Differences in accumulation and phosphorylation of proteins in vegetatively growing and aggregation-competent cells of Dictyostelium discoideum.

A comparison of proteins from whole cell lysates of vegetative amoebae and aggregation-competent cells by high-resolution two-dimensional gel electrophoresis coupled with a sensitive silver staining method revealed distinct differences. In aggregation-competent cells, 16 proteins present in the vegetative amoebae disappeared, and 25 new proteins appeared. A few other proteins showed quantitative variation during the transition of vegetative amoebae to aggregation competence. Identification of phosphoproteins by in vivo labeling with [32P]orthophosphate showed that none of the developmentally regulated cellular proteins were modified. Phosphorylation was observed in four proteins. One protein was phosphorylated exclusively in aggregation-competent cells. The phosphorylation level of two other proteins was higher in aggregation-competent cells compared with vegetative amoebae. The data suggest that phosphorylation of cellular and certain ribosomal proteins may be regulated coordinately in Dictyostelium discoideum.     

Protein kinases of Dictyostelium discoideum, strain AX-2.

In cell homogenates of Dictyostelium discoideum, strain AX-2, four major soluble protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) and one membrane-associated protein kinase activity were identified. The enzymes showed high affinity for casein. One of the enzymes was purified by affinity chromatography on casein-coated Sepharose. The soluble high molecular weight enzymes phosphorylated histones, whereas the low molecular weight enzymes did not. The same protein kinase species were present in vegetative and aggregation-competent cells. Their specific activity, however, changed during the development to aggregation competence. None of the enzymes was stimulated by cyclic AMP or cyclic GMP, regardless of their origin from vegetative or aggregation-competent cells.     

Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium discoideum

Membrane interaction in aggregating cells of Dictyostelium discoideum can be blocked by univalent antibodies directed against specific membrane sites. Using a quantitative technique for measuring cell association, two classes of target sites for blocking antibodies were distinguished and their developmental dynamics studied. One class of these sites is specific for aggregation-competent cells, their quantity rising from virtually 0-level during growth, with a steep increase shortly before cell aggregation. The serological activity of these structures is species specific; they are not detectable in a nonaggregating mutant, but present in a revertant undergoing normal morphogenesis. Patterns of cell assembly in the presence of antibodies show that selective blockage of these membrane sites abolishes the preference for end-to-end association which is typical for aggregating cells. A second class of target sites is present in comparable quantities in particle fractions from both growth-phase and aggregation-competent cells. Blockage of these sites leads to aggregation patterns in which the side-by-side contacts of aggregating cells are abolished. The target sites of aggregation-inhibiting antibodies are suggested to be identical or associated with the molecular units of the cell membrane that mediate cell-to-cell contacts during aggregation. The results indicate that in one cell, two independent classes of contact sites can be simultaneously active.     

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.     

AGGREGATELESS MUTANT DEFECTIVE IN SIGNALING AND RELAYING IN THE CELLULAR SLIME MOLD, DICTYOSTELIUM DISCOIDEUM

An aggregateless mutant (HT41), isolated from D. discoideum NP14, was examined for the parameters involved in cell aggregation. HT41 cells were induced to express some of these parameters by cyclic AMP pulses. Spontaneous oscillations in light scattering were not observed in the suspension of the mutant cells. A pulse of cyclic AMP caused a monophasic decrease in light scattering, which was similar to the response of pre-aggregation cells of the wild-type. When cyclic AMP pulses were applied on a cell layer, HT41 cells aggregated without forming the streams. These results suggest that HT41 is a mutant defective in signal emission and relay.     

