Mobility Of Cell Surface Receptors Research Articles

Analysis of Human Hyaluronan Synthase Gene Transcriptional Regulation and Downstream Hyaluronan Cell Surface Receptor Mobility in Myofibroblast Differentiation.

The ubiquitous extracellular glycosaminoglycan hyaluronan (HA) is a polymer composed of repeated disaccharide units of alternating D-glucuronic acid and D-N-acetylglucosamine residues linked via alternating β-1,4 and β-1,3 glycosidic bonds. Emerging data continue to reveal functions attributable to HA in a variety of physiological and pathological contexts. Defining the mechanisms regulating expression of the human hyaluronan synthase (HAS) genes that encode the corresponding HA-synthesizing HAS enzymes is therefore important in the context of HA biology in health and disease. We describe here methods to analyze transcriptional regulation of the HAS and HAS2-antisense RNA 1 genes. Elucidation of mechanisms of HA interaction with receptors such as the cell surface molecule CD44 is also key to understanding HA function. To this end, we provide protocols for fluorescent recovery after photobleaching analysis of CD44 membrane dynamics in the process of fibroblast to myofibroblast differentiation, a phenotypic transition that is common to the pathology of fibrosis of large organs such as the liver and kidney.

Analysis of human hyaluronan synthase gene transcriptional regulation and downstream hyaluronan cell surface receptor mobility in myofibroblast differentiation.

The ubiquitous extracellular glycosaminoglycan hyaluronan (HA) is a polymer composed of repeated disaccharide units of alternating D-glucuronic acid and D-N-acetylglucosamine residues linked via alternating β-1,4 and β-1,3 glycosidic bonds. Emerging data continue to reveal functions attributable to HA in a variety of physiological and pathological contexts. Defining the mechanisms regulating expression of the human hyaluronan synthase (HAS) genes that encode the corresponding HA-synthesizing HAS enzymes is therefore important in the context of HA biology in health and disease. We describe here methods to analyze transcriptional regulation of the HAS and HAS2-antisense RNA 1 genes. Elucidation of mechanisms of HA interaction with receptors such as the cell surface molecule CD44 is also key to understanding HA function. To this end, we provide protocols for fluorescent recovery after photobleaching analysis of CD44 membrane dynamics in the process of fibroblast to myofibroblast differentiation, a phenotypic transition that is common to the pathology of fibrosis of large organs such as the liver and kidney.

Microtubule inhibitors differentially affect translational movement, cell surface expression, and endocytosis of transferrin receptors in K562 cells

We used quantitative fluorescence microscopy and fluorescence photobleaching recovery techniques to investigate the translational movement, cell surface expression, and endocytosis of transferrin receptors in K562 human erythroleukemia cells. Receptors were labeled with fluorescein-conjugated transferrin (FITC-Tf). Coordinated decreases in surface fluorescence counts, the photobleaching parameter K, and transferrin receptor fractional mobility were observed as FITC-Tf was cleared from the cell surface by receptor-mediated endocytosis. Based on the kinetics of decrease in these parameters, first order rate constants for FITC-Tf uptake at 37 degrees C and 21 degrees C were calculated to be 0.10-0.15 min-1 and 0.02-0.03 min, respectively. K562 cells were treated with colchicine or vinblastine to investigate the role of microtubules in transferrin receptor movement and endocytosis. Treatment of cells for 1 hr with a microtubule inhibitor prevented transferrin receptor endocytosis but had no effect on the translational mobility of cell surface receptors. In contrast, drug treatment for 3 hr caused translational immobilization of cell surface receptors as well as inhibition of endocytosis. These effects were not produced by beta-lumicolchicine, an inactive colchicine analog, or by cytochalasin, a microfilament inhibitor. The effect of microtubule inhibitors on transferrin receptor mobility was reversed by pretreating cells with taxol, a microtubule-stabilizing agent. Microtubule inhibitors had no effect on the translational mobility of cell surface glycophorins or phospholipids, indicating that intact microtubules were not required for translational movement of these molecules. We conclude that the translational movement of cell surface transferrin receptors is directed by a subpopulation of relatively drug-resistant microtubules. In contrast, transferrin receptor endocytosis depends on a subpopulation of microtubules that is relatively sensitive to the action of inhibitors. These results appear to demonstrate at least two functional roles for microtubules in receptor-mediated transferrin uptake in K562 cells.

Effect of tricyclic antidepressant drugs on lymphocyte membrane structure.

