Calcium-dependent adhesion of Drosophila embryonic cells

Summary By using an in vitro functional assay, we have shown that Drosophila embryonic cells possess Ca²⁺-dependent adhesive sites, which resemble in many respects those described for vertebrate cells and tissues. The cells, obtained by mechanical disruption of gastrulastage embryos, form aggregates within 30 min when maintained under constant rolling. The aggregation is completely dependent on the presence of Ca²⁺ in the medium. In its absence, the cells remain dispersed but the process is reversible by readdition of Ca²⁺. In addition the aggregation is temperature-dependent. No aggregation occurs at 4° C but it can be restored by raising the temperature to 25° C. These properties are characteristic of these cells: established cell lines do not aggregate under the same conditions and mixing of cell lines and embryonic cells does not result in chimeric aggregates, thus pointing towards cell-type selectivity with respect to aggregability. Observations in electron microscopy have shown that the embryonic cells in the aggregates tightly adhere to one another and form, as early as after 30 min, maculae adherens junctions. Drosophila embryonic cells have adhesion sites that are protected from trypsin proteolysis in the presence of Ca²⁺ and sensitive in its absence. The cells' aggregation can be inhibited by a mouse antiserum directed against cell-surface components and a good correlation exists between neutralization of the inhibitory activity of the antiserum and the presence of trypsin-sensitive sites on the cells. These data are in favour of cell-cell adhesion mediated by specific adhesion proteins.     

Cadherins Promote Skeletal Muscle Differentiation in Three-dimensional Cultures

The cell–cell adhesion molecule N-cadherin, with its associated catenins, is expressed by differentiating skeletal muscle and its precursors. Although N-cadherin's role in later events of skeletal myogenesis such as adhesion during myoblast fusion is well established, less is known about its role in earlier events such as commitment and differentiation. Using an in vitro model system, we have determined that N-cadherin– mediated adhesion enhances skeletal muscle differentiation in three-dimensional cell aggregates. We transfected the cadherin-negative BHK fibroblastlike cell line with N-cadherin. Expression of exogenous N-cadherin upregulated endogenous β-catenin and induced strong cell–cell adhesion. When BHK cells were cultured as three-dimensional aggregates, N-cadherin enhanced withdrawal from the cell cycle and stimulated differentiation into skeletal muscle as measured by increased expression of sarcomeric myosin and the 12/101 antigen. In contrast, N-cadherin did not stimulate differentiation of BHK cells in monolayer cultures. The effect of N-cadherin was not unique since E-cadherin also increased the level of sarcomeric myosin in BHK aggregates. However, a nonfunctional mutant N-cadherin that increased the level of β-catenin failed to promote skeletal muscle differentiation suggesting an adhesion-competent cadherin is required. Our results suggest that cadherin-mediated cell–cell interactions during embryogenesis can dramatically influence skeletal myogenesis.     

Intercellular adhesion in butyrate-treated HeLa S3 cultures : Flow cytometric analysis

The application of DNA flow cytometry (FCM) for analysis of sodium butyrate-induced intercellular adhesion in human carcinoma (HeLa S3) cell cultures is described. To prepare cell suspensions for FCM, the monolayers of cells were treated with medium containing 10% serum, 0.2% non-ionic detergent Triton X-100 and 1 μg/ml DNA fluorochrome 4,6′-diamidino-2-phenylindole (DAPI). Total numbers of single cells, and aggregates containing two, three, four or more cells, were determined from DNA histograms. In cultures treated with 5 mM butyrate for 16 h, more than 80% of the cells were aggregated. Intercellular adhesion began to appear 8 h after addition of butyrate, was maximal at 16–24 h and stable in the presence of butyrate, but disappeared 24 h after its removal. Treatment with EDTA (0.2%) dissociated only 50%, whereas trypsin (0.1%) separated all cell aggregates into single cells. Actinomycin D (actD) (0.5 μg/ml) prevented cell adhesion while blocking of cells in S phase with 250 μM 5-fluorouracil or 10 μM methotrexate did not interfere with aggregation. The number of cell aggregates estimated from DNA histograms of butyrate-treated HeLa S3 cultures was the same after staining with DAPI in the presence of Triton X-100 or after vital staining with Hoechst 33342. The DNA content was used as a marker to estimate the cellular composition of aggregates in mixed cultures of HeLa S3 cells and human fibroblasts (U cells). Intercellular adhesion in these cultures was seen only between HeLa S3 cells, indicating specificity of butyrate-induced cell aggregation. FCM provides fast automatic measurement of cell aggregate formation, estimates frequency of aggregates containing different cell numbers, shows participation of cells at different cycle phases in aggregates, and allows the detection of homotypic from heterotypic cell aggregates if the interacting cells have different DNA ploidy.     

