The role of microvilli in the agglutination of cells by concanavalin A

Morphological correlates of lectin agglutinability were examined in eight cell lines of varying sensitivity to agglutination by concanavalin A (ConA). The number of microvilli on the surface of cells growing in monolayers was positively correlated with agglutinability. However, when cells were brought into suspension, they all developed numerous microvilli which persisted when the cells were treated with ConA regardless of whether or not they were agglutinated by the lectin. Treatment of cells with dibutyryl cyclic AMP (db-cAMP) and theophylline caused a parallel decrease in agglutinability and numbers of microvilli in monolayer cultures, but suspended cells from control and treated cultures were identical in appearance in the absence or presence of ConA. The surface morphology of cells agglutinated by ConA was very similar to that of cells that spontaneously agglutinated in the absence of the lectin, and surface bound ConA was rapidly withdrawn from microvilli on all cell types. Neither the morphology of cells nor the surface distribution of ConA can explain observed differences in agglutinability.     

Reversible inhibition of cAMP-induced axon formation in neuroblastoma cells by concanavalin a and vinblastine : Relationship to microtubules and cytotoxicity

The formation of axons induced by dibutyryl-adenosine 3′,5′-cyclic monophosphate (db-cAMP) in neuroblastoma cells was inhibited by concanavalin A (ConA) and vinblastine. These compounds also caused the retraction of existing axons. After removal of ConA or vinblastine, addition of db-cAMP again resulted in axon formation. The cytotoxicity of ConA and vinblastine for neuroblastoma cells was reduced when cell multiplication was inhibited by db-cAMP. Linearly growing normal fibroblasts were also more sensitive to the cytotoxic effect of ConA than confluent non-multiplying fibroblasts. The effects of ConA and vinblastine were additive both in their effects on axon formation and cytotoxicity. Wheat germ agglutinin (WGA) and lumicolchicine did not affect axon formation or reduce cell viability. It is suggested that ConA bound to the cell surface can interfere with the assembly of cytoplasmic microtubules involved in axon formation and cell division.     

Reaction of lectins with human erythrocytes : III. Surface charge density and agglutination

A number of commercially available enzymes were used to modify the cell surface of human erythrocytes to varying degrees. In protease-treated erythrocytes the decrease in surface charge (determined by cell electrophoresis or analysis of sialic acid content) correlates with an increase in agglutinability with concanavalin A (ConA) and wheat germ agglutinin (WGA). On the other hand, treatment with neuraminidase leads to very large decrease in surface charge with only an intermediate increase in agglutinability with both lectins. Subsequent protease treatment of these cells enhances their agglutinability appreciably without further altering their surface charge. It is concluded that the increased agglutinability following protease treatment is due both to a decrease in the net negative charge and a removal of peptides and glycopeptides from the cell surface that may sterically hinder the agglutination reaction.     

Relationship between cyclic AMP, microtubule organization, and mammalian cell shape : Studies on Chinese hamster ovary cells and their variants

Treatment of Chinese hamster ovary cells with N⁶,2′-O-dibutyryl adenosine cyclic 3′,5′-monophosphate (db-cAMP) and hormones converts their shape from a knobbed, epithelial-like morphology to a smooth fibroblast-like form. Ultrastructural studies demonstrate an increased number of microtubules and their arrangement in parallel array along the long axis of the cell after treatment with these agents. Although an epithelial-like variant, when treated with db-cAMP, shows an increase in the number of microtubules; these microtubules remain in a disorganized nonparallel array. The numerous long microtubules which are already present in a fibroblast-like variant become further elongated when the cells are treated with db-cAMP. These experiments establish the relationship between cAMP level, assembly and organization of microtubules, and cell shape in cultured Chinese hamster cells.     

The chronic effects of db-cAMP on cell cycle parameters and cell size in asynchronous culture of Chinese hamster ovary cells

N⁶,O^2′-dibutyryl adenosine 3′,5′-cyclic-phosphate (db-cAMP) has been shown to convert Chinese hamster cells of ovarian origin (CHO-K₁) from compact, randomly oriented cells growing in multilayers to elongated fibroblast-like cells which grow in monolayers. This compound also has been reported to have a variety of effects on the cell cycle. Most such studies have employed synchronized cells to determine cell cycle effects, and consequently have been limited to the short-term effects of the compound. We have looked for chronic effects on the cell cycle in cultures exposed continuously to db-cAMP from the initiation of the cultures until they had reached or approached the plateau phase. This was done by combined autoradiography and Feulgen microspectrophotometry plus measurements of the protein content of mitotic cells to detect any influence on cell size. The overall results were that continuous exposure to db-cAMP had at most only minor effects on the cell cycle and cell size when the culture medium was renewed daily. Somewhat greater effects were found on plateau-phase cells in cultures in which the medium was not renewed. In this case fewer cells appeared to remain in the cell cycle in the cultures with db-cAMP. Comparison with our earlier results with Chinese hamster V79 cells led to the conclusions that cell cycle parameters and cell size at mitosis were less altered during culture growth in CHO cells, but that CHO cells seemed to be less able to maintain cells in the cell cycle in crowded cultures.     

CYCLIC AMP MEDIATES THE CONCANAVALIN A AGGLUTINABILITY OF MOUSE FIBROBLASTS

We have devised a quantitative way to measure the agglutination of cells which utilizes the size discrimination feature of an automatic particle counter. With this method we have studied the agglutinability by concanavalin A of 3T3 cells, a mutant of 3T3 cells (3T3cAMP^tcs) in which cyclic AMP levels fall when the cells are subjected to temperature change or fresh serum, and L929 cells. We find with 3T3cAMP^tcs cells that low levels of cyclic AMP correlate with increased agglutinability and that high levels of cyclic AMP correlate with decreased agglutinability. Prior treatment of these cells with a cyclic AMP phosphodiesterase inhibitor or Bt₂cAMP blocks the increase in agglutinability induced by temperature change. When 3T3 cells are treated with fresh serum, their agglutinability also increases although to a much smaller extent than with 3T3cAMP^tcs cells. Cells change their agglutinability very rapidly. Treatment of L929 cells for 15 min with 1-methyl-3-isobutyl xanthine at 1 mM decreases their agglutinability to the level of normal 3T3 cells. We conclude that in normal and transformed cells the level of cyclic AMP regulates agglutinability.     

Modulation of agglutinability by alteration of the surface topography in mouse ascites tumor cells

Factors involved in controlling agglutinability of cells with plant lectins include number, distribution, availability and mobility of cell surface lectin receptor sites. We have examined the concanavalin A (ConA)-mediated agglutination of mouse sarcoma 180 ascites tumor cells in the presence or absence of cytochalasin B (CB) using a quantitative electronic particle counter assay. These cells become substantially more agglutinable after brief treatment with low concentrations of CB. Scanning and transmission electron microscopy indicate that CB causes formation of large, broad, cell surface ruffles and loss of narrow projections that appear to be microvilli. Studies using fluorescent ConA suggest that lectin receptor sites concentrate on these ruffles and that the ruffles seem to directly mediate increased agglutinability in this system. Electron spin resonance studies suggest that CB does not alter lipid “fluidity” in these cells. The results indicate that the gross cell surface topography favoring high agglutinability is one displaying broad ruffles, not numerous narrow projections.     

Cell surface microvilli and cell agglutinability.

Detached cells of some transformed mouse fibroblast lines have a villous surface whereas similarly treated cells of other lines are relatively smooth. These differences in surface morphology of detached cells are not reflected in their agglutinability with ConA and they cannot unambigously be explained from their morphology in situ. Treatments of normal and transformed Swiss mouse fibroblasts that induce marked changes in agglutinability with ConA do not cause equivalent changes in surface morphology. It is, therefore, unlikely that agglutinability of mouse fibroblasts by ConA is determined by the number of microvilli on the cell surface.     

Multiple modes of dibutyryl cyclic AMP-induced process formation by clonal nerve and glial cells

Exposure of clonal nerve and glial cells to dibutyryl adenosine-3′,5′-monophosphate (db-cAMP) induced processes. These cellular processes were generated either by the extension of a process (growth) or by retraction of parts of an extended cytoplasm (shrinkage). In all cases, these cells from the central nervous system rounded up in response to db-cAMP, rather than spread on the substratum.     

Modulation of serum-stimulated DNA synthesis in cultured human fibroblasts by cAMP

Density-dependent inhibition of growth of cultured human fibroblasts was associated with a 3- to 4-fold rise in the intracellular concentration of cyclic AMP (cAMP). Serum lowered cAMP levels in 2–5 min, with the low levels persisting for several hours. When quiescent fibroblast cultures were treated with 10% serum, the incorporation of [³H]TdR into DNA increased after a 10–16 h lag, reaching a peak by 20–24 h. Dibutyryl cyclic AMP (db-cAMP), when present throughout serum treatment, produced a dose-dependent inhibition of [³H]TdR incorporation. Half-maximal inhibition was seen with 0.1 mM db-cAMP. When db-cAMP or another cyclic nucleotide phosphodiesterase inhibitor, l-methyl-3-isobutylxanthine (SC-2964), was added together with serum to maintain elevated cAMP levels and after 4 h was replaced with fresh serum-containing medium, the wave of DNA synthesis induced by serum was not delayed. This implied that stimulation by serum could occur without an initial decrease in cAMP concentration. In contrast, db-cAMP added 8 h later than serum and not removed, inhibited [³H]TdR incorporation at the peak to the same extent as db-cAMP added together with serum. The inhibition decreased progressively when db-cAMP was added more than 8 h after serum. These results suggested that a cAMP-sensitive step occurred approx. 8 h after the addition of serum in mid-G1 of the cell cycle. Results obtained using fibroblasts synchronized at the G1/S boundary with hydroxyurea or exposed to db-cAMP for 24 h suggested that db-cAMP also inhibited TdR incorporation at the G1/S interphase or during S phase. Thus, whereas reduced cAMP concentrations did not appear to serve as an initial trigger for serum-stimulated DNA synthesis in human fibroblasts, db-cAMP and SC-2964, presumably by elevating cAMP levels, appeared to act in mid-G1 and possibly at the G1/S boundary or within S phase to inhibit thymidine incorporation.     

Change of concanavalin A induced agglutinability during preimplantation mouse development

Mouse embryos during early cleavage (zygote to eight-cell stage) were agglutinable with a low concentration (10 μg/ml) of concanavalin A (ConA). This agglutinability was reduced during the first mitotic division. Morulae were agglutinable with a slightly higher concentration (100 μg/ml), whereas blastocysts were not agglutinable even with ConA at a concentration of 5000 μg/ml; however, isolated inner cell masses agglutinated readily at 10 μg/ml of ConA. Embryos grown in vitro behaved as did those isolated directly from the genital tract. Treatment with proteolytic enzymes did not induce agglutinability of mouse blastocyst. The change in agglutinability of trophoblastic cells reflects dramatic changes in the cell surface.     

The repair of the surface structure of animal cells

Experiments were performed to determine if animal cells in culture possess specific mechanisms to repair surface molecules damaged by enzymes. The surface membranes of a primary cell culture, chick fibroblasts, a permanent hamster cell line, BHK₂₁/C₁₃, and its virally transformed counterpart, C₁₃/B₄ were damaged by exposure to trypsin or to neuraminidase. Following digestion with trypsin, the incorporation of radioactive amino acids or sugars into purified surface membrane of cells was monitored. No differences were noted in rates of incorporation when control and trypsin-damaged cells were compared. Neuraminidase damage to the surface of BHK₂₁/C₁₃ and C₁₃/B₄ cells was evidenced by altered gel filtration profiles of surface glycopeptides, i.e., delayed elution because of reduction in size. By labelling cells with ¹⁴C-L-fucose prior to neuraminidase treatment and following the incorporation of ³H-L-fucose into cell surface glycopeptides after neuraminidase digestion, we were able to monitor the synthesis and turnover of fucose-containing glycopeptides in the same cells. Gel filtration profiles indicated that little or no desialylated glycoproteins were resialylated (repaired) by specific replacement of sialic acid. Comparing neuraminidase-digested and control cells we observed no difference in rates of ³H-L-fucose incorporation or of ¹⁴C-L-fucose loss from these cells; nor did we find differences in the rate of incorporation of isotopic glucosamine into sialic acid. Neuraminidase treatment failed to alter the rate of cell growth or the pattern of isotopic incorporation into various cell surface components. These results support the suggestion that return of sialic acid (repair) was effected by turnover which serves as a non-specific repair mechanism to replace damaged cell surface molecules (Warren and Glick '68; Warren, '69).     

