A fine structural study of adhesive cell junctions in heterotypic cell aggregates.

Mixed suspensions of cells obtained by dissociation of 7 day chicken embryo heart and pigmented retina were allowed to reaggregate in tissue culture. The reaggregates which resulted contained both kinds of cells. Establishment of homogeneous tissues by cell sorting out in these reaggregates was advanced by 20 hr in culture and was complete within 2 days. When sorting out was advanced, heterotypic aggregates were fixed, sectioned, and examined in the electron microscope. Particular attention was paid to the morphology of regions of contact between cells. No qualitative differences were observed in the contact junctions between like cells (heart-heart or pigmented retina-pigment retina junctions) and unlike cells (heart-pigmented retina junctions). Broad areas of undifferentiated cell contact with cell membranes separated by a 100-200 A gap were formed regardless of cell type. Specialized junctions of the fascia and macula adherens type were also present, not only between like cells but also between unlike cells.

Cell sorting in the presence of cytochalasin B.

The ability of cytochalasin B to inhibit ruffled membrane activity and cellular locomotion of vertebrate cells in monolayer culture prompted its use to study the necessity for this kind of active cellular locomotion in cell sorting in heterotypic cell aggregates. Cell sorting was inhibited in chick embryo heart-pigmented retina aggregates but a remarkable degree of sorting did occur in neural retina-pigmented retina aggregates. In these experiments, the levels of cytochalasin B employed (5 or 10 microg/ml) are sufficient to inhibit completely locomotion of these cell types in monolayer culture. It is proposed that the degree of cell movement achieved during sorting in neural retina-pigmented retina aggregates in the presence of cytochalasin B is the result of changes in cell contact resulting from adhesive interaction of cells. The effect of cytochalasin B on the initial aggregation of dissociated cells was also tested. With the cell types used in this study (chick embryo neural retina and limb bud), aggregation was not affected for a period of several hours.

An instructive role for the interstitial matrix in tissue patterning: tissue segregation and intercellular invasion.

Intercellular invasion is the intrusion of the cells of one tissue into space occupied by a second tissue. The alternative situation to invasion, one characteristic of most coherent tissues, is segregation, with identifiable boundaries existing between contiguous tissues. The interfaces between mesenchymal and myocardial tissues in the developing avian heart show a profoundly different character in different regions of the heart: the interface between epicardial mesenchyme and heart wall myocardium is planar, without intermingling of the two cell types, whereas the interface between endocardial cushion mesenchyme and myocardium is diffuse, with extensive invasion of both tissue types across the border to produce intermingling of the two tissues. Thus, invasion and tissue segregation coexist in different regions of the mesenchyme-myocardium contact zone. Investigation of the involvement of the interstitial matrix in invasion and segregation has been conducted by maintaining the two tissues in mutual contact in organ culture. Investigation of the mechanisms by which the two cell types sort out in randomized chimeric tissue reaggregates has provided insight into the conditions for tissue segregation. We have modeled invasion in organ culture by fusing aggregates of myocardial cells with aggregates of cardiac mesenchymal cells. Cells of both tissues invaded the partner aggregate during a period of 1-3 d of coculture. Both invasion and segregation in the aggregates appear to depend on the presence or absence of a fibronectin-rich interstitial matrix elaborated by the cardiac mesenchyme. During sorting, the matrix appears selectively in regions occupied by the mesenchyme. Under conditions of culture that are nonpermissive for matrix deposition, sorting fails to occur. Stimulation of matrix deposition by addition of serum, transforming growth factor beta, or isolated matrix itself is accompanied by sorting out of the two tissues. Sorting out is blocked reversibly by inclusion of the fibronectin adhesion site peptide, GRGDSP. Invasion of fused aggregates is preceded by a redistribution of the fibronectin-containing matrix of the mesenchymal aggregate such that matrix-poor regions come to occupy the interface with the myocardial partner aggregate. The invasion that ensues involves mesenchymal cells emigrating from, and myocardial cells intruding into, matrix-poor regions of the mesenchymal aggregate.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane protein redistribution during Xenopus first cleavage.

