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D A Rees

Publications and source records attributed to D A Rees.

At least 55 records · Page 3Linked to original sources
PubMed1985

Coupling of cytoskeleton functions for fibroblast locomotion.

Using a chick cell phenotype specialised for locomotion with morphometric measurements made possible by modern instrumentation technology, we have reinvestigated motile functions in fibroblast locomotion. Quantitative analysis of rapid fluctuations in cell form and organelle distribution during locomotion showed many significant correlations between different parts of the cell despite much irregularity in individual displacements over the time scale of the order of one second. These broke down when external perturbations caused changes in shape or direction. Partial energy deprivation caused the cells to lose control of shape and organelle distribution even though forward protrusion continued unaffected. Cytoplasmic displacements shown by marker mitochondria correlated with adjacent fluctuations at the leading edge, and drug treatments which increased the amplitude of mitochondrial movements caused visible protrusions in projected positions at the leading edge. We conclude that fibroblast locomotion may be driven coordinately by a common set of motility mechanisms and that this coordination may be lost as a result of physical or pharmacological disturbance. Taking our evidence with results from other Laboratories, we propose the following cytoskeleton functions. (i) Protrusive activity, probably based on solation--gelation cycles of the actin based cytoskeleton and membrane recycling which provides cellular and membrane components for streaming through the cell body to the leading edge; this is Ca++ sensitive but relatively energy insensitive. (ii) Constraining activity on the cell membrane and on certain organelles to maintain shape and so facilitate directionality and the drawing along of the trailing body; this is Ca++ insensitive but relatively energy sensitive. (iii) Channeling function of microtubules to direct the flow towards multiple foci on the leading edge, and so determine cell polarity. Such a mechanism of locomotion for fibroblasts has many features consistent with evidence for other cell types, especially amoebae and leukocytes.

Animals↗
PubMed1984

Mechanism and dynamics of conformational ordering in xanthan polysaccharide.

The thermally induced order-disorder transition of xanthan (extracellular bacterial polysaccharide from Xanthomonas campestris) has been investigated by optical rotation, differential scanning calorimetry, stopped-flow reaction kinetics and low-angle laser light scattering, and the results have been analysed in terms of Zimm -Bragg helix-coil transition theory. The reciprocal of the transition midpoint temperature (Tm) varies linearly with the logarithm of cation (K+) the salt dependence of Tm, is in agreement with Manning polyelectrolyte theory the ordered structure. The associated increase in cation binding, calculated from the salt dependence of tm, is in agreement with the Manning polyelectrolyte theory for one of the candidate structures from X-ray diffraction, a 5(1) single helix stabilized by packing of side-chains along the polymer backbone, but not for the alternative double-helix structure that has also been proposed. At each salt concentration, the two fundamental parameters of the Zimm -Bragg theory, s and sigma, were calculated. The equilibrium constant for growth of the ordered structure (s) is derived directly from calorimetric measurement of transition enthalpy (delta Hcal ), and sigma, which quantifies the relative instability of the helix nucleus, is derived from the ratio of delta Hcal to the apparent transition enthalpy (delta Happ ) obtained by van't Hoff analysis of the optical rotation data. The temperature course of conformational ordering calculated theoretically is in good quantitative agreement with experimental results from both optical rotation and scanning calorimetry. The calculated average length of stable, ordered chain-sequences increases with decreasing temperature, but equals or exceeds the total chain length from light scattering only at temperatures more than approximately equal to 70 K below Tm, suggesting that ordered and disordered regions may co-exist within the same xanthan molecule. Consistent with this interpretation, the observed rate of conformational ordering increases sharply under conditions where the starting solution for dynamic measurements is partially ordered, suggesting that ordered sequences within each chain may act as helix nuclei for adjacent disordered regions, so that helix growth, rather than the slower nucleation process, becomes rate limiting.

Calorimetry, Differential Scanning↗
PubMed1984

Requirements for cell spreading on polyHEMA coated culture substrates.