Tricyclic antidepressant-induced perturbations of murine splenic lymphocyte membranes and cell surface concanavalin A receptor mobility have been investigated using the fluorescent probes diphenylhexatriene and fluorescein-conjugated concanavalin A. Results of these studies illustrate the possible relationship between tricyclic antidepressant-induced membrane perturbations and tricyclic antidepressant-induced suppression of the normal murine lymphocyte mitogen response. Tricyclic antidepressant effects on murine splenic lymphocyte membranes are dose-, time- and temperature- dependent. Murine lymphocyte concanavalin A cell surface receptor mobility is not apparently altered by the tricyclic antidepressants.

Isolation of actin-containing transmembrane complexes from ascites adenocarcinoma sublines having mobile and immobile receptors.

The molecular nature of the cell surface-cytoskeleton interaction in microvilli isolated from ascites 13762 rat mammary adenocarcinoma sublines with immobile (MAT-C1) and mobile (MAT-B1) receptors was investigated by extraction and fractionation studies on the microvillar membranes. Extraction of membranes from MAT-C1 cells with Triton X-100-containing buffers gave insoluble residues showing three major components by NaDodSO(4)/polyacrylamide gel electrophoresis: actin, a 58,000-dalton polypeptide, and a cell surface glycoprotein of 75,000-80,000 daltons. The ratio of these components in Triton X-100-insoluble residues, as determined by scintillation counting of bands from gels of [(3)H]leucine-labeled microvillar membranes, approached equimolar, suggesting a specific complex of the components. The three components of the putative complex cosedimented on sucrose density gradients of Triton X-100/buffer-treated membranes. Gel filtration on Sepharose 2B gave a peak included in the column that contained only the glycoprotein, actin, and 58,000-dalton polypeptide by one-dimensional NaDodSO(4) electrophoresis and by two-dimensional isoelectric focusing/NaDodSO(4) electrophoresis. The glycoprotein-actin association could be disrupted only under strongly denaturing conditions. Complex prepared from MAT-B1 microvillar membranes by Sepharose 2B gel filtration in Triton X-100-containing buffers contained actin and the glycoprotein but no 58,000-dalton polypeptide. From these results we propose that the cell surface-cytoskeleton interactions in the 13762 tumor cell microvilli involve direct association of actin with the cell surface glycoprotein. We further suggest that the 58,000-dalton polypeptide stabilizes the association of this complex with the microfilaments in the MAT-C1 microvilli, thereby stabilizing the microvilli and restricting cell surface receptor mobility.

Differences in the response of several cell types to inhibition of surface receptor mobility by local concanavalina binding

Concanavalin A (conA) modulates the lateral mobility of cell surface receptors differently on different cell types. This was demonstrated by using fluorescence photobleaching recovery (FPR) to measure the inhibition of the lateral mobility of conA receptors by localized binding of conA on lymphocytes, fibroblasts, and macrophages. On mouse spleen lymphocytes, binding of conA platelets above a threshold coverage (about 12% of the upper cell-surface area) reduced the diffusion coefficient of mobile TMR-SconA-receptor complexes from 3.0×10 −10 cm 2/sec to 0.6× 10 −10 cm 2/sec (a 5-fold decrease), and the fraction of mobile receptors was concomitantly reduced from 0.4 to 0.11. Below the threshold occupancy, no effect on either parameter was detected. On 3T3 cells, a qualitatively similar threshold phenomenon was observed: coverage of over 9% of the upper cell surface by conA platelets induced a 3-fold reduction in the diffusion coefficient of TMR-SconA-receptor complexes from 5×10 −10 cm 2/sec to 1.7× 10 −10 cm 2/sec. However, no effect on the mobile fraction (about 0.4) was observed. In contrast, neither the diffusion coefficient nor the mobile fraction of TMR-SconA-receptor complexes on mouse peritoneal macrophages (both resident and thioglycolate-stimulated) or on the mouse macrophage cell line P388D 1 were affected by the binding of conA platelets in amounts covering over 50% of the upper cell surface (approx. 4.6× 10 −10 cm 2/sec and 0.5 for the diffusion coefficient and mobile fraction, respectively). These differences are correlated to the different cytoskeletal functions of the various cell types studied, and are discussed regarding the mechanism of the conA-induced modulation.

Interferon increases the abundance of submembranous microfilaments in HeLa-S3 cells in suspension culture.

Human beta (fibroblast) interferon inhibits the proliferation of human HeLa-S3 carcinoma cells in suspension culture. Accompanying this effect, the lateral mobility of cell surface receptors for concanavalin A is decreased and the rigidity of the plasma membrane lipid bilayer is increased. The present findings show a marked increase in the number of polymerized actin-containing microfilaments 3 days after treatment of HeLa-S3 cells with beta-interferon (640 units/ml). The cortical region of the treated enlarged cells contains a thick and dense meshwork of 40-70 A microfilaments. The actin nature of the filaments was verified by their ability to bind heavy meromyosin. These results support the concept that beta-interferon induces a coordinated response in the plasma membrane and the underlying microfilaments in both tumor and normal cells.

Role of cell surface receptor mobility in concanavalin A-induced agglutination of Novikoff hepatoma and normal rat liver cells.

The relationshio between Con A-receptor mobility and Con A-induced agglutination of Novikoff hepatoma and normal rat liver cells was investigated. Novikoff cells, incubated with fluorescein-labelled Con A at 3 degrees C displayed uniform, ring-like surface fluorescence. Increasing the temperature of the cells to 37 degrees C caused capping of Con A receptors in approximately 65% of the cells, a phenomenon that could be prevented by prefixing the cells with glutaraldehyde. In spite of these variations in Con A-receptor distribution, Con A-induced agglutination was remarkably constant over a temperature range from 3 to 37 degrees C. In contrast to Novikoff cells, normal hepatocytes displayed a uniform, ringlike surface fluorescence at both 3 and 37 degrees C. No capping was observed. However, hepatocytes, similar to Novikoff cells, were agglutinable by low concentrations of Con A. These findings indicate that, in this model system, Con A-induced cytoagglutination is not dependent upon long-range lateral mobility of Con A receptors. The qualitative differences in the lateral mobility of cell-surface Con A receptors of normal and malignant rat liver cells may represent a marker for neoplastioc transformation during hepatocarcinogenesis, adaptation to growth in ascitic form, or progression of a tumour to a more malignant state.

Independence of normal phenotypic properties and cell surface receptor mobility in variant cell lines.

We report the use of three classes of variants from the long-established, malignantly transformed LM cell line to demonstrate that the apparent mobility of cell surface receptors need not be dependent on the expression of the transformed phenotype in vitro.

Receptor diffusion on cell surfaces modulated by locally bound concanavalin A.

In order to test the anchorage modulation hypothesis, the fluorescence photobleaching recovery method was used to measure the global inhibition of cell surface receptor mobility induced in 3T3 mouse fibroblasts by local binding of platelets labeled with concanavalin A (Con A). By measuring the diffusion of antibody-labeled cell surface receptors at various points on the cell surface, two states, immobile and mobile, were distinguished in the receptor population. Bound Con A-platelets, occupying between 4% and 30% of the cell surface, decreased the diffusion coefficient of the mobile population by a factor of 6. The magnitude of this effect was independent of distance from the sites of the bound Con A-platelets, demonstrating the propagated and nonlocal properties of the modulation effect. The immobile fraction of the population was not changed by Con A-platelet binding. Modulation of the diffusion constant of mobile receptors was partially reversed by treatment with microtubule-disrupting agents such as Colcemid and Vinca alkaloids. High doses of soluble Con A induced even higher levels of modulation than Con A-platelets, but reversal by microtubule-disrupting drugs was observed. These experiments provide additional support for the anchorage modulation hypothesis and provide a measure of the nature and degree of mobility at the molecular level. They also put important constraints on the hypothesized interactions among submembranous components (microtubules and microfilaments) of surface modulating assemblies.

Cell shape changes and transmembrane receptor uncoupling induced by tertiary amine local anesthetic.

Tertiary amine local anesthetics (dibucaine, tetracaine, procaine, etc.) modify cell morphology, concanavalin A (Con A)-mediated agglutinability and redistribution of Con A receptors. Con A agglutination of untransformed mouse 3T3 cells was enhanced at low concentrations of local anesthetics, and the dynamics of fluorescent-Con A indicated that ligand-induced clustering was increased in the presence of the drugs. In contast, these drugs inhibited Con A-induced receptor capping on mouse spleen cells. These effects can be duplicated by combinations of vinblastine (or colchicine) and cytochalasin B suggesting that local anesthetics act on microtubule and microfilament assemblies which are involved in the trans-membrane control of cell surface receptor mobility and distribution. It is proposed that tertiary amine local anesthetics displace plasma membrane-bound Ca2+, resulting in disengagement of microfilament systems from the plasma membrane and increased cellular Ca2+ concentration to levels which disrupt microtubular organization. The possible involvement of cellular Ca2+ in cytoskeletal destruction by local anesthetics was investigated utilizing Ca2+-specific ionophores A23187 and X537A. In media containing Ca2+ and cytochalasin B these ionophores caused effects similar to tertiary amine local anesthetics.