A large increase in surface area must accompany formation of the amphibian embryo first cleavage furrow. The additional membrane for this areal expansion has been thought to be provided entirely from cytoplasmic stores during furrowing. We have radioiodinated surface proteins of fertilized, precleavage Xenopus laevis embryos and followed their redistribution during first cleavage by autoradiography. Near the end of first cleavage, membrane of the outer, pigmented surface of the embryo and a short band of membrane at the leading edge of the furrow displayed a high silver grain density, but the remainder of the furrow membrane was lightly labeled. The membrane of the cleavage furrow is thus mosaic in character; the membrane at the leading edge originates in part from the surface of the zygote, but most of the membrane lining the furrow walls is derived from a source inaccessible to surface radioiodination. The furrow membrane adjacent to the outer, pigmented surface consistently showed a very low silver grain density and was underlain by large membranous vesicles, suggesting that new membrane derived from cytoplasmic precursors is inserted primarily in this location, at least during the later phase of cleavage. Radioiodinated membrane proteins and surface-attached carbon particles, which lie in the path of the future furrow, contract toward the animal pole in the initial stages of cleavage while markers in other regions do not. We suggest that the domain of heavily labeled membrane at the leading edge of the definitive furrow contains the labeled elements that are gathered at the animal pole during the initial surface contraction and that they include membrane anchors for the underlying contractile ring of microfilaments.

Studies of intercellular invasion in vitro using rabbit peritoneal neutrophil granulocytes (PMNS). I. Role of contact inhibition of locomotion.

Intercellular invasion is the active migration of cells on one type into the interiors of tissues composed of cells of dissimilar cell types. Contact paralysis of locomotion is the cessation of forward extension of the pseudopods of a cell as a result of its collision with another cell. One hypothesis to account for intercellular invasion proposes that a necessary condition for a cell type to be invasive to a given host tissue is that it lack contact paralysis of locomotion during collision with cells of that host tissue. The hypothesis has been tested using rabbit peritoneal neutrophil granulocytes (PMNs) as the invasive cell type and chick embryo fibroblasts as the host tissue. In organ culture, PMNs rapidly invade aggregates of fibroblasts. The behavior of the pseudopods of PMNs during collision with fibroblasts was analyzed for contact paralysis by a study of time-lapse films of cells in mixed monolayer culture. In monolayer culture, PMNs show little sign of paralysis of the pseudopods upon collision with fibroblasts and thus conform in their behavior to that predicted by the hypothesis.

Transepithelial invasion and intramesenchymal infiltration of the chick embryo chorioallantois by tumor cell lines.

The ability of cultured tumor cell lines to invade across epithelia was studied by placing 10(4) to 10(6) dispersed cells on the chorionic epithelium (CE) of the chorioallantoic membrane of the 10-day chick embryo. Tumor lines included Walker carcinosarcoma, F87 cl 6T2 and B16-BL6 melanomas, and KiSV-NIH, 3B77SC4, and HT 1080 sarcomas. The CE is a bilayer of cells with a superficial periderm overlying a basal layer. Invasion across an intact CE was very weak (limited to the formation of "microtumors" by a small fraction of the inoculated cells in 5 to 50% of the embryos) but was massive (most or all of the inoculated cells invaded in over 99% of the embryos) if the chorioallantoic membrane was traumatized in a fashion which disrupted the periderm but left the basal layer intact. Normal fibroblasts also invaded across the traumatized CE. The histological picture of invasion suggests that cells inoculated on the traumatized CE induced large-scale active retraction of the basal layer, resulting in the formation of large gaps in its continuity. Migration into the subjacent mesoderm occurred through these gaps. The nodules formed by both tumorigenic and normal cells became extensively vascularized within 3 days.

Intercellular invasion and the organizational stability of tissues: a role for fibronectin.

