Comparison of molecular mechanisms mediating cell contact phenomena in model developmental systems:...

Comparison of molecular mechanisms

mediating cell contact phenomena in model

developmental systems: an exploration

of universality

Vivienne M. Bowers-Morrow1, Sinan O. Ali1 and Keith L. Williams2

1 Department of Biological Sciences, Macquarie University, Sydney, NSW, 2109, Australia2 Proteome Systems Limited, Locked Bag 2073, North Ryde, 1670, NSW, Australia (E-mail : [email protected])

(Received 11 May 2001; revised 9 September 2003; accepted 10 September 2003)

ABSTRACT

Are there universal molecular mechanisms associated with cell contact phenomena during metazoan onto-genesis? Comparison of adhesion systems in disparate model systems indicates the existence of unifyingprinciples.

Requirements for multicellularity are (a) the construction of three-dimensional structures involving a crucialbalance between adhesiveness and motility ; and (b) the establishment of integration at molecular, cellular, tissue,and organismal levels of organization. Mechanisms for (i) cell–cell and cell–substrate adhesion, (ii) cell movement,(iii) cell–cell communication, (iv) cellular responses, (v) regulation of these processes, and (vi) their integration withpatterning, growth, and other developmental processes are all crucial to metazoan development, and must havebeen present for the emergence and radiation of Metazoa. The principal unifying themes of this review are thedynamics and regulation of cell contact phenomena.

Our knowledge of the dynamic molecular mechanisms underlying cell contact phenomena remains frag-mentary. Here we examine the molecular bases of cell contact phenomena using extant model developmentalsystems (representing a wide range of phyla) including the simplest i.e. sponges, and the eukaryotic protistDictyostelium discoideum, the more complex Drosophila melanogaster, and vertebrate systems. We discuss cell contactphenomena in a broad developmental context.

The molecular language of cell contact phenomena is complex ; it involves a plethora of structurally andfunctionally diverse molecules, and diverse modes of intermolecular interactions mediated by protein and/orcarbohydrate moieties. Reasons for this are presumably the necessity for a high degree of specificity of inter-molecular interactions, the requirement for a multitude of different signals, and the apparent requirement foran increasingly large repertoire of cell contact molecules in more complex developmental systems, such as thedeveloping vertebrate nervous system. However, comparison of molecular models for dynamic adhesion insponges and in vertebrates indicates that, in spite of significant differences in the details of the way specificcell–cell adhesion is mediated, similar principles are involved in the mechanisms employed by members ofdisparate phyla. Universal requirements are likely to include (a) rapidly reversible intermolecular interactions ;(b) low-affinity intermolecular interactions with fast on–off rates ; (c) the compounding of multiple inter-molecular interactions ; (d) associated regulatory signalling systems. The apparent widespread employment ofmolecular mechanisms involving cadherin-like cell adhesion molecules suggests the fundamental importanceof cadherin function during development, particularly in epithelial morphogenesis, cell sorting, and segregationof cells.

Key words : cell adhesion molecules, cell movement, dynamics, cadherins, molecular mechanisms, development,model organisms.

Biol. Rev. (2004), 79, pp. 611–642. f Cambridge Philosophical Society 611DOI: 10.1017/S1464793103006389 Printed in the United Kingdom

CONTENTS

I. Introduction ................................................................................................................................................. 612II. Multicellularity : making connections between cells ............................................................................... 615

(1) Cadherins : fundamental cell adhesion molecules ............................................................................ 615(a) Vertebrate classic cadherins .......................................................................................................... 615(b) Structurally and functionally analogous cadherin-dependent supramolecular

complexes in vertebrate and Drosophila melanogaster development ............................................. 620(c) Putative cadherins in less complex metazoans ............................................................................ 620(d ) A putative cadherin, and b-catenin homologue in Dictyostelium discoideum ............................... 620

(2) Filopodia-based mechanisms for intercellular junction formation ................................................. 621(3) Clustering of cadherins : adhesion, anchorage, junction formation and signalling ...................... 621(4) Actin cytoskeletal dynamics in promotion of epithelial cell adhesion ........................................... 622(5) Mediation of cell aggregation during Dictyostelium discoideum morphogenesis ................................ 623

(a) Homophilic protein–protein binding mechanisms ..................................................................... 623(b) Role for cell extensions, and changing distribution of two cell adhesion molecules .............. 623(c) Role of cell adhesion molecules in morphogenesis ..................................................................... 623(d ) Physical properties of intermolecular binding facilitate de-adhesion and motility ................. 623(e) Clustering of cell adhesion molecules ........................................................................................... 624(f ) Comparison of adhesion in Dictyostelium discoideum with that in metazoans ............................. 624

(6) Sponge aggregation factors : mediation of adhesion via supramolecular intercellularextracellular matrix complexes ............................................................................................................ 624(a) Cell rearrangements ........................................................................................................................ 624(b) The molecular basis of cellular interactions ................................................................................ 626(c) Role for cell extensions ................................................................................................................... 628(d) Comparison with cadherin-mediated systems and unifying principles .................................... 628

III. The balance between adhesive and motile cellular phenotypes : mechanisms for theregulation of cell–cell adhesion .................................................................................................................. 628(1) Changing expression patterns ............................................................................................................. 629(2) Quantitative differences in cadherin expression ............................................................................... 629(3) Homeodomain transcription factor-mediated regulation of cell adhesion molecules ................. 629

(a) Vertebrate Hox and Pax proteins ................................................................................................. 629(b) Vertebrate and invertebrate Otx proteins ................................................................................... 629(c) Murine Msh genes ........................................................................................................................... 630(d) C. elegans ceh-43 gene ........................................................................................................................ 630

(4) Intracellular modulation: catenins and Rho GTPases .................................................................... 630(a) Catenins ............................................................................................................................................ 630(b) Rho GTPases ................................................................................................................................... 631(c) IQGAP1 ........................................................................................................................................... 631(d ) p120-catenin ..................................................................................................................................... 631(e) Recycling of E-cadherin ................................................................................................................. 632

(5) Concerted activities of different types of cell adhesion molecules and adhesive systems ............ 632(a) Migrating neural crest cells ............................................................................................................ 632(b) A regulatory module in D. discoideum ............................................................................................ 632

IV. Conclusions .................................................................................................................................................. 635V. Acknowledgements ...................................................................................................................................... 636VI. References .................................................................................................................................................... 636

I. INTRODUCTION

‘… the phenomenon of cellular adhesion is the prerequisite for the

evolution and ontogenesis of multicellular organisms. ’(Townes & Holtfreter, 1955)

The aim of this review is to consider the followingquestion: are there universal molecular mechanisms under-lying cell contact phenomena fundamental to metazoan

ontogenesis? Metazoan development involves the processesof morphogenesis, and pattern formation; the former gen-erates three-dimensional form, while the latter results inthe division of undifferentiated regions of tissue into areasof specific morphogenetic fate. Accumulating evidence sug-gests that many of the molecular building blocks, and muchof the biochemical/molecular ‘machinery’ required for de-velopment, are conserved among disparate phyla (Giudice,2001). For example, the basic body plan of most (if not all)

612 Vivienne M. Bowers-Morrow, Sinan O. Ali and Keith L. Williams

metazoans is defined by patterns of homeotic Hox geneexpression. Crucial to metazoan development are the fol-lowing cell contact phenomena: (a) cell–cell recognition andadhesion; (b) cell-substrate adhesion; (c) the dynamic inter-relationship between cell movements and cell adhesion; (d )cell–cell signalling, and intracellular signal transduction ; (e)regulation, coordination, and integration of these processes ;and ( f ) integration of them with other processes such as celldifferentiation. In this review, comparisons will be madebetween dynamic adhesive mechanisms in various develop-mental systems to determine whether similar problems aresolved in similar ways.

Only a few organisms have been extensively used asmodels for investigating cell contact phenomena; fortu-nately, they are widely distributed across the ‘phylogenetictree ’ of metazoans (Fig. 1). (Note that the Lophotrochozoansare not well represented.)

These organisms have been used by experimentalists asdevelopmental models because they can be readily ma-nipulated. Drosophila melanogaster (fruit fly), and Caenorhabditiselegans (nematode worm) are the focus of most investigationat the present time, largely because much is known abouttheir developmental genetics, they are amenable to geneticanalysis, they can be genetically modified, and they can betransformed to make transgenics. The mouse also has theseadvantages, and many developmental mutations have beenidentified. This organism is regarded as a good model formammalian development, including human development.A newly exploited model is the zebrafish Brachydanio reriowhich has great potential for genetic investigations. Amphib-ian models such as Xenopus laevis are advantageous for theanalysis of cell behaviours in morphogenetically relevantcontexts, at the cell population level, because the embryonictissue can be explanted and grafted (Keller et al., 1985;Keller & Winklbauer, 1992).

The C. elegans genome has been completed (Science, 11December 1998), as has that for D. melanogaster (Science, 24March 2000). Details of the mouse genome are also avail-able (http://www.celera.com/). As complete genome cataloguesbecome available for model organisms, the approach involv-ing systematic interrogation and manipulation of genomeswill be increasingly exploited in the near future. The rela-tively new science of proteomics (Wilkins et al., 1997), whichwill eventually enable an overview of the characteristicsand activity of every protein that an organism synthesizes (itsproteome), promises new insights with a variety of modelorganisms. At this stage, however, our understanding ofadhesion in metazoans remains fragmentary, and manydevelopmental systems still await investigation.

Since the phylogenetic history of metazoans and theprocesses of development are inextricably linked, the emerg-ence of a ‘ founder ’ set of molecules and molecular mech-anisms for dynamic cellular adhesion and other cell contactphenomena must have been essential for the transitionduring evolution from single-celled organisms to metazoanswith three-dimensional body plans. Cellular slime moulds(represented by D. discoideum) are Mycetazoans which belongto the ‘shady underworld’ of living organisms (Bonner,1993). While these eukaryotes have some animal-likecharacteristics, they also have certain features similar to

those of plants, fungi, and protozoa. Taxonomic classifi-cation of D. discoideum is difficult (Loomis & Smith, 1995;Mutzel, 1995; Kessin, 1997). Phylogenetic analyses basedon molecular data place Mycetazoans as emerging amongthe multicellular eukaryotes ; there is tentative support fortheir being more closely related to animals+fungi thanare green plants (Baldauf & Doolittle, 1997). This intriguingorganism, called appropriately, and colourfully by the French‘amibes sociales ’, has unicellular and multicellular phasesduring its asexual life cycle ; starving amoebae can surviveby forming communities. Multicellularity is achieved viaaggregation of unicellular amoebae to form a ‘slug ’ whichis subsequently transformed into a fruiting body carryingspores. In spite of the fact that D. discoideum is probablynot ancestral to members of extant metazoan phyla, andmay have ‘ invented ’ an independent means to achievemulticellularity (no growth is required), investigation ofthe molecular bases of cell contact phenomena crucial toD. discoideum development may provide clues to universalmolecular mechanisms required for the transition fromsingle-celled to multicellular organisms, and for metazoandevelopment.

As well as having mechanical functions, the majority ofcell adhesion molecules (CAMs) have also been found toserve as adhesion receptors, and signal transducers (Pigott &Power, 1993). [Note that the distinction between CAMsand signalling molecules has become blurred because manysignalling molecules have also been found to have adhesiveactivity.] Cell adhesion influences all the basic cellularprocesses governing the chain of events involved in tissueformation, i.e. proliferation, differentiation, migration, andapoptosis. The molecular language is complex. Themediation of cell–cell and cell–substrate adhesion and com-munication in metazoans such as vertebrates and D. mela-nogaster involves a plethora of structurally and functionallydiverse CAMs, substrate adhesion molecules (SAMs),and other associated molecules. Reasons for this are pre-sumably the necessity for a high degree of specificity ofintermolecular interactions, the requirement for a multitudeof different signals, and the apparent requirement for anincreasingly large repertoire of adhesive molecules in morecomplex developmental systems such as that of the devel-oping vertebrate nervous system. Diverse modes of specificintermolecular interactions mediated by protein and/orcarbohydrate moieties are involved. However, adhesionmolecules are classified as belonging to a small number ofgene families, the most important for development beingthe cadherin superfamily (Ranscht & Dours-Zimmerman,1991; Buxton & Magee, 1992; Vestal & Ranscht, 1992;Sacristan et al., 1993; Koch & Franke, 1994; Kirkpatrick &Peifer, 1995; Takeichi, 1995; Marrs & Nelson, 1996;Hatzfeld, 1999; Takeichi et al., 2000; Angst, Marcozzi &Magee, 2001), the immunoglobulin (Ig) superfamily(Williams, 1987; Williams & Barclay, 1988; Brummendorf& Rathjen, 1994), the integrins (Hynes, 1992; Burke, 1999),and the proteoglycans (Selleck, 2000). Analyses of D. mela-nogaster and C. elegans genomic sequences, using a largenumber of vertebrate sequences, has indicated a highdegree of evolutionary conservation in cell–cell, and cell-extracellular matrix (ECM) adhesion. The evolution of

Molecular mechanisms mediating cell contact phenomena 613

M.

L. K. J.

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Eukaryoticprotists

Unicellularprokaryotes

Mycetozoa

PoriferaCnidariaCtenophora

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BrachiopodaMolluscaAnnelidaNemertineaPlatyhelminthes

Ecdysozoans

Protostomes

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DeuterostomesEchinodermataHemichordataChordata

Fig. 1. Metazoan phylogeny, extant phyla, and model organisms. Schematic diagram (compiled from a variety of sources) showinga contemporary, broad phylogenetic framework for considering the relationships among metazoan phyla. A monophyletic origin ofMetazoa is indicated. The body plans of the major model organisms, and the phyla they represent are shown.

There is general agreement that metazoans must have evolved from ancestral protists (Wainright et al., 1994). The protists area group of miscellaneous organisms which ‘do not constitute a cohesive historical assemblage ’ (Erwin, 1993). According to Kessin (1997),the Protista are best viewed as having long, independent lines of descent, with an ancient divergence. Dictyostelium discoideum isa eukaryotic protist of the phylum Mycetozoa.

In constructing an accurate representation of evolution, the aim is to identify true clades of metazoans (groups derived from acommon ancestor). This diagram represents a consensus of 18S ribosomal RNA phylogenies generated by several authors, andpresented by Adoutte et al. (1999). The protostomes are shown as being monophyletic, and are subdivided into two large branches :the lophotrochozoans (who share a similar ciliated larva), and the ecdysozoans (characterized, in part, by moulting of an exoskeleton)(see Aguinaldo et al., 1997; Martindale & Kourakis, 1999; Adoutte et al., 2000). De Rosa et al. (1999) have examined the genomes ofsome key metazoan taxonomic units for Hox genes ; the identification of distinct Hox genes which are shared by some, but not all,protostome phyla, supports the division of protostomes into the lophotrochozoans and ecdysozoans. Recent palaeontologicaldocumentation of Cambrian fauna has led to phylogenetic analyses which also are broadly congruent with this division (seeMartindale & Kourakis, 1999; Shu et al., 1999).

Model organisms include : A, PhylumMycetozoa: cellular (dictyostelid) slime moulds ; Dictyostelium discoideum ; B, Phylum Porifera :sponges, Microciona prolifera and Geodia cydonium ; C, Phylum Cnidaria, Class Hydrozoa Hydra magnipapillata ; D, Phylum Nematoda:Caenorhabditis elegans ; E, Phylum Arthropoda, Class Insecta : fruit fly, Drosophila melanogaster ; F, Phylum Arthropoda, Class Insecta :grasshoppers, Schistocerca sp ; G, Phylum Mollusca, Class Gastropoda: snail, Aplysia californica ; H, Phylum Echinodermata, ClassEchinoidea. sea urchins, e.g. Lytechinus variegatus ; I, Phylum Chordata, subphylum Vertebrata, Class Osteichthyes : zebra fish,Brachydanio rerio ; J, Phylum Chordata, subphylum Vertebrata, Class Amphibia : African clawed toad, Xenopus Laevis ; K, PhylumChordata, subphylum Vertebrata, Class Aves : chicken, Gallus gallus domesticus ; L, Phylum Chordata, subphylum Vertebrata, ClassMammalia : mouse, Mus musculus ; M, Phylum Chordata, subphylum Vertebrata, Class Mammalia : man, Homo sapiens.


metazoans from single cells would have involved the evol-ution of new protein adhesion domains ; for example, im-munoglobulin domains, cadherins, and integrins are absentfrom yeast. However, once the domains and genes evolved,they have been used repeatedly (Hynes & Zhao, 2000).While the signalling pathways of the various CAMs are notcompletely understood, one general principle that hasemerged is that adhesion receptors are closely linked toprotein kinases and phosphatases (Ruoslahti & Obrink,1996).

Cellular adhesion is characterized by transient contacts,and a dynamic balance exists between cell adhesion andmotility. Grant (1826) wrote colourfully about Spongillafriabilis that it ‘quickly diffuses among the saliva leaving only someearthy particle between the teeth ’ and further, agitation of ‘ thegelatinous matter ’ of the sponge led to it ‘ resolving itself almostentirely into minute … granules, which have a singular tendency toreunite ’. After a few hours ‘ they unite into a compact … velvetymembrane … and attach themselves to the bottom of the vessel. ’Further, a few placed in a watchglass with water ‘ form them-selves into minute spheres, being constantly rolled to and fro by theanimalcules ’, and the smallest of the ‘granular bodies ’ were seento be locomotive. Long before there was any understandingat a molecular level, Holtfreter (1943, p. 311) emphasizedthe need for us to ‘ look more for the factors making for instabilitythan for those tending towards stability, for it is these continuousdeviations from an equilibrium … which are the essential features ofmorphogenesis. ’

The transient nature of cell adhesion is exemplified bytwo disparate organisms: D. discoideum, and X. laevis. Even acursory viewing of a time-lapse video of elongated, stream-ing amoebae during the aggregation stage of D. discoideumdevelopment shows that the end-to-end contacts are brokenand rejoined constantly (Bozzaro & Ponte, 1995). Because ofthe transient nature of these cell–cell contacts, individualamoebae can enter cell streams by literally pushing in, andmaking contact [see images of fluorescently labelled stream-ing amoebae at http://sdb.bio.purdue.edu/QTmovies/Knecht_wt.mov (Shelden and Knecht, 1995)]. Winklbaueret al. (1993), using time-lapse video recordings, have inves-tigated the movement of mesodermal cells on the innersurface of the blastocoel roof during X. laevis gastrulation.In the gastrula, the mesoderm moves as a compact andcoherent multilayered cell mass ; any part of the mesoderm,if excised, will move as a coherent piece of tissue. Cellsmove by extending filopodia and lamellipodia. While smallaggregates of cells can move coherently, contacts areformed and broken continuously, and cells can moverelative to each other, changing positions. The cells thatcomprise metazoan cell collectives and tissues exist in twobasic states, epithelial or mesenchymal. These differ in theirtype, activity and distribution of cell–cell and cell–ECMadhesion molecules, and also in their propensity for celllocomotion. During dynamic processes such as gastrulation,and neural crest cell migration, interconversion occurs be-tween epithelium and mesenchyme (usually of mesodermalorigin) [epithelial to mesenchymal transition, EMT]; suchtransitions are bidirectional, and can drive tissue remodel-ling. Mesenchymal cells are motile, and can invade andmigrate through the ECM; they can aggregate and form

an epithelium. The earliest example of an EMT duringembryonic development is the generation of the third germlayer, the mesoderm, from the epiblast (primitive ecto-derm), which marks the beginning of gastrulation (Viebahn,1995; Viebahn, Mayer & Miething, 1995). For example,gastrulation in sea urchins begins with an EMT, a processinvolving loss of adhesion. This process allows cells tomigrate (via the use of many filopodia) into the blastocoel,as single cells [Hay, 1995; Hay & Zuk, 1995; Wolpert et al.,1998].

The dynamic balance between cell adhesion and cellmovement is arguably the most important universal fea-ture of adhesion in development. Morphogenetic processesare characterized by: (a) cells moving as individuals ; (b) cellsmoving as loose clusters, or cell streams; (c) the dynamicarrangement and rearrangement of cells and cell layers ; (d )the spreading and folding of sheets of cells, with componentcells in close contact, and the sheet moving as a unit(Schoenwolf & Alvarez, 1992; Bronner-Fraser, 1993; New-green & Tan, 1993; Winklbauer et al., 1993; Duband et al.,1995). In this review we examine what is known about the‘make and break’ mechanisms involved in morphogeneticprocesses.

II. MULTICELLULARITY: MAKING

CONNECTIONS BETWEEN CELLS

Here we compare the means by which connections aremade between cells in different contexts : (i) during morpho-genesis in vertebrates, D. melanogaster, and less complexmetazoans, and in D. discoideum via cadherins and adherensjunctions ; (ii) during the aggregation stage of morphogenesisin D. discoideum via two different CAMs; and (iii) duringcellular aggregation in sponges via multimolecular ECMcomplexes.

(1 ) Cadherins: fundamental celladhesion molecules

(a ) Vertebrate classic cadherins

The dominant means to achieve multicellularity has beenvia cell division and growth. Metazoan tissue can be formedfrom homotypic founder cells which are prevented fromseparating from one another by ECM macromolecules,and by the formation of specialized cell junctional, supra-molecular complexes such as adherens junctions (Fig. 2).Adherens junctions serve to mediate cell–cell adhesion,anchor the actin-based cytoskeleton, and provide an organ-izing centre for signal transduction molecules, and signallingpathways (Kirkpatrick & Peifer, 1995; Marrs & Nelson,1996; Yap et al., 1997). Adherens junctions can thus func-tion as scaffolding for a variety of adhesive and signallingelements, and serve as the focus of diverse elements involvedin cell contact phenomena, and their regulation. The mol-ecular architecture of the undercoat of the adherens junc-tion comprises (i) protein–protein interactions between thecytoplasmic domain of cadherin molecules, and cytoplasmicproteins ; (ii) protein–protein interactions between catenins


and cytoskeletal elements (See Fig. 3C). Adherens junctionslink cells together, and connect their cytoskeletal elements ;they are dynamic, semistable structures which are repeat-edly assembled and disassembled in vivo (Tsukita et al., 1992).

Cells forming epithelial cell sheets are held together inthis way. Cadherins and integrins are arguably the keyCAMs required for the emergence of metazoans. They arethe major mediators of the assembly of epithelial cells invertebrates. Cadherins mediate assembly of the cells lat-erally, while integrins attach them basally to a subepithelialbasement membrane (Hagios, Lochter & Bissell, 1998) (seeFig. 2). The ability of epithelial tissues to form variousshapes and to compartmentalize the metazoan body hasbeen fundamental for the evolution of complex body plans.The formation of an epithelial sheet typically involvesthe reorganization of a cluster of mesenchymal cells into amonolayer of tightly adhering polarized cells.

Cadherins are crucial in the determination of celladhesion specificity for most types of vertebrate cells. Invertebrates, cadherin-based multiprotein adhesion com-plexes are apparently ubiquitous, fundamental elementsin adhesion systems operating during development ; cad-herin activity exists in the cells contributing to the formationof all solid tissues, in tissues derived from all three germlayers, and at all stages of development. We focus here onthe classic cadherins ; the mammalian subclasses E-cadherin,and N-cadherin are the best characterized (Takeichi,1995). These molecules establish adhesion between thecells that comprise epithelial and neuroepithelial layers.

In epithelial tissues, cadherins are required for the as-sembly of cells into multiple layers, and the establishmentand maintenance of the epithelial phenotype. Epithelialcells adhere tightly to their neighbours ; this is essential forfunctions such as the provision of a barrier, and polarizedsecretion.

Classic cadherins maintain the integrity of cell groupsthrough the formation of adherens junctions, and drivemorphogenetic movements. They play essential roles in :(a) mediating the mechanical connection of homotypic cells ;(b) maintaining tissue integrity ; (c) supramolecular cell–celljunction (adherens junctions) assembly and disassembly;(d ) establishment and maintenance of the structural andfunctional organization of cells and tissues ; (e) the structuraland functional organization of membrane domains inpolarized cells ; and ( f ) the generation of specific trans-membrane signals.

The modulation of cadherin-based adhesion is thoughtto control various dynamic morphogenetic events includingepithelial–mesenchymal conversions, and tubulogenesis.[For reviews of experimental evidence demonstrating vari-ous functions of cadherins during development see Marrs &Nelson (1996) and Simonneau et al. (1995).]

Classic cadherins are integral membrane glycoproteins.They typically have an extracellular domain composed offive tandemly repeated subdomains (EC1–EC5) (Fig. 3A, B),a single membrane-spanning segment, and a highly con-served cytoplasmic region (see Fig. 3C). There are sitesin the extracellular N-terminal domain for adhesive

Tight junctions Apical surface

Nucleus

Integrins

Basement membrane

Adherens junctionsCytoskeleton

Signals

Fig. 2. Architecture of epithelial tissue. Crucial to epithelial sheet formation are cell–cell, and cell-(extracellular matrix) adhesionmechanisms associated with (i) the formation of supramolecular intercellular junctions such as adherens junctions at the apical-lateral interface, and (ii) the formation of junctions mediating cell–ECM interactions which are involved in cell anchorage to thebasement membrane. In this schematic diagram, the location of integrin-mediated adhesion to the matrix is shown, as well as themediation of annular cell–cell adhesion via adherens junctions. Adapted from Hagios et al. (1998, p. 859). Reprinted with permissionfrom The Royal Society.


recognition, calcium binding, membrane integration,cytoskeletal interactions, and post-translational processing(e.g. glycosylation, phosphorylation, and proteolytic cleav-age). Sites for N-linked glycosylation have been mappedto the second, third, and fifth EC domains. The amino-terminal EC subdomain (EC1) is the most conserved domainof the extracellular subdomains across cadherin subclasses,and between species ; this subdomain has been implicated inmediating selectivity of binding. Binding sequences arethought to be located in the N-terminal EC subdomainEC1; the histidine-alanine-valine (HAV) motif, and alsoamino acids flanking this motif have been implicated.(Takeichi, 1988, 1990, 1991, 1995; Kemler, 1992; Tsukitaet al., 1992; Grunwald, 1993; Gumbiner & McCrea, 1993;Nagafuchi, Tsukita & Takeichi, 1993; Nathke, Hinck &Nelson, 1993; Pigott & Power, 1993; Sacristan et al., 1993;Behrens, 1994; Brar, 1994; Dalseg, Gaardsvoll & Bock,1994; Ranscht, 1994; Broders & Thiery, 1995; Kirkpatrick& Peifer, 1995; Shapiro et al., 1995a, b ; Gumbiner, 1996;Marrs & Nelson, 1996; Steinberg, 1996.)

Classical cadherins interact across the intercellular gaphomophilically. Stable cadherin-mediated cell–cell adhesiondepends on specific homophilic binding sites in the extra-cellular region of the molecule. Note that cell–cell contactsare not achieved by interaction between single molecules.The formation of higher-order complexes with high avidityis generally required. Two alternative models are depictedin Fig. 3A and B.