Induction of cell-specific ribosomal proteins in aggregation-competent nonmorphogenetic Dictyostelium discoideum

Vegetatively growing amoebae, if shaken in a starvation (nonnutrient) buffer, acquired aggregation competence, but do not embark on a morphogenetic program. The quantitative variation of ribosomal proteins in vegetative and aggregation-competent cells was compared by labeling the different cell types with [35S]methionine. Vegetative cells were examined at various phases of the growth cycle. No changes could be detected in the content of ribosomes or the apparent stoichiometry of ribosomal proteins in growing cells. In stationary phase cells, the net ribosome content declined to 15% of that observed in logarithmic phase, but the relative amounts of individual ribosomal proteins were not altered. Although aggregation-competent cells contained 30% less ribosomes compared with logarithmic phase cells, the total fraction of newly made ribosomal proteins was the same in both. In contrast to vegetative cells, distinct changes were induced in the ribosomal proteins of aggregation-competent cells. The composition of ribosomes in aggregation-competent phase resembled in every respect that observed in spore cells. As reported earlier, changes were found in all 12 of the developmentally regulated ribosomal proteins. For the majority of newly made ribosomal proteins during aggregation competence, the stoichiometry was similar to that in logarithmically growing cells. However, the relative synthesis of some was particularly higher (13- to 46-fold for A and L; 3- to 8-fold for D, E, S24, L3, S6, and L4) compared with logarithmic phase cells. About 18 proteins, which included the cell-specific ribosomal proteins L18, S10, S14, S16, and L11, were synthesized in lesser amounts than in logarithmic phase cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adhesion of Dictyostelium discoideum cells to carbohydrates immobilized in polyacrylamide gels. II. Effect of D-glucoside derivatives on development

In an accompanying report (Bozzaro, S., and Roseman, S. (1983) J. Biol. Chem. 258, 13882-13889), evidence is presented that the slime mold Dictyostelium discoideum contains three cell surface receptors specific for D-glucose, D-mannose, and N-acetyl-D-glucosamine, respectively. The synthetic probes used for these studies consisted of the sugars covalently linked to polyacrylamide gels. In the present experiments, starved cells were placed on these and other immobilized sugars to determine whether the sugar derivatives influenced normal development in this organism. When D. discoideum cells are on a solid surface under water, they form aggregation centers and strands of cells (which radiate from the center), send "signals" i.e. pulses of cyclic AMP from the center down the strands, and finally, after cells in the strands migrate to the center, form tight aggregates. These results were obtained on all polyacrylamide gel derivatives tested except one class, derivatives of D-glucose (O- and S-glucosides, cellobiosides, and maltosides). On these gels, aggregation centers and strands formed normally, but at a certain point stopped "signaling" and suddenly dissociated, with the cells rapidly migrating away from one another by negative chemotaxis (see Appendix to this report). Furthermore, a simultaneous dissociation of several centers was often observed. Following a brief period of random movement after dissociation, aggregation centers once again formed and the cycle was repeated. This cycle was repeated as often as 30 times or more over a 24-h period. The cells on the glucoside gels became aggregation-competent at the same time as the control cells, and the adhesion-dissociation cycle appeared to have no effect on the synthesis of some developmentally regulated proteins, such as UDP-glucose pyrophosphorylase. Interpretations of the phenomenon and its potential for studying gene regulation in this organism are discussed.     

Migratory behavior of cells on embryonic retina basal lamina

In order to study cell translocation in vitro on a physiological substrate a novel cell migration assay was developed using the inner limiting membrane of the avian embryonic retina. The matrix sheet consists of a laminin-rich basal lamina covered by a dense layer of neuroepithelial endfeet. The retina basal lamina does not contain fibronectin. Cells translocating on this substrate displace the neuroepithelial endfeet, leaving behind tracks in the endfeet monolayer. Motility of cells and the relative forward to lateral migration can be quantitated by measuring lengths, widths, and areas of the tracks. Using this assay system, the conditions and patterns of cell migration for a variety of cells have been examined. In the absence of serum all cell types show only minor migratory activity and addition of serum to the culture medium always enhances the rate of cell migration in a saturable, dose-response manner. The serum cannot be replaced by fibronectin or vitronectin (serum spreading factor). For maximum cell migration, serum has to be constantly present in the medium; however, 58% cell migration is obtained in serum-free medium when the matrix is preincubated with serum. According to the area and linearity of the tracks, the migratory behavior of the different cells can be classified into three groups: (i) fibroblasts and the nonpigmented Bowes melanoma cells form straight and long tracks; (ii) glioma, sarcoma, and carcinoma cells from straight but short tracks, and (iii) neuronal tumor cells, epithelial cells, and pigmented B16 melanoma cells form wide and short tracks. Comparative studies with low and high metastatic clones of tumorgenic cell lines show that migratory activity and metastatic potential of cells do not necessarily correlate. Finally, we show that fibroblasts deposit fibronectin fibrils on their paths as they migrate on the basal lamina. Fibronectin trails are also seen when fibroblasts are cultured on plain basal laminae that are pretreated with detergent to remove the endfeet monolayer. Likewise, when fibroblasts are cultured in the presence of antifibronectin antibodies, the fibronectin secreted by cells is detectable. Due to antibody treatment the cellular fibronectin is precipitated and its normal fibril formation is inhibited; however, the translocation of fibroblasts is not impaired.     