Inhibition of mobility of cell-surface receptors by factors which mediate specific cell-cell interactions.

PREVIOUS studies on embryonic chick neural retina cells1 have demonstrated that cells in intact tissues or single cells prepared by mechanical dispersion have relatively little ability to redistribute their surface receptors into caps. In contrast, cells prepared by trypsinisation and dissociation of intact tissues were found to exhibit lateral redistribution of surface receptors even in the absence of a multivalent ligand. After 4 h culture, however, cells dissociated by trypsinisation have lost the capability for both spontaneus and lectin-induced rearrangements of surface components. Mobility of cell-surface receptors thus seems to be related to, or determined by, trypsin-labile components at the cell surface. One kind of component which seems to be trypsin labile is also important in intercellular recognition and adhesion2,3. We now report that the binding of tissue-type specific cell-surface ligands which seem to mediate recognition and adhesion causes inhibition of directed rearrangement of plant lectin receptors in the plane of the membrane.

Modulation of lymphocyte mitogenesis.

Comparisons of the stimulation of normal lymphocytes and lymphoma cells by tetrameric concanavalin a (Con ta) and dimeric succinyl-Con A suggest that both stimulatory and inhibitory signals operate to modulate mitogenesis. Synergistic effects can be obtained for the stimulatory event using lectins, phorbol esters, and calcium ionophores, all of which are independently mitogenic for lymphocytes. The inhibitory effects of high doses of Con A could be mimicked by the simultaneous addition of the phorbol ester and Con A under conditions in which both reagents are optimally mitogenic when used alone. No inhibition of stimulation was found, however, when succinyl-Con A was used with phorbol ester under the same conditions. Moreover, when lymphocytes were cultured with Con A in the presence of succinyl-Con A, the inhibitory effect of the native lectin was seen at lower doses than in the absence of the derivative. These observations suggest that the stimulatory and inhibitory portions of the dose-response curve can be manipulated independently and may be mediated by two distinct signals. It is likely the signal for the inhibition of cell proliferation is regulated by the same cell surface modulating assembly that controls the mobility of cell surface receptors.

Structure and function of concanavalin A.

Lectins have been extensively used to analyze a variety of fundamental processes in cell biology. In conjuntion with our studies on the cell surface and mitosis, we have determined the amino acid sequence and three-dimensional struction of concanavalin A (Con A), the mitogenic lectin from the jack bean. Knowledge of the structure has been helpful in interpreting experiments on lymphocyte mitogenesis and the effects of Con A on cell surface receptor mobility. Con A subunits for molecular weight 25,500 are folded into dome-like structures of maximum dimensions 42 times 40 times 39 A. The domes are related by 222 symmetry to form roughly tetrahedral tetramers. Each subunit contains two large antiparallel pleated sheets, and subunits are joined to form dimers and tetramers by interactions involving one of these pleated sheets. We have examined the binding of a variety of carbohydrates to Con A and have obtained preliminary data which suggest that there are differences in the saccharide-binding behavior of Con A in solution and in the crystalline state. Dimeric chemical derivatives of Con A have been prepared and shown to have biological activities different from those of the native tetrameric protein. Under different conditions, native Con A exhibits two antagonistic activities on the lymphoid cell surface: the induction of cap formation by its own receptors and the inhibition of the mobility of a variety of receptors, including its own receptors. The dimeric derivative, succinyl-Con A, is just as effective a mitogen as the native lectin, but it lacks the ability to modulate cell surface receptor mobility. The data suggest that neither extensive immobilization of cell surface receptors nor cap formation is required for cell stimulation. Further studies on modulation of receptor translocation suggest that hypothesis that there exists a connecting network of colchicine-sensitive proteins that links receptors of different kinds and mediates their rearrangement. The degree of connectivity of this postulated network appears to be altered by changes in the state of attachment of various surface receptors to the network. Thus the network might provide the cell with a means of transmitting signals such as the stimulus for mitosis by lectins or antigens.