Cell aggregation on agar as an indicator for cell-matrix adhesion: effects of opioids

The slow aggregation assay is generally used to study the functionality of cell–cell adhesion complexes. Single cells are seeded on a semisolid agar substrate in a 96-well plate and the cells spontaneously aggregate. We used HEK FLAG-MOP cells that stably overexpress the mu opioid receptor and the mu-opioid-receptor-selective agonists DAMGO and morphine to study whether other factors than functionality of cell–cell adhesions complexes can contribute to changes in the pattern of slow aggregation on agar. HEK FLAG-MOP cells formed small compact aggregates. In the presence of DAMGO and morphine, larger and fewer aggregates were formed in comparison to the vehicle control. These aggregates were localized in the center of the agar surface, whereas in the vehicle control they were dispersed over the substrate. However, in suspension culture on a Gyrotory shaker, no stimulation of aggregation was observed by DAMGO and morphine, showing that opioids do not affect affinity. A dissociation experiment revealed that HEK FLAG-MOP aggregates formed in the absence or presence of opioids are resistant to de-adhesion. We demonstrated that the larger aggregates are neither the result of cell growth stimulation by DAMGO and morphine. Since manipulations of the substrate such as increasing the agar concentration or mixing agar with agarose induced the same changes in the pattern of slow aggregation as treatment with opioids, we suggest that cell–substrate adhesion may be involved in opioid-stimulated aggregation.     

Inhibition of desmosome formation in chick cell aggregates.

Desmosomes (macula adherens) have been associated with the function of adhesion. Their possible role in aggregation and sorting of chick and mouse epithelial cells has been investigated. Treatment of aggregates with 2-5 microgram/ml of actinomycin D which inhibited RNA synthesis also inhibited both desmosome formation and aggregation if administered at the beginning of the aggregation process. In contrast, if the drug was administered at six hours, when the cells had recovered from the process of dissociation, then aggregation over the following six hours appeared normal from observation of living samples. Such aggregates incorporated leucine-3H at roughly 85% of the control level. A quantitative comparison was made of desmosome formation in aggregates treated with actinomycin D for hours 6-12 and those cultured in normal medium. Desmosome formation was inhibited by the drug, although aggregation could proceed. Combinations of chick corneal and mouse skin cells sorted out in the presence of actinomycin D to the same extent as controls. Thus desmosome formation, which normally occurs during aggregation of the epithelial cells studied here, is not coupled with the aggregation or cell sorting process in these cells of stratified epithelia. When cells were treated with cycloheximide (100 muM) both desmosome formation and the progressive rounding up of aggregates was inhibited.     

Inhibition of fibronectin receptor function by antibodies against baby hamster kidney cell wheat germ agglutinin receptors

Previous studies suggest that the baby hamster kidney (BHK) cell fibronectin receptor is also a wheat germ agglutinin receptor (WGA-R). To analyze this possibility further, IgG and Fab fragments of antibodies produced against a BHK cell WGA-R preparation were tested to determine their effects on cell adhesion mediated by fibronectin, wheat germ agglutinin, concanavalin A, and polycationic ferritin. The WGA-R preparation was isolated by octylglucoside extraction of BHK cells followed by chromatography of the extract on WGA-agarose. The antibodies against the WGA-R preparation reacted primarily with polypeptides of molecular weights 48, 61, 83, 105, 120, 165, 210, and 230 kilodaltons (kdaltons). It was concluded that the antibodies interfered with BHK cell fibronectin receptors on the basis of the ability of anti-WGA-R IgG or Fab fragments to (a) inhibit cell spreading on fibronectin-coated substrata; (b) cause rounding and detachment of cells previously spread on fibronectin-coated substrata; and (c) inhibit binding of fibronectin-coated latex beads to the cells. Antibody activity was blocked by treatment of anti-WGA-R with the WGA-R preparation or by absorption of anti-WGA-R with intact BHK cells. The antibodies also appeared to prevent coupling of ligand-receptor complexes (involving concanavalin A or polycationic ferritin) with the cytoskeleton. Finally, cell rounding and detachment caused by the antibodies were found to require metabolic energy since it did not occur in the presence of azide or at 4 degrees C.     

