cell adhesion play a major role in the organization and homeostasis of tissues. Here, we review the intracellular molecules and regulations controlling the formation of cell-cell contacts initiated by homophilic interactions of cadherin ectodomain. These regulations involve proteins associated to cadherin cytoplasmic tail, named catenins, their association to the actin cytoskeleton and the stability of these complexes at the cell membrane. The underlying molecular mechanisms, which participate in the formation of dynamic cell-cell contacts, are intensively investigated.     

The cadherins: cell-cell adhesion molecules controlling animal morphogenesis

Cadherins are a family of glycoproteins involved in the Ca2+-dependent cell-cell adhesion mechanism which is detected in most kinds of tissues. Inhibition of the cadherin activity with antibodies induces dissociation of cell layers, indicating a fundamental importance of these molecules in maintaining the multicellular structure. Cadherins are divided into subclasses, including E-, N- and P-cadherins. While all subclasses are similar in molecular weight, Ca2+- and protease-sensitivity, each subclass is characterized by a unique tissue distribution pattern and immunological specificity. Analysis of amino acid sequences deduced from cDNA encoding these molecules showed that they are integral membrane proteins of 723-748 amino acids long and share common sequences; similarity in the sequences between subclasses is in a range of 50-60% when compared within a single animal species. L cells, with very little endogenous cadherin activity, transfected with the cadherin cDNA acquired high cadherin-mediated aggregating activity. Their colony morphology was altered by the ectopic expression of cadherins from the dispersed type to the compact type, providing direct evidence for a key role of cadherins in cell-cell adhesion. It has been suggested that cadherins bind cells by their homophilic interactions at the extracellular domain and are associated with actin bundles at the cytoplasmic domain. It appears that each cadherin subclass has binding specificity and this molecular family is involved in selective cell-cell adhesion. In development, the expression of each cadherin subclass is spatiotemporally regulated and associated with a variety of morphogenetic events; e.g. the termination or initiation of expression of a cadherin subclass in a given cell collective is correlated with its segregation from or connection with other cell collectives. Antibodies to cadherins were shown to perturb the morphogenesis of some embryonic organs in vitro. These observations suggest that cadherins play a crucial role in construction of tissues and the whole animal body.     

Calcium-dependent cell-cell adhesion molecules (cadherins): subclass specificities and possible involvement of actin bundles

Cadherins are a family of cell-cell adhesion molecules and are divided into subclasses with distinct adhesive specificities and tissue distribution. Here we examined the distribution of cadherins at contact sites between cells expressing the same or different cadherin subclasses. Each cadherin was concentrated at the boundary between cells expressing an identical cadherin subclass, irrespective of the cell types connected. However, such localization decreased or disappeared at the boundary between cells containing different cadherin subclasses. We also found that the localization of cadherins precisely coincided with that of actin bundles; both were detected at the apical region of cell sheets. This co-localization was retained even after cells were either treated with cytochalasin D or extracted with the detergent NP40. These results suggest that each cadherin subclass preferentially interacts with its own molecular type at intercellular boundaries, and that cadherin molecules may be associated with actin- based cytoskeletal elements.     

EndoCAM: a novel endothelial cell-cell adhesion molecule

Cell-cell adhesion is controlled by many molecules found on the cell surface. In addition to the constituents of well-defined junctional structures, there are the molecules that are thought to play a role in the initial interactions of cells and that appear at precise times during development. These include the cadherins and cell adhesion molecules (CAMs). Representatives of these families of adhesion molecules have been isolated from most of the major tissues. The notable exception is the vascular endothelium. Here we report the identification of a cell surface molecule designated "endoCAM" (endothelial Cell Adhesion Molecule), which may function as an endothelial cell-cell adhesion molecule. EndoCAM is a 130-kD glycoprotein expressed on the surface of endothelial cells both in culture and in situ. It is localized to the borders of contiguous endothelial cells. It is also present on platelets and white blood cells. Antibodies against endoCAM prevent the initial formation of endothelial cell-cell contacts. Despite similarities in size and intercellular location, endoCAM does not appear to be a member of the cadherin family of adhesion receptors. The serologic and protease susceptibility characteristics of endoCAM are different from those of the known cadherins, including an endogenous endothelial cadherin. Although the precise biologic function of endoCAM has not been determined, it appears to be one of the molecules responsible for regulating endothelial cell-cell adhesion processes and may be involved in platelet and white blood cell interactions with the endothelium.     

