A Fine Structural Study of Adhesive Cell Junctions in Heterotypic Cell Aggregates (original) (raw)
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The formation of gap and tight junctions between retinal pigment cells in cell cultures
Journal of Neurocytology, 1979
The reformation of the junctional complex of retinal pigment cells was studied after trypsin disaggregation and in vitro reaggregation. Control specimens show a zonula occludens (tight junction) with integrated gap junctions and very large macular gap junctions. Isolation after trypsination results in disaggregation of the large gap junctions and fragmentation of the tight junctions with disaggregation of their integrated gap junctions. After two to four days of incubation the restoration of the zonula occludens is complete. After approximately five days of incubation, large gap junctions are found with a patchy arrangement of particles similar to that seen in vivo.
FUNCTIONAL CHARACTERIZATION OF AN ADHESIVE COMPONENT FROM THE EMBRYONIC CHICK NEURAL RETINA
1979
We have developed a quantitative assay for tissue-specific adhesive components which is based on the agglutination of glutaraldehyde-fixed cells. At least 2 components are required for fixed-cell agglutination: a cell-surface ligand which is obtained from tissue culture-conditioned medium, and a soluble 'agglutinin' which accumulates in conditioned medium from monolayer cultures. Our results suggest that the surface-binding ligand and the agglutinin interact directly, resulting in tissue-specific agglutination of cells. The agglutination reaction exhibits divalent cation, temperature, and pH dependence. Several models of cell adhesion are described; the simplest of these which can account for the data is a multicomponent model in which the 2 adhesive components have structural roles.
Experimental Eye Research, 1997
For most epithelial cells, the adherens junction protein E-cadherin is an epithelial morphogen, inducing the development of an epithelial phenotypein vitroafter cell contact at confluency. Here retinal pigment epithelial cells (RPE), which lack E-cadherin but express a cadherin that is also found in many non-epithelial cells (N-cadherin), were examined for the ability to produce an epithelial phenotypein vitro. Subpopulations of grossly epithelioid or fusiform cells were selected for analysis from RPE cultures derived from adult human donors. After confluency, epithelioid RPE cells were observed to undergo time-dependent changes that were similar to those previously found in epithelial cells expressing E-cadherin: the cadherin gradually developed a zonular distribution of detergent-resistant protein that co-localized with forming circumferential actin bundles; Na/K ATPase accumulated at cell contact sites, then polarized to its tissue-specific domain (the apical membrane for RPE); the cells formed elevated domes on the impermeant culture substrate. In contrast to cells expressing E-cadherin, these events in RPE required weeks rater than days at confluency. Additional proteins were examined in epithelioid RPE cells revealing that cytokeratins reorganized after confluency producing a zonular array, and several other adhesion proteins (α5β1 integrin, ICAM-1, PECAM-1, NCAM) became enriched at cell–cell contact sites, each developing a distinct pattern at a distinct postconfluency interval. In contrast to epithelioid RPE, in fusiform RPE the adhesion molecules did not develop discrete distribution patterns after confluency, although the same complement of adhesion proteins was expressed. In cells expressing E-cadherin, the absence of epithelial properties is often due to underexpression of the cadherin or of the catenins, adherens junction proteins that link the cadherin to actin. Fusiform RPE, however, were not deficient in these proteins, expressing amounts of N-cadherin, α-catenin, β-catenin, plakoglobin, p120, α-actinin and vinculin that were equivalent to epithelioid cells. It appears, therefore, that a subset of epithelial cells that express N-cadherin can produce a highly-developed epithelial phenotypein vitrothrough a slow morphogenetic process. However, the expression alone of adhesion molecules, including those with a morphoregulatory function in other cells, is insufficient to produce an epithelial phenotype in all cells derived from the pigment epithelium.