CHANGES IN CHARGED GROUPS ON THE CELL SURFACE DURING DEVELOPMENT OF THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM: AN ELECTRON MICROSCOPIC STUDY

The distribution of charged groups on the surface of Dictyostelium cells and their change during development were examined by electronmicroscopy using cationic and anionic ferritins. The number of anionic sites on the cell surface decreased greatly during the course of development. The whole surface of vegetative cells stained strongly with cationic ferritin (CF). On the other hand, the surface of aggregation-competent cells had fewer negative charges and these were unequally distributed, the surface of the advancing area (lobopodial region) being devoid of anionic sites. The number of anionic sites on the cell surface decreased progressively during further development, and the suface of slug cells did not stain at all with CF. The cell surface did not stain with anionic ferritin at any developmental stage, indicating the absence of detectable cationic sites. The biological significance of these findings is discussed in connection with cell adhesiveness and movement.     

Dictyostelium discoideum surface changes elicited by high concentrations of cAMP

The effects of high concentrations of cAMP on both morphological and biochemical development of Dictyostelium discoideum amebae are reported. Observations using light and scanning electron microscopy (SEM) indicate that the cells' response to such treatment varies with the length of time they had been starved prior to cAMP addition. Vegetative and early developmental amebae become rounded within a short period after treatment. Such cells are capable of undertaking a normal aggregation after a delay of a few hours. A substantial induction of phosphodiesterase activity is elicited from these cells by cAMP treatment but their levels of cAMP surface binding sites remain low. cAMP addition to aggregation competent cells causes amebae first to flatten and then to retract into spherical forms and group into small aggregates. No induction of phosphodiesterase activity is observed in such cells and the levels of cAMP binding sites present on the amebae decrease rapidly. The data are discussed in terms of the different states of cAMP-sensitivity between vegative and aggregation-competent amebae.     

Analysis of migratory behavior of neural crest and fibroblastic cells in embryonic tissues

The precise migration of neural crest cells is apparently controlled by their environment. We have examined whether the embryonic tissue spaces in which crest cells normally migrate are sufficient to account for the pattern of crest cell distribution and whether other migratory cells could also distribute themselves along these pathways. To this end, we grafted a variety of cell types into the initial crest cell migratory pathway in chicken embryos. These cell types included (a) undifferentiated neural crest cells isolated from cultured neural tubes, intact crest from cranial neural folds, and crest derivatives (pigment cells and spinal ganglia); (b) normal embryonic fibroblastic cells from somite, limb bud, lateral plate, and heart ventricle; and (c) a transformed fibroblastic cell line (Sarcoma 180). Crest cells or their derivatives grafted into the crest migratory pathway all distributed normally, although in contrast to the result when neural tubes were graftedin situ, fewer cells were observed in the epithelium and few or none were localized in the nascent spinal ganglia. Grafted quail somite cells contributed to normal somitic structures and did not migrate extensively in the chicken host. Other fibroblasts did not migrate along cranial or trunk crest pathways, or invade adjacent tissues, but remained intact at the graft site. Sarcoma 180 cells, however, distributed themselves along the normal trunk crest pathway. Cranial and trunk crest cells and crest derivatives grafted ectopically in the limb bud or somite also dispersed, and were found along the ventral migratory pathway. Fibroblastic cells grafted into ectopic sites again remained intact and did not invade host tissue. We conclude (1) that neural crest cells and their derivatives are highly motile and invasive in their normal pathway, as well as in unfamiliar embryonic environments; and (2) that the crest pathway does not act solely to direct neural crest cells, since at least one transformed cell can follow the crest migratory route.     

Direct and Indirect Track Features: What Sediment Did a Dinosaur Touch?

Studies of dinosaur tracks have benefited from a distinction between true tracks and those made in subsurface layers—undertracks. However, the straightforward definition of true tracks becomes problematic when dealing with deep tracks, which often perforate or incise surface layers rather than simply distort them. Deep tracks from the Late Triassic of Greenland were made by theropods moving their feet through a volume of sediment along a complex three-dimensional trajectory. I suggest that designating different portions of the track as direct or indirect features is fruitful for reconstructing foot motion. Identifying which sedimentary grains, rather than which layers, were touched by the foot avoids dismissing deep tracks as undertracks and overlooking a valuable source of kinematic data.     

Cranial nerve growth in birds is preceded by cholinesterase expression during neural crest cell migration and the formation of an HNK-1 scaffold

Summary The expression of the neural crest cell (NCC) markers acetylcholinesterase (AChE) and the HNK-1-epitope is compared from the emigration of cephalic NCC until the formation of the cranial nerves V-X in chicken and quail hindbrain. We show that NCC transiently express acetylcholinesterase (AChE) activity during their emigration; NCC migrate into butyrylcholinesterase (BChE)-positive areas of the cranial mesenchyme. Along these migratory tracks that foreshadow the course of later projecting cranial nerves, BChE increases strongly in cells that may represent immature Schwann cells. Both AChE and BChE, but not HNK-1, are expressed in the ectodermal placodes. In NCC, HNK-1 is expressed strongly only when they approach their destination sites. Their intense expression of HNK-1 then leads to the establishment of tunnel-shaped HNK-1 matrices, within which G4-positive cranial neurites begin to extend. We conclude that AChE and HNK-1 expression in cephalic NCC serve different functions, since AChE is related to their migration, and HNK-1 to their aggregation and the formation of an extracellular neurite scaffold.     

Inhibition of cell adhesion in Dictyostelium discoideum by tunicamycin is prevented by leupeptin

The carbohydrate requirement for cell adhesion of aggregation-competent cells of Dictyostelium discoideum has been examined by use of a selective glycosylation inhibitor of N-glycosyl protein, tunicamycin (TM). TM completely inhibited EDTA-stable cell adhesion and glycosylation of some membrane glycoproteins in aggregation-competent cells of D. discoideum (Yamada, H., et al. (1982) J. Biochem. 92, 399-406). The present study showed that the inhibition of EDTA-stable cell adhesion by TM was prevented significantly when the cells were treated with TM in the presence of a protease inhibitor, leupeptin (LP), whereas the inhibition of glycosylation by TM was not prevented. The cell extract of aggregation-competent cells contained acid proteases, and LP strongly inhibited acid protease from D. discoideum in vitro. On analysis by SDS-polyacrylamide gel electrophoresis (PAGE), many protein bands present in the membrane fraction of control cells disappeared or decreased on TM treatment of the cells in the absence of LP, however, some of these proteins were restored when the cells were treated with TM in the presence of LP. These results strongly support an idea that EDTA-stable cell adhesion characteristic to aggregation-competent cells is mediated by glycoproteins with asparagine-linked carbohydrate. However, the requirement for the carbohydrate moiety of the glycoprotein in cell adhesion appears to be indirect in that it acts to protect the protein moiety from proteolytic degradation.     

Capillary endothelial cell cultures: phenotypic modulation by matrix components

Capillary endothelial cells of rat epididymal fat pad were isolated and cultured in media conditioned by bovine aortic endothelial cells and substrata consisting of interstitial or basement membrane collagens. When these cells were grown on interstitial collagens they underwent proliferation, formed a continuous cell layer and, if cultured for long periods of time, formed occasional tubelike structures. In contrast, when these cells were grown on basement membrane collagens, they did not proliferate but did aggregate and form tubelike structures at early culture times. In addition, cells grown on basement membrane substrata expressed more basement membrane constituents as compared with cells grown on interstitial matrices when assayed by immunoperoxidase methods and quantitated by enzyme-linked immunosorbent inhibition assays. Furthermore, when cells were grown on either side of washed, acellular amnionic membranes their phenotypes were markedly different. On the basement membrane surface they adhered, spread, and formed tubelike structures but did not migrate through the basement membrane. In contrast, when seeded on the stromal surface, these cells were observed to proliferate and migrate into the stromal aspect of the amnion and ultimately formed tubelike structures at high cell densities at longer culture periods (21 d). Thus, connective tissue components play important roles in regulating the phenotypic expression of capillary endothelial cells in vitro, and similar roles of the collagenous components of the extracellular matrix may exist in vivo following injury and during angiogenesis. Furthermore, the culture systems outlined here may be of use in the further study of differentiated, organized capillary endothelial cells in culture.     

Importance of cell-aggregation during induction of neural differentiation in PCC-7 embryonal carcinoma cells.

The importance of cell-aggregation during retinoic acid-induced neural differentiation of embryonal carcinoma cells was studied on the PCC-7 cell line. These cells were chosen as they display low tendency for spontaneous aggregation, and they develop preferentially to neurons upon induced in vitro differentiation. Forced aggregation of these cells, in the absence of retinoic acid, did not result in development of neuron- or glial-like cells. Application of retinoic acid prior to or after the cell-aggregation did not result in neural tissue-like differentiation, either. Irreversible induction of neural development was achieved if cell-aggregation and retinonic acid acted simultaneously, and for a period longer than 48 h. Retinoic acid, on the other hand, was found to be toxic on non-aggregated PCC-7 cells. Our data suggest that cell to cell contacts alter the response of these cells to retinoic acid, and their close apposition is a prerequisite for the retinoic acid-induced neural differentiation.     

Periodic organization of the contractile apparatus in smooth muscle revealed by the motion of dense bodies in single cells

To study the organization of the contractile apparatus in smooth muscle and its behavior during shortening, the movement of dense bodies in contracting saponin skinned, isolated cells was analyzed from digital images collected at fixed time intervals. These cells were optically lucent so that punctate structures, identified immunocytochemically as dense bodies, were visible in them with the phase contrast microscope. Methods were adapted and developed to track the bodies and to study their relative motion. Analysis of their tracks or trajectories indicated that the bodies did not move passively as cells shortened and that nearby bodies often had similar patterns of motion. Analysis of the relative motion of the bodies indicated that some bodies were structurally linked to one another or constrained so that the distance between them remained relatively constant during contraction. Such bodies tended to fall into laterally oriented, semirigid groups found at approximately 6-microns intervals along the cell axis. Other dense bodies moved rapidly toward one another axially during contraction. Such bodies were often members of separate semirigid groups. This suggests that the semirigid groups of dense bodies in smooth muscle cells may provide a framework for the attachment of the contractile structures to the cytoskeleton and the cell surface and indicates that smooth muscle may be more well-ordered than previously thought. The methods described here for the analysis of the motion of intracellular structures should be directly applicable to the study of motion in other cell types.     

Innate structure of DNA foci restricts the mixing of DNA from different chromosome territories

The distribution of chromatin within the mammalian nucleus is constrained by its organization into chromosome territories (CTs). However, recent studies have suggested that promiscuous intra- and inter-chromosomal interactions play fundamental roles in regulating chromatin function and so might define the spatial integrity of CTs. In order to test the extent of DNA mixing between CTs, DNA foci of individual CTs were labeled in living cells following incorporation of Alexa-488 and Cy-3 conjugated replication precursor analogues during consecutive cell cycles. Uniquely labeled chromatin domains, resolved following random mitotic segregation, were visualized as discrete structures with defined borders. At the level of resolution analysed, evidence for mixing of chromatin from adjacent domains was only apparent within the surface volumes where neighboring CTs touched. However, while less than 1% of the nuclear volume represented domains of inