Cadherins and their connections: adhesion junctions have broader functions.

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

Human autoantibodies against a desmosomal core protein in pemphigus foliaceus.

Pemphigus foliaceus (PF) is a human autoimmune disease in which antibodies are directed against the cell surface of epidermal cells with resultant blister formation. The histopathology of these blisters indicates that cells have detached from each other, and electron microscopy of early blisters shows diminished numbers, to complete loss, of desmosomes as well as abnormalities of the tonofilament-desmosome complex. In this study we demonstrate that autoantibodies from certain PF patients bind to a desmosomal core glycoprotein called desmoglein (DG) I. Proteins in extracts of normal human epidermis were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), then transferred to nitrocellulose or 2-aminophenylthioether paper for immunoperoxidase staining. Results of these immunoblots indicated that sera from 6 of 13 PF patients specifically and intensely stained an approximately 160,000 mol wt polypeptide, "PF antigen". Such staining was not seen with normal human sera or sera from patients with pemphigus vulgaris or bullous pemphigoid, two autoimmune blistering skin diseases that are clinically, histologically, and immunochemically distinct from PF. However, rabbit antiserum directed against DGI, that was isolated from bovine muzzle desmosomes, stained a polypeptide band which co-migrated with PF antigen. Furthermore, when proteins from extracts of normal human epidermis were electrophoresed in two dimensions (isoelectric focusing, then SDS-PAGE) before transfer to nitrocellulose for immunoperoxidase staining, PF antibodies and antibodies to DGI stained identical spots. Finally, PF sera as well as PF IgG that was affinity purified with PF antigen from normal human epidermis, both selectively bound to DGI extracted from bovine muzzle desmosomes. These studies demonstrate that the human autoantibodies from certain patients with PF, a disease of epidermal cell adhesion, are directed against a desmosomal core protein.

Isolation of the intercellular glycoproteins of desmosomes.

To characterize the desmosome components that mediate intercellular adhesion and cytoskeletal-plasma membrane attachment, we prepared whole desmosomes and isolated desmosomal intercellular regions (desmosomal "cores") from the living cell layers of bovine muzzle epidermis. The tissue was disrupted in a nonionic detergent at low pH, sonicated, and the insoluble residue fractionated by differential centrifugation and metrizamide gradient centrifugation. Transmission electron microscopic analyses reveal that a fraction obtained after differential centrifugation is greatly enriched in whole desmosomes that possess intracellular plaques. Metrizamide gradient centrifugation removes most of the plaque material, leaving the intercellular components and the adjoining plasma membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with methods that reveal carbohydrate-containing moieties on gels demonstrate that certain proteins present in whole desmosomes are glycosylated. These glycoproteins are specifically and greatly enriched in the desmosome cores of which they are the principal protein constituents, and thus may function as the intercellular adhesive of the desmosome.

Do morphogenetic tissue rearrangements require active cell movements? The reversible inhibition of cell sorting and tissue spreading by cytochalasin B.

Previous studies have indicated that cell sorting and tissue spreading are caused by cell combination-specific differences in intercellular adhesive energies, acting in a system of motile cells. We wished to determine whether these adhesive energies could drive cell rearrangements as well as guide them. Accordingly, aggregates of intermixed embryonic cells were cultured in solutions of the drug cytochalasin B (CCB) at a concentration shown to inhibit the locomotion of cells on a solid surface. In addition, spherical aggregates of several kinds were cultured in mutual contact under similar conditions. Both cell sorting and tissue spreading were found to be inhibited. The prompt release of this inhibition upon removal of the CCB showed that the inhibited cells were not merely injured. Moreover, aggregation experiments showed that CCB did not prevent cells of several kinds from initiating mutual adhesions. In fact, heart cell aggregation was enhanced by CCB. We conclude that interfacial forces, originating outside the cell, act together with forces originating inside it in bringing about the morphogenetic movements of cell sorting and tissue spreading. We propose the term "cooperative cell locomotion" to describe translational movements of cells arising from such a combination of intrinsic and extrinsic forces.

