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D Thom

Publications and source records attributed to D Thom.

30 records · Page 2Linked to original sources
PubMed1979

Mechanisms of cellular adhesion. IV. Role of serum glycoproteins in fibroblast spreading on glass.

We have investigated the exogenous factors required for the transition from the round shape of suspended fibroblasts to the characteristic spread shape on serum-coated glass. Following the evidence of others that the transition is facilitated by adsorbed component(s) related to CIG/LETS (cold-insoluble globulin/large external transformation-sensitive) proteins, we have isolated 2 such preparations from chick serum. Their influence has been investigated on fibroblast adhesion, spreading and growth and they have been characterized by gel electrophoresis, immunological cross-reactivity, amino acid and carbohydrate residue analysis, sedimentation velocity behaviour and circular dichroism spectroscopy. The preparations have molecular weights of 225/215,000 and 140,000 Daltons respectively and are closely similar in composition and secondary structure. The 225/215 000 Dalton doublet is probably a product of limited proteolysis which almost certainly occurred in the avian circulation. For cells seeded on glass precoated in different ways and in different supplemented media we could detect no change in the extent of attachment but there were profound influences on cell shape following this initial adhesion. We confirm that prior adsorption of either CIG-related preparation to glass does indeed promote fibroblast spreading in the absence of other serum components and that CIG is the sole serum component with this type of activity. We now add 2 important qualifications: (i) the presence of substrate-adsorbed serum CIG is not essential for spreading when other serum components are present in the medium; and (ii) the adhesive organization shown by interference reflexion microscopy is incompletely formed unless the additional serum components are present in the medium. We therefore conclude that 16C fibroblasts have the ability when given the stimulus of soluble serum components other than CIG, but not otherwise, to synthesize all the components necessary for the highly organized contacts with glass, including endogenous CIG/LETS proteins.

Amino Acids↗
PubMed1979

Co-operativity of lectin binding to fibroblasts and its relation to cellular actomyosin.

We have investigated the binding of 125I-concanavalin A (125I-Con A) and 125I-succinyl concanavalin A (125I-s-Con A) to rat fibroblasts (16C line) as a function of the concentration of added lectin, and the alterations to this binding behaviour caused by drugs which modify the cytoskeleton. The changes in cell behaviour which occur at different levels of binding have also been studied. As shown previously for some other systems, the binding of Con A is complex and partly co-operative. Three phases can be distinguished in our system: (i) pre-nucleation binding, (ii) binding which shows a small positive slope in a Scatchard plot and a Hill coefficient greater than unity, and which therefore is incipiently co-operative, and (iii) post-co-operative binding. The co-operative phase of binding is paralleled by progressive inhibition of EGTA-mediated cell detachment from substrata, with inhibition being complete when this phase of binding is complete. Likewise, the phagocytosis of latex spheres is progressively inhibited up to a threshold which coincides with the completion of co-operative binding. Thirdly, cells pretreated with Con A round up with colchicine (10(-5) M) if co-operative finding is complete, but adopt broad epithelial shapes if it is not. s-Con A does not show cooperative binding, and correspondingly does not inhibit EGTA-mediated cell detachment, or show a distinct threshold in the inhibition of phagocytosis, or promote the 2 types of shape change with colchicine. The pattern of Con A binding is drastically altered by pretreatment of cells with cytochalasin B or azide. The Scatchard and Hill plots show that the co-operative phase remains and is complete at about the same level of binding, but that it is more readily nucleated and takes place against a changed number and/or distribution of receptors. Pretreatment of cells with colchicine causes changes in the pattern of binding which are different from those observed with cytochalasin B or azide and are more difficult to interpret. We conclude that a reciprocal relationship exists between the cellular actomyosin and the state of cell surface receptors. Perturbation of actomyosin by cytochalasin B or azide can enhance the freedom of some receptors to participate in a co-operative rearrangement which facilitates the binding of further molecules of lectin. Vice versa, the co-operative event has a feedback influence on the cellular actomyosin to cause alterations of cellular response.

Actomyosin↗
PubMed1977

Rapid isolation of plasma membranes in high yield from cultured fibroblasts.

1. A method was developed which allows the rapid preparation of pure plasma membranes in high yield from cultured fibroblasts. 2. Cells are lysed in hypo-osmotic borate/EDTA and, after differential centrifugation, the membranes collected by centrifugation on a sucrose barrier. 3. Electron microscopy of the isolated material shows large membrane vesicles essentially free from contaminating organelles. 4. There is no detectable activity of the endoplasmic-reticulum enzyme marker, NADH2--lipoamide oxidoreductase (EC 1.6.4.3), and that of succinate dehydrogenase (EC 1.3.99.1), a marker for mitochondria, is substantially decreased. Chemical compositions are in good agreement with previous observations. 5. This study confirms the usefulness of applied isotopic markers for isolating plasma membranes.

Borohydrides↗
PubMed1977

Control of grip and stick in cell adhesion through lateral relationships of membrane glycoproteins.

Although cell adhesion to substrate must ultimately depend on 'sticking' by physical forces at the outer surface, we suggest that control is exercised through the 'grip' of the cytoskeleton. Our experiments indicate that grip can be relaxed by agents which release surface glycoproteins from some form of side-to-side organisation, and can be consolidated by suitable cross-linking to augment this organisation.

Animals↗