Junctions between Living Cells
Scientific American, 1978
Where the cells of certain key animal tissues meet they do not simply touch. They are linked by specialized structures, the architecture of which is revealed by electron microscopy by 1. Andrew Staehelin and Barbara E. Hull
AN ASSAY FOR INTERCELLULAR ADHESIVE SPECIFICITY
Journal of Cell Biology, 1971
A modification of an assay for intercellular adhesive specificity is described . The method involves the collection of radioactively labeled cells by aggregates of the same (isotypic aggregates) or different (heterotypic aggregates) types of tissue and determination of the number of cells collected by liquid scintillation counting. The use of 32P to label the tissues permitted a much more rapid estimation of cell collection than was obtained previously. With the use of chick embryo neural retina, liver, forebrain, pectoral muscle, and heart ventricle tissue, it was shown that isotypic was always greater than heterotypic collection . Labeled neural retina cell collection by neural retina aggregates was studied as a function of time, cell suspension density, aggregate diameter, temperature, and aggregate number . Neural retina aggregates were treated with certain enzymes in an attempt to determine whether specific changes on the surface of the aggregates would interfere with labeled neural retina cell collection . Of the various proteases and glycosidases tested, only ß-galactosidase rendered the surface more nonspecific .
Cell adhesion: the molecular basis of tissue architecture and morphogenesis
Cell, 1996
Cell adhesion is crucial for the assembly of individual the plasma membrane, cell adhesion receptors associcells into the three-dimensional tissues of animals. Cells ate with cytoplasmic plaque or peripheral membrane do not simply "stick" together to form tissues, but rather proteins. Cytoplasmic plaque proteins serve to link the are organized into very diverse and highly distinctive adhesion systems to the cytoskeleton, to regulate the patterns. A variety of cell adhesion mechanisms are functions of the adhesion molecules, and to transduce responsible for assembling cells together and, along signals initiated at the cell surface by the adhesion rewith their connections to the internal cytoskeleton, deceptors. These adhesion protein complexes will genertermine the overall architecture of the tissue. Thus, cell ally be considered as functional units in this review. The adhesion systems should be regarded as mechanisms biochemical properties of these classes of proteins and that help translate basic genetic information into the the diversity of roles of the various families of adhesion complex three-dimensional patterns of cells in tissues. receptors will not be described here in detail; for such The goal of this review is to illustrate the roles of information, the reader is referred to several excellent adhesion mechanisms in the generation of tissue archireviews (Bernfield et al., 1993; Gumbiner, 1993; Hynes, tecture. To understand tissue morphogenesis, it is es-1992; Hynes and Lander, 1992; Mosher et al., 1992; sential to know the endpoint of the process, and there-Springer, 1994; Turner and Burridge, 1991). fore we will first consider the molecular basis of cell adhesion in fully formed tissues, that is, what maintains the structure at steady state once the tissue has devel-Stable Connections between Cells and the oped. In the second part, we will consider how these Maintenance of Tissue Structure cell arrangements arise during tissue development,
Cell Structure and Function, 1977
The adhesive property was studied of Chang conjunctiva cells of human epithelial line. Cells dissociated with only EDTA aggregated in Ca2+-free and Mg2+-free conditions, whereas cells dissociated by trypsin did not aggregate under the same conditions. Cells dissociated with trypsin solution containing Ca2+ aggregated in the presence of Ca2+ and/or Mg2+. Cells dissociated by trypsin without Ca2+ showed less aggregation in the presence of divalent cations. In cases of cell adhesion to non-cellular substrates, the particular method of cell dissociation did not produce different results. Only a small fraction of inoculated cells attached onto plastic coated with bovine serum albumin or gelation. Many inoculated cells attached rapidly and spread onto plastic coated with collagen, when Mg2+ was present in the medium. These results were discussed with the hypothesis that Ca2+ and Mg2+ act differently in cell adhesion.
Tight Junction Formation in Cultured Epithelial Cells (MDCK
Synthesis and assembly of tight .junctions are studied in monolayers of MDCK cells plated at a density sufficient for confluence, allowed to attach for 1 hr, and transferred to fresh media without cells containing or not Ca ;+. 20 hr later, while monolayers with Ca ,-+ have fully developed junctions that confer an electrical resistance across of 346 • 51 D, cm 2, those without Ca 2 § have a negligible resistance. If at this time Ca :~ is added, junctions assemble and seal with a fast kinetics, that can be followed through the development of electrical resistance, penetration of ruthenium red, and electron microscopy. Drugs that impair synthesis, maturation and transport of proteins (cyclohex-imide, tunicamycin, monensin) indicate that protein components are synthesized early upon plating, do not seem to require N-glycosylation, and are stored in the Oolgi compartment. Upon addition of Ca 2+ they are transferred to the membrane with the participation of microfilaments but not of microtubules. These components seem to insert directly in the position they occupy in the strands, and the cell circles its perimeter with one strand as early as 15 rain, even if in some segments it only consists of a row of particles. New strands develop in association with previous ones, and the pattern completes in 4 to 6 hr. Ca 2. is required for the maintenance of the assembly and also for the sealing with neighboring cells. These processes cannot occur below 25~ Serum is not required. Polarized distribution of intramembrane particles (IMP) in apical and basolateral regions follows the same time course as junction formation, in spite of the fence constituted by those strands that are already assembled. This suggests that IMP do not redistribute by lateral displacements in the plane of the membrane, but by removal and insertion in the apical and basolateral domains. Key Words epithelial monolayers 9 MDCK cells 9 tight junctions 9 calcium 9 biosynthesis of junctions-junctional assembly 9 apical/basolateral polarization
Neuroscience Letters, 1984
We report here that, in comparison to aggregates from retinal cells alone, addition of pigmented epithelial cells to retinal cells in rotary culture results in a pronounced increase of spatial order. A particularly high level of organization is found in about 15-20°70 of the aggregates. In these 'retinoids' the main layers characteristic of developing in vivo retinae can be distinguished in correct sequential arrangement on the basis of morphological criteria and by using acetylcholinesterase histochemistry [5, 6, 15], peanut agglutinin-lectin binding [11] and Lucifer Yellow staining [7-9]. Dissociated single cells from embryonic chick retinae reaggregate quickly and form relatively organized histotypic structures in a rotary culture system. Reaggregation of dissociated single cells starts with a random alignment of retinal cells, followed by the formation of primary rosettes and by the incorporation of additional cells into the aggregate [1, 4, 10-14]. We studied the internal order of these aggregates upon prolonged incubation of 2-3 weeks. Fujisawa [3] described specific layers which are formed upon aggregation of retinal cells on the chorioallantoic membrane. We were able to demonstrate a similar high degree of laminar organization in a pure in vitro system of aggregates derived from mixed cultures of retinal and pigmentepithelial (PE) cells. Methodologically, the neuroretinae and pigmented epithelia from E6 chicken embryos (White Leghorn) were dissected and washed with Hank's solution. Pigmented epithelia were first incubated in 1 mg/ml collagenase and 300 U/ml hyaluronidase (both from Boehringer) in Eagle's Minimal Essential Medium (Ea-MEM) for 10 min at 37°C, washed once in Hank's solution and treated with 1 mg/ml trypsin (Difco) for 20 min at 37°C. Isolated retinae were digested only by trypsin (10 min, 20°C and 10 min, 37°C). Then the tissues were rinsed in Ea-MEM and mildly dissociated into single cells in the presence of 0.05 mg/ml DNAase. After a 3-fold wash in Ea-MEM the cells were resuspended in aggregation medium (10070 fetal calf serum, 2°70 chicken serum, 1 070 glutamine, 0.1 070 penicillin/streptomycin and 0.02 mg/ml gentamycin in Ea-MEM). A 2 ml suspension of cells consisting either of 2-5 x 106 retina
Neuroscience Letters, 1986
Key words." chick retina aggregate-lamination pigment epithelium Wc report here a striking age dependency for tissue reconstruction capacity in rotatmg cultures of chick retinal cells, in particular in mixtures of retinal and pigment epithelial cells. The comparison of sections after acetylcholinesterase histochemistry delivered the following results. (1) In aggregates from retinal cells, sorting out is clearly observable, but only a minor age-dependent effect is detectable, e.g. the laminar order of aggregates derived from 6-day-old cells is increased compared to aggregates from other stages. (2) In aggregates from mixtures of retinal and pigment epithelial cells, the degree of laminar order is highest with cells from 5-day-old embryos and decreases significantly at older ages. The highest number of aggregates with the reconstruction of all main retinal layers was obtained by the reaggregation of retinal cells of embryonic day 5 (25%). (3) To achieve laminar order in aggregates, at all ages, the addition of pigment epithelial cells seems to be a prerequisite.