Measurement of tumor cell cohesion and suppression of invasion by E- or P-cadherin.

Invasiveness of carcinomas was connected early to decreased cohesiveness and has more recently been associated with loss or decreased activity of E-cadherin. In the first thermodynamic measurements of cohesive intensities among malignant cells, we here find the cohesive intensities of Lewis lung carcinoma cells to fall within the range measured previously for cells from a series of noninvasive embryonic tissues. Thus, too-low cohesiveness is itself an insufficient explanation for invasiveness. Nevertheless, transfection-mediated cadherin expression sufficient to increase cohesiveness by as little as 26% suffices to greatly reduce invasion of aggregates of Lewis lung carcinoma cells into Matrigel. This property is not restricted to E-cadherin but is shared by P-cadherin. The same cadherin-transfected cells do not display this invasion suppression when plated sparsely, indicating that invasion-suppression activity of cadherins requires cell-cell contact. These facts are consistent with the invasion-suppression activity of cadherins resulting either from the physical restraint of increased cohesion per se or from another cadherin activity mediated through cell-cell contact.

Dexamethasone up-regulates cadherin expression and cohesion of HT-1080 human fibrosarcoma cells.

The synthetic glucocorticoid dexamethasone markedly decreases the invasiveness of HT-1080 human fibrosarcoma cells. We show here that dexamethasone treatment of HT-1080 cell aggregates more than doubles their cohesivity from 3.9 to 9.7 dyne/cm. Western blot analysis shows a corresponding increase in cadherin expression. This was accompanied by an increase in the rate of calcium-dependent aggregation. Dexamethasone-treated aggregates spread to form a monolayer in Matrigel spreading assays, but the cells remained much more contiguous than their untreated counterparts. Invasion-suppression by dexamethasone may therefore be due, at least in part, to a previously unsuspected increase in cadherin-mediated cohesion.

Purification of desmoglein II: a method for the preparation and fractionation of desmosomal components.

We have developed rapid and efficient methods for the isolation of desmosomes and the fractionation of their components. These methods involve the use of 6 M guanidine HCl to isolate the desmosomes from bovine epidermis, followed by hydroxyapatite column chromatography in the presence of SDS to fractionate the desmosomal components. All of the desmoplakins and desmogleins were purified at least partially by these procedures, and desmoglein II was purified to apparent homogeneity. We expect these procedures to facilitate a detailed biochemical analysis of the molecular components of desmosomes. In addition, these methods may be applicable to the purification of other plasma membrane domains involved in cell adhesion.

Morphogenesis of the axolotl pronephric duct: a model system for the study of cell migration in vivo.

Pronephric duct (PND) morphogenesis is a critical early event in the development of the vertebrate excretory system. This structure is the exit channel for both pronephric and mesonephric filtrate, forms the ureteric bud of the metanephros and gives rise to the ductus deferens of the testis. In addition, the PND and ureteric bud epithelia induce terminal differentiation of the mesonephric and metanephric mesenchyme, respectively. Elongation of the PND in all vertebrates involves active cell migration of the primordium. In urodele embryos--unlike in some anuran, avian and mammalian embryos--elongation of the PND occurs solely by cell migration. In the axolotl embryo, the PND primordium segregates as an ovoid tissue mass from the anterodorsal flank mesoderm directly beneath somites 3-7. The primordium then extends caudally along the ventral border of the developing somites until it reaches the cloaca. The ease with which these embryos can be manipulated microsurgically makes the PND system ideal for the study of the mechanisms controlling cell migration in vivo. This review summarizes the progress that has been made in characterizing the environmental cues and the cell surface recognition systems that drive this tightly regulated migration event.

Desmosomal antigens are not recognized by the majority of pemphigus autoimmune sera.

Sera from 7 patients with pemphigus vulgaris and both mouse and rabbit antisera against bovine epidermal desmosomes contained antibodies that bound to cell surface components of the spinous layer of bovine epidermis. The antidesmosomal sera showed significant binding to purified desmosomal proteins in an enzyme-linked immunosorbent assay (ELISA). Two of 7 pemphigus sera bound to desmosomal protein-coated microtiter plates at low dilution titers. Two of 6 normal human sera also bound to desmosomal protein-coated microtiter plates at titers comparable to those of the pemphigus sera. Indirect immunofluorescent labeling of frozen sections of monkey esophagus revealed striking differences in the distribution of pemphigus antigens and desmosomal constituents. Pemphigus antisera produced rather uniform fluorescence around the borders of spinous cells of the esophageal epithelium, while anti-desmosomal antibodies bound in a punctate pattern. Anti-desmosomal antibodies labeled cells of the basal layer in a strongly punctate pattern. Only 1 pemphigus serum appreciably labeled basal cells. Two of 3 anti-desmosomal antisera bound avidly in the upper differentiating layers of the epithelium. Pemphigus antibodies did not. Pemphigus sera that reacted with desmosomal proteins in ELISA were absorbed by affinity chromatography on immobilized desmosomal proteins. This treatment did not alter the immunofluorescent labeling patterns produced by these sera. From these results we conclude that the pemphigus autoantibodies studied here bind to epithelial cell surface antigens which are distinguishable from the structural components of desmosomes.

Electron microscopic localization of specific carbohydrate groups in thin sections of tissues embedded in a hydrophilic resin: concanavalin A receptors in mouse spleen.

An improved postembedding method for electron microscopic localization of concanavalin A (Con A) receptors in ultrathin sections is reported. Materials are embedded in a hydrophilic resin, glycol methacrylate copolymerized with glutaraldehyde and urea, using a precisely controlled polymerization schedule that preserves ultrastructural integrity. Ferritin-Con A binds specifically in ultrathin sections of mouse spleen tissue and of Sephadex beads embedded in this resin. Quantitative investigations of procedures for decreasing nonspecific labeling demonstrate that preincubation of sections with bovine serum albumin reduces background to a very low level. This high-resolution method allows macromolecular affinity probes access to receptors in all cellular locations, while maintaining good morphological preservation.

Adhesion in development: an historical overview.

My purpose here is to provide brief historical overviews of three related subjects conceptually fundamental to the broader subject of "cell adhesion in development." These subjects are (1) the evolution of our present understanding of how animal cells cohere; (2) the question of what principles underlie the ability of embryonic cell populations to organize themselves into anatomically correct structures; and (3) the ongoing effort to understand the origins of the "recognition specificity" evinced in the latter phenomenon. Because this review must be brief, it is not possible to mention all of the significant advances, many of which will be referenced in a recent more detailed review of this subject (Grunwald, 1991). For the same reason, the important work on cell adhesion in nonvertebrate systems is not included.

An immunoelectron microscopic comparison of desmosomal constituents and hemidesmosomal ones originating from the same tissue of the same animal.

The molecular constituents of desmosomes and hemidesmosomes were compared by examining bovine muzzle epidermis under immunoelectron microscopy using a postembedding method, first with antibodies prepared to four desmosomal antigens (DP1/2, DP3, DG1, DG2/3), followed by protein A-gold (PAG) complexes. The four antibodies showed almost negative labeling at hemidesmosomes as compared with the labeling observed at the desmosomes in the same tissue. By counting the number of PAG particles/200 millimicrons at hemidesmosomes and desmosomes, the above qualitative observation was confirmed quantitatively. These results support a new concept which has recently been proposed by several researchers that hemidesmosomes and desmosomes are immunochemically distinct.

Do rates of intercellular adhesion measure the cell affinities reflected in cell-sorting and tissue-spreading configurations?

