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D R Garrod

Publications and source records attributed to D R Garrod.

At least 91 records · Page 5Linked to original sources
PubMed1986

Calcium-induced desmosome formation in cultured kidney epithelial cells.

Previous work has shown that cultured keratinocytes do not form desmosomes at low [Ca2+] (less than 0.1 mM) but may be induced to do so by raising [Ca2+] to physiological levels (1.8-2 mM). Here, fluorescent antibody staining with specific anti-desmosomal antibodies and electron microscopy have been used to determine whether Ca2+-induced desmosome formation also occurs in simple epithelial cells. Both Madin-Darby canine and bovine kidney cells (MDCK and MDBK) exhibit Ca2+-induced desmosome formation, but there are significant differences between them. MDCK cells resemble keratinocytes in showing rapid desmosome formation characterized by the simultaneous appearance of four desmosomal antigens at the cell periphery within 15-20 min of raising the [Ca2+]. In contrast MDBK cells take between 7 and 8 h to form desmosomes after Ca2+ switching, and this is characterized by slow appearance of two desmosomal antigens, the 175-164(X 10(3)) Mr glycoprotein and desmoplakin, at the cell periphery. Differences in the pattern of staining for desmosomal antigens between the two cell types in low and high [Ca2+] are described and discussed in relation to desmosome formation and internalization. Triton X-100 extractability of desmosomal antigen staining is also considered. While most is non-extractable, staining for the glycoproteins known as desmocollins is completely extractable from MDCK cells in low [Ca2+], but that which reaches the cell periphery after Ca2+ switching becomes non-extractable. Although neither cell type forms desmosomes in low [Ca2+], both possess zonulae adhaerentes, suggesting a difference in Ca2+ requirement for formation of these two junctions.

Animals↗
PubMed1985

Mutual desmosome formation between all binary combinations of human, bovine, canine, avian and amphibian cells: desmosome formation is not tissue- or species-specific.

Our previous work has suggested that the molecular components of desmosomes are highly conserved between different tissues and different vertebrate species. In order to determine whether the adhesion recognition mechanism of desmosomes is also conserved we have examined the specificity of desmosome formation between different epithelial cell types by co-culturing binary combinations of cells from different species and from epidermal and non-epidermal origin. The following cell types were used: human (HeLa, cervical carcinoma), bovine (Madin Darby bovine kidney, MDBK), canine (Madin Darby canine kidney, MDCK), avian (chick embryonic corneal epithelium) and amphibian (Rana pipiens, adult corneal epithelium). Different cells in co-culture were identified on the basis of at least one of the following criteria: (1) morphology by phase-contrast microscopy; (2) presence or absence of staining of cytokeratin with monoclonal antibody LE61; (3) morphology at the electron microscope level. Mutual desmosome formation between different cell types was assessed using fluorescent antibody staining with anti-desmoplakin antibodies and confirmed using electron microscopy. We have found that mutual desmosome formation occurred between all binary combinations of human, bovine, canine, avian and amphibian cells. Thus there is complete non-selectivity of desmosome formation between five different epithelial cell types from three vertebrate classes. Our results suggest that desmosome formation is not tissue- or species-specific and that the mechanism for intercellular binding involved in desmosomal adhesion is highly conserved.

Animals↗
PubMed1984

Calcium-induced reorganization of desmosomal components in cultured human keratinocytes.

We used antibodies raised against individual desmosomal components to study calcium-induced desmosome formation in human keratinocytes. When keratinocytes are forced to grow as a monolayer by reducing the level of calcium ions in the culture medium, there is little contact between adjacent cells. Raising the level of calcium ions rapidly induces desmosome formation, and stratification occurs within 24 h. We found that before addition of calcium the 115,000- and 100,000-mol-wt core glycoproteins were distributed over the entire cell surface, whereas the plaque proteins (205,000 and 230,000 mol wt), the 82,000- and 86,000-mol-wt proteins, and the 150,000-mol-wt glycoprotein were located throughout the cytoplasm. 15 min after increasing the calcium ion concentration, all of these molecules appeared at the cell margins. The intensity of peripheral staining increased over the next 2 h and during this time the distribution of keratin filaments changed from predominantly perinuclear to extend throughout the cytoplasm. Keratinocytes could be dissociated with EDTA for up to 2 h after exposure to calcium. After 3 h of exposure to calcium the cells were no longer susceptible to EDTA dissociation and staining for desmosomal plaque antigens persisted in regions of intercellular contact. Desmosomal staining in stratified cultures became greatly reduced within 24 h of lowering the calcium ion concentration again. We have preliminary evidence that stratification occurs by breakdown of desmosomes at lateral surfaces and reformation at surfaces of contact between basal and suprabasal cells, rather than by rearrangement of existing desmosomes. Involucrin-positive cells in the monolayer appeared to contain more 205,000- and 230,000-mol-wt proteins free in the cytoplasm than involucrin-negative cells.

Antigens↗
PubMed1984

Organization of extracellular matrix by chick embryonic corneal epithelial cells in culture and the role of fibronectin in adhesion.

Corneal epithelial cells from 15-day chick embryos produce an extensive extracellular matrix when cultured for up to 7 days on plastic, glass, and gelatin-coated substrata. Immunofluorescence studies revealed the presence of a fibrillar fibronectin matrix that became more extensive with time in culture. When cells were cultured in fibronectin-depleted medium little or no fibronectin was evident in the first 24 h, although the cells attached and spread normally when compared with controls. Considerably more fibronectin was associated with the cells after 48 h. Incubation of cells with [35S]methionine, followed by immunoprecipitation of cell extracts by specific anti-fibronectin antibody, confirmed that the cells synthesize fibronectin. Cells cultured in fibronectin-coated substrata were able to reorganize the fibronectin into fibrillar form as well as to endocytose fibronectin. It is suggested that cells 'comb' fibronectin from the substratum and organize it into fibrils. Interference reflection microscopy revealed no simple correlation between the distribution of fibronectin and focal contacts. However, some focal contacts seemed to lack fibronectin. In the presence of anti-fibronectin immunoglobulin G cells initially attached but failed to spread, and subsequently detached from glass and fibronectin-coated substrata. However, they attached and spread on collagen substrata. The results suggest that fibronectin is unnecessary for initial cell attachment but may be important in maintaining cell adhesion and spreading on certain substrata. However, it is not necessary for attachment and spreading on collagen. Cells on collagen may attach and spread directly on the substratum via specific collagen receptors or by using some other type of attachment factor(s). Electron microscopy revealed a densely staining fibrillar matrix between the basal cell layer and substratum as well as in the intercellular spaces between cells. Components of this matrix and the cell surface were strongly stained by ruthenium red, which indicates the presence of acidic groups such as glycosaminoglycans.

Animals↗
PubMed1984

Role of glycosaminoglycans and collagen in the development of a fibronectin-rich extracellular matrix in cultured embryonic corneal epithelial cells.

Corneal epithelial cells from 15-day chick embryos produce a fibronectin-rich extracellular matrix when cultured on glass, plastic and fibronectin-coated substrata. Cell culture in the presence of Streptomyces hyaluronidase or chondroitinase ABC resulted in considerable reduction of the matrix; collagenase had a lesser effect but nevertheless also reduced the matrix. In all enzyme treatments the cells attached and spread to form characteristic epithelial cell islands, but the marginal cells of these islands showed a marked reduction in the number of lamellipodia and focal contacts. Also, the immunofluorescent staining pattern for fibronectin was considerably reduced. Control cells cultured on a fibronectin-coated surface were able to reorganize the fibronectin into fibrils, whereas cells cultured in enzymes showed little or no ability to do so. The cellular reorganization of fibronectin could also be inhibited by the addition of L-azetidine-2-carboxylic acid (LACA), an inhibitor of collagen secretion. Cells plated out in the presence of LACA spread much better on collagen substrata than on plastic, glass or fibronectin. However, in all cases very little fibronectin matrix was detectable in the epithelial islands. The results suggest that components of the extracellular matrix (ECM) such as collagen, hyaluronic acid and chondroitin sulphates are not essential for the initial attachment and spreading of corneal epithelial cells in culture, but are important in the development of the ECM, and in maintaining a flattened morphology and spreading behaviour. It is suggested that fibronectin plays an important role in these interactions and that the ability of cells to organize fibronectin into fibrils is dependent on the presence of other ECM components such as glycosaminoglycans and collagen.

Animals↗
PubMed1984

Chick embryonic pigmented retina is one of the group of epithelioid tissues that lack cytokeratins and desmosomes and have intermediate filaments composed of vimentin.

Using sodium dodecyl sulphate/polyacrylamide gels to analyse detergent-insoluble residues, and indirect immunofluorescence, we have found that the major protein of intermediate filaments in cultures and freshly explanted fragments of chick embryonic retinal pigment epithelium (RPE) is vimentin. Moreover, these cells also fail to stain with antibodies against cytokeratins and most components of true desmosomes (maculae adhaerentes). Staining with anti-vinculin antibody suggests that the principal intercellular junction is the zonula adherens. Thus although RPE is an epithelium according to all other criteria, it belongs to a group of tissues (including vascular endothelium, iris and lens-forming epithelium) that have intermediate filaments composed of vimentin and possess neither cytokeratins nor desmosomes. That a tissue can be fully epithelial by other criteria, whilst lacking these components, is in agreement with other work, which has shown a lack of effect of micro-injection of antibodies to cytokeratin, and of the suppression of desmosome formation, on epithelial organization in culture. Although our observations were made solely on chick embryonic tissue, we suggest that published ultrastructural studies are consistent with the possibility that RPE of other species, including human, may lack true desmosomes.

Animals↗
PubMed1984

Anchoring filaments of the amphibian epidermal-dermal junction traverse the basal lamina entirely from the plasma membrane of hemidesmosomes to the dermis.

An electron microscopical study of the epidermal-dermal junction in the axolotl and adult Rana pipiens has been carried out. This shows that filaments of about 12nm in diameter, known as anchoring filaments, pass from the hemidesmosomes at the base of the epidermal cells across the basal lamina to the dermis. There they may unite to form broader fibres, known as anchoring fibrils, or may simply form bundles. In the axolotl, particularly, the anchoring fibrils or bundles of anchoring filaments, enmesh with the collagen fibres of the dermis. Removal of epidermal cells with EDTA results in separation along a plane in the lamina rara of the basal lamina, i.e. between the plasma membrane of the cells and the lamina densa. The anchoring filaments remain inserted into the lamina densa. Hemidesmosomal plaques are no longer visible in regions of the plasma membrane that have been separated from the basal lamina by EDTA, and no evidence was found that plaques are engulfed by the cells. It is proposed that the hemidesmosome-anchoring filament system provides a structural link between the collagenous filament system of the dermis and the intracellular cytokeratin filament system of the epidermis, which, in turn, is linked between cells by desmosomes.

Ambystoma↗
PubMed1983

Antibodies to epithelial desmosomes show wide tissue and species cross-reactivity.

Many workers regard cell adhesion as a highly specific phenomenon, believing that different molecular mechanisms are involved in the adhesion of cells of different tissues and different species. We believe that the evidence from cell behaviour is against this view and that cells share common adhesion mechanisms (for reviews see refs 1, 2); however, molecular evidence is lacking. As an approach to providing such evidence we have begun to study desmosomes, the cell-surface organelles responsible for strong intercellular adhesion in epithelia. We have raised antisera against each of five high-molecular weight (MW) desmosomal components. Having determined the specificity of our antisera by immunoblotting, we show here that each gives a staining pattern corresponding to the distribution of desmosomes in a range of tissues from different vertebrate species, demonstrating that desmosomal components are widely shared and highly conserved.

Animals↗
PubMed1983

Identification of the cohesion molecule, contact sites B, of Dictyostelium discoideum.

Polyspecific antibodies were raised against vegetative cells of Dictyostelium discoideum, strain Ax2. Monovalent (Fab') fragments of antibodies CMC 1, 5, 7 and 12 blocked completely the cohesion of vegetative cells. Antibody CMC 1 was studied in detail. The Fab' of this blocked the cohesion of aggregation-competent cells by 40%. It also caused some loss of cell contact in aggregation streams. In so doing the contacts that remained were mostly at the ends of the cells. Immunofluorescence showed that CMC 1 Fab' bound to both the cytoplasm and the surface of fixed cells. It also bound to the surface of live cells. A control (N Fab') also bound to the cell surface but did not block vegetative cell cohesion. An extract of vegetative cells was obtained using the detergent Triton X-100. D. discoideum proteins were immunoprecipitated from this extract using protein A-Sepharose and CMC 1 immunoglobulin G (IgG). These immobilized proteins absorbed the cohesion-blocking activity of CMC 1 Fab'. About 30 proteins were obtained when the Triton-soluble fraction was immunoprecipitated with IgG of CMC 1, 5, 7 and 12. Five of these were found to be cell surface proteins by the technique of lactoperoxidase-catalysed radio-iodination. These proteins had molecular weights of 178 000, 166 000, 126 000 and 64 000. CMC 12 IgG immunoprecipitated an additional cell surface protein of 46 000 molecular weight. Slices of polyacrylamide gel containing each of the five proteins identified as possible contact sites were fixed, washed and incubated with CMC 1 Fab'. Gel that contained protein of 178 000, 166 000 and 64 000 molecular weight had no effect on the activity of CMC 1 Fab'. However, Fab' that had been incubated with gel containing protein of 126 000 molecular weight no longer blocked cell cohesion.

Dictyostelium↗
PubMed1982

Fibronectin, intercellular junctions and the sorting-out of chick embryonic tissue cells in monolayer.

A hierarchy of relative cohesiveness in monolayer of four different embryonic chick tissues was determined in a previous study. The hierarchy is: corneal epithelium congruent to liver parenchyma greater than pigmented epithelium greater than limb bud mesenchyme. The purpose of this paper is to describe the correlation between these adhesive relationships and, firstly, the amount of the adhesive glycoprotein, fibronectin, associated with the cells and, secondly, the morphology of their intercellular contacts. Fluorescent antibody staining of the cells with anti-fibronectin antibody showed that limb bud mesenchyme cells, the most weakly cohesive, had much more fibronectin than the other cell types. Thus there was a negative correlation between the amount of fibronectin and cellular cohesiveness. Analysis of intercellular contacts by electron microscopy showed that the most strongly cohesive cell types, corneal epithelium and liver parenchyma, were also those that possessed desmosomes.

Animals↗
PubMed1979

A ligand-receptor model for the cohesive behaviour of Dictyostelium discoideum axenic cells.

Axenically grown cells of D. discoideum Ax-2 harvested in the log phase of growth, cohere rapidly when shaken in phosphate buffer. After 3.5 days in the stationary phase of growth, cells become completely non-cohesive. Although they do not stick to each other, stationary phase cells do stick to both log phase cells and aggregation-competent cells. The cohesion of stationary phase cells with these other 2 cell types is inhibited by both EDTA and the low-molecular-weight factor which we have previously demonstrated in stationary-phase growth medium. There is a decline in the sensitivity of slime mould cell cohesion to the low-molecular-weight inhibitory factor as the cells become aggregation-competent. This effect parallels the developmentally-regulated decline in sensitivity to EDTA. The low-molecular-weight inhibitor is not a chelating agent, however. The effect of the inhibitor seems to be specifically against contact sites-B mediated cohesion. We suggest that the simplest cohesive mechanism which can explain our results, is that the EDTA-sensitive cohesion of log phase cells could be dependent on a ligand-receptor system.

Adhesiveness↗
PubMed1979

The sorting out of embryonic cells in monolayer, the differential adhesion hypothesis and the non-specificity of cell adhesion.

It has been reported previously that sorting out of chick embryonic liver parenchyma and limb bud mesenchymal cells would take place in monolayer culture. The distribution of cell types obtained (liver formed the internal, discontinuous phase) was interpreted in terms of the differential adhesion hypothesis. It was suggested that, in monolayer, liver cells were more cohesive than limb bud cells. In this paper we set out to extend the previous observations with 2 particular questions in mind: (i) Is sorting out in monolayer a general phenomenon occurring between a wider range of cell types? (ii) Can evidence be provided for or against the interpretation of results in terms of the differential adhesion hypothesis? Sorting-out experiments were conducted on circular hydrophilic islands, on an otherwise hydrophobic substratum. Under these conditions, sorting-out in monolayer was obtained with binary combinations of 4 chick embryonic tissue types: liver parenchyma, limb bud mesenchyme, pigmented epithelium of the eye and corneal epithelium. With every combination but one, the cells of one type surrounded the cells of the other type, generating what we have called a 'circle-within-a-circle' configuration. With the remaining combination, liver parenchyma and corneal epithelium, only localized sorting was obtained. The 'circle-within-a-circle' configuration is consistent with an interpretation in terms of the differential adhesion hypothesis, according to which the distribution of cells is determined by the relative strengths of cohesions between their lateral surfaces. In direct support of this is the finding from plating the different cell types at sub-confluent density on hydrophilic substrata that limb bud is the cell tye having the weakest lateral cohesion in monolayer. Limb bud surrounded the other 3 tissues on hydrophilic island. A hierachy of lateral cohesiveness between the 4 cell types has been constructed. It is unlikely that the results can be explained in terms of specific cohesion. When plated together at subconfluent density, the 3 epithelial cell types aggregate together to form mixed monolayered islands, suggesting that they share common adhesive mechanisms.

Animals↗
PubMed1979

Effect of isolated plasma membranes on cell cohesion in the cellular slime mould.

The effects of isolated plasma membranes on cohesion of Dictyostelium discoideum Ax-2 cells have been studied. Membranes isolated from cells in the log phase of growth gave complete inhibition of log-phase cell cohesion. This effect was specific for the cohesion log-phase cells mediated by contact sites B, since the cohesion of aggregation-competent cells which have acquired contact sites A was only partially inhibited by log-phase plasma membranes. Membranes isolated from stationary phase cells gave partial inhibition of log-phase cell cohesion, while membranes from aggregation-competent cells gave complete inhibition of log-phase cell cohesion but at much higher concentration than log membranes. Treatment of log-phase cells with cycloheximide for 8 h rendered them completely non-cohesive. Membranes from cycloheximide-treated cells had no effect on log-phase cell cohesion. Log-phase membranes gave complete inhibition of cohesion of 4 slime mould species. The results are discussed in terms of our ligand-receptor model of log-phase cell cohesion.

Cell Cycle↗
PubMed1978

Mutual cohesion and cell sorting-out among four species of cellular slime moulds.

Interspecific cell cohesion among 4 species of cellular slime moulds, Dictyostelium discoideum, D. mucoroides, D. purpureum and Polysphondylium violaceum has been studied. Binary mixtures of aggregation-stage cells of the 4 species were shaken in suspension, one species of each pair being labelled with [3H]thymidine. Cell aggregates were sampled at intervals over 24 h and their composition examined by autoradiography. The following results were obtained: (i) Cells of each species were capable of cohesion with those of the other 3 species. (ii) In general cells of both species in any mixture were present in aggregates after 1 h, but were not localized according to species. (iii) Within 8-h aggregates cells of different species were regionally localized, i.e. sorting-out appeared to have taken place. (iv) 24-h aggregates were more varied: in mixtures of Dictyostelium species, the different species were localized within the aggregates; in mixtures of Dictyostelium species with Polsphondylium, there was a tendency for cells of the different species to become segregated into completely separate aggregates. The significance of these results in relation to both previous descriptive work and recent biochemical studies on the mechanism of slime mould cell cohesion is discussed.

Adhesiveness↗
PubMed1977

An inhibitor of cell cohesion from axenically grown cells of the slime mould, Dictyostelium discoideum.

Medium from a stationary phase culture of axenically grown D. discoideum cells contains an inhibitor of cohesion of log phase cells. The inhibitor is a heat-stable, low molecular weight substance. Its biological effects include inhibition of cohesion of aggregation-competent cells, of cells of other slime mould species, the blocking of development on Millipore filters and a reduction in adhesiveness of slime mould cells to glass. Present evidence suggests that the inhibitor may bind to the cell surface.

Adhesiveness↗