We have investigated the topography of polyHEMA coated culture substrates by scanning electron microscopy, and quantitatively assessed their effect upon the spreading activity of mammalian cells. Results indicate a clear correlation between cell spreading activity and polymer film discontinuity. Preparation of polyHEMA films on modified tissue culture substrates has allowed direct investigation of the role of the underlying substrate in regulating cell spreading, and confirms that apparent modulation of cell spreading by polyHEMA reflects increasing expression of the coated surface. We have further employed a spinning technique by which films of precise thickness, down to 0.01 micron, may be produced on coverslips. All polyHEMA coatings prepared in this way are smooth and complete. They do not allow cell attachment at any thickness. We conclude that polyHEMA is non-adhesive for mammalian cells.

Animals↗
PubMed1984

Relationship of heparan sulfate proteoglycans to the cytoskeleton and extracellular matrix of cultured fibroblasts.

The distribution of heparan sulfate proteoglycans (HSPG) on cultured fibroblasts was monitored using an antiserum raised against cell surface HSPG from rat liver. After seeding, HSPG was detected by immunofluorescence first on cell surfaces and later in fibrillar deposits of an extracellular matrix. Cell surface HSPG aligned with microfilament bundles of rat embryo fibroblasts seen by phase-contrast microscopy but was diffuse on transformed rat dermal fibroblasts (16C cells) which lack obvious stress fibers. Focal adhesions isolated from either cell type and monitored by interference reflection microscopy showed a concentration of HSPG labeling with respect to the rest of the membrane. Increased labeling in these areas was also seen for fibronectin (FN) by using an antiserum that detects both plasma and cell-derived FN. Double immunofluorescent staining of fully adherent rat embryo fibroblast cells showed some co-distribution of HSPG and FN, and this was confirmed by immunoelectron microscopy, which detected HSPG at localized areas of dorsal and ventral cell membranes, overlapping cell margins, and in the extracellular matrix. During cell shape changes on rounding and spreading, HSPG and FN may not co-distribute. Double labeling for actin and either HSPG or FN showed a closer correlation of actin with HSPG than with FN. The studies are consistent with HSPG being closely involved in a transmembrane cytoskeletal-matrix interaction; the possibility that HSPG coordinates the deposition of FN and other matrix components with cytoskeletal organization is discussed.

Animals↗
PubMed1983

Redistribution of microfilament-associated proteins during the formation of focal contacts and adhesions in chick fibroblasts.

The roles of the microfilament-associated proteins vinculin, alpha-actinin, myosin and filamin have been studied by immunofluorescence and double fluorescence in conjunction with interference reflection microscopy (IRM), during the development of focal contacts and focal adhesions in a chick fibroblast system which initially has no such adhesion specializations but then develops them sequentially over a 48 h period. Without exception, all focal contacts and focal adhesions contain both vinculin and alpha-actinin at every stage that we can detect by IRM or by double staining to reveal the associated microfilament bundles. Indeed the appearance of small bodies containing alpha-actinin and vinculin is shown to precede focal contact formation in our model system and such structures (not visible by IRM) are proposed to be the precursors of focal contacts and adhesions. Myosin and filamin are distributed generally with some reticular patterning in the early motile cells which lack the focal specializations, but as focal contacts and adhesions form these proteins become progressively recruited into the associated microfilament bundles. Only then do we see the marked depletion that has been reported earlier of diffusely distributed myosin and filamin in the leading lamella. Although this is not initially associated with any change in the motile status of the cells, the recruitment of these microfilament-associated proteins into stress fibres is proposed to occur in preparation for anchorage and bracing of cells to the substratum when they later become stationary.

Actinin↗
PubMed1983

Distribution and number of epidermal growth factor receptors in skin is related to epithelial cell growth.

Epidermal growth factor (EGF), a low-molecular-weight polypeptide (G. Carpenter and S. Cohen, 1979, Annu. Rev. Biochem. 48, 193-216), stimulates the proliferation and keratinisation of cultured embryonic epidermis (S. Cohen, 1965, Dev. Biol. 12, 394-407) and promotes epidermal growth, thickening, and keratinisation when injected into neonatal mice (S. Cohen and G.A. Elliott, 1963, J. Invest. Dermatol, 40, 1-5). We have determined the distribution of the available receptors for epidermal growth factor in rat skin using autoradiography following incubation of explants with 125I-labelled mouse EGF. EGF receptors are detected on the epithelial cells overlying the basement membranes of the epidermis, sebaceous gland, and regions of the hair follicle all of which have proliferative capacity. In marked contrast, tissues which have started to differentiate and lost their growth potential, carry either an undetectable or sharply reduced number of EGF receptors. The EGF receptor number and receptor affinity of epidermal basal cells freshly isolated from rats of increasing age has also been determined. We find that receptor affinity remains unchanged (3.3 nM) but that basal cell surface receptor number decreases markedly with age. This decrease in receptor number is similar in trend to the known drop in basal cell [3H]thymidine labelling index which occurs over the same time period. The data suggest that the distribution of EGF receptors and EGF cell surface receptor number in skin are important in the spatial and temporal control of epithelial proliferation.