These experiments constitute the first experimental test of the hypothesis that the rates of adhesion between cells measure the intensities of adhesion or tissue affinities that could explain cell sorting and tissue spreading. For any set of relative adhesive intensities between cells in a heterogeneous population, a corresponding minimal free energy configuration can be calculated. This is the cell distribution toward which both cell sorting and tissue spreading should lead. Equilibrium configurations were determined for combinations of 7-day embryonic retina (R) with liver (L) and heart (H), both of which became completely enveloped by R. To produce these results, the adhesive intensities would have to fall in the sequences: L-L > L-R > R-R; and H-H > H-R > R-R. To determine whether the rates of adhesion fall into these same sequences, we have devised a new technique which measures the rates of adhesion between pairs of already-formed cell aggregates of like and unlike kinds. These fall in the sequence L-L > or = H-H > L-H > R-R > H-R > L-R. If these rates paralleled the corresponding intensities of adhesion at configurational equilibrium, both L and H should have become only partially enveloped by R. Thus the rates at which adhesions are initiated do not predict the relative adhesive intensities that could explain the observed tissue configurations.

Strategies for specifying form and pattern: adhesion-guided multicellular assembly.

We define a material pattern as a particular arrangement of material elements in space. We then make an effort to categorize the developmental strategies that underlie the emergence of multicellular patterns. These strategies are divided into three broad categories according to whether cell position influences or is influenced by cell fate. In that category of strategies in which cell fate influences cells to move to particular positions, we focus our attention upon morphogenetic and patterning phenomena that appear to be determined by adhesion-mediated interactions of cells with each other and with their surroundings. The differential adhesion hypothesis details how cellular adhesive properties can guide tissue movements and specify patterns of cell association. Motile, adhesive cells will naturally tend to group so as to maximize their adhesive interactions (minimize interfacial free energy). A homogeneous population of uniformly adhesive (isotropic) cells will tend toward spherical form. Cell surface adhesive anisotropies can determine other most-stable (equilibrium) configurations of the population, such as cell sheets, tubes and vesicles. Heterogeneous cell populations may preferentially either intermix or sort out, depending upon the balance of adhesive forces between like and unlike elements. The precise configuration adopted will depend upon the particular adhesive relationships that prevail. Both this end state and the approach toward it arise from the adhesive relationships among the interacting cells. Such morphogenetic phenomena as tissue spreading and the segregation of organ primordia are probably brought about in this way. We outline here some results of our recent experiments on the morphogenesis of the salamander pronephric duct. These illustrate the reality of emergent adhesion-generated tissue immiscibility as a cause of organ segregation and point toward a craniocaudally travelling adhesion gradient as the information that guides the migrating pronephric duct to the cloaca.

Elongation of axolotl tailbud embryos requires GPI-linked proteins and organizer-induced, active, ventral trunk endoderm cell rearrangements.

Application of phosphatidylinositol-specific phospholipase C to early tailbud stage axolotl embryos reveals that a specific subset of morphogenetic movements requires glycosylphosphatidylinositol (GPI)-linked cell-surface proteins. These include pronephric duct extension, "gill bulge" formation, and embryonic elongation along the anteroposterior axis. The work of Kitchin (1949, J. Exp. Zool. 112, 393-416) led to the conclusion that extension of the notochord provided the motive force driving anteroposterior stretching in axolotl embryos, elongation of other tissues being a passive response. We therefore conjectured that axial mesoderm cells might display the GPI-linked proteins required for elongation of the embryo. However, we show here that removal of most of the neural plate and axial and paraxial mesoderm prior to neural tube closure does not prevent elongation of ventrolateral tissues. Tissue-extirpation and tissue-marking experiments indicate that elongation of the ventral trunk occurs via active, directed tissue rearrangements within the endoderm, directed by signals emanating from the blastopore region. Extension of both dorsal and ventral tissues requires GPI-linked proteins. We conclude that elongation of axolotl embryos requires active cell rearrangements within ventral as well as axial tissues. The fact that both types of elongation are prevented by removal of GPI-linked proteins implies that they share a common molecular mechanism.