Intracellular invasion is the movement of cells of one type into the fabric of other, contiguous tissues. Invasion is a signature behavior of the malignant tumor and also is found as part of the normal behavior of inflammatory blood cells and tissues engaged in the morphogenetic movements of normal embryogenesis and in a number of instances of normal and pathological tissue remodeling in the adult. Informed by the view that the underlying mechanisms of invasion will be similar for tumor cells and invasive blood and embryonic cells, this review adopts a comparative approach to the analysis of invasion. Invasion results in the development of a diffuse interface between contiguous tissues. Its alternative is the maintenance of stable, planar tissue boundaries. This is the more usual condition for contiguous tissues in the animal. This review will focus on the processes that, on the one hand, stabilize planar contact interfaces between tissues, and, on the other, promote the destabilization of tissue integrity by fostering intercellular invasion. Particular attention is devoted to a role for adhesive interactions mediated by the matrix adhesion molecule, fibronectin. In certain instances, fibronectin in the matrix promotes invasion whereas in others, the presence of fibronectin prevents invasion. The distinction appears to depend on whether the invasive tissue is migrating into an acellular extracellular matrix or whether invasion involves densely cellular tissues. In the first instance, fibronectin promotes invasion, whereas in the second, it stabilizes the interface of the contacting tissues and prevents invasion.

Proteinase inhibitory activity released from the horseshoe crab blood cell during exocytosis.

The blood cell of the horseshoe crab, Limulus, is packed with granules that can be stimulated to release their contents by exocytosis. We have identified a family of proteinase inhibitors in the released materials. Included is a factor similar to the alpha 2-macroglobulin homologue present in the plasma and acid-stable and acid-instable active-site inhibitors. The acid-stable factor is active against both serine (trypsin, chymotrypsin) and metal (thermolysin) proteinases. The trypsin- and chymotrypsin-inhibitory activity has a molecular weight of 6100, as determined by gel-filtration chromatography.

Involvement of alpha 2-macroglobulin and C-reactive protein in a complement-like hemolytic system in the arthropod, Limulus polyphemus.

Homologues of two plasma proteins of vertebrates, alpha 2-macroglobulin and C-reactive protein, participate in a hemolytic system of the ancient arthropod, Limulus polyphemus. C-reactive protein, which can under the appropriate circumstances activate the classical pathway of the mammalian complement system, is an essential element of the hemolytic system of Limulus. The selective removal of C-reactive protein from the plasma with phosphorylethanolamine-agarose inactivated hemolysis. Addition of affinity-purified C-reactive protein to inactive plasma restored activity. Exposure of plasma to phosphorylethanolamine in solution potentiated hemolysis. alpha 2-Macroglobulin is a member of the same protein family as the complement protein C3 and both require an intact thiol ester for activity. Treatment of Limulus plasma with methylamine under conditions that inactivate thiol-ester-containing proteins reduced the hemolytic activity of some plasma preparations. Addition of purified Limulus alpha 2-macroglobulin to the methylamine-treated plasma restored hemolytic activity. However, alpha 2-macroglobulin is not necessary for hemolysis since the hemolytic activity of some pooled plasma preparations was insensitive to methylamine treatment under conditions that inactivated alpha 2-macroglobulin. Purified C-reactive protein was hemolytic in the absence of alpha 2-macroglobulin. These observations suggest that the proteins in Limulus plasma that participate in hemolysis represent the components of an ancient invertebrate defense system with distant evolutionarily affinities to the vertebrate complement system.

Alpha2-macroglobulin does not function as a C3 homologue in the plasma hemolytic system of the American horseshoe crab, Limulus.

A major problem of comparative immunology is the characterization of the internal defense systems that lyse foreign cells, such as bacteria and other microbial pathogens that have gained entry into the body. The plasma cytolytic system of the American horseshoe crab, Limulus polyphemus, is sensitive to treatment with methylamine, which inactivates the abundant plasma defense protein alpha2-macroglobulin. This has been interpreted to mean that alpha2-macroglobulin plays an important role in hemolysis, analogous to the role of complement component C3 of the mammalian complement system (Enghild et al., 1990). Sensitivity to methylamine has been suggested to reflect an evolutionary homology with the plasma cytolytic system of mammals, in which the complement system is inactivated by the reaction of methylamine with complement components C3 and C4. C3, C4 and alpha2-macroglobulin contain an internal thiol ester bond linking cysteinyl and glutamic acid residues and methylamine inactivates all three proteins by reaction with the thiol-esterified glutamic acid. However, we have recently shown that the principal effector of hemolysis in Limulus is the plasma lectin, limulin (Armstrong et al., 1996). In this article we show that native, unreacted alpha2-macroglobulin is not involved directly in hemolysis but instead that methylamine-reacted alpha2-macroglobulin inhibits the hemolytic activity of limulin. Thus the thiol ester proteins alpha2-macroglobulin and C3 operate very differently in the hemolytic systems of Limulus and mammals and are not functionally homologous. Limulus alpha2-macroglobulin functions indirectly in hemolysis: its inactivation yields an inhibitory molecule for limulin-mediated hemolysis.