Intracellular degradation of hemoglobin transferred into fibroblasts by fusion with red blood cells

Hamster fibroblast protein and rabbit hemoglobin were labelled by incubation of fibroblasts (BHK21) or reticulocytes with [³H]leucine. Alternatively, human or rabbit hemoglobin was labelled by carbamoylation of erythrocytes with K¹⁴CNO. The labelled hemoglobins were introduced into fibroblasts by virus-mediated fusion between the blood cells and fibroblasts. The hemoglobins became uniformly distributed throughout the cytoplasm. Degradation was assessed from release of acid-soluble radioactivity into the medium. Radioactivity from [¹⁴C]-carbamoylhemoglobin was released as carbamoylvaline and homocitrulline, and these compounds were not metabolized or reincorporated by the cells. Intermediate degradation products could not be detected. The degradation of hemoglobin followed first-order kinetics. The half-life of both carbamoylated and native rabbit hemoglobin in hamster fibroblasts was 28 h, and the half-life of carbamoylated human hemoglobin was about 150 h in fibroblasts from hamster (BHK21), mouse (Balb/3T3), and man (MRC 5), corresponding to that of the more stable endogenous proteins. Phenylhydrazine increased the intracellular degradation of carbamoylated human hemoglobin about 13 times, whereas the degradation of endogenous proteins was little affected. Hemoglobin was degraded in homogenates at 31% h^?1 at pH 5 and 0.3% h^?1 at pH 7.4. Phenylhydrazine increased these rates to 45% h^?1 and 9.7% h^?1, respectively. Growing hamster fibroblasts, which are brought into quiescence by serum deprivation or by high culture density, increase the degradation of endogenous protein and of hemoglobin in parallel.     

Flow cytometric analysis and modeling of cell-cell adhesive interactions: the neutrophil as a model

The immune function of granulocytes, monocytes, lymphocytes, and other specialized cells depends upon intercellular adhesion. In many cases the molecules mediating leukocyte cell adhesion belong to the Leu-CAM superfamily of adhesive molecules. To elucidate the events of homotypic aggregation in a quantitative fashion, we have examined the aggregation of neutrophils stimulated with formyl peptides, where aggregate formation is a transient reversible cell function. We have mathematically modeled the kinetics of aggregation using a linear model based on particle geometry and rates of aggregate formation and breakup. The time course was modeled as a three-phase process, each phase with distinct rate constants. Aggregate formation was measured on the flow cytometer; singlets and larger particles were distinguished using the intravital stain LDS-751. Aggregation proceeded rapidly after stimulation with formyl peptide (CHO-nle-leu-phe-nle-tyr-lys). The first phase lasted 30-60 s; this was modeled with the largest aggregation rate and smallest rate of disaggregation. Aggregate formation plateaued during the second phase which lasted up to 2.5 min. This phase was modeled with an aggregation rate nearly an order of magnitude less than that of the initial fast phase, whereas the disaggregation rate for this phase did not change significantly. A third phase where disaggregation predominated, lasted the remaining 2-3 min and was modeled with a four to fivefold increase of the disaggregation rate. The mechanism of cell-cell adhesion in the plateau phase was probed with the monoclonal antibody IB4 to the CD18 subunit of the adhesive receptor CR3. Based on these studies it appears that new aggregates do not form to a large degree after the first phase of aggregate formation is complete. However, new adhesive contact sites may form within the contact region of these adherent cells to keep the aggregates together.     

Abnormal response of glycolipid synthesis to temperature in transformed BHK cells thermosensitive for growth control.

In DMN4B cells, a line of chemically mutagenized BHK hamster cells which exhibit transformed behavior at 38.5°C but not at 32°C, [¹⁴C]-palmitate incorporation into mono-, di-, and trihexosylceramides was unimpaired at 32°C when compared with incorporation rates in untransformed BHK cells. At 38.5°C, labeling of these glycolipids increased greatly in the BHK cells, but failed to increase comparably in the DMN4B cells. Assay of cell-free preparations of the galactosyltransferase which catalyzes trihexosylceramide synthesis revealed a stimulatory effect of increased temperature on activity of the BHK enzyme, but not the DMN4B enzyme. The results suggest that transformation can result from a mutation affecting glycolipid synthesis.     

Adipocyte insulin receptor. Generation of a cryptic domain of the alpha-subunit during internalization of hormone-receptor complexes.

The dynamics of the internalization of photoaffinity-labelled insulin-receptor complexes was investigated in isolated rat adipocytes by using tryptic proteolysis to probe both the orientation and cellular location of the labelled complexes. In cells that were labelled at 16 degrees C and not prewarmed, 150 micrograms of trypsin/ml rapidly degraded the labelled 125 kDa insulin-receptor subunit into a major proteolytic fragment of 70 kDa and minor amounts of 90- and 50-kDa fragments. With milder trypsin treatment conditions (100 micrograms of trypsin/ml, 15 s at 37 degrees C), the 90 kDa peptide (different from the 90 kDa beta-subunit of the insulin receptor) appeared as a major intermediate proteolytic product, but this species was rapidly and completely converted into the 70- and 50-kDa fragments with continued exposure to trypsin, such that it did not accumulate to appreciable amounts in cells that were not prewarmed before trypsin exposure. By contrast, trypsin treatment of cells prewarmed to 37 degrees C for various times showed that: first, a proportion of the labelled 125 kDa receptors was internalized (became trypsin-insensitive); secondly, the 90 kDa tryptic peptide was formed in large amounts, with proportionate decreases occurring in the amounts of the 70- and 50-kDa tryptic peptides. The increased accumulation of the 90 kDa tryptic peptide from cells preincubated at 37 degrees C, but not at 16 degrees C, indicated that trypsin cleavage sites within the 90 kDa segment of the insulin-receptor alpha-subunit that were exposed at 16 degrees C were made inaccessible by incubation at 37 degrees C, a finding that is consistent with generation of a cryptic domain of the receptor subunit. The tryptic generation of the 90 kDa peptide at 37 degrees C was rapid, becoming half-maximal in 4.4 +/- 0.6 min and maximal in 15-20 min, preceded the intracellular accumulation of labelled receptors (half-maximal in 12.6 +/- 0.7 min and maximal in 30-40 min), was highly correlated with receptor internalization, and was not observed in cultured IM-9 lymphocytes, a cell line in which photolabelled insulin receptors are primarily lost by shedding into the incubation media. These results show that, in adipocytes incubated at 37 degrees C, rapid masking of a previously (at 16 degrees C) accessible domain of the insulin-receptor alpha-subunit occurs and that this dynamic process happens at an early stage in the internalization of insulin-receptor complexes.     

Aggregate formation by ERp57-deficient MHC class I peptide-loading complexes

The endoplasmic reticulum (ER)-resident proteins TAP, tapasin and ERp57 are the core components of the major histocompatibility complex (MHC) class I peptide-loading complex and play an important role in peptide loading by MHC class I-beta(2)microglobulin dimers. ERp57 and tapasin form a stable disulfide-linked heterodimer within the peptide-loading complex. We demonstrate that ERp57-deficient loading complexes, obtained by expression in a tapasin-negative cell line of a tapasin mutant (C95A) that is not able to form a disulfide bond with ERp57, are prone to aggregation. We studied the assembly, stability and aggregation of the core loading complex using cell lines stably expressing fluorescently tagged tapasin (wild type or C95A mutant) and TAP1. Part of the loading complexes containing the tagged C95A tapasin and TAP1 were sequestered in the ER, without change of their ER transmembrane topology, and were surrounded by a mesh of filaments at the cytosolic side, resulting in formation of protein aggregates with characteristic morphology. Protein aggregates were associated with changes in ER protein turnover but did not affect the cell viability and did not induce the unfolded protein response. Fluorescence resonance energy transfer analysis of the aggregate-free ER fraction revealed that lack of ERp57 did not affect the stoichiometry or stability of tapasin-TAP1 interactions in the assembled 'soluble' core loading complexes. We conclude that the presence of ERp57 is important for the stability of core loading complexes, and that in its absence, the core loading complexes may form stable aggregates within the ER.     

Intercellular adhesion

Summary A quantitative procedure for determining the early kinetics of cell aggregation (adhesion) is described. The cells used for this study were obtained by dissociation of 8-day-old embryonic chicken neural retina with crude trypsin. The method is based on determining the decrease in single cells in an aggregating population with the Coulter electronic particle counter. A variety of experiments show that the method is reproducible and capable of detecting relatively small changes in the rate of aggregation. Using a number of criteria, the loss of single cells from the population with increasing time of incubation was shown to result from the formation of aggregates, and not from other phenomena such as cell death or changes in cell permeability. The intercellular adhesions formed under these conditions were stable to mechanical shear and to ethylenediaminetetraacetate, and were partially resistant to crude trypsin. The logarithm₁₀ of the number of single cells in the population was found to be directly related to the time of incubation. The slope of the resultant straight lines could be used as a measure of the rate of aggregation. No lag in aggregation was demonstrable under the standard assay conditions. the rate was affected by the initial cell density, speed of rotation during aggregation, temperature, and by Ca²⁺ and Mg²⁺. It was not affected by inhibitors of protein synthesis, metabolic inhibitors, ATP, ADP, cyclic-AMP, or horse serum at 37 °C. The quantitative method for determining the initial rate of adhesion should be applicable to studies on the chemistry of this process.Contribution No. 557 from the McCollum-Pratt Institute.     

Surface immunoglobulin on rabbit lymphoid cells. I. Ultrastructural distribution and endocytosis of b4 allotypic determinants on peripheral blood lymphocytes

Immunoglobulin (Ig) b4 allotypic determinants are detected on the surface membrane of rabbit peripheral blood lymphocytes by an indirect immunoferritin labeling technique. Cells coated with antiallotype antibodies are labelled with soluble complexes of ferritin and rabbit antiferritin of a given allotype. At 0 °C a patchy distribution of labeled surface immunoglobulin is visualized on 80% of the lymphocytes examined. Warming of the cells for 1–5 min at 37 °C causes rapid endocytosis of surface label in a perinuclear fashion. Cap formation is not observed. Cross-linking of immunoferritin labelled surface determinants with sheep anti-rabbit Ig (SARG) inhibits endocytosis and promotes aggregation of small surface patches. Indirect evidence suggests that sloughing and/or stripping of labelled surface Ig can occur after this aggregation. These surface changes may be the first step in the induction of lymphocyte activation.     

Keratinization and the effect of vitamin A in aggregates of a squamous carcinoma cell line NBT II

The terminal differentiation, keratinization, of a rat bladder tumor cell line, NBT II, occurred in multicellular aggregates. After aggregation, these cells did not undergo a round of mitosis before keratinization. 5-Bromodeoxyuridine added to the monolayer cell culture 2 days before aggregation completely prevented this differentiation; it was ineffective when added at the time of cell aggregation. Vitamin A prevented the keratinization of NBT II cells in aggregates but did not inhibit aggregate formation; it enhanced the number of cells engaged in DNA synthesis. This model appears to be very useful for analyzing the mechanisms of terminal differentiation and its modulation by vitamin A in tumor cells.     

Protein kinase Cepsilon binds peripherin and induces its aggregation, which is accompanied by apoptosis of neuroblastoma cells

A hallmark of the afflicted nervous tissue in amyotrophic lateral sclerosis is the presence of protein aggregates, which to a large extent contain the intermediate filament protein peripherin. Here we show that activation of protein kinase C (PKC) or overexpression of PKCepsilon induces the aggregation of peripherin in cultured neuroblastoma cells with elevated amounts of peripherin. The formation of aggregates was coupled to an increased apoptosis, suggesting a functional link between these events. Both induction of aggregates and apoptosis were suppressed in cells that had been transfected with small interfering RNAs targeting PKCepsilon. PKCepsilon and peripherin associate as shown by co-immunoprecipitation, and the interaction is dependent on and mediated by the C1b domain of PKCepsilon. The interaction was specific for PKCepsilon since corresponding structures from other isoforms did not co-precipitate peripherin, with the exception for PKCeta and -, which pulled down minute amounts. PKCepsilon interacts with vimentin through the same structures but does not induce its aggregation. When the PKCepsilon C1b domain is expressed in neuroblastoma cells together with peripherin, both phorbol ester-induced peripherin aggregation and apoptosis are abolished, supporting a model in which PKCepsilon through its interaction with peripherin facilitates its aggregation and subsequent cell death. These events may be prevented by expressing molecules that bind peripherin at the same site as PKCepsilon.     

Cell-to-cell adhesion of dissociated embryonic brain cells; the effects of metabolic inhibitors and temperature

Brain cells from 16 to 18-day-old mice embryos were dissociated by mild trypsinization and rotated for 120 min. The area and density of of the adhesive complexes formed were registered using the method described previously. The adhesiveness of dissociated embryonic brain cells (measured during the 120 min of rotation) was diminished in the presence of inhibitors of protein synthesis (puromycin, cycloheximide and inhibition of mRNA synthesis actinomycin D). The inhibition was, however, not distinct, because 1 microgram/ml of cycloheximide and actinomycin was without any significant effect, and the degree of inhibition evoked by 10 micrograms/ml and 25 micrograms/ml of puromycin bordered on significance. However, protein synthesis inhibitors in long-term aggregation experiments had a pronounced inhibitory effect and/or induced destruction of the aggregates. Metabolic inhibitors (KCN and NaN3) caused an inhibition at the lowest level of significance (p less than 0.05) 10(-3) mol/l KCN reduced the final adhesive product significantly. Cells rotated at room temperature and at +5 degrees C adhere to the same extent as in control experiments (37 degrees C). The adhesion was significantly inhibited at +60 degrees C and also after freezing at -80 degrees C with subsequent thawing. The adhesion of cells exposed for 30 min to between +80 degrees C and 100 degrees C was completely abolished. The process of embryonic brain cell adhesion requires a low energy supply, and is relatively independent of biosynthetic processes and of temperature changes between +5 degrees C and +50 degrees C.     

Growth and survival signalling in B16F10 melanoma cells in 3D culture

The two‐way communication between the ECM (extracellular matrix) and the cytoplasm via the integrins has many functions in cancer cells, including the suppression of apoptosis. As cells in a 3D (three‐dimensional) architecture resemble the in vivo situation more closely than do cells in more conventional 2D cultures, we have employed a substratum that prevents cell adhesion and induces cell aggregation to determine why highly metastatic B16F10 melanoma cells resist anoikis. We compared the behaviour of B16F10 cells in 2D [on tPS (tissue culture polystyrene)] and 3D culture {on polyHEMA [poly(2‐hydroxyethylmethacrylate)]} configurations. For this, we analysed cell morphology, proliferation, apoptosis and the activation status of several proteins involved in cell proliferation and survival [RhoA, FAK (focal adhesion kinase), Akt, ERK1/2 (extracellular‐signal‐regulated kinase 1/2)]. B16F10 cells in 3D architecture were able to proliferate as cell aggregates for 3 days, after which the number of cells decreased. The normal Swiss 3T3 cells used as an anoikis‐sensitive control did not proliferate on the anti‐adhesive substratum. Rho A was activated in B16F10 aggregates throughout their time in culture, whereas it was not in Swiss 3T3 aggregates. An absence of apoptotic activity was correlated with the proliferation of B16F10 cells in aggregates: caspase 3 was significantly activated only after 3 days in culture on polyHEMA. FAK and Akt were transiently activated, and their inactivation was correlated with the induction of apoptosis. ERK1/2 were activated throughout the 3D culture. No survival protein was activated in Swiss 3T3 aggregates. Data obtained from cells in 3D culture suggest that B16F10 cells are resistant to anoikis through the activation of the FAK and Akt signalling pathways.     

Inhibition of intercellular adhesion by concanavalin A is associated with concanavalin A-mediated redistribution of surface receptors

The inhibition of adhesion between aggregates and layers of embryonic retinal cells by concanavalin A (Con A) and Con A-mediated rearrangements of Con A receptors on retinal cells were studied. A short incubation of aggregates and layers with 10 micrograms/ml Con A substantially reduced aggregate-to-layer adhesion in a subsequent assay without soluble lectin present. This effect of Con A was dose-dependent, temperature-sensitive, involved events subsequent to Con A binding, and was reduced by cytochalasin B. The inhibition produced by succinylated Con A was substantially increased by incubation with antibody to Con A. Visualization of ConA- receptor complexes by fluorescence microscopy revealed that binding of Con A induced clearing of Con A receptors from filopodia, flattened regions of growth cones, and the edges of axons. This clearing reaction was prevented by the same agents that reduced Con A's inhibition of cell adhesion: low temperature, succinylation of Con A, or cytochalasin B. Aggregate-layer adhesion was restored by releasing Con A at 37 degrees C. Inhibitors of protein and ATP synthesis did not prevent recovery of ability to make adhesions. However, release of Con A at lowered temperatures did not prevent recovery. The results suggest that intercellular adhesion is inhibited by events associated with redistribution of Con A-receptor complexes on retinal cells.     

Regulation of gene expression in Dictyostelium discoideum cells exposed to immobilized carbohydrates

When amoebae of Dictyostelium discoideum develop on gels of polyacrylamide that are derivatized with glucosides, they become capable of aggregation at the same time as cells not exposed to glucosides. However, the aggregation centers and streams of adherent cells formed on immobilized glucosides suddenly disintegrate. The cells repeatedly re-aggregate, but never form tight aggregates as they do on other substrata. Tight aggregates formed in the absence of glucosides disperse after their transfer to glucoside gels, and the cells undergo aggregation-disaggregation cycles. The formation of tight aggregates is correlated with the expression of specific post-aggregative poly(A)⁺ RNAs. These RNAs are not expressed in cells developing on glucoside gels, and the dispersal of tight aggregates on such gels is accompanied by the almost complete loss of these RNAs. A developmentally regulated membrane glycoprotein called contact site A, which is a marker of aggregation-competent cells, is normally expressed on glucoside gels. Cyclic AMP is also produced, indicating that the strong increase of adenylate cyclase activity during the preaggregation phase is not affected. In conclusion, cell contact with immobilized glucosides specifically inhibits postaggregative gene expression and arrests development at the aggregation stage.     

The role of C-4-substituted mannose analogues in protein glycosylation. Effect of the guanosine diphosphate esters of 4-deoxy-4-fluoro-D-mannose and 4-deoxy-D-mannose on lipid-linked oligosaccharide assembly.

The effects of the guanosine diphosphate esters of 4-deoxy-4-fluoro-D-mannose (GDP-4FMan) and 4-deoxy-D-mannose (GDP-4dMan) on reactions of the dolichol pathway in chick-embryo cell microsomal membranes were investigated by studies with chick-embryo cell microsomal membranes in vitro and in baby-hamster kidney (BHK) cells in vivo. Each nucleotide sugar analogue inhibited lipid-linked oligosaccharide biosynthesis in a concentration-dependent manner. GDP-4FMan blocked in vitro the addition of mannose to Dol-PP-(GlcNAc)2Man from GDP-Man (where Dol represents dolichol), but did not interfere with the formation of Dol-P-Man, Dol-P-Glc and Dol-PP-(GlcNAc)2. Although GDP-4FMan and Dol-P-4FMan were identified as metabolites of 4FMan in BHK cells labelled with [1-14C]4FMan, GDP-4FMan was a very poor substrate for GDP-Man:Dol-P mannosyltransferase and Dol-P-4FMan could only be synthesized in vitro if the chick-embryo cell membranes were primed with Dol-P. It therefore appears that the inhibition of lipid-linked oligosaccharide formation in BHK cells treated with 4FMan [Grier & Rasmussen (1984) J. Biol. Chem. 259, 1027-1030] is due primarily to a blockage in the formation of Dol-PP-(GlcNAc)2Man2 by GDP-4FMan. In contrast, GDP-4dMan was a substrate for those mannosyltransferases that catalyse the transfer of the first five mannose residues to Dol-PP-(GlcNAc)2. In addition, GDP-4dMan was a substrate for GDP-Man:Dol-P mannosyltransferase, which catalysed the formation of Dol-P-4dMan. As a consequence of this, the formation of Dol-P-Man, Dol-P-Glc and Dol-PP-(GlcNAc)2 may be inhibited through competition for Dol-P. In BHK cells treated with 10 mM-4dMan, Dol-PP-(GlcNAc)2Man9 was the major lipid-linked oligosaccharide detected. Nearly normal extents of protein glycosylation were observed, but very little processing to complex oligosaccharides occurred, and the high-mannose structures were smaller than in untreated cells.