Cadherin-based cell adhesion in neuromuscular development

The organisation and differentiation of striated skeletal muscles and their innervation is a particularly complex process implicating cells of mesodermic (myoblasts and fibroblasts) and neuroectoderrmic origin (neurons and glial cells). Myogenic and motor neuron precursors, the two major cell types participating in the formation of the neuromuscular axis, migrate, segregate, reassociate and differentiate in a coordinated fashion. The subsequent organisation of muscle cells and the establishment of muscle innervation rely on a complex tissular and cellular architectural organisation, which cannot be understood without taking into account juxtacrine cell interactions, and especially cell adhesion. Cell adhesion receptors of the cadherin family are widely expressed and dynamically regulated in space and time throughout neuromuscular development. A single cell expresses in general more than one cadherin at its surface and it is the combination of these molecules and their level of expression that determine their action within a given cell population. We focused in this review on the expression and roles of classical cadherins in relation to muscle cell and motoneuron differentiation. We also review the latest results on the mode of action of cadherins allowing to propose cellular and molecular cues on the mechanisms by which these cell adhesion receptors control muscle and neuronal cell shape, migration and differentiation.     

Direct evidence that neural cell adhesion molecule (NCAM) polysialylation increases intermembrane repulsion and abrogates adhesion

Molecular force measurements quantified the impact of polysialylation on the adhesive properties both of membrane-bound neural cell adhesion molecule (NCAM) and of other proteins on the same membrane. These results show quantitatively that NCAM polysialylation increases the range and magnitude of intermembrane repulsion. The repulsion is sufficient to overwhelm both homophilic NCAM and cadherin attraction at physiological ionic strength, and it abrogates the protein-mediated intermembrane adhesion. The steric repulsion is ionic strength dependent and decreases substantially at high monovalent salt concentrations with a concomitant increase in the intermembrane attraction. The magnitude of the repulsion also depends on the amount of polysialic acid (PSA) on the membranes, and the PSA-dependent attenuation of cadherin adhesion increases with increasing PSA-NCAM:cadherin ratios. These findings agree qualitatively with independent reports based on cell adhesion studies and reveal the likely molecular mechanism by which NCAM polysialylation regulates cell adhesion and intermembrane space.     

Cadherin-mediated cell sorting not determined by binding or adhesion specificity.

Cadherin adhesion molecules play important roles in the establishment of tissue boundaries. Cells expressing different cadherins sort out from each other in cell aggregation assays. To determine the contribution of cadherin binding and adhesion specificity to the sorting process, we examined the adhesion of cells to different purified cadherin proteins. Chinese hamster ovary cell lines expressing one of four different cadherins were allowed to bind to the purified cadherin extracellular domains of either human E-cadherin or Xenopus C-cadherin, and the specificity of adhesion was compared with cell-sorting assays. None of the different cadherin-expressing cells exhibited any adhesive specificity toward either of the two purified cadherin substrates, even though these cadherins differ considerably in their primary sequence. In addition, all cells exhibited similar strengthening of adhesion on both substrates. However, this lack of adhesive specificity did not determine whether different cadherin-expressing cells would sort from each other, and the tendency to sort was not predictable by the extent of sequence diversity in their extracellular domains. These results show that cadherins are far more promiscuous in their adhesive-binding capacity than had been expected and that the ability to sort out must be determined by mechanisms other than simple adhesive-binding specificity.     

Cadherin-mediated cell-cell adhesion: sticking together as a family

The cadherins comprise a family of single-pass transmembrane proteins critical for cell-cell adhesion in vertebrates and invertebrates. The recently determined structure of the whole ectodomain from C-cadherin suggests that the adhesion of cadherins presented by juxtaposed cells is mediated by a strand-swapped dimer in which core hydrophobic elements are exchanged between the partner molecules. Sequence analysis suggests that several cadherin subfamilies share this adhesive mechanism. Recent work has shed new light on the molecular basis of cadherin adhesion, although understanding the specificity of these interactions remains a major challenge.     

Cadherins and their connections: adhesion junctions have broader functions.

Cadherins - a family of cell-cell adhesion molecules - are linked to the actin cytoskeleton via intervening proteins. Recent results address molecular explanations for observed cadherin behavior, point to signals that regulate adhesion by modulating elements of the cadherin-associated complex, challenge the belief that different cadherins generally cannot cross-adhere, and highlight instructive roles for cadherins in cell signaling and differentiation.     

Retinal patterning by Pax6‐dependent cell adhesion molecules

Long‐standing evidence gained from Pax6 mutant embryos pointed to an involvement of Pax6‐dependent cell adhesion molecules in patterning the central nervous system and, in particular, the retina. However, direct evidence for such pathways remained elusive. We here present direct evidence that knockdown of Pax6 expression by morpholino antisense molecules in Xenopus embryos and knockdown of maternal N‐cadherin (mNcad), N‐cadherin (Ncad) and neural cell adhesion molecule (NCAM) produce similar phenotypes. Eye formation is reduced and retinal lamination is heavily disorganized. In Pax6 knockdown embryos, the levels of mRNAs coding for these cell adhesion molecules are markedly reduced. Overexpression of Pax6 efficiently rescues the phenotype of Pax6 knockdown embryos and restores expression of these putative target genes. Rescue of Pax6‐deficiency by the putative target gene mNcad moderately rescues eye formation. The promoters of the genes coding for cell adhesion molecules contain several putative Pax6 binding sites, as determined by computer analysis. Chromatin immunoprecipitation shows that, in embryonic heads, Pax6 binds to promoter regions containing such predicted binding sites. Thus, several cell adhesion molecules are direct target genes of Pax6 and cooperate in retinal patterning. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 764–780, 2010     

Desmoglein shows extensive homology to the cadherin family of cell adhesion molecules.

Desmoglein is a major adhesive component of the desmosome. It is also at least one of the antigenic targets of pathogenic antibodies circulating in the sera of patients with the blistering disease Pemphigus foliaceus. To examine the molecular basis of desmosomal adhesion and to further our understanding of its disruption in various bullous disorders we have cloned cDNAs encoding four of the extracellular domains of desmoglein. The predicted amino acid sequence of these clones shows extensive homology with the cadherin class of calcium-dependent cell adhesion molecules. Desmoglein represents a novel subtype of this family.     

Transmembrane control of cadherin-mediated cell-cell adhesion

The cadherin family of cell-cell adhesion molecules plays a central role in organization of cells into multicellular structures. An important feature of the action of cadherins is that they form a complex with cytoskeletal proteins, and the formation of this complex is crucial for their adhesive function. Cadherin-mediated cell adhesion is thus controlled through the interaction with cytoplasmic proteins, and, for such control, phosphorylation of these proteins and also cadherins themselves might be involved. This regulatory mechanism of cell adhesion is perhaps fundamental to a variety of morphogenetic processes.     

The roles of catenins in the cadherin-mediated cell adhesion: functional analysis of E-cadherin-alpha catenin fusion molecules

The carboxyl terminus-truncated cadherin (nonfunctional cadherin) has no cell adhesion activity probably because of its failure to associate with cytoplasmic proteins called alpha and beta catenin. To rescue this nonfunctional cadherin as adhesion molecules, we constructed three cDNAs for fusion proteins between nonfunctional E-cadherin and alpha catenin, nE alpha, nE alpha N, and nE alpha C, where the intact, amino- terminal and carboxy-terminal half of alpha catenin, respectively, were directly linked to the nonfunctional E-cadherin, and introduced them into mouse L cells. The subcellular distribution and cell adhesion activity of nE alpha and nE alpha C molecules was similar to those of intact E-cadherin transfectants: they bound to cytoskeletons, were concentrated at cell-cell adhesion sites and showed strong cell adhesion activity. nE alpha N molecules, which also bound to cytoskeletons, showed very poor cell adhesion activity. Taken together, we conclude that in the formation of the cadherin-catenin complex, the mechanical association of alpha catenin, especially its carboxy- terminal half, with E-cadherin is a key step for the cadherin-mediated cell adhesion. Close comparison revealed that the behavior of nE alpha molecules during cytokinesis was quite different from that of intact E- cadherin, and that the intercellular motility, i.e., the cell movement in a confluent sheet, was significantly suppressed in nE alpha transfectants although it was facilitated in E-cadherin transfectants. Considering that nE alpha was not associated with endogenous beta catenin in transfectants, the difference in the nature of cell adhesion between nE alpha and intact E-cadherin transfectants may be explained by the function of beta catenin. The possible functions of beta catenin are discussed with a special reference to its role as a negative regulator for the cadherin-mediated cell adhesion system.     

MMP inhibitors augment fibroblast adhesion through stabilization of focal adhesion contacts and up-regulation of cadherin function

Increased pericellular proteolysis due to an imbalance between MMPs (matrix metalloproteinases) and TIMPs (tissue inhibitors of metalloproteinases) promotes early stages of tumorigenesis. We have reported that TIMP-1 down-regulation confers tumorigenicity on immortal Swiss 3T3 fibroblasts. In pursuit of the mechanism involved in this transformation, we asked whether MMP inhibitors modulate contact inhibition and cell adhesion, because the dysregulation of these events is essential for cellular transformation. Using both genetic and biochemical means, we demonstrate that MMP inhibitors regulate fibroblast cell adhesion. TIMP-1 down-regulated cells formed dense, multilayered colonies, suggesting a loss of contact inhibition. Recombinant TIMP-1 and synthetic MMP inhibitors (MMPi) restored normal cell contact and density of these cells in a dose-dependent manner. Consequently, the effect of MMPi on both cell-extracellular matrix (ECM) and cell-cell adhesion were investigated. Upon MMPi treatment, p125(FAK) was redistributed, together with vinculin, to points of cell-ECM contact. Furthermore, phosphorylation of p125(FAK) was restored to levels similar to that of wild type. In parallel, MMPi treatment increased cadherin levels and stabilized cadherin-mediated cell-cell contacts. Moreover, enhanced cadherin function was evident as increased calcium-dependent cell-cell aggregation and co-localization of cadherin and beta-catenin at the cell membrane. We also obtained independent evidence of altered cadherin function using timp-1(-/-) mouse embryonic fibroblasts. Our data provide provocative evidence that increased pericellular proteolysis impacts cell adhesion systems to offset normal contact inhibition, with subsequent effects on cell transformation and tumorigenesis.     

Cell Signalling: Do adhesion molecules signal via FGF receptors?

Increasing evidence suggests that cadherin and immunoglobulin-family cell adhesion molecules can activate FGF receptors. This interaction may be crucial to developmental processes that are regulated by adhesion.     

Cell adhesion: More than just glue (Review)

The ability of cells to interact with each other and their surroundings in a co-ordinated manner depends on multiple adhesive interactions between neighbouring cells and their extracellular environment. These adhesive interactions are mediated by a family of cell surface proteins, termed cell adhesion molecules. Fortunately these adhesion molecules fall into distinct families with adhesive interactions varying in strength from strong binding involved in the maintenance of tissue architecture to more transient, less avid, dynamic interactions observed in leukocyte biology. Adhesion molecules are extremely versatile cell surface receptors which not only stick cells together but provide biochemical and physical signals that regulate a range of diverse functions, such as cell proliferation, gene expression, differentiation, apoptosis and migration. In addition, like many other cell surface molecules, they have been usurped as portals of entry for pathogens, including prions. How the mechanical and chemical messages generated from adhesion molecules are integrated with other signalling pathways (such as receptor tyrosine kinases and phosphatases) and the role that aberrant cell adhesion plays in developmental defects and disease pathology are currently very active areas of research. This review focuses on the biochemical features that define whether a cell surface molecule can act as an adhesion molecule, and discusses five specific examples of how cell adhesion molecules function as more than just 'sticky’ receptors. The discussion is confined to the signalling events mediated by members of the integrin, cadherin and immunoglobulin gene superfamilies. It is suggested that, by controlling the membrane organization of signalling receptors, by imposing spatial organization, and by regulating the local concentration of cytosolic adapter proteins, intercellular and cell-matrix adhesion is more than just glue holding cells together. Rather dynamic ‘conversations’ and the formation of multi-protein complexes between adhesion molecules, growth factor receptors and matrix macromolecules can now provide a molecular explanation for the long-observed but poorly understood requirement for a number of seemingly distinct cell surface molecules to be engaged for efficient cell function to occur.     

Identification of a cadherin cell adhesion recognition sequence

The molecular mechanisms by which the cadherins interact with one another to promote cell adhesion have not been elucidated. In particular, the amino acid sequences of the cadherin cell adhesion recognition sites have not been determined. Here we demonstrate that synthetic peptides containing the sequence HAV, which is common to all of the cadherins, inhibit two processes (compaction of eight-cell-stage mouse embryos and rat neurite outgrowth on astrocytes) that are known to be mediated by cadherins. The data suggest that the tripeptide HAV is a component of a cadherin cell adhesion recognition sequence.