The linear zipper model (Fig. 3A) has been proposed[based on nuclear magnetic resonance (NMR), and X-raycrystallographic studies of E-cadherin, and N-cadherin]for cadherin-mediated adherens junctions. (Takeichi, 1990,1991, 1995; Tsukita et al., 1992; Grunwald, 1993; Stappert& Kemler, 1993; Takeichi et al., 1993; Behrens, 1994;Dalseg et al., 1994; Hulsken, Birchmeier & Behrens,1994a, b ; Pavalko & Otey, 1994; Overduin et al., 1995;Patel & Gumbiner, 1995; Shapiro et al., 1995a, b ; Weis,1995; Gumbiner, 1996; Marrs & Nelson, 1996; Nagar et al.,1996.) It is proposed that the cadherin dimerises laterallyat the cell surface.

It is thought that : (i) the building block of any higherorder structural organization is a parallel cadherin dimer;(ii) this structure is promoted by, and dependent on boundcalcium ions ; (iii) the known calcium requirement for theintegrity of cell junctions is, at least in part, explained bythe mechanical stabilization of the cadherin moleculeby calcium-mediated dimerization, and rigidification (Nagaret al., 1996).

The cadherin dimers make head-to-head contact acrossthe intercellular gap to form a so-called adhesive zipper. It isthought that the extracellular zipper-like structure interactswith a superstructure of cytoplasmic components of the ad-herens junction. Weis (1995) has proposed mechanismsfor the assembly and disassembly of cell–cell adhesion zones,and for the dynamics of cadherin-mediated cell–cell ad-hesion. The zipper can be viewed as an oligomeric interac-tion at the cell surface in which multiple, weakly interactingcadherins cooperate to form a strongly adhesive cell–celljunction. It is postulated that shifting the equilibrium be-tween strand dimers, and higher order multimers, would

result in zippering/unzippering of the cell junctionalcomplex.

An alternative model : the ‘deep intercalation model ’(Fig. 3B) was proposed by Chappuis-Flament et al. (2001)based on a structure-function analysis of the homophilicbinding properties of the soluble C-cadherin ectodomain.It is hypothesized that cadherins are fully intercalated, withinteractions between EC1 and EC5, and possibly betweenEC2 and EC4, and EC2 and EC3. There can also be sig-nificant interactions between domains EC1 and EC4, andpossibly between EC1 and EC2, and EC3. It is thoughtthat, while the EC1 domain is required for the formationof an effective adhesive bond, a minimum of three of theEC domains is required for effective homophilic bindingand adhesion. The authors suggest that an element of thezipper model may still be important : cis-dimerization couldendow the cadherin molecule on one cell with more thanone adhesive site. With multiple EC domains, the bindinginteractions of each dimer could potentially occur in manydifferent orientations resulting in the formation of a two-dimensional lattice instead of a linear zipper. This wouldbe consistent with the concept of a mutivalent low-affinityinteraction between cadherin dimers.

The conclusions of Chappuis-Flament et al. (2001) areconsistent with the deletion mutant studies of Renaud-Young & Gallin (2002), and also with direct molecular forcemeasurements between cadherin ectodomains that demon-strate multiple adhesive interactions (Sivasankar et al., 1999;Sivasankar, Gumbiner & Lechband, 2001).

Also crucial to the stability of cadherin-mediated cell–celladhesion are interactions with the actin cytoskeleton viathe cytoplasmic domain. The carboxy-terminal cytoplasmicdomain (Fig. 3C) is essential to cadherin function (Nagafuchi& Takeichi, 1988; Imamura et al., 1999). It mediates con-nections with cytoskeletal elements such as actin filaments,in adherens junctions, and associates with at least fourcytoplasmic proteins called catenins : a-catenin, b-catenin,plakoglobin (c-catenin), and p120ctn (Takeichi, 1995). Thereare two subtypes of a-catenin: a-E-catenin, and a-N-catenin (Takeichi et al., 1993; Shimamura et al., 1994; Wa-tabe et al., 1994). A cadherin molecule forms a complexwith a member from each of two sets of catenins : one setis comprised of b-catenin and plakoglobin; the other set iscomprised of aE- and aN-catenin (Shimamura et al., 1994).b-catenin and plakoglobin form mutually exclusive com-plexes with E-cadherin (Barth, Nathke & Nelson, 1997;Hatzfeld, 1999). b-catenin is the linker protein between E-cadherin and a-catenin ; binding sites for b-catenin existin the cytoplasmic domain of E-cadherin, and in the amino-terminal domain of a-catenin (Jou et al., 1995). Alpha-catenin mediates interactions with the cytoskeleton viaeither a-actinin and vinculin, or via a direct interaction withactin filaments (Hatzfeld, 1999).

The formation of cadherin/catenin complexes is a pre-requisite for the adhesive function of the cadherins ; cadherinmolecules lacking the catenin-binding sites cannot bind tothe cytoskeleton or mediate cell–cell adhesion (Hirano et al.,1992; Kirkpatrick & Peifer, 1995; Tsukita et al., 1992).Further, cells which express cadherins and b-catenin nor-mally, but which lack a-catenin, cannot adhere nor build


adhesive cell junctions (Nagafuchi & Takeichi, 1989; Hiranoet al., 1992). Deletion mutant studies and cell dissociationassays have indicated that the interaction of a-catenin with

the actin-based cytoskeleton through a functional domaincalled Z0-1 is required for a stronger state of E-cadherin-based cell adhesion activity (Imamura et al., 1999).

Plasma membrane Transmembrane domain

EC5

EC4

EC3

EC2

EC1

Plasma membrane Transmembrane domain

N-terminalHAV motif

Stranddimer

Adhesiondimer

Calcium ion

A

Fig. 3. Schematic diagrams representing models for classic cadherin-mediated cell–cell interactions. (A, B) Mediation of dynamiccell–cell interactions via intermolecular interactions between extracellular domains of cadherins. Two cadherin dimers are shownmaking contact across the intercellular gap. The extracellular domains consist of five paired subdomains (EC1–5). They also have asingle membrane-spanning segment and a highly conserved cytoplasmic region (C). (A) The linear zipper model. The primary areasof contact are located near the amino terminus and the histidine-alanine-valine (HAV) motif is located on the adhesive inter-face. Multiple regions of the amino-terminal domain, including the HAV flanking region, may be involved in cadherin–cadherininteractions. (B) The deep intercalation model of Chappuis-Flament et al. (2001). Interactions between multiple EC domains canoccur, creating the potential for the formation of a two-dimensional lattice rather than a linear zipper. (C) Multiple protein–proteininteractions mediated by the cytoplasmic domain of cadherins. The carboxy terminal cytoplasmic domain of cadherins mediatesconnections with cytoskeletal elements such as actin filaments and associates with cytoplasmic proteins called catenins-three areshown here : a-catenin (a-cat), b-catenin (b-cat) and p120ctn. Asterisks indicate sites of potential regulation. See text for furtherdetails. B is adapted from Chappuis-Flament et al. (2001). Reprinted with permission from The Rockefeller University Press. C isadapted from Lilien et al. (2002). Reprinted by permission of Wiley & Sons, Inc.


Members of the p120ctn-related family of catenins alsointeract (independently) with classical cadherins. p120ctn

colocalizes with E-cadherins at adherens junctions. Directassociation with cadherins is mediated via a central ‘arm

repeat ’ region. p120ctn co-exists with b-catenin and plako-globin in the same E-cadherin complex. As for b-cateninand plakoglobin, p120ctn associates with E-, N-, and P-cadherin. p120ctn does not associate with a-catenin ; hence,

Cadherincytoplasmicregion

Actin

Cytoskeleton

Transmembrane regionof cadherin dimer

Plasma membrane

A. Binding via multiple EC domains(potential 2D lattice)

C

B

Fig. 3. (Cont.)


p120ctn does not directly link cadherin molecules to the cyto-skeletal network. p120ctn has several splice variants whichshow cell-type-specific expression patterns. p120ctn is phos-phorylated in response to various receptor tyrosine kinases(Shibamoto et al., 1995; Daniel & Reynolds, 1997; Hatzfeld,1999).

Beta-catenin also binds to a cytoskeletal complex con-taining APC (adenomatous polyposis coli protein), andmicrotubules. Cadherins and APC compete for binding sites,and form mutually exclusive complexes with catenins (Barthet al., 1997).

The supramolecular complex discussed above formsan intercellular adhesion complex : the adherens junction,which assembles around a cadherin. The supramolecularorganization of cadherins appears to be essential for strongcell–cell adhesion. This reflects the weak intrinsic head-to-head affinity of the N-terminal domains. Cooperativeinteraction is required to generate sufficient adhesivestrength to maintain adhesion (Angst et al., 2001).

(b ) Structurally and functionally analogous cadherin-dependentsupramolecular complexes in vertebrate and Drosophilamelanogaster development

There are significant structural and functional similaritiesbetween cadherin-based epithelial supramolecular adhesioncomplexes in vertebrates and in D. melanogaster ; the com-plexes appear to be analogous (Peifer et al., 1993;Takeichi, 1995; Pai et al., 1996; Daniel & Reynolds, 1997).Similarities between vertebrate and D. melanogaster systemsinclude :

(a) DE-cadherin, the major epithelial cadherin inD. melanogaster, is associated, via the cytoplasmic domain,with D. melanogaster homologues of the vertebrate cateninsa-catenin and b-catenin: Da-catenin, and Armadillo,respectively. Further, Da-catenin forms a complex withDE-cadherin together with Armadillo. Dp120ctn is also acomponent of the D. melanogaster cadherin-catenin complex(Tepass et al., 2001).

(b) Armadillo and b-catenin are similar in the followingways : (i) there exists amino acid sequence homology; (ii)each is a component of a multiprotein complex containing acadherin, and a-catenin ; (iii) both are localized in epithelialadherens junctions.

(c) Cell aggregation assays involving the formation ofgreen fluorescent protein (GFP)-positive and -negative cellaggregates indicate that DE-cadherin and DN-cadherinbind selectively to like cadherin subclasses as is found invertebrates (Oda & Tsukita, 1999).

(d ) Multiple cadherins are expressed in a tissue-specific,or region-specific way in D. melanogaster. DE-cadherin iscontinuously expressed in ectodermal epithelia ; it isexpressed mostly in epithelial tissues at the apical portionof cell–cell contacts in differentiated epithelial layers. Therestricted distribution and polarised pattern of expression ofDE-cadherin is similar to that of vertebrate E-cadherin.

(e) The main roles of DE-cadherin, like E-cadherin,are in epithelial morphogenesis. Evidence suggests a role inmaintaining epithelial structures. With the possible excep-tion of the blastoderm, the D. melanogaster cadherin-catenin

complex is essential to maintain adhesion and tissuearchitecture of primary and secondary epithelia (Tepasset al., 2001).

( c ) Putative cadherins in less complex metazoans

Cadherin-dependent control of morphogenesis probablyexists in a diverse range of metazoan phyla. Cadherins, in-cluding classic cadherin-like molecules, have been identifiedin vertebrates (mice, chickens, Xenopus laevis, and zebrafish)and invertebrates [including D. melanogaster, echinoderms(sea urchins), C. elegans (Costa et al., 1998), and Hydra magni-papillata (Hobmayer et al., 1996)].

There are at least two ‘conventional ’ cadherins insea urchin embryos of molecular weight 140 and 125 kDa.The 140 kDa protein is the equivalent of vertebrateE-cadherin, and D. melanogaster DE-cadherin. During gastru-lation, the 140 kDa cadherin appears to become graduallyrestricted to the invaginating epithelial cells of the gutrudiment, while the 125 kDa cadherin is expressed inmost cells of the embryo. It is suggested that this differentialdistribution might result in the creation of two groupsof cells (Ghersi et al., 1993). Thus, dynamic regulation ofcadherin localization may play a role in sea urchinmorphogenesis.

As in vertebrates and D. melanogaster, cadherins and cate-nins appear to play important roles in dynamic morpho-genetic processes involving transient cell–cell contactformation in sea urchin development. Beta-catenin has beencloned in the sea urchin Lytechinus variegatus, and its distri-bution examined in cells involved in morphogenetic move-ments. From the cleavage stage, b-catenin is associatedwith lateral cell–cell contacts, and accumulates at adherensjunctions. At gastrulation, changes in junctional b-cateninlocalization are associated with epithelial–mesenchymalconversion, and convergent–extension movements. Epi-thelial–mesenchymal conversion coincides with a rapid lossof b-catenin from adherens junctions. In epithelial cellsundergoing convergent–extension movements, there is asignificant decrease in b-catenin associated with adherensjunctions. It is hypothesized that b-catenin may play a rolein regulating cell–cell adhesion, and the function of ad-herens junctions during gastrulation of sea urchins (Miller &McClay, 1997a, b).

(d ) A putative cadherin and b-catenin homologue inDictyostelium discoideum

Siu et al., have found a putative cadherin involved in med-iating cell–cell adhesion in D. discoideum at the aggregationstage of its life cycle : DdCAD-1, which shares a significantdegree of sequence similarity to vertebrate E-cadherin(Brar, 1994; Sesaki & Siu, 1996; Wong et al., 1996; Yanget al., 1997). It may be a primitive member of the cadherinfamily (C.-H. Siu, personal communication). Evidence fromin vitro studies indicates that it mediates a homophilicbinding mechanism. A Ca2+-dependent adhesion system isinvolved, with inhibition of the Ca2+-binding sites resultingin loose aggregates, and loss of compacted cellular organ-ization. DdCAD-1 can form a complex with several


membrane and cytoplasmic elements, e.g. calmodulin ; thecomponents of this complex may play a role in the dynamicsof the cytoskeleton and in signal transduction.