The ultrastructure of process formation following treatment with db-cAMP of a Chinese hamster ovary X Chinese hamster brain cell hybrid

Scanning (SEM) and transmission (TEM) electron microscopy studies were performed on a hybrid, resulting from the Sendai-virus fusion of a Chinese hamster ovary (CHO) Glycine A (Gly⁻A) auxotrophic mutant cell [1,2] with a freshly-biopsied suspension of Chinese hamster cerebral cortex cells. In normal growth medium the hybrid differs from the CHO parental cell in displaying a squamous, polygonal, epithelioid appearance with sparse microvilli and ribosome-filled knobs (blebs). Slender filopodia, which sometimes reach a length of 25 μm, extend from interphase cells. Bundles of microfilaments (6 nm diameter) are observed closely associated with the cell membrane and the perinuclear region, arranged more or less in parallel to the glass substrate. The untreated hybrid has a relatively unpatterned arrangement of microtubules and reveals desmosomes at points of cell contact. When treated with N⁶,O²-dibutyryl adenosine 3′:5′-cyclic monophosphoric acid (db-cAMP) plus the Synergist testololactone, the response of the hybrid differs markedly from the fibroblastic habitus assumed by CHO [3, 4, 5]. The hybrid cells become stellate, forming processes or cytoplasmic extensions which radiate from a central, microvillus-covered, rounded, cell body. The arborizing processes number 2–8 per cell and form a contiguous network between cells of a colony. Desmosomes are seen at these points of process-to-cell body junctions. Parallel microtubules, 10 nm filaments, and 6 nm microfilaments, as well as organelles of the unstimulated cytoplasm such as free ribosomes, lipid granules, mitochondria, rough ER, and myelin figures are present in the nerve-like extensions. Knobs disappear completely following cAMP treatment. On removal of the db-cAMP, disappearance of the processes is apparent in 1–2 h so that the cell returns to its original morphology. This reversal is accompanied by ruffling activity at the cell borders. The central, rounded portion of the cell returns to the former flattened state somewhat more slowly. These studies demonstrating a cAMP-induced change in morphology and microtubule arrangement, produced in CHO X brain cell hybrids support the previous proposals that: (1) cAMP action is necessary for organization of cellular microtubules to form a pattern; (2) this pattern is a function of the cellular differentiation state and is determined by genetic or epigenetic factors.     

Culture of cardiac muscle cells in serum-free media

Cardiac muscle cells from neonatal rats have been cultured in completely defined serum-free media. The most successful system consists of precoating culture flasks with fibronectin at a concentration of 5 μg/cm² of surface area and adding fetuin and either dibutyryl cyclic AMP (db-cAMP), cholera toxin, epidermal growth factor or insulin plus dexamethasone to the medium. In order to define a serum- and a hormone or growth factor-free medium, cardiac muscle cells were grown in the presence of fibronectin, fetuin and db-cAMP for 4 days, after which time db-cAMP was omitted from the medium. Under these conditions the cells continue to maintain their differentiated morphology for at least 4 days thereafter. These morphological studies demonstrate that dissociated neonatal cardiac muscle cells are able to grow and differentiate in a chemically defined medium in the absence of animal serum.     

Differential effects of various cAMP derivatives on the morphological and electrical maturation of neuroblastoma X glioma hybrid cells

The influence of adenosine 3′: 5′-cyclic monophosphate (cAMP) and some of its derivatives on the morphological differentiation and on the expression of electrical activity was investigated in neuroblastoma X glioma hybrid cells. This permanent cell line constitutes a well established culture system for studying neuronal properties in vitro. cAMP (1 mM) caused cell death. With 8-bromo-cAMP (0.1–1 mM) present giant multinuclear cells appeared, which were more obvious at 0.1 than at 1 mM 8-bromo-cAMP. 8-p-chlorophenylthio-cAMP (0.1–1 mM) induced an extension of neurites. These cellular processes were comparable to those elicited in the presence of db-cAMP (1 mM). However, only the cells treated with db-cAMP, but not those exposed to 8-p-chlorophenylthio-cAMP, were found to generate action potentials upon electrical stimulation. Neither dexamethasone nor carboxymethylcellulose, nor 8-bromo-cAMP could elicit the formation of processes in hybrid cells.     

Glycopeptide fractions prepared from purified central and peripheral rat myelin

Myelin was purified from rat brain and sciatic nerve after invivo labeling with [³H]fucose and [¹⁴C]glucosamine to provide a radioactive marker for glycoproteins. The glycoproteins in the isolated myelin were digested exhaustively with pronase, and glycopeptides were isolated from the digest by gel filtration on Bio-Gel P-10. The glycopeptides from brain myelin separated into large and small molecular weight fractions, whereas the glycopeptides of sciatic nerve myelin eluted as a single symmetrical peak. The large and small glycopeptide fractions from central myelin and the single glycopeptide fraction from peripheral myelin were analyzed for carbohydrate by colorimetric and gas liquid chromatographic techniques. The glycopeptides from brain myelin contained 2.4 μg of neutral sugar and 0.59 μg of sialic acid per mg total myelin protein, whereas sciatic nerve myelin glycopeptides contained 10 μg of neutral sugar and 3.8 μg of sialic acid per mg total protein. Similarly, the gas-liquid chromatographic analyses showed that the glycopeptides from peripheral myelin contained 4- to 7-fold more of each individual per mg total myelin protein than those from central myelin. Most of the sialic acid and galactose in the glycopeptides from central myelin were in the large molecular weight fraction, and the small molecular weight glycopeptides contained primarily mannose and $\text{N-}\text{acetylglucosamine}$. The considerably higher content of glycoprotein-carbohydrate in peripheral myelin supports the results of gel electrophoretic studies, which indicate that the major protein in peripheral myelin in glycosylated while the glycoproteins in purified central myelin are quantitatevely minor components.     

Binding of I-Concanavalin a and agglutination of embryonic neural retina cells : Age-dependent and experimental changes

Embryonic chick neural retina cells dissociated from retina tissue by treatment with EGTA (a calcium chelator) show an age-dependent decline in ability to agglutinate with concanavalin A (ConA). This developmental change in cell surface properties is not due to loss of ConA-binding sites, since mature retina cells can be rendered agglutinable by mild trypsinization. It is also not due to masking of ConA receptors, or to a decrease in their amount, since retina cells from late embryos (19 days) bind four times as much ¹²⁵I-ConA as cells from early embryos (8 days). Our findings lead us to suggest that, as the retina differentiates the lateral mobility of ConA receptors in the cell membrane decreases resulting in a reduction of cell agglutinability; trypsinization of late embryo retina cells increases the mobility of the receptors and thereby facilitates their clustering by the lectin into a configuration conducive to cell agglutination.The ability of late embryo (19 day) retina cells dispersed with EGTA to agglutinate with ConA could be increased by still other treatments: by pre-incubation of the cell suspension in Tyrode's balanced salt solution (1 h, 37 °C); and by brief pre-exposure to glutaraldehyde. These two treatments did not enhance cell agglutination with wheat germ agglutinin (WGA). Glutaraldehyde treatment of trypsinized cells made them agglutinable with ConA also at 4 °C; cells treated otherwise agglutinated only at higher temperature. Surface-saturation of monodispersed retina cells with ConA at 37 °C—but not at 4 °C—prevented their agglutination with this lectin, but not with WGA; this inhibition was reversible by methyl a-D-glucopyranoside (αMG).     

The measurement of cyclic GMP and cyclic AMP phosphodiesterases

Methods are described for measuring phosphodiesterases for cGMP and cAMP in the range of activity yielding 10⁻¹² to 10⁻⁸ mol of product. The 5′-GMP formed is measured by conversion to GDP with guanylate kinase. Amounts of GDP greater than 10⁻¹⁰ mol are measured directly with an enzyme system which results in stoichiometric oxidation of NADH. This is either determined by the decrease in fluorescence or the excess NADH is destroyed with acid and the NAD⁺ measured by its fluorescence in strong NaOH. With smaller amounts of GDP, sensitivity is amplified 1000-fold with the succinic thiokinase-pyruvate kinase cycle. In the case of cAMP diesterase, larger amounts of 5′-AMP are measured in the same way as 5′-GMP, except that adenylate kinase is substituted for guanylate kinase. With smaller amounts, the 5′-AMP is converted to ATP, and sensitivity is amplified with the adenylate kinase-pyruvate kinase cycle. As little as 20 ng dry weight of average brain is sufficient for accurate assay of the diesterase activity toward either cAMP or cGMP. When there is danger of significant destruction of AMP or GMP by tissue 5′-nucleotidase, this is prevented by adding GMP to the cAMP reagent, AMP to the cGMP reagent, or 5′-UMP to either reagent.     

Purification of plasma membranes of rat mammary gland : Comparisons of subfractions with rat milk fat globule membrane

Partially purified plasma membranes of rat mammary gland, obtained as light (F₁) and heavy (F₂) fractions by flotation on a discontinuous sucrose density gradient, were further fractionated by density perturbation flotation using digitonin to shift the density of the cholesterol-rich portion of the membranes. The shifted fraction (F₁F₃) of digitonin-treated F₁ was highly enriched in 5′-nucleotidase, cholesterol and sialic acid, but free of galactosyltransferase, suggesting that it contained highly purified plasma membranes. The unshifted fraction (F₁DF₁) was enriched in galactosyltransferase and depleted in nucleotidase, cholesterol and sialic acid, suggesting that it contained Golgi fragments. The F₂ fraction shows substantially different behavior. Part of it re-equilibrates to the F₁ position upon reflotation. When treated with digitonin, part of F₂ is shifted to a higher density (F₂DF₃). F₂DF₃ is enriched in 5′-nucleotidase, cholesterol, sialic acid and galactosyltransferase. These properties suggest that this subfraction comes from a plasma membrane containing galactosyltransferase.The sialoglycoproteins of the various fractions were compared with those of rat milk fat globule membrane, which is derived in part from the apical surface of the mammary secretory cell. Dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals two major glycoprotein bands (GP-II and GP-III) in F₁DF₃. F₂DF₃ contains these and an additional band of lower mobility (GP-I). Both crude and purified MFGM contain all three bands. Comparisons of peanut lectin receptors by autoradiography of polyacrylamide gels run in SDS and then treated with [¹²⁵I]peanut lectin also suggest that F₂DF₃ is more similar to the milk fat globule membrane than is F₁DF₃. However, analysis of the membrane polypeptides and concanavalin A (ConA) receptors shows no obvious relationship between milk fat globule membrane and any of the isolated mammary membrane fractions. These results indicate that the relationship between the milk fat globule membrane and mammary membranes is complex, possibly involving components not associated with the mammary plasma membrane or only selected components of the plasma membrane.     

Cyclic AMP is one of the intracellular signals during the mating of Chlamydomonas eugametos

Chlamydomonas eugametos gametes of opposite mating type make cell-cell contact via their flagellar surfaces. This contact triggers an increase in the intracellular level of cyclic AMP (cAMP) and several cellular responses which are necessary for cell fusion. Here, we show that wheat-germ agglutinin, which binds to the flagellar surface and induces all mating responses, also increased the intracellular cAMP level. Dibutyryl-cAMP added to non-mating gametes induced flagellar twitching, cell-wall lysis, mating-structure activation, flagellartip activation and an increase in agglutinability. It did not induce agglutinin transport to the flagellar tip (tipping) and may not be the direct cause of flagellar twitching and flagellar-tip activation. In non-illuminated cells, dibutyryl-cAMP was far more effective in evoking mating reactions than in illuminated cells. Light induced a 50% decrease in the cAMP level within 1 min. Adenylate cyclase was found to be associated with cell membranes but only 8% of the total was present in the gamete flagella.Abbreviations db-cAMP dibutyryl-cAMP - FTA flagellar tip activation - Mab monoclonal antibody - mt ^–/mt⁺ mating-type minus/plus - WGA wheat-germ agglutinin We gratefully acknowledge the fruitful discussions with Dr. Rainer Gilles of the Department of Biochemistry at the University of Cologne (FRG), and the advice generously given by Dr. Roel van Driel of the Department of Biochemistry at the University of Amsterdam (The Netherlands).