Localisation of the major reactive lysine residue involved in the self-crosslinking of proteinase-activated Limulus alpha 2-macroglobulin.

When alpha 2-macroglobulin (alpha 2M) from the American horseshoe crab, Limulus polyphemus, reacts with proteinases, its thiol esters, like those of other alpha-macroglobulins, become activated, leading to the formation of covalently crosslinked species that can be detected as high molecular weight bands in reducing SDS-PAGE. While other alpha-macroglobulins extensively form crosslinks to the reacting proteinase, Limulus alpha 2M does not. It rather becomes internally crosslinked. It was found from N-terminal sequence analysis of purified [14C]carboxymethylated peptides from Limulus alpha 2M-trypsin complexes that an isopeptide bond formed in approx. 60% yield from the thiol esterified Gln-1002 specifically to Lys-254 in the opposing monomer of the disulphide bridged dimer is the main cause of the internal crosslinking.

Role of endogenous proteinase inhibitors in the regulation of the blood clotting system of the horseshoe crab, Limulus polyphemus.

Blood clotting in Limulus is dependent on the activity of a proteinase which converts the zymogen, coagulogen, into a form that undergoes polymerization to form the clot. The abilities of a series of recently discovered endogenous proteinase inhibitors to inhibit this enzyme and thereby serve as potential regulators of its activity were explored. The blood plasma of Limulus contains a single inhibitor that is functionally and structurally homologous to vertebrate alpha 2 macroglobulin. During exocytosis, the blood cells (amebocytes) release a series of inhibitors, including small quantities of the alpha 2 macroglobulin homologue; a low molecular weight, acid-and heat-stable inhibitor; and an acid acid-labile activity. Of the three inhibitory activities, only the cell-released, acid-labile inhibitor is capable of inhibiting the clotting enzyme.

Alpha2-macroglobulin: an evolutionarily conserved arm of the innate immune system.

All animals and plants have immune systems that protect them from the diversity of pathogens that would otherwise threaten their survival. The different components of the immune system may inactivate the pathogens themselves or promote the inactivation and clearance of toxic products produced by the pathogens. An important category of virulence factors of bacterial and prokaryotic pathogens are the proteases, which act to facilitate the invasion of the pathogens and to promote their destructive growth in the host organism. The present review concentrates on the comparative biology of an evolutionarily conserved arm of the immune system, the protein, alpha2-macroglobulin. alpha2-Macroglobulin is an abundant protein of the plasma of vertebrates and members of several invertebrate phyla and functions as a broad-spectrum protease-binding protein. Protease-conjugated alpha2-macroglobulin is selectively bound by cells contacting the body fluids and alpha2-macroglobulin and its protease cargo are then internalized and degraded in secondary lysosomes of those cells. In addition to this function as an agent for protease clearance, alpha2-macroglobulin binds a variety of other ligands, including several peptide growth factors and modulates the activity of a lectin-dependent cytolytic pathway in arthropods.

Transforming growth factor-beta 1 localized within the heart of the chick embryo.

Transforming growth factor-beta 1 is a pleiotropic peptide mediator of growth, differentiation, and extracellular matrix synthesis. In the embryonic chick heart prior to the formation of the endocardial cushions, evidence from in vitro experiments suggests that transforming growth factor-beta 1 may be an inducer of the differentiation of atrioventricular endothelial cells into endocardial cushion mesenchyme. Further in vitro evidence suggests that the factor stimulates mesenchymal cell proliferation, and, thus, growth of the cushions. Using an antibody made against a peptide duplicating the aminoterminal 30 amino acid sequence of transforming growth factor-beta 1, we stained sections of stage 11, 18, 23, 26, and 36 chick hearts by an in situ immunofluorescence technique. Transforming growth factor-beta 1 staining localized to the endocardial surface and epicardial surface of the stage 11 heart, but it decreased from these locations in later stages. The cardiac jelly (stage 11), endocardial cushions (stage 18, 23, and 26), and, subsequently, the heart valve leaflets (stage 36) stained intensely for the growth factor.