Age Factors↗
PubMed1983

Adhesion, growth, and matrix production by fibroblasts on laminin substrates.

Human embryonic skin fibroblasts have been shown to attach and spread on laminin substrates in the absence of protein synthesis and presence of fibronectin-depleted serum and anti-fibronectin antibodies. Rates of attachment and the type of spreading are virtually identical on fibronectin and laminin-coated substrates with the development of microfilament bundles and focal adhesions. Antibodies to laminin, but not fibronectin, will prevent or reverse fibroblast adhesion to laminin, whereas antibodies to fibronectin but not laminin will give similar results on fibronectin-coated substrates. These and other results indicate that fibroblasts possess distinct receptors for laminin and fibronectin which on contact with suitable substrates promote adhesion through interaction with common intermediates. This type of adhesion is compatible with subsequent growth and extracellular matrix production.

Antibodies↗
PubMed1983

Stages in specialization of fibroblast adhesion and deposition of extracellular matrix.

Fibroblast cells seeded on a serum glycoprotein shown previously to mediate a spread shape without focal adhesions or microfilament bundles (Stage 1 spread) are now shown to have substratum contacts in which coated pits are abundant and associated with small globular deposits of glycocalyx bridging to substratum and staining for fibronectin and acidic glycoconjugates. After stimulation with serum or fibronectin to form focal adhesions and microfilament bundles (Stage 2 spread), clathrin-based structures remain at the cell underside but no longer in conspicuously higher concentration than on the dorsal surface; extracellular material at adhesions is now as regular strands which stain for acidic glycoconjugates but (as reported earlier by Chen and Singer) not always for fibronectin. During these stages of adhesion, striking changes are seen in the cellular display of fibronectin monitored by immunofluorescence. In rounded cells this is granular and cytoplasmic, concentrated around the submembranous cortex; on spreading to Stage 1, it remains granular and intracellular but is now oriented strongly towards the lower cell surface; only in Stage 2 does externalisation proceed to deposit fibrillar fibronectin on the substratum. While cytoplasmic orientation of matrix precursors can be determined by cell contact, organised externalisation is therefore coupled to fully developed adhesion status.

Animals↗
PubMed1982

Analysis of the proteins, glycoproteins and glycosaminoglycans of fibroblast adhesions to substratum.

The focal adhesion preparations which remain attached to a glass substratum when fibroblast bodies are removed by a gentle stream of buffer have been analysed by gel electrophoresis coupled with other selective methods of analysis. The results are consistent with the presence of three classes of macromolecular components. (i) Muscle and associated proteins amongst which actin was abundant with significant amounts of tropomyosin, some myosin and traces of alpha-actinin. Some vimentin was present but no vinculin. We detected a major new protein component, as yet unidentified, with a molecular weight in the region of 50000-55000 which is not desmin or tubulin and could have an important function at the focal adhesion. (ii) Glycoproteins which are a specialised subset of those in the whole plasma membrane and included a family which bind ricin and therefore contain beta-galactose end groups, together with a series having carbohydrate chains which bound neither ricin nor concanavalin A. The relative proportion of ricin-binding glycoproteins compared to concanavalin A-binding glycoproteins was higher than in whole plasma membranes. (iii) Glycosaminoglycans, with hyaluronate identified as the major component by column chromatography and its susceptibility to Streptomyces hyaluronidase.

Animals↗
PubMed1982

Fibronectin has a dual role in locomotion and anchorage of primary chick fibroblasts and can promote entry into the division cycle.

Fibronectin (FN), which is already known to be a natural factor for fibroblast spreading on substrata, has now been shown to be essential for two distinct types of adhesion with different biological functions in chick heart fibroblasts, namely adhesion directed toward locomotion and toward stationary anchorage for growth. Manipulation of culture conditions and the use of antisera of differing specificities has demonstrated that both exogenous and cell-derived FN are important in each process. The organization of the fibronectin-containing matrix differs between the two states. Immunoelectron microscopy with a colloidal gold marker reveals the presence of small membrane-associated plaques of fibronectin in motile cells with associated submembranous specialization. A fibrillar matrix containing fibronectin is dominant in nonmotile, growing fibroblasts. The development of focal adhesions for stationary anchorage can be dramatically enhanced by addition of cell-derived FN at an appropriate stage, and this promotes entry into the growth cycle. New macromolecular synthesis in addition to FN is necessary for focal adhesion development but not for locomotion.

Animals↗
PubMed1982

Concentration dependence of the order-disorder transition of carrageenans. Further confirmatory evidence for the double helix in solution.

The temperature-induced changes in optical rotation which are associated with the order-disorder transition of carrageenans have been analysed to investigate the concentration dependence and hence to attempt to derive the number of polysaccharide chains involved in the ordered species at equilibrium. The consequences of the known polydispersity have been analysed by computer-modelling and it is shown that this need not cause misleading results. For iota-carrageenan segments of 0.1 M NaCl and 0.1 M KCl solution the results point to a two-chain state and therefore support the proposed double helix model. For chi-carrageenan segments the apparent average number of molecules involved in the ordered state at equilibrium is 3.7, suggesting that if double helices form here they aggregate further. The method of investigation may be generally applicable to measuring the number of chains which associate in ordered states in aqueous solutions of polysaccharides.

Carrageenan↗
PubMed1982

Fragmentation and modification of iota-carrageenan and characterisation of the polysaccharide order-disorder transition in solution.

A procedure is described in detail by which the algal polysaccharide, iota-carrageenan, can be modified and fragmented to products of more regular structure and shorter chain length. Since most chains of this product can form uninterrupted helices they undergo the disorder-order transition without the gelation that is a feature of the behaviour of the natural polymer. The disorder-order transition becomes much sharper and occurs at higher temperatures with increasing chain length. The sign and magnitude of the accompanying shift in optical rotation can be estimated satisfactorily from the solid-state geometry of the helix using our semi-empirical method for correlating optical rotation with the conformations at glycosidic linkages. In solutions of the helical form, but not the coil form, cationic dyes such as methylene blue or acridine orange show induced optical activity and a changed visible spectrum which we attribute to binding and stacking on the helix. This also causes a shift in the temperature of the helix-coil transition. We discuss the possibility that the tendency of dyes to stack could drive polysaccharides into conformations that they would not otherwise adopt, with implications for their use as 'conformational probes'.

Carrageenan↗
PubMed1982

Organelle-cytoskeleton relationships in fibroblasts: mitochondria, Golgi apparatus, and endoplasmic reticulum in phases of movement and growth.

When fibroblasts first emerge from explants of embryonic chick heart, most mitochondria are clustered tightly around the nucleus, with very few extending towards the leading lamella. Although cytoplasmic microtubules are well displayed, mitochondria do not obviously codistribute with them. As the fibroblasts cease locomotion and adapt to growth, however, the mitochondria become dispersed through the cytoplasm and clearly codistributed with the microtubules and centrioles but not with microfilament bundles or 10 nm filaments. This rearrangement occurs in concert with the other changes we have shown previously - including the development of pronounced microfilament bundles and of stable and well-defined focal adhesions - and appears to be related to changes in the motility status of the cells rather than to alterations in growth or synthetic capability. Mitochondrial mobility is strongly reduced by the actions of both colchicine and dihydrocytochalasin B showing that orientation and translocation depend on a co-ordinate interaction of microtubules and microfilamentous meshwork around the centrioles as origin. The Golgi apparatus and endoplasmic reticulum do not rearrange dramatically during the phenotypic conversion, although the locomotory cells are characterized by a zone close to the leading lamella, which is completely free from these organelles. The form and distribution of the Golgi apparatus, but not the endoplasmic reticulum, was sensitive to microtubule disruption but was also shown to have direct functional associations with the centriolar region. The relative distributions of the three types of organelle during the phases of cell movements and cell growth, are consistent with their biochemical functions in cellular activity.

Animals↗