Cell cycle changes during neural crest cell differentiation in vitro.

Chick trunk neural tubes containing neural crest cells were cultured in vitro. Cell outgrowth from these neural tube explants consists primarily of a small stellate cell population. After 3 days in culture the small stellate cell population undergoes a remarkable change in morphology that is characterized by a more refractile appearance in the phase contrast microscope. Subsequent to this change in morphology, pigment granules become visible in the cytoplasm after 4 days in culture. After 6 days in culture, virtually all of the small stellate cells are pigmented. The cell cycle parameters of the small stellate cell population are: S = 4.4 ± 1.2 hr (SD). G2 = 1.5 ± 1.0 hr (SD). M = 1.7 ± 0.6 hr (SD). and Gl = 3.8 ± 1.0 hr (SD). Continuous label experiments demonstrate that (G1+G2+M) increases from 7 hr in Day 4 cells, as yet unpigmented, to 12 hr in Day 5 cells that have become pigmented. This change is consistent with an increase in G1 and/or G2 that is closely correlated with the appearance of pigment granules. It is of interest that this cell cycle change is correlated with a rather late event in the developmental program of these neural crest cells rather than with the earlier morphological change observed after 3 days in culture.     

Cell surface changes during myoblast differentiation: preparation and carbohydrate composition of plasma membranes.

A technique for the preparation of plasma membrane from a skeletal muscle myogenic cell culture is described. Electron microscopic and enzymatic studies indicate that the preparation has kept its morphological integrity and has negligeable amount of cellular contiaminants. Carbohydrate composition studies have shown that the differentiated cells contain less hexosamine and sialic acid but accumulate glucose and galactose; the latter increase reflects the presence of a glucose-galactose-hydroxylysine unit which appears at the cell surface when myoblasts reach confluency.     

Cell surface changes during the differentiation of Dictyostelium discoideum : Interaction of cells with Concanavalin A

The parameters affecting the agglutination of cells of Dictyostelium discoideum by Concanavalin A (ConA) have been investigated. Under the incubation conditions employed, incubation time does not markedly affect agglutination, but there are distinct optima for cell density and gyration speed. Agglutination does not occur at low temperatures, but the transition temperature between the unagglutinated and fully agglutinated states is markedly influenced by ConA concentration. The rate of aggregation of strain NC-4 is considerably reduced by ConA. In contrast, the differentiation of strain Ax-2 in the presence of ConA is either unaffected or only slightly inhibited, depending on the incubation conditions. Succinylated-ConA binds to the same sites as the unmodified lectin, but has no effect on the differentiation of strain NC-4, suggesting that ConA binding sites are not directly involved in cell-cell contacts vital to the differentiation of D. discoideum. There is a gradual decrease in the susceptibility of cells of D. discoideum to agglutination by ConA as the cells pass from exponential growth phase to stationary growth phase in axenic medium and from vegetative amoebae to aggregates on a solid substratum. These results provide quantitative evidence for a gradual change in carbohydrate containing binding sites during differentiation.     

Use of a cDNA library for the study of mRNA changes during muscle differentiation

A cDNA library was used to measure changes in many individual mRNAs during muscle differentiation in culture. A library of 1000 clones was constructed from total myofiber poly(A) RNA. About 23% of these clones gave a detectable colony hybridization signal using end-labeled myofiber mRNA, the remainder containing muscle sequences too rare to be detected with this assay. The 230 positive clones were grouped into four classes based on relative visual intensity. Reconstruction experiments using pure globin mRNA enable us to determine the approximate percentage of total RNA made up by each mRNA hybridizing to a cDNA clone. Those clones containing sequences complementary to developmentally regulated mRNAs were identified by a differential hybridization procedure. The cDNA library was screened with end-labeled mRNA from both undifferentiated myoblasts and differentiated myofibers. Although the bulk of the clones hybridized essentially the same with both RNA populations, several dozen were found which hybridized differentially. Some clones contained sequences which were not present at all in myoblasts and present in very high quantities in myofibers. Others contained sequences found in both myoblasts and myofibers but in increased quantities in the differentiated cells. Still others contained sequences which decreased in quantity during muscle differentiation. The clones in the first group were chosen for immediate analysis since they likely contain contractile protein mRNA sequences. However, all the characterized cDNA clones can now be used as probes to study the chromosomal organization and developmental expression of genes active during muscle differentiation.     

Cell surface changes during preimplantation development in the mouse

Scanning electron microscopy reveals microvilli on all preimplantation stages, indicates that their number and length may be dependent on embryo size, and provides examples of regional alterations in their number. Cellular adherence, as evidence by interactions of microvilli, migration of cellular processes, and junctional complexes, increases during development and is accompanied by changes in the shapes of cells and embryos. Cell surfaces bordering the blastocoel differ markedly from the outer cell surfaces of the embryo.     

Quantitation of changes in cell surface determinants during skeletal muscle cell differentiation using monospecific antibody

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate anti-genie changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS-1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty-one clones were obtained which produced antibodies specific for myogenic cells. Thirty-five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.     

Cell interactions and the regulation of cholinergic enzymes during neural differentiation in vitro.

Seven-day-old chick embryo neural retina (NR), telencephalon (T), optic lobe (OL), and rembencephalon (Ro) were dissociated, and the resulting cell suspensions were allowed to reaggregate in vitro during 3 days either independently or in different binary combinations. Interactions could be detected by the comparison of the activity of the enzymes of the cholinergic system, choline acetyltransferase (CAT) and acetylcholinesterase (ACE), in “pure” and “combined” aggregates.The results clearly show that the activity of both enzymes in embryonic neural cells can be modified selectively by interactions between different cell populations. Thus, combined NR-OL aggregates show an increase in CAT without changes in ACE, NR-T an increase in CAT and a decrease in ACE, T-Ro a decrease in both CAT and ACE, and OL-T no changes at all. Experiments in which NR and OL cells were combined in different proportions indicate that the interactions require the presence of defined numbers of cells from each kind. Isochronous and heterochronous combinations of 7- and 10-day-old NR and OL cells show that the interactive capacities of the cells change with development.     

Remodelling of the nuclear periphery during muscle cell differentiation in vitro

We have examined the composition and ultrastructure of the nuclear periphery during in vitro myogenesis of the rat myoblast cell line, L6E9. Immunofluorescence labelling and immunoblotting showed that lamins A/C and B were all present in undifferentiated cells, but that they increased significantly before extensive cell fusion had occurred, with lamins A/C increasing proportionately more. Electron microscopic observations were consistent with these results, showing an increase in the prominence of the lamina during differentiation. On the other hand, immunofluorescence labelling suggested that the P1 antigen began to disappear from the nuclear periphery as the cells were fusing, after the increase in lamin quantity, and was no longer detectable in multinucleated cells. Unexpectedly, however, P1 was readily detected in isolated nuclei, whether prepared from myoblast or differentiated cultures, as well as in both myoblast and myotube nuclear matrices. It appears probable, therefore, that the fading of P1 labelling is due to masking of the epitope by a soluble factor recruited to the nuclear periphery as cells differentiate. These data, together with evidence that the genome is substantially rearranged during L6E9 myogenesis [Chaly and Munro, 1996], suggest that L6E9 cells are a useful model system in which to study the interrelationship of nuclear envelope organization, chromatin spatial order, and nuclear function. © 1996 Wiley-Liss, Inc.     

Alterations in purine nucleotide metabolism during muscle differentiation in vitro

Pathways of purine nucleotide metabolism affecting the availability of ATP in the muscle tissue were studied in differentiating rat muscle cultures. The rate of de novo purine nucleotide synthesis and of AMP deamination were found to increase markedly with cell differentiation, but the rate of IMP dephosphorylation was similarly low in both myoblasts and contracting fibers. The above differentiation-associated alterations in purine nucleotide metabolism conform with the greater need for ATP as a source of energy in the contracting myotubes.     

Passive cation movements in the Ehrlich ascites tumor cell: the effects of 2,4,6-trinitrobenzene sulfonic acid.

We have investigated the effects of 2,4,6-trinitrobenzene sulfonic acid (TNBS), an amino reactive reagent, on passive cation movements in Ehrlich ascites tumor cells. Incubation of tumor cells with TNBS (3 mM) results in a two phase association of TNBS with the cells. An initial, rapid phase, presumably at the level of the membrane, is independent of temperature, while the second phase increases linearly in time and is temperature dependent. Kinetic analyses of Na+ movements indicate that TNBS: (1) inhibits Na+ movement from a slowly exchanging cellular compartment, but is without effect on a more rapidly exchanging compartment; (2) does not alter net Na+ accumulation in transport-inhibited cells; and (3) is without effect on non-exchange Na+ efflux at 0 degrees C. The actions of TNBS on K+ movements depend upon temperature and the continued presence of TNBS in the environment. At 22 degrees C two minute exposure of the cells to TNBS leads to 77% inhibition of K+ efflux. With continued exposure to TNBS, the inhibition is only 42%. Reduction of the temperature to 0 degrees C decreases K+ efflux in control cells by 82%. Two minute exposure to TNBS enhances K+ efflux by 50%, while continuous exposure increases it by 144%. These results suggest: (1) TNBS interacts with several classes of membrane sites which are involved with the regulation of passive cation movements; and (2) passive Na+ and K+ movements across the cell membrane proceed by different pathways.     

A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation

A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha- smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha- smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm- 1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha- smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti- desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin- negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.     

Cell surface immunoglobulin XXI. appearance of IgD on murine lymphocytes during differentiation.

The differentiation of Ig-bearing lymphocytes in adult mice was studied by monitoring the appearance of IgD relative to IgM on the surface of splenocytes obtained from lethally irradiated animals reconstituted for various periods of time with adult bone marrow cells, neonatal splenocytes, or Ig- adult splenocytes. It was found that IgM appears before IgD on differentiating lymphocytes. Furthermore, the rate of appearance of IgD during differentiation of adult cells is similar to that observed with neonatal cells.     

Membrane microenvironmental changes during activation of human blood platelets by thrombin. A study with a fluorescent probe.

Membrane microenvironmental changes associated with thrombin-induced platelet activation were followed by fluorescence intensity and polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled human platelets. The labeling of washed platelets with DPH did not alter platelet intactness and morphology. In response to thrombin, DPH-labeled platelets exhibited reduced serotonin release, yet aggregation was barely inhibited. Shape change induced by thrombin or ADP was indistinguishable in control and in DPH-labeled platelets. During platelet aggregation induced by thrombin, fluorescence intensity increased by about 14%, which may indicate a more hydrophobic exposure of the probe. However, no change in fluorescence was detected during platelet shape change, induced either by thrombin in presence of EDTA or by ADP. Thrombin-activated platelets exhibited an increase in values of fluorescence polarization (P) during the stages of shape change and secretion, which further increased during aggregation. A similar pattern of increase in P values characterized platelet shape changes, caused either by thrombin in the presence of EDTA or by ADP. Changes in individual platelets are discernible from the alterations of the aggregating cells. These results may indicate that platelet activation is accompanied by an increase in rigidity of the membrane lipids. Functionally, the elevated "microviscosity" may reflect a primary role of membrane lipids in modulating the process of platelet activation or secondary transitions in lipids due to membrane events mediated by proteins.     

Cell surface interactions between Trypanosoma congolense and macrophages during phagocytosis in vitro.

Trypanosoma congolense bloodstream forms preincubated with a high titer of anti-variant surface antigen (VSG)-specific antibody, a low amount of anti-VSG plus complement-active mouse serum (MS), MS alone, and trypsin were cocultivated with mouse peritoneal macrophages in vitro. Immunofluorescence as well as transmission and scanning electron microscopy revealed that upon attachment to the macrophages' surface, trypanosomes opsonized with anti-VSG/MS formed opsonized filopodia, which were rapidly internalized by the phagocytes. Although these cells attached as frequently as anti-VSG or trypsin-pretreated parasites, the rate of phagocytosis of anti-VSG/MS pretreated trypanosomes was reduced significantly. Trypanosomes pretreated with high antibody titers alone were lysed on the surface of the macrophages before phagocytosis was completed. Parasites opsonized with complement alone adhered only occasionally and were rarely phagocytosed. Trypsin-treated trypanosomes, which served as positive control cells, rapidly attached and remained intact until ingulfment by the macrophages was completed. Untreated control parasites did not attach to the macrophages and were not phagocytosed. Cocultivation of macrophages with anti-VSG/MS-opsonized trypanosomes caused internalization of the flagellum by membrane fusion. Filopodia formation by T. congolense is thus correlated with a marked reduction in phagocytosis even in the presence of only a sublytic antibody titer.     

Calcium and the control of muscle-specific creatine kinase accumulation during skeletal muscle differentiation in vitro.