Another intriguing discovery is a complex ultrastruc-ture in the D. discoideum developing fruiting body: actin-associated intercellular junctions are located in a constrictedregion of the stalk tube (Grimson et al., 2000; Coates &Harwood, 2001). The morphology of the cell junctions andtheir association with actin filaments gives them the ap-pearance of metazoan adherens junctions. A gene (aardvark)encoding a b-catenin homologue [Aardvark (Aar)] has beenidentified, and found to be located at the cell junctions.Aardvark shows a potential a-catenin binding site in the firstArm repeat, an equivalent position to that present in b-catenin. Also, aardvark contains a cluster of potential glyco-gen synthase kinase (GSK-3) phosphorylation sites in theamino terminus, as does b-catenin. Ablation of the aardvarkgene results in the formation of mechanically weak fruitingbodies. In aardvarkmutants, the constriction of the stalk tube,the intercellular junctions, and the actin ring are absent.Expression of the aardvark cDNA restores adherens junc-tions, and overexpression results in the formation of ectopicjunctional complexes in the prestalk cells that surroundthe developing stalk tube. Thus, aardvark is required for thestability of adherens junctions.

In addition to its structural role, Aar has a signallingfunction that appears to be independent of its structuralrole (Grimson et al., 2000; Coates & Harwood, 2001) ; itplays a role in the induction of prespore genes. Interestingly,one of these is the gene encoding a putative CAM, prespore-specific antigen (PsA) ; we have recently reported the firstevidence implicating this CAM in cell–cell adhesion at theslug stage of development (Bowers-Morrow, Ali & Williams,2002). GskA activity (the protein kinase which, in meta-zoans, phosphorylates b-catenin) positively regulates Aar toelicit psA expression.

It is likely that cadherin-mediated homophilic bindingmechanisms, the posession of adherens junctions andcatenins, and the molecular systems required to regulatethe actin cytoskeleton all evolved before the emergence ofmetazoans. Thus, these are all potential universal molecularmechanisms underlying cell contact phenomena.

(2 ) Filopodia-based mechanisms for intercellularjunction formation

On a macro-scale, the first step in cell–cell interaction, inmany situations, is via the physical contact made betweenfilopodia of apposing cells. Filopodia are found at the lead-ing edge of various types of migrating cells. Filopodia, whichare dynamic structures appearing as thin cylindrical exten-sions of a cell’s membrane, contain long actin filamentsorganized as a tight bundle with their barbed (fast-growing)ends pointing towards the direction of protrusion; theyextend through actin polymerization at the tips of the actinfilaments (Wood & Martin, 2002).

Based on studies with primary mouse keratinocytes,Vasioukhin & Fuchs (2001) have proposed a model for afilopodia-driven process. When grown on ECM, these cellsextend numerous filopodia. Upon contact, the filopodia

from two neighbouring cells slide along each other andproject into the apposing cell’s membrane. The filopodiaare rich in F-actin. Embedded tips of filopodia are stabilizedby puncta comprising transmembrane clusters of adherensjunction proteins. This process brings regions of two cellsurfaces together ; they are then clamped by desmosomes.There is a reorganization of actin fibres at filopodia tips.Actin polymerization is initiated at stabilized puncta cre-ating the force needed to push and merge puncta into asingle line. Actin-based movement physically brings re-maining regions of apposing membranes together ; they arethen sealed into epithelial sheets.

During development there are many morphogeneticevents which require two free epithelial edges to fuse andcreate a continuous epithelium. Filopodia play importantroles in two similar tissue movements involving zipperingcell sheets together : (a) dorsal closure in Drosophila melano-gaster (sealing of the dorsal epidermis along the midline ofthe embryo), and (b) ventral enclosure in C. elegans. Duringdorsal closure, the leading edge of advancing epithelialfronts extends filopodia ; when apposing epithelial frontsmeet along the mid-line, these filopodia contact and engage,and in this way the epithelial faces are zippered together.During ventral enclosure in C. elegans, the most anterior cellsin the epidermis initiate closure by extending filopodiatowards the ventral mid-line. E-cadherin complexes clusterat the tips of the filopodia forming weak adhesions whichevolve into mature adherens junctions. The rapid formationof junctions requires the function of cadherin molecules,and homologues of a-catenin and b-catenin. A dynamicfilopodia-driven, epithelial-cell adhesion process is alsodescribed for thorax closure during metamorphosis inDrosophila melanogaster. In addition, primary and secondarymesenchyme cells of sea urchin embryos extend filo-podia as they move, making contacts with the ectoderm.Filopodia also participate in DdCAD-1-mediated cell–celladhesion in D. discoideum, as discussed below (this involves aputative cadherin) (Raich, Agbunag & Hardin, 1999;Jacinto et al., 2000; Martin-Blanco, Pastor-Pareja & Garcia-Bellido, 2000; Simske & Hardin, 2001; Vasioukhin &Fuchs, 2001; Wood &Martin, 2002). Thus, the involvementof filopodia in initial cell–cell contact is a widespreadphenomenon.

(3 ) Clustering of cadherins: adhesion, anchorage,junction formation and signalling

The concentration of cadherins at cell–cell adhesion sitesis essential for generating strong adhesion. Cadherin-dependent epithelial morphogenesis is exemplified by theprocess of compaction which results in the segregation ofcells ; the outer layer is characterized by tightly packed,polarized epithelial cells, while cells within the inner cellmass stay loosely connected (Stappert & Kemler, 1993;Gumbiner, 1996; Marrs & Nelson, 1996). E-cadherin-dependent changes in cell–cell adhesion are required forcompaction of preimplantation eutherian embryos. Beforecompaction, E-cadherin is uniformly distributed on the cellsurface, and is not adhesive, resulting in the blastomeresbeing loosely adherent. With the onset of compaction,


E-cadherin becomes concentrated in cell–cell contact zones.Loosely organized mesenchymal-like cells surrounded byECM condense together and form extensive contacts alongtheir surfaces. The cells form tightly adherent polarisedepithelial cell sheets with epithelial cell junctions. Blasto-meres on the outer surface of early morula show increasedcell–cell adhesion.

Adherens junctions contain clusters of cadherins linkedto the cytoskeleton and to intracellular signalling cascades.Movement and concentration of cadherin molecules withinthe cell membrane is the first step in the assembly processresulting in the formation of adherens junctions at contactsites (Kusumi, Suzuli & Koyasako, 1999).

While the mechanisms underlying the concentration ofcadherin molecules are not yet understood, Baumgartneret al. (2000) suggest that adhesion might be regulated viatethering of cadherin cytoplasmic domains to the actin fila-ment cytoskeleton. The results of studies involving singleparticle tracking and optical tweezers support a model inwhich the movements of E-cadherin in the plasma mem-brane are regulated via the cytoplasmic domain by tetheringto actin filaments via catenin(s), and ‘corralling ’ by themembrane skeleton/cytoskeleton meshes (Sako et al., 1998;Kusumi et al., 1999). Tethering would restrain lateralmobility. It would also increase the probability of aparticular strand dimer rebinding after dissociation ratherthan moving apart via lateral diffusion. Further, tetheringfavours both assembly and stability of clustered higher-ordercomplexes. Signalling mechanisms which can change cyto-skeletal tethering of cadherins would have an importantimpact on the lateral mobility of cadherins, and thus, on thenumber of adhesive bonds (Baumgartner et al., 2000).

Clustering may be important for the initiation ofcadherin-mediated signalling, and assembly of cell–celljunctions. The results of experiments involving the reactionof cells expressing N-cadherin with beads covalently linkedto the extracellular domain of N-cadherin, indicated thatclustering induced specific global enhancement of adherensjunction assembly (Katz et al., 1998). Further experimentswith chimeric molecules demonstrated that the signal forlong-range adherens junction assembly can be triggeredby experimental aggregation of the cytoplasmic tail only.Signals triggered by clustering resulted in (a) the recruitmentof components into pre-existing adherens junctions ; (b)enhancement of the extent of assembly of these sites ; and(c) reversal of dominant negative effects of overexpression oftail chimera on adherens junctions. It is concluded thatthe cytoplasmic tail contains information required to directthe cadherin molecule to the adherens junction (Katz et al.,1998). Murase et al. (2000) constructed various mutants ofcadherin-4 (Cdh4; R-cadherin), and examined the adhesionproperties of the transfectants. They have concluded thatCdh4 has intrinsic activity of cluster formation, and that thisis involved, at least in part, in the process of concentrationof Cdh4 at cell–cell contact sites which generates cell–celladhesion activity in a mutant lacking the entire cytoplasmicdomain.

While the underlying mechanisms remain to be un-ravelled, the lateral clustering of adhesion proteins may bethe most important factor in the regulation of cell–cell

adhesion. The clustering of cadherins into adherens junc-tions would be a way to achieve cumulation of relativelyweak-affinity adhesive interactions resulting in high aviditybinding (Hatzfeld, 1999).

(4) Actin cytoskeletal dynamics in promotion ofepithelial cell adhesion

The actin cytoskeleton is crucial in intercellular adhesion.Data suggesting a role for the actin cytoskeleton in facilitat-ing the process that brings apposing cell membranestogether and stabilizing them once junction formation hasbeen initiated were discussed above. One model proposesthat this stabilization is achieved by the deposition ofcadherin-catenin complexes (Vasioukhin & Fuchs, 2001).

Adams et al. (1998), using a functional protein comprisingE-cadherin fused to green fluorescent protein (EcadGFP)stably expressed in Madin–Darby canine kidney (MDCK)epithelial cells, have monitored E-cadherin and actin cyto-skeletal dynamics during epithelial sheet formation. A modelis proposed for the transformation from the migratoryphenotype of a single cell to a sedentary phenotype in amulticellular monolayer:

(i ) Cell–cell adhesion is initiated by weak binding betweenextracellular domains of E-cadherin present in a mobilepool at the plasma membrane. At, or close to, the same time,E-cadherin-catenin complexes attach to actin filamentswhich branch off from actin cables which circumscribeindividual migratory cells. These processes convert a diffusepool of mobile E-cadherin clusters into immobile punctateaggregates along developing contact zones. Multiple punctaloosely hold contacting cells together.

(ii ) A second phase involves much larger E-cadherinclusters being formed (‘plaques ’) at the edges of the contact,and the rearrangement of circumferential actin cables.The coordinated reorganization of E-cadherin and the actincytoskeleton leads to compaction of contacting cells, result-ing in strong cell–cell adhesion and maximization of thearea of membrane contact between cells. An actin cablewhich circumscribes the free edges of the contacting cells isproduced, and is embedded into either side of a plaque atthe margins of contact.

(iii ) A third phase (condensation) occurs once anothercell adheres to a two-cell colony. Additional cells adhereto larger cell colonies via the same mechanisms. Furtherreorganization of puncta and the circumferential actin cyto-skeleton occurs. This is initiated by the lateral translocationof plaques on one side of the colony towards each other untilthey coalesce. Plaques cinch together to form multicellularvertices. Contractility within the circumferential actin cablebrings plaques from adjacent cells together.

Alpha-catenin is the only catenin that can directly bindto actin filaments. There is strong evidence that clusteringof the E-cadherin-catenin complex and cell–cell adhesionrequires a-catenin. This molecule associates with b-catenin,f-actin, and other proteins, some of which are actin-bindingproteins themselves. The role of a-catenin may be toorganize a multiprotein complex with multiple actin-bind-ing, bundling and polymerization activities (Vasioukhin &Fuchs, 2001).


(5 ) Mediation of cell aggregation duringDictyostelium discoideum morphogenesis

(a ) Homophilic protein–protein binding mechanisms

Cell adhesion studies using D. discoideum have, to date,been concentrated on unravelling the molecular basis ofcell contact phenomena associated with the aggregationof motile, free-living, single-celled amoebae with filopodia,to form a multicellular slug. This dynamic, morphogeneticprocess involves CAM-mediated cell–cell contact, adenosine3k,5k-cyclic monophosphate (cAMP)-mediated cell–cell sig-nal propagation, directed chemotactic migration of cells,and cell-substrate adhesion. Cell ‘ streams’ are formed byend-to-end, and side-to-side contacts between amoebae.The directed movement of amoebae towards ‘aggregationcentres ’ via chemotaxis is followed by the formation of a cellaggregate which becomes a three-dimensional ‘mound’.

Two CAMs are known to be involved in mediatingdynamic cell contact phenomena at the chemotactic mi-gration, streaming, and aggregation stages of morpho-genesis : (a) the putative cadherin DdCAD-1 (gp24) [relativemolecular mass (M(r)) 24 000] ; (b) a lipid glycan-anchoredglycoprotein, contact sites A or csA [also referred to as gp80;M(r) 80 000].

While these two CAMs are very different structurally,according to current models they both mediate homotypiccell–cell interactions via homophilic protein–protein bindingmechanisms (Choi & Siu, 1987; Siu, Lam & Wong, 1988a ;Siu et al., 1988b, c ; Brar & Siu, 1993; Brar, 1994; Desbarats,Brar & Siu, 1994; Sesaki & Siu, 1996; Wong et al., 1996;Yang et al., 1997; Coates & Harwood, 2001).

(b ) Role for cell extensions, and changing distribution oftwo cell adhesion molecules

Cells initially make contact via DdCAD-1-mediated bindingof F-actin-containing filopodia. As neighbouring cells drawtheir filopodia back into their cell bodies more extensivecontacts are made, these being mediated by csA. At theonset of chemotactic migration, DdCAD-1 becomes local-ized mainly at the cell periphery, and there is enrichment ofthis molecule on membrane ruffles. DdCAD-1 also becomesassociated with lamellipodia and filopodia. When cellsare forming streams, filopodia may act as ‘ feelers ’ thatexplore the cell’s environment. DdCAD-1 may play a rolein the recruitment of cells into streams. During chemotacticmigration, DdCAD-1 exists mainly on cells at the tip, andon the outer margins of cell streams. During cell streamingthere is a dramatic, and rapid redistribution of DdCAD-1as cells move towards the aggregation centre ; the level ofDdCAD-1 decreases in contact zones, and csA becomesthe dominant CAM. csA molecules are most abundant at thetwo polar contact zones of migrating cells, and preferentialassociation with filopodia is observed.

Coates & Harwood (2001) compare the pattern ofassembly of adhesive contacts to the sealing of epithelial cellsheets during development in metazoans D. melanogasterand C. elegans ; in both species the cells project filopodiawhich extend in front of the moving cell sheet. The pro-cesses in D. discoideum, D. melanogaster, and C. elegans share

some events, e.g. the initiation of contact, rearrangementof the cytoskeleton, and the formation of more extensiveadhesive contacts.

( c ) Role of cell adhesion molecules in morphogenesis

Ponte et al. (1998) found that csA is required for normaldevelopment. Furthermore, csA expression confers a selec-tive advantage to populations of wild-type cells comparedwith csA-null mutants which show reduced cell–cell ad-hesion, increased cell–substrate adhesion, and decreasedmotility. As a result of these differences, csA-null cells sortout from aggregating wild-type cells. Many csA mutantsfail to enter the multicellular phase of development. Theimportance of strength of adhesion during aggregation isalso demonstrated by smlA mutants which show reducedexpression of csA and DdCAD-1 during streaming and earlyaggregation. The resultant reduction in cell–cell adhesioncauses streams to break up, and form aggregates smallerthan those of the wild-type. On the other hand, mutationsin countin (a factor which limits aggregation size) result inincreased DdCAD-1 expression and cell–cell adhesion, andto the formation of huge aggregates and fruiting bodies(Coates & Harwood, 2001).

(d ) Physical properties of intermolecular binding facilitatede-adhesion and motility

Benoit et al. (2000) have quantified, in vivo, de-adhesionforces at the resolution of single csA molecules by controllingthe interactions between single cells and combining single-molecule force spectroscopy with genetic manipulation.Adhesion between two adjacent cell surfaces was found toinvolve discrete interactions characterized by an unbindingforce of 23¡8 pN, measured at a rupture rate of 2.5¡0.5 mm sx1. This de-adhesion force is small in comparisonwith those for most antibody–antigen or lectin–sugar inter-actions, which frequently exceed 50 pN at comparablerupture rates. This is consistent with the ability of motilecells to glide against one another as they become integratedinto a multicellular structure.

An emerging concept is that of transient cell–cell contactsmediated by weak-affinity intermolecular interactions withfast on–off rates. Based on affinity and kinetic analyses usingbiosensor technology, it has been suggested that CAM in-teractions which mediate transient cell adhesion may havelow affinities, and extremely fast dissociation rate constants(van derMerwe & Barclay, 1994, 1996; van der Merwe et al.,1994). The nature of csA–csA binding may be a factor thatfacilitates transient cell–cell contact formation; it is thoughtcsA may mediate cooperative, multiple, weak-affinity inter-actions (Stein & Gerisch, 1996). Relatively low affinity fortheir ligands is a feature shared by most, if not all, adhesionproteins. For example, the dissociation constant (Kd) forhomophilic binding of the neural CAM polysialatedN-CAM (mediated via ionic interactions in the same way ascsA–csA interactions) is of the order of 10x6 M (Ruoslahti &Obrink, 1996). A major advantage of polyvalent low-affinityadhesion for dynamic (transient) cell adhesion over mono-valent and higher-affinity adhesive interactions is that


reversibility of adhesion (without destruction or inter-nalization of adhesion molecules, and their receptors)could be achieved through dynamic ‘on and off’ binding oflow-affinity single sites ; this would facilitate cell motility.

( e ) Clustering of cell adhesion molecules

Cell–cell adhesion is generally mediated by large adhesioncomplexes assembled from clustered receptors that link tothe cytoskeleton via cytoplasmic adapter proteins (e.g.catenins in the case of cadherin-based complexes). Withinthe complexes, cis- and trans-oligomers of adhesion mole-cules can assemble into zippers and lattices which providestrong adhesion through enhanced binding avidity (Harriset al., 2003).

csA is an analogue of glycosyl phosphatidylinositol-anchored proteins (GPI-APs). The question of how a CAMlike csA which has no transmembrane domain, can mediatesignalling is an intriguing one; there is substantial evidencesupporting the hypothesis that GPI-APs are clustered insphingolipid-sterol membrane microdomains or ‘ lipid rafts ’(Simons & Ikonen, 1997; Friedrichson & Kurzchalia, 1998;Varma & Mayor, 1998; Chatterjee & Mayor, 2001).

Like many vertebrate GPI-APs, csA is a major componentof Triton X-100-insoluble floating fractions (TIFFs).Further, Triton X-100-insoluble contact regions have beenfound to be a cytoskeleton-associated form of TIFFs, andlarge domains containing TIFF proteins and lipids localizeto csA-mediated cell–cell contacts. Thus, a role for TIFF isimplicated in csA-mediated adhesion (Harris et al., 2001a ;Harris, Ravandi & Siu, 2001b).

Harris et al. (2001b, 2003) and Harris & Sui (2002)have investigated the assembly and organization of csAmolecules in raft-like membrane domains as csA-mediatedcell–cell contacts formed during development. csA wasfound to oligomerize preferentially in these domains bydirect cis-interactions between their extracellular domains.[These interactions are distinct from the trans-homophilicbinding mechanism discussed above.] csA oligomers havealso been detected on live cells. A model has been proposedfor the assembly of adhesion complexes (see Fig. 4) : (i)membrane rafts enriched with sterols and phospholipid-anchored proteins exist before csA is expressed; (ii) csAmolecules are recruited into rafts while most transmem-brane proteins are excluded; (iii) the overall level of csAexpression increases, and the elevated concentration of csAmolecules in rafts leads to local cis-interactions via theirextracellular domains forming stable oligomeric moleculeswith multiple GPI anchors (detected in 50–70 nm clusters) ;(iv) the affinity of csA for rafts is increased upon oligomer-ization; (v) the oligomers accumulate as development pro-ceeds ; (vi ) the presentation of cis-oligomers in rafts facilitatesthe avid trans-interactions required for cell–cell adhesion,and the combination of trans and cis interactions promotesthe cross-linking and coalescence of adhering rafts into largestable domains ; (viii ) association of the cytoskeleton withthe enlarged rafts contributes to the further expansion of theadhesion complexes. csA recruits the actin-binding proteinponticulin (an atypical membrane protein containing trans-membrane domains and a phospholipid anchor) which

mediates interaction between the adhesion complex and thecytoskeleton.

( f ) Comparison of adhesion in Dictyostelium discoideumwith that in metazoans

The factors involved in mediating cell–cell adhesion duringthe dynamic aggregation stage of D. discoideum probably in-clude some of those important during phases of increasedadhesion in morphogenesis in metazoans: (a) the employ-ment of filopodia, and associated cytoskeletal rearrange-ments ; (b) adhesion mediated by a cadherin-like moleculeinvolved in homophilic interactions ; (c) the clustering ofCAMs; (d ) the interaction between multiple CAMs andassociated proteins ; (e) the formation of multimolecularadhesion complexes ; and ( f ) the involvement of multiplelow-affinity interactions between CAMs.

(6) Sponge aggregation factors: mediation ofadhesion via supramolecular intercellularextracellular matrix complexes

Sponges (Porifera) represent the oldest and simplest extantmetazoans. Most molecular, morphological, and develop-mental evidence available supports the view that the king-dom Animalia is of monophyletic origin (Morris, 1993;Wainwright, Patterson & Sogin, 1994; Muller, 1995, 1997,1998; Muller & Schacke, 1996; Conway Morris, 1998;Muller et al., 1999, 2001). Sponges have many characteristicmetazoan molecules required for tissue formation (Muller,1997, 1998).

(a ) Cell rearrangements

Sponge cell behaviour is of considerable relevance to thestudy of metazoan developmental processes involving cellcontact phenomena, because cellular mobility and cellularinteractions are important phenomena in all phases ofthe sponge life cycle. A low degree of cell differentiationcombined with a high degree of cell motility characterizesponge ‘chronic morphogenesis ’ (Gaino & Magnino, 1999).Sponges show the same dichotomy of epithelial cell sheets,and motile mesenchymal cells seen in metazoan embryos(Morris, 1993). Beneath the pinacoderm (the outer epithelialcell sheet) is the mesohyl comprised of the most activesponge cells loosely embedded in a voluminous lectin matrix(Bond, 1992). On the inner side of the mesohyl is a layerof choanocytes. Sponge tissue structure is constantly beingremodelled via cell rearrangements. Mesohyl cells andchoanocytes continuously move and rearrange. The cellsmove in patterns that restructure the organism’s simpleanatomy. Choanocyte chambers interact with pinacocytesand mesohyl cells to form excurrent channels ; these chan-nels continually move, fuse with one another, and branchfrom one another. The positioning of cells within the or-ganism is controlled by coordinated cell–cell and cell–ECMmatrix interactions (Bond, 1992.)

Dissociated sponge cells reaggregate, and sort in a species-specific way via cell–cell interactions (Wilson, 1907, 1910;Humphreys, 1970). The marine sponges Microciona prolifera(red beard sponge), and Geodia cydonium have been used


Cytoskeleton

(csA)

gp80

Ponticulin

Sterol

Lipidanchor

Fig. 4. Model for the assembly of contact sites A (csA), also referred to as gp80, adhesion complexes. The schematic drawing depicts a csAraft (shaded box), and csA–csA interactions leading to the assembly of adhesion complexes. (1) Membrane rafts enriched with sterols andphospholipid-anchored proteins exist prior to the expression of the cell adhesion molecule (CAM) csA. (2) csA molecules are recruited intorafts via interactions between their phospholipid anchor and the sterol-rich domains. (3) As the overall level of csA increases, the elevatedconcentration of csA molecules in rafts leads to local cis-interactions via their extracellular domains. (4) Upon oligomerization, the affinity ofcsA for rafts increases. (5) As development proceeds, csA cis-oligomers accumulate. (6) The presentation of cis-oligomers facilitates trans-dimerization required for cell–cell adhesion. The combination of trans- and cis-interactions promotes the crosslinking and coalescence ofadhering rafts. (7) Association of the cytoskeleton with the enlarged rafts contributes to further expansion of csA adhesion complexes. csArecruits the actin-binding protein ponticulin which mediates interactions between the adhesion complex and the cytoskeleton. Kinases areactivated. (Reprinted from Journal of Biological Chemistry with permission from The American Society for Biochemistry and MolecularBiology.)


extensively for in vitro and in vivo investigations of the associ-ated cell contact phenomena (see Galtsoff, 1925a, b ; Burgeret al., 1975; Muller et al., 1990; Misevic & Burger 1990a, b,1993; Muller, 1995; Spillmann et al., 1995; Varner, 1995,1996; Fernandez-Busquets, Kammerer & Burger, 1996;Wagner-Hulsmann et al., 1996).

(b ) The molecular basis of cellular interactions

In the sponge ECM, glycosaminoglycans, fibrous proteins,and adhesion glycoproteins interact with cells. These

structural elements allow sheet-forming epithelial cells,and motile mesenchymal cells to differentiate. There existcalcium-mediated, lectin-mediated, and transmembraneprotein receptor-mediated molecular interactions (Kruseet al., 1994, 1996; Schacke et al., 1994; Muller & Schacke,1996; Kruse, Muller & Muller, 1997; Muller, 1997; Panceret al., 1997). According to Morris (1993) ‘ there are too manyprecise similarities in the composition, structure, and function of extra-cellular matrices of sponges and other animals to infer that this majoradaptive complex has evolved more than once. ’ Further work isrequired to determine whether these sponge molecules are

A

B

Cell

Cell

rrAF

AF

g-6g-200

g-200

g-6

MAFp4 carrying g-6glycan

MAFp3 carrying g-200glycan

Fig. 5. Model for the cell-adhesion system ofMicrociona prolifera. (A) Atomic force microscope image of nativeMicrociona aggregationfactor (MAF) showing the localization of MAFp3 in the ring (black circumferences) and MAFp4 in the ‘arms’. MAF–MAF self-interaction is the main element of the cell-adhesion system. Native MAF is comprised of two N-glycosylated glycoproteins :MAFp3 carries the g-200 glycan and MAFp4 the g-6 glycan. (B) Cell–cell adhesion is mediated via : (i ) homophilic, Ca2+-dependent,carbohydrate–carbohydrate interactions (g-200–g-200) ; the high repetitivity of the adhesion epitope (2500 sites) may be essentialfor establishing strong MAF–MAF binding. AF indicates the MAF core protein. (ii ) Heterophilic, Ca2+-independent,protein–carbohydrate interactions : ([MAFp4]-[g6]-[plasma membrane receptor]). MAF–MAF association mediates cell aggre-gation only when MAF is anchored to the cell membrane via the MAF-cell receptor which is proteinaceous (PMR, labelled r inthe diagram). The cell-binding domain which is Ca2+-independent, provides very high affinity (association constantKa=4.5r1010 Mx1), and species-specificity in MAF-cell receptor association. One MAF molecule has approximately 1000 repeatsof the glycan g-6. The species specificity, and high affinity of MAF-cell interactions are the result of the cooperative binding of theseg-6 glycans to cell surface receptors. Polymerization of the protein could result in the carbohydrate polyvalency required for cellaggregation.

Adapted from Jarchow et al. (2000) ; see also Varner (1995, 1996) ; Fernandez-Busquets et al. (1996, 1998) ; Fernandez-Busquets &Burger (1997) ; Blumbach et al. (1998). A and B are reprinted from the Journal of Structural Biology with permission from ElsevierScience. (C) A proposed model for MAF-mediated sponge cell adhesion (Fernandez-Busquets & Burger, 2003). For simplification,only half of MAFp4 and of the g-200 chains are represented in each MAF molecule. A : Ca2+-dependent self-interactions of theMAFp3-bound g-200 glycan mediate self-binding of two aggregation factor complexes. B : MAFp3 and MAFp4 interact via anhyaluronin (HA)-like molecule. C : Binding of MAFp4 via the g-6 glycan to cell receptors (AR) probably involves other pericellularproteins such as (for M. prolifera) p68 and p210. D : A putative membrane-linked MAFp3-MAFp4 monomer (there may be directinteractions between MAFp3 and MAFp4). The inset at the bottom right shows an atomic force microscopy image of an MAFp3/MAFp4 unit. The two chains protruding from MAFp3 are thought to be the g-200 glycan. Reprinted from Fernandez-Busquets &Burger (2003) with permission from Birkhauser Publishing Ltd.


employed for the same functions they serve in more complexmetazoans. Nevertheless, as Pancer et al. (1997) conclude,sponges use the structural elements required for regulatedcell–cell, and cell–ECM interactions.

Fig. 5 shows models for the molecular basis of cell–cellinteractions in M. prolifera. For a comprehensive review ofadhesion in marine sponges, see Fernandez-Busquets &Burger (2003). Cell–cell adhesion in marine sponges is

C

(g-200)

g200

MAFp3

MAFp3

MAFp3

MAFp3

g200

Ca2+ Ca2+

MAFp3

MAFp4domains

HA-like

AR

Signaltransduction

cascade

g6

g6AR p68

p210

g6

(g-6)

MAFp4domain

MAFp4

Fig. 5. (Cont.)


mediated by a new family of modular proteoglycans whosegeneral supramolecular structure resembles that of hya-lectans ; these proteoglycans have been called ‘ spongicans ’(Fernandez-Busquets & Burger, 2003). In M. prolifera, celladhesion is mediated by one of these spongican molecules,a circular proteoglycan-like ‘aggregation factor ’ (MAF)which is a supramolecular, intercellular ECM complex.

Carbohydrates form a major part of the MAF molecule ;it is composed of glucuronic acid, fucose, mannose, galac-tose, and N-acetylglucosamine (GlcNAc). No informationis available yet on the overall structure of MAF glycans.MAF molecules interact with each other in a homophilic,divalent cation-dependent, and oligosaccharide-dependentmanner (Misevic & Burger, 1990 a, b, 1993; Spillmann et al.,1995; Varner, 1995; Fernandez-Busquets & Burger, 2003).

Adhesion occurs through a two-step process involving (i )a Ca2+-dependent self-aggregation of the negativelycharged MAF complexes, and (ii ) a Ca2+-independentbinding to cell surface receptors which are nonintegralmembrane proteins (Fernandez-Busquets & Burger, 2003).

Twenty units from each of two N-glycosylated glycopro-teins, MAFp3 and MAFp4, form the central ring andradiating arms of MAF, respectively ; this is stabilised viaa hyaluronidase-sensitive component ( Jarchow et al., 2000;see Fig. 5A). MAF interacts with a proteinaceous cell-surface receptor (PMR) through a small glycan, g-6, carriedby MAFp4, and also a larger glycan, g-200, carried byMAFp3. g-200 is a highly fucosylated, acidic, negativelycharged, N-linked, glucuronic-acid-rich polysaccharide(see Fig. 5B). The binding through the g-6 glycan to PMRprobably involves other pericellular proteins such as p68and p210. Each MAFp3 ring unit carries one or two copiesof the g-200 glycan, while each MAFp4 unit carriesapproximately 50 copies of the g-6 glycan (see Fig. 5C)(Fernandez-Busquets & Burger, 2003).

The models for the M. prolifera adhesion system are basedon the polyvalency of carbohydrate determinants. Carbo-hydrates are the most exposed and highly polyvalentcomponents of the cell surface and of the ECM. MAF–MAFself-interaction has been shown to be based on carbo-hydrate–carbohydrate interactions mediated by g-200. Thisself-interaction is polyvalent and cooperative (Fernandez-Busquets & Burger, 2003). The cooperative effect of mul-tiple low-affinity interactions required for MAF-mediatedaggregation might be based on the cross-linking of a smallglycosylated protein into a large polymer with active carbo-hydrate sites appropriately exposed (Misevic & Burger,1986; Fernandez-Busquets et al., 1996).

( c ) Role for cell extensions

Gaino & Magnino (1999) have used Clathrina (C. cerebrum andC. clathrus) as a model system for examining the behaviour ofdissociated sponge cells. Following dissociation, choanocyteswhich settle onto a surface in vitro undergo a de-differen-tiation process during which cytoplasmic extensions usedin cell–cell and cell–ECM interactions are produced (sclero-podia and lamellipodia). These two types of extension canbe present in the same cell. Lamellipodia, which are broad,flat sheet-like extensions showing microspikes and ruffles

at their edge last 3–15 min before being completely with-drawn. Their main component is actin, arranged as either ameshwork of filaments or a system of bundles. Lamellipodiaconstitute the leading support for cell locomotion. Sclero-podia, which have an actin-containing core, are stiff, thinmicroextensions. During cell locomotion scleropodia appearto have an exploratory function, are continuously protrudedand retracted, and can make intercellular contacts favouringaggregation. Scleropodia can emerge from lamellar borders.The leading edge of lamellipodia, and the tips of scleropodiaare the preferential sites of cell-substrate attachment.

(d ) Comparison with cadherin-mediated systems andunifying principles

There appear to be vast differences in the adhesionmechanisms used by sponges in comparison with previouslydiscussed models for invertebrates and vertebrates. Forsponges, ECM-cell interactions form the basis of adhesion,while junctional complexes such as adherens junctions areapparently not involved. While protein–protein interactionsform the basis of adhesion in cadherin-mediated systems,the sponge cell adhesion system involves carbohydrate–carbohydrate interactions. However, important principlesare the same. Motile, actin-containing cell extensions prob-ably initiate cell contact, with subsequent stabilizationinvolving multimolecular complexes and low-affinity inter-actions, and, finally, the achievement of sufficiently strongadhesion via the compounding of multiple interactions. Also,connections are made which enable signalling to the cellinterior to occur.

While the ‘ jigsaw’ depicted here has many missing pieces,the impression is that cadherins would have played a fun-damental role in mediating cell–cell adhesion in at leastsome of the earliest metazoans. Cadherins, cytoplasmicassociates, and cadherin-mediated cell junctional supra-molecular complexes are probably almost universal re-quirements for building connections between cells duringmetazoan development.

Founding adhesive mechanisms may have involved justone or two CAMs (perhaps cadherins) sufficient to initiatecell–cell contact. Concentration of CAMs in cell contactzones probably allowed sufficiently strong adhesion to holdcells together. A set of interacting proteins involving extra-cellular, cytoplasmic, and cytoskeletal components mayhave represented the minimal unit able to mediate cell–celladhesion within a multicellular structure.

III. THE BALANCE BETWEEN ADHESIVE AND

MOTILE CELLULAR PHENOTYPES:

MECHANISMS FOR THE REGULATION OF

CELL–CELL ADHESION

The physical and molecular dynamics of cell–cell adhesionduring tissue formation in vivo is still an embryonic areaof research. However, we are able to glean some insightsfrom studies on the regulation of cadherin-mediatedsystems. One type of mechanism for the regulation of cad-herin-mediated adhesion, during dynamic processes such as


cell sorting and cell segregation, involves the specific bindingproperties of the cadherin molecules. Another type ofmechanism is physiological regulation of cadherin activity(Takeichi et al., 2000). Below is a discussion of the likely rolesof (i) changing expression patterns of cadherins, and quan-titative differences in cadherin expression; (ii) homeodomaintranscription factors, catenins, and Rho GTPases. Theimportance of the concerted activities of different types ofCAMs and adhesive systems is also discussed.

(1 ) Changing expression patterns

Each classic cadherin subclass is characterized by a uniquetissue distribution pattern, and immunological specificity.The cell-surface expression of cadherins, like other CAMs,is developmentally regulated; cadherins are dynamicallyexpressed at critical times, and places, with specific spatialand temporal distributions in embryos, and in developingtissues and organs (Cunningham, 1991; Takeichi, 1995).In their interactions, each cadherin subtype preferentiallybinds to the like subtype (Takeichi, 1995). A particular celltype generally coexpresses multiple subclasses of cadherins,and their combination varies with the cell type. Thus, thecombinatory action of multiple cadherin subtypes is likelyto be involved in the determination of adhesive specificityof cells ; in this way a wide range of adhesive specificitiescould be created (Steinberg & Takeichi, 1994; Broders &Thiery, 1995; Takeichi, 1995).

Neural crest cells escape from the neural tube prior toneural crest cell migration to various destinations necessi-tating the regulation of cell–cell adhesion between like andunlike cells. Takeichi et al. (2000) have provided evidencesupporting the hypothesis that cadherin subtype switchingplays a role in neural crest cell emigration from the neuraltube. Studies involving overexpression of various cadherinconstructs in neural-crest-generating cells suggest thatthe regulation of cadherin expression, or its activity at theneural-crest-forming area play a vital role in the emigrationof neural crest cells from the neural tube. In the chickenembryo, two cadherins are expressed in the early neuraltube: N-cadherin, and cadherin-6B; these are expressed inthe dorsal-most area where neural crest cells are generated.Migrating neural cells from the neural tube start to expressa different cadherin : cadherin-7, while N-cadherin andcadherin-6B are downregulated. Many crest cells fail toleave the neural tube if N-cadherin or cadherin-7 is over-expressed causing crest-cell segregation from neuroepithelialcells to be blocked (Nakagawa & Takeichi, 1998; Takeichiet al., 2000).

Thus, it may be that qualitative changes in cadherinexpression, involving changing patterns of adhesive specifi-cities, play a role in the segregation of cell populations invarious developmental contexts. The underlying mechan-isms involve control of the spatial and temporal expressionof cadherins with varying adhesive specificities.

(2 ) Quantitative differences in cadherin expression

Under certain conditions, a quantitative difference in cad-herin expression is involved in cell sorting. Steinberg’s

‘differential adhesion hypothesis ’ attempts to explain cellsorting by assuming only quantitative differences in ad-hesiveness between cells due to different levels of expressionof a particular CAM (Steinberg, 1996). Steinberg’s math-ematical computations predict that two motile cell types,differing only in the level of expression of a single adhesionsystem, should separate from each other, and sort out sothat less cohesive cells envelop a core of the more cohesiveones.

In order to determine the accuracy of Steinberg’s pre-dictions, Takeichi differentially expressed a single homo-philic adhesion system in L-cells. Cells were transfectedwith P-cadherin cDNA, and two cell populations expressingsubstantially different amounts of P-cadherin were mixed.The results confirmed Steinberg’s predictions, and showedthat : (i ) the cells of the two intermixed cell populationssorted out, resulting in the segregation of unlike cells ; (ii ) onetissue selectively spread over the surface of the other ; and(iii ) specific inside/outside tissue stratification occurred, withthe less cohesive cell populations enveloping the more co-hesive cell populations. It is not yet known whether differ-ences in adhesive site frequency are actually used in extantin vivo developing systems as a means of control (Steinberg& Takeichi, 1994; Steinberg, 1996, 1978).

Thus, in summary, it is likely that both qualitative, andquantitative differences in cadherin expression contributeto cell sorting, and segregation of cells (Takeichi, 1991,1995; Takeichi et al., 1993; Steinberg & Takeichi, 1994).

(3 ) Homeodomain transcription factor-mediatedregulation of cell adhesion molecules

(a ) Vertebrate Hox and Pax proteins

There is evidence supporting the hypothesis that homeoboxand Pax genes play roles in regulating cell–cell adhesion.Edelman & Jones (1998) have explored, in the context ofneural morphogenesis, the relationship between these genes(expressed in spatiotemporal sequences, and known to in-fluence morphology) and CAMs (responsible for the deter-mination of tissue boundaries and the local patterning ofcellular processes). According to the ‘morphoregulatorhypothesis ’ regulation of particular CAMs by spatially re-stricted transcription factors such as Hox and Pax proteinswould result in the morphogenetic patterning of the ner-vous system. Promotors for a number of neural CAM genescontain binding sites for Hox and Pax proteins, and appearto be authentic downstream targets of regulation by theseproteins (Edelman & Jones, 1998; Bellipanni et al., 2000).A specific example of transcription factor-mediated CAMregulation is the control of R-cadherin by Pax6 in the mouseforebrain ; Pax6 is required for the expression of R-cadherinin specific regions of the forebrain, and for the formation ofa normal boundary between cortical and striatal regionsof the telencephalon (Bellipanni et al., 2000).

(b ) Vertebrate and invertebrate Otx proteins

Bellipanni et al. (2000) have investigated, in zebrafishembryos, the effect of specific transcription factors (zOtxproteins) on cell–cell interactions. Members of the Otx


homeodomain family are transcription factors identifiedin the vertebrate anterior brain. Conservation of functionof this family is evidenced by the presence in Drosophilamelanogaster of the homologue otd, a gene required for theformation of the embryonic anterior head and other neuralstructures. Experiments were performed by following theprogeny of injected single blastomeres of zebrafish embryos ;descendants of such blastomeres usually spread and mixthroughout the embryo, and are not restricted to specifictissues. Ectopic zOtx1 expression was found to cause em-bryonic cell aggregation; the aggregates retained coherenceduring subsequent developmental stages. The Otx domainis necessary for the aggregation to occur and seems to besufficient when substituted for the homeodomain of anunrelated homeodomain protein. When cells containinginjected zOtx1 RNA were limited to the zone normally fatedto become the anterior brain and neural retina, the inducedaggregates contributed to anterior brain and retina tissues.By contrast, in many other regions which do not expressendogenous zOtx1, aggregates did not become integratedwith developing tissues. The results strongly suggest a rolefor Otx proteins to maintain the coherence of prospectiveanterior brain cells within the ectoderm. It is thoughtthat the results are most consistent with a model involvingpromotion of selective cell adhesion.

( c ) Murine Msh genes

Lincecum et al. (1998) have investigated the potentialrelationship between murine Msx-1 and Msx-2 expression,and cadherin-mediated cell adhesion and cell sorting. TheMsx-1 and Msx-2 genes are two closely related homeoboxgenes (of the Msh class) which are expressed in cephalicneural crest tooth buds, the optic cup endocardial cushions,and the developing limb. These locations correspond toregions of active cell sorting and proliferation, particularly atepithelial–mesenchymal junctions ; this suggests that Msx-1and Msx-2 regulate cell–cell interactions. Lincecum et al.(1998), investigated whether the expression of Msx genes issufficient to alter cell adhesion and cell sorting. Total proteinlevels of cadherins and catenins are not altered by Msxgene expression. The studies involved in vitro dissociation/reaggregation and cell sorting assays, using fluorescentlylabelled cells from murine cell lines. It was found that : (a)expression of Msx-1 alters cadherin-mediated adhesion,while Msx-2 has no such effect ; (b) Msh gene expressionis sufficient to confer differential cell sorting properties toaggregated cells ; (c) homotypic combinations of Msx-1 cellsor control cells results in a randomly mixed aggregate ; (d )heterotypic combinations of Msx cells with control cellsshow specific sorting with the control cells forming a centralcore, and the Msx cells forming a ‘shell ’ around the core ; (e)heterotypic combinations of Msx-1 and Msx-2 cells resultin the formation of a core of Msx-1 cells, and a shell ofMsx-2 cells.

Thus, sorting behaviour is specific for Msx-1 cells. It issuggested that : (a) the Msx-1-mediated decrease in Ca2+-dependent adhesion is consistent with the idea that changesin the cellular localization of cadherins may act as a rapidregulatory mechanism during cell sorting ; (b) intracellular

pools of cadherins may be maintained while their transportto the cell surface is blocked; (c) Msh gene expression mayregulate (e.g. via phosphorylation) or modify the stabilityof cadherin/catenin interactions ; (d) Msh genes may actas rapid regulators of epithelial–mesenchymal interactionsby modulating cadherin-mediated adhesion which resultsin cell sorting (Lincecum et al., 1998).

(d ) Caenorhabditis elegans ceh-43 gene

The C. elegans CEH-43 belongs to a family of homeodomaintranscription factors, the Distal-less (DII) class, that is in-volved in appendage development in metazoans of manyprotostome and deuterostome phyla. The ceh-43 gene isknown to be required specifically for closure or stability ofthe anterior-most head hypodermis. The function of thisgene has been investigated by RNA-mediated interference(RNAi), a means to disrupt gene function (Aspock & Burglin,2001). It is thought that hypodermal lesions in ceh-43(RNAi)are probably due to defects in adhesive properties of oneor more anterior hypodermal cells amongst themselves. Atthe beginning of morphogenesis, ceh-43 (RNAi) embryos startto lose cells through a hole in the head hypodermis ; theyleak out without contact to each other. Resultant pheno-types suggest a role for ceh-43 in development of adhesiveproperties in the head hypodermis which connects the epi-thelia of the skin with the digestive tract.

While there is some evidence for the involvement ofhomeodomain transcription factors in regulation of cellularinteractions, data are lacking for most developmental sys-tems. Nevertheless, since homeodomain transcription fac-tors are universal participants in metazoan development,it will be of great interest to determine how widespreadare direct connections between the function of these proteinsand regulation of cell adhesion systems.

(4) Intracellular modulation: catenins andRho GTPases

With respect to the cadherin cytoplasmic domain, the for-mation of cadherin-mediated adhesive contacts is thoughtto involve (i ) the formation of cis dimers ; (ii ) associationwith the actin cytoskeleton; and (iii ) actin-driven cadherinclustering. The protein–protein interactions involved ateach step are sites of potential regulation of cadherin func-tion (Lilien et al., 2002) (see Fig. 3C).

(a ) Catenins

Catenins mediate the linkage of cadherin-receptor bindingin the cell membrane, and cytoskeletal actin/intermediatefilament fibres in the cytoplasm (Fig. 3C). The cytoplasmicdomain has been implicated in control of cell adhesionby providing a linkage of cadherin molecules to corticalactin filaments (Nagafuchi & Takeichi, 1988, 1989; Hiranoet al., 1992; Kemler, 1992; Nagafuchi et al., 1993; Nathkeet al., 1993; Stappert & Kemler, 1993). Various intracellularmolecules have been implicated in regulation of the dy-namics of cadherin-mediated adhesion (Takeichi, 1993;Brady-Kalnay & Tonks, 1995; Shibamoto et al., 1995; Weis,


1995; Barth et al., 1997; Daniel & Reynolds, 1997; Brady-Kalnay et al., 1998; Hatzfeld, 1999; Takeichi et al., 2000).

b-catenin acts as the bridge between the cytoplasmicdomain of cadherins and the actin-containing cytoskeleton.There is considerable evidence supporting a role for b-catenin phosphorylation in regulating its interaction withcadherin and hence cadherin-mediated adhesion. Disrup-tion of the cadherin-cytoskeletal connection has the poten-tial to execute very rapid changes in cadherin function.Regulation of b-catenin’s phosphotyrosine content is afunction of the accessibility of b-catenin tyrosine kinases andphosphatases and their activity. Several kinases and phos-phatases have been shown to have the potential to alterthe phosphotyrosine content of b-catenin. It is likely thatmany kinases and phosphatases regulate cadherin function.However, it is not yet possible to define a role for the func-tional inactivation of cadherins via tyrosine phosphorylationof b-catenin during development (Lilien et al., 2002). b-catenin associates with E-cadherin soon after cadherinsynthesis, and mutants deficient for b-catenin binding areretained within the endoplasmic reticulum and rapidly de-graded. This may form a basis for modulation of cadherin-mediated adhesion. The cytoplasmic domain of a cadherinmolecule is not structured in the absence of b-catenin. Ithas been shown that most cadherin cytoplasmic domainscontain PEST sequences, motifs associated with ubiquitin/proteosome degradation. These PEST sequences overlapthe b-catenin binding site. It is hypothesized that the PESTsequences may function as signals for degradation of un-complexed cadherins. Binding of cadherins to b-cateninmay prevent recognition of degradation signals that areexposed in the unstructured cytoplasmic domain; thiswould favour a cell surface population of catenin-boundcadherins capable of participating in cell adhesion. Cad-herins that are not associated with b-catenin may betargeted for degradation, reducing the population of cate-nin-free cadherins at the cell surface. This represents apossible mechanism for regulating cadherin turnover (Huberet al., 2001).

(b ) Rho GTPases

The Rho family of low molecular mass guanosinetriphos-phatases (GTPases) (e.g. RhoA, Rac1 and Cdc42) appearsto be involved in cadherin function, but their roles arenot yet understood (Anastasiadis & Reynolds, 2000, 2001;Vasioukhin & Fuchs, 2001; Van Aelst & Symons, 2002).Rho GTPases cycle between a GDP-bound (inactive) stateand a GTP-bound (active) state. In the active state, theseGTPases can relay signals from adhesion molecules toregulate various processes such as actin cytoskeleton organ-ization (Van Aelst & Symons, 2002). Various data supporta critical role for Rho GTPases in mediating the EMT(‘cell scattering’) (Van Aelst & Symons, 2002). Duringsuch transitions cells lose many of their distinctive epithelialcharacteristics and take on a more mesenchymal fibroblasticphenotype. An important model for studying the role ofRho GTPases in this process involves the scattering ofMadin–Darby canine kidney (MDCK) epithelial cells in-duced by hepatocyte growth factor (HGF). HGF induces

multiple effects ; these include triggering the activation ofCdc42 and Rac, accompanied by the formation of filopodiaand lamellipodia. The potential roles of the Rho GTPasesin the disruption of cell–cell junctions requires clarification.Studies involving normal human keratinocytes have indi-cated that both Rho and Rac are required for the regulationof cadherin-dependent cell–cell adhesion (Braga et al., 1997).Further data suggests that Rac activation can specificallydestabilize cadherin receptors from mature cell–cell junc-tions in such keratinocytes. It is hypothesized that Rac isable to activate specific signalling pathways that perturbstability (Braga et al., 2000).

( c ) IQGAP1

IQGAP1, which is a target of the Rho family of GTPaseshas been shown to interact with the cytoplasmic portionof E-cadherin as well as with b-catenin. IQGAP1 anda-catenin compete for an overlapping binding site inb-catenin, which can result in dissociation of a-catenin fromthe adhesive complex. In this way the Rho GTPases regu-late cell–cell adhesion, and linkage of cadherins to the actinfilament network. IQGAP1 also binds to calmodulin ; dis-ruption of this interaction enhances association of IQGAP1with the cadherin-b-catenin complex, resulting in impairedE-cadherin-mediated adhesion. Since IQGAP1 can bothpromote adhesion or inhibit it, it can act as a sensitiveregulator of cadherin-mediated cell–cell adhesion (Angstet al., 2001).

(d ) p120-catenin

Available data strongly suggest a role for p120-catenin(p120ctn), a catenin which directly binds the cytoplasmicjuxtamembrane domain (JMD) of cadherin molecules. TheJMD has been implicated in regulating cell motility, andalso in regulating adhesion (Anastasiadis & Reynolds, 2000;Noren et al., 2000; Paulson et al., 2000). There is consider-able controversy concerning the role of p120ctn, and under-lying mechanisms have yet to be elucidated. It would appearthat p120ctn can both promote and inhibit adhesion de-pending on signalling cues. Conflicting results have beenobtained (perhaps due to the use of different cell types), andare not yet reconciled. For example, one study has shownthat the JMD supports cadherin clustering, and promotesstrong adhesion (Yap, Nessen & Gumbiner, 1998), whileothers have suggested that p120ctn can negatively regulateadhesion. Further complicating matters is the existenceof cell-type-specific expression of different p120ctn isoforms(implying functional differences), and increasing evidencethat multiple signalling events regulate p120ctn activity. Im-portant candidates for the regulation of p120ctn are tyrosineand serine/threonine kinases (Anastasiadis & Reynolds,2000; Thoreson et al., 2000). Thus, current models are veryfragmented.

Anastasiadis & Reynolds (2000) have proposed a ‘p120ctn-centric ’ model for cadherin-mediated adhesion. Thisinvolves the following sequence of events : (i ) formation oflateral (cis) cadherin dimers ; (ii ) formation of adhesive (trans)dimers between cadherins on adjacent cells resulting in


activation of p120ctn ; (iii ) p120ctn-facilitated clustering ofadhesive dimers at regions of cell–cell contact to establishstrong cell–cell adhesion, this process involving direct/indirect p120ctn dimerization, or cadherin-mediated dimer-ization triggered by binding of activated p120ctn ; and (iv)actin crosslinking, coordinated with clustering, but furtherrequiring the catenin-binding domain, Rac1 and Cdc42,and direct association with the actin cytoskeleton.

Analysis of cells expressing human E-cadherin mutantconstructs has indicated that p120ctn is required for the E-cadherin-mediated transition from loose to tight adhesion(the process of compaction). p120ctn-uncoupled E-cadherinmutants were found to be deficient in the reorganizationof E-cadherin and actin that would normally result in thecondensation of adjacent cells into compact epithelial col-onies. Each cell within a colony retained its own individualcircumferential actin ring ; normally, an actin cable wouldcircumscribe the entire colony, resulting in a seamlessconnection between adjacent cells. It has been postulatedthat p120ctn is required for the consolidation of looseadhesion (mediated in part by homotypic association ofcadherin extracellular domains) into tight adhesion me-diated by additional rearrangement and attachment of theactin cytoskeleton. It is noted that because the mutantsused did not associate with p120ctn there exists the possi-bility that in the context of a cadherin complex, p120ctn

might also negatively regulate adhesion (Thoreson et al.,2000).

A p120ctn-dependent inhibition of the cadherin-mediatedadhesion system in colon carcinoma cells has been found,leading to a model being proposed for a signalling systemto regulate p120ctn function. The model involves phos-phorylation of p120ctn, and the inhibition of unknownkinases. The p120ctn-dependent process is the type ofmechanism which would allow tumour cells to destabilizetheir own adhesion, detach, and metastasize (Takeichi et al.,2000).

There is strong evidence indicating that p120ctn actsthrough regulation of Rho GTPases. It has been foundthat p120ctn can inhibit RhoA and activate Rac and Cdc42(reviewed by Anastasiadis & Reynolds, 2001). For example,Noren et al. (2000) provide evidence that p120ctn contributesto the cross-talk between cell–cell junctions and the motilemachinery of cells. They present a model for how p120ctn

may regulate the activity of RhoA, Rac1, and Cdc42; thisinvolves the shuttling of p120ctn between a cadherin-boundstate and a cytoplasmic pool in which it can interact withregulators of Rho family GTPases.

( e ) Recycling of E-cadherin

Fujita et al. (2002) have provided evidence that the E-cadherin complex in epithelial cells is ubiquinated andendocytosed following activation of the tyrosine kinase.A novel E-cadherin binding protein has been identified;‘Hakai ’ is an E3 ubiquitin-ligase which mediates ubiquiti-nation of the E-cadherin complex. Hakai interacts withE-cadherin in a tyrosine phosphorylation-dependent man-ner, inducing ubiquitination of the E-cadherin complex.Expression of Hakai disrupts cell–cell contacts, enhances

endocytosis of E-cadherin, and enhances cell motility. TheE-cadherin complex is in a ubiquitination-de-ubiquitinationequilibrium, and Hakai appears to affect this equilibrium;the molecular mechanisms involved are not yet known.Hakai can thus play a role in modulating cell adhesion. Itmay play a role in the regulation of epithelial-mesenchymaltransitions.

Thus, in summary, while some of the potential playersinvolved in the regulation of cadherin function have beenidentified, the underlying mechanisms remain sketchy. Ingeneral terms, regulation of cadherin function involves thecadherin cytoplasmic domain, the catenins, the actin cyto-skeleton, and a network of intracellular protein–proteininteractions, and signalling events.

(5) Concerted activities of different types ofcell adhesion molecules and adhesive systems

(a ) Migrating neural crest cells

It is thought that cell adhesion specificity is acquired by theconcerted activities of a number of CAMs and adhesivesystems. For example, during vertebrate neural develop-ment a plethora of types of CAMs are employed, includingmultiple cadherin types, members of the Ig superfamily,integrins, and ECM molecules such as proteoglycans. Thereare 30 or more known cadherins alone. The developingnervous system is characterized by a great degree of mol-ecular complexity, and diverse adhesive interactions be-tween similar and dissimilar cell types (Ranscht, 1991;Redies, Englehart & Takeichi, 1993; Redies & Takeichi,1993a, b ; Dalseg et al., 1994; Redies, 2000).

Consider the migration of vertebrate neural crest cells.Regulation of adhesive interactions during cell migrationrequires the coordination of a multiplicity of signals ; regu-latory phenomena must be coordinated in space and time.As in dynamic sponge cell interactions, coordinated inter-play is required between cell–cell, and cell–ECM adhesionsystems. Mechanisms for cell detachment, as well as mech-anisms for a change from a cell–cell mode of adhesion topreferential adhesion to ECM elements, are essential formigration to occur (Duband et al., 1995).

Migrating neural crest cells adhere to the ECM proteinfibronectin in an integrin-dependent process. It is thoughtthat, in migrating neural crest cells, beta 1 and beta 3 in-tegrins are involved in triggering a signalling cascade whichcontrols the surface distribution, and activity of N-cadherin(Fig. 6). [Such a direct linkage of CAM receptors via intra-cellular signals may be important for the coordinated ‘cross-talk ’ between cell–cell and cell–ECM adhesion processes(see Monier-Gavelle & Duband, 1997).] Subsequently,reinitiation of cadherin expression in target tissues allowsadhesion at target locations where neural crest cells expresscadherins of the same type present on the target tissue(Marrs & Nelson, 1996).

(b ) A regulatory module in D. discoideum

There exists a potential link between DdCAD-1-mediatedcell–cell contact and: (a) a cAMP-mediated signal relay


pathway; (b) a pathway which regulates gene expression,including csA (gp80) gene expression. Thus, two distinctcontact-triggered signalling pathways form a ‘regulatorymodule’ (Fig. 7). The two pathways are connected, at leastat the level of cAMP receptor 1 protein (cAR1). The acti-vation of the G-protein receptor Ga2 may be a shareddownstream event (Brar & Siu, 1993; Brar, 1994; Desbaratset al., 1994; Sesaki & Siu, 1996; Wong et al., 1996). Notethat cells lacking cAR1 neither aggregate nor express de-velopmentally regulated genes (Soede et al., 1994).

This regulatory module incorporates elements at : (a)molecular and cellular levels of organization; (b) differenttime-scales : (i ) cell movement : order of seconds ; (ii ) cAMPrelay : order of minutes ; (iii ) gene expression: order of hours ;(c) different stages of development : pre-aggregation, andaggregation.

Evidence suggests [DdCAD-1]–[DdCAD-1] interactionsmay initiate a signalling pathway that results in the upre-gulation of csA. DdCAD-1-mediated homotypic cell–cellcontact at the pre-aggregation stage of development plays

a role in the regulation of expression of the gene for csAat the aggregation stage. The gene encoding csA is first ex-pressed at a basal level ; later chemotactically responsivecells secrete cAMP, and transcription of csA is greatly aug-mented by the extracellular cAMP.

Exogenous cAMP pulses result in a ‘precocious ’ andenhanced level of csA expression at the transcription level.The disruption of earlier binding sites (mediated byDdCAD-1) results in an inhibition of the cAMP-induced,high-level expression of csA; this inhibition cannot be res-cued by exogenous pulses of cAMP. However, exogenouscAMP pulses can rescue csA expression if the formationof cell contacts is permitted to occur. Thus, previous cell–cellcontact is required for the activation of the cAMP-mediatedsignal transduction pathway leading to the production ofhigh levels of csA expression (Desbarats et al., 1994).

DdCAD-1 forms a complex with several membrane andcytoplasmic elements, which may play a role in the dy-namics of the cytoskeleton and signal transduction (Des-barats et al., 1994). It is likely that there exists some form of

Source tissue

IntegrinsN-cadherindownregulation

Dissociation of cells

Cell migration Cell-ECMadhesion

CELL-CELL ADHESION

Target tissues

Cadherin Aexpression

Cadherin Bexpression

Cadherin Cexpression

CELL-CELL ADHESION

Fig. 6. Regulation of cadherins during neural crest cell migration via integrin-mediated signalling. The emigration of neural crestcells from the neural tube involves (a) downregulation of N-cadherin expression allowing dissociation of cells from the original tissue.(b) Integrin-mediated signalling which regulates the surface distribution, and activity of N-cadherin. (c) Adhesion of migrating cells tothe extracellular matrix (ECM) protein fibronectin in an integrin-dependent process. (d ) Reinitiation of cadherin expression in targettissues allowing adhesion where neural crest cells express cadherins of the same type present on target tissue.


Pathway A

Cell 2

cAR1

cAMPcAMP

cAR1

Cell 1

Pulsatilesecretion

Cadherin-mediatedcell-cellcontact

Extracellularspace

Activation of adenylate cyclasevia a G-protein-dependent signal

transduction pathway

Pulsatile cAMP synthesis

Pathway B

Extracellularspace

cAR1

cAMP

Preaggregationphase

Cadherin-mediated

cell-cell contact

Aggregationphase

csA-mediatedcell-cell contact

High levels ofcell-surface

csA expression

Cytoplasm

Nucleus

Gα2-dependent signaltransduction pathway

Gene regulation pathways

Differential activation of aggregation-stagespecific genes

Upregulation of csA expression

Fig. 7. Model for the coupling of two cell–cell contact-triggered signalling pathways in a regulatory module in Dictyosteium discoideum.(A) An adenosine 5k,3k-cyclic monophosphate (cAMP) signal relay pathway; (B) a gene regulation pathway. (A) The aggregation ofamoebae is mediated, in part, by cAMP which serves as a chemoattractant, and plays a key role in oscillatory cell–cell signalling. ThecAMP signal relay pathway involves cAMP binding to a cell-surface cAMP receptor (cAR1), and G-protein-coupled activation ofadenylate cyclase resulting in the production of intracellular cAMP; this cAMP is secreted, and binds to cAR1 receptors on adjacent


communication between DdCAD-1 and cytoplasmic com-ponents of a signalling pathway that leads to csA expression(Brar, 1994).

It appears that the number of different CAMs (and theirreceptors) expressed depends on the level of complexityof the structure to be formed. Perhaps, during the courseof evolution, the emergence of new CAMs (includingnew cadherins) paralleled increasing complexity of bodyplan, and of physiology. However, as in the neural crestexample, aggregation in D. discoideum involves more thanone CAM type, and also multiple signalling pathwayswithin an integrated module for the regulation of cell–celladhesion.

IV. CONCLUSIONS

(1) The dynamic balance between cell adhesion and cellmotility and the molecular means to regulate this balanceare universal, and arguably the most important featuresof adhesion during metazoan development. While currentmodels for the molecular bases of dynamic cell phenomenaare, at this stage, fragmentary, we have gleaned some in-sights into universality by comparing models for members ofdisparate phyla such as the eukaryotic protist D. discoideum,and the metazoans : Porifera, C. elegans, D. melanogaster, andvarious vertebrates ; and by considering the adhesion re-quirements for the evolution of multicellularity.

(2) We have considered how cells make initial contact.At a macro-level, dynamic processes driven by motile, actin-containing cell extensions such as filopodia occur withinvery different developmental systems. These include theaggregation of amoebae in D. discoideum ; the adhesion ofdissociated sponge cells ; the migration of cells during seaurchin gastrulation; the movement of mesodermal cellsduring gastrulation in X. laevis ; and the creation of con-tinuous epithelia in D. melanogaster and C. elegans. Certainfeatures and events are often shared: cell extensions providethe means for exploration by a moving cell, and for initialcell–cell contact and molecular recognition. Further, re-arrangement of the cytoskeleton, and the formation of in-creasingly extensive adhesive contacts between cells oftenoccur. We conclude that it is likely that the mediation ofexploratory cell movement, and initial cell–cell contact byactin-containing cell extensions facilitated the emergence ofmulticellularity.

(3) For the task of mediating the formation of transientcellular contacts, or maintaining contact within stable two-dimensional and three-dimensional structures, there exists a

potentially bewildering array of structurally and functionallydiverse molecular mediators of cell–cell and cell–ECMinteractions. Further, there appears to be a requirement foran increasingly large repertoire of cell contact moleculesin the more complex developmental systems such as thedeveloping nervous system, compared with sponge andD. discoideum adhesion systems.

In spite of the apparent complexity, CAMs share manystructural features, and have been classified as belonging toa small number of gene families, the most important fordevelopment being the cadherin superfamily, the integrins,the Ig superfamily, and the proteoglycans.

We have focussed on the classic cadherins. Examinationof cadherin-based adhesion systems has provided a frame-work for us to compare the details of adhesion systemsoperating in different organisms, and in different develop-mental contexts.

(4) Cadherin-dependent homophilic binding mechan-isms, the ability to organize catenins and their cytoplasmicassociates, and adherens junctions associated with actin,the molecular systems required to regulate the actin cyto-skeleton, and cadherin-dependent control of morphogenesisexist in most, if not all, metazoan phyla, as well as inD. discoideum. We conclude that the molecular means toregulate cadherin-mediated adhesion probably evolvedbefore the emergence of metazoans, and that cadherins arefundamental adhesion molecules. Further, cadherins musthave been crucial to the evolution of epithelial tissue, andhence to the complexity of multicellular organization.

(5) Relatively low affinity for their ligands is a featureshared by most, if not all, adhesion proteins. Thus, CAM-mediated cell–cell adhesion involves weak-affinity inter-molecular interactions. Dynamic ‘on and off’ binding oflow-affinity sites would facilitate the de-adhesion/adhesioncycle required for transient cell contact formation, and alsocell motility.

Stabilization and strength of cell adhesion, whererequired, can be achieved, at least in part, by the com-pounding of multiple, weak-affinity interactions. We havediscussed how this can be achieved via dissimilar adhesionsystems with different modes of interaction between cellcontact molecules : protein–protein interactions are involvedin vertebrate cadherin-mediated systems, while the spongeadhesion system involves the interaction between highlypolyvalent carbohydrate molecules. The molecular meansto facilitate ‘collective adhesive strength ’ while at the sametime facilitating motility must be universal.

(6) The building of macromolecular assemblies (as insponges), and of higher order structures such as complex celljunctions (adherens junctions) in D. discoideum, sea urchins,

cells. cAMP is produced and secreted in a pulsatile manner (Desbarats et al., 1994; Fontana, 1995; Milne et al., 1997). (B) Anotherpathway leads to the differential activation of some aggregation-stage-specific genes including the regulation of contact sites A (csA)expression. This pathway is triggered by the transient activation of a cell-surface cAMP receptor (probably cAR1). The receptor iscoupled to the G-protein receptor Ga2. It is thought that this pathway results in the interaction of DNA-binding protein(s) withcAMP-response elements in the DNA upstream of the csA gene (Desbarats et al., 1994). Pathways A and B are linked via the[cAMP]-[cAR1] complex. Thus cAMP has been co-opted for use in two different regulatory signalling pathways, both of whichare associated with cell–cell adhesion.


D. melanogaster, and in vertebrates probably forms the foun-dation for achieving localized strength of adhesion, and thefacilitation of CAM-mediated intracellular signalling cas-cades involved in the regulation of adhesion/de-adhesionand other cell functions.

(7) The movement (laterally) and concentration of CAMsand associated molecules in the cell membrane at sites ofadhesion, and rearrangements of the actin cytoskeleton asseparate, apposing cells make membrane contact are likelyto be universal requirements for achieving strength of ad-hesion. The lateral clustering of cadherins, and tetheringto the actin filament cytoskeleton favour cumulation ofrelatively weak interactions to form strong bonds, theassembly of junctions and higher-order structures, andcadherin-mediated signalling.

(8) Our examination of adhesion in D. discoideum and incadherin-based adhesion systems leads us to suggest that themechanisms for regulating, in vivo, the balance between celladhesion and motility during morphogenesis in most or-ganisms are likely to include: (a) qualitative and quantitativechanges in CAM expression, involving control of the spatialand temporal expression of CAMs with different adhesivespecificities ; (b) regulation of CAM function via a set ofregulatory intracellular cytoplasmic protein–protein inter-actions. In metazoan cadherin-based systems, componentsof a regulatory network involved in positively and negativelyregulating cell adhesion are likely to include catenins suchas b-catenin, a-catenin, and p120ctn ; the actin cytoskeleton;and kinases, phosphatases, and Rho GTPases ; (c) the con-certed activities of a number of different CAMs and typesof adhesive systems, involving interactive signalling systems,and a high level of integration. This is crucial in the ad-hesion systems of very disparate phyla : during neural crestmigration during vertebrate development, and also in theestablishment of ‘ streams’ during D. discoideum development.

(9) A more informed evaluation of universality requiresthat the molecular mechanisms underlying many moreadhesion systems be unravelled. While many regulatorysignalling components are known, their interactions andprecise roles need to be determined. Studies of the ways inwhich CAMs become concentrated in the cell membraneat adhesion sites will be of great interest, as will adhesionstudies using relatively simple organisms which provideclues to features important for the emergence of multi-cellularity.

V. ACKNOWLEDGEMENTS

We thank Dr Richard Morrow (Morrow Corona Solutions Pty Ltd)for assistance with editing, and production of the figures. We alsothank Prof. Jack Bassett for helpful comments.

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