A polyacrylamide gel separation method for creatine kinase (CPK) isoenzymes is described, and its use to determine muscle-specific CPK (M-CPK) levels in skeletal muscle cultures is illustrated. In cultures in which cell fusion has been prevented by very low Ca²⁺ concentrations, the increases in M-CPK after 96 hr are similar to those in control cultures. Slightly higher concentrations of Ca²⁺, however, inhibit both cell fusion and M-CPK accumulation. As the calcium concentration is gradually increased further, cell fusion is permitted, followed, at even higher Ca²⁺ levels, by M-CPK accumulation. These effects can be obtained both by adding EGTA to the culture medium and by using Ca²⁺-free culture medium and varying the Ca²⁺ concentration directly. The latter method has the advantage that deleterious effects of EGTA on cell attachment and cell numbers do not occur, even at the lowest Ca²⁺ concentrations. By revealing dramatic effects on CPK levels of small changes in external Ca²⁺ concentrations, these observations may resolve conflicting data in the literature on the question of whether cell fusion is a prerequisite for muscle-specific protein synthesis. Possible mechanisms for the two effects of Ca²⁺ on CPK specific activity (permissive at very low, but inhibitory at intermediate, concentrations) are considered, including membrane mediation, mediation by changes in ionized cytoplasmic Ca²⁺ levels, and possible involvement of cyclic nucleotides.     

Anion transport in the Ehrlich ascites tumor cell: the effect of 2,4,6-trinitrobenzene sulfonic acid

We have investigated the effects of the amino reactive reagent, 2,4,6-trinitrobenzene sulfonic acid (TNBS) on anion transport (chloride and sulfate) and on the K⁺ content of Ehrlich ascites tumor cells. Incubation of tumor cells with TNBS (3 mM or 10 mM) results in a time dependent uptake of this molecule. Tightly bound TNBS caused a loss of K⁺ as well as inhibition of sulfate uptake. Although sulfate transport was inhibited by tightly bound TNBS (40% inhibition with 20 nmoles bound per 10⁷ cells), reversibly bound TNBS exerted much greater inhibition. Kinetic analysis of sulfate transport in the presence and absence of TNBS suggests that: (1) tightly bound TNBS exerts a competitive inhibition by occupying membrane sites remote from the specific transport site, (2) TNBS reversibly interacts with a separate site also in a competitive fashion. Increasing amounts of tightly bound TNBS resulted in an enhanced chloride influx. However, reversibly bound TNBS was without effect. These results are in contrast to the effect of TNBS on sulfate transport and show that TNBS, at least in this cell type, is not a general inhibitor of anion transport.     

Cell surface modifications in the epithelium of rat ventral prostate during adaptation to in vitro conditions: An ultrastructural study

Summary Sequential changes in epithelial cells of collagenase-dissociated rat ventral prostate were studied by thin-section and freeze-fracture electron microscopy. Epithelial cells did not attach to the substrate for 48 h. Pelleted cells obtained 1, 24, and 48 h after dissociation were assigned to three categories depending on morphology and cellular associations. (a) Solitary epithelial cells degenerated as determined by extensive vacuolization in the cytoplasm and aggregation of intramembranous particles (IMP). (b) Epithelial clusters consisted of a homogeneous population of well-maintained, closely packed cells. Aggregation of IMP was minimal. Tight junctions that formed between cells at the periphery of the clusters appeared normal and provided an effective permeability barrier demonstrated by the exclusion of ruthenium red tracer. (c) Tissue fragments were comprised of varying combinations of epithelial, endothelial, and smooth muscle cells as well as fibroblasts and erythrocytes. Maintenance of tissue fragments was variable. Plasma membranes often displayed aggregated IMP and proliferated tight junctional strands. An effective permeability barrier was absent. After the 48 h “latent period”, epithelial cells in the clusters lost interdependence, disassociated from one another, and attached to the substrate. These isolated cells, which did not display aggregated IMP, retained the ability to form an effective permeability barrier upon reaching confluency. During the first 48 h, epithelial cells did not tolerate solitary existence, yet as participants in clusters they were well maintained. After this interval, they no longer required interactions with neighbors in order to survive. These results indicate that under our experimental conditions, an adaptation period is required by prostatic epithelial cells. The enhanced quality of maintenance associated with epithelial clusters suggests that control over the internal microenvironment, provided by a tight junctional barrier, may be important during the initial period of adaptation in vitro. This work was supported by funds from NIH Grants CA 26063, 29513, and CA 15776; National Cancer Institute; DHHS; and Charlton Fund, Tufts University School of Medicine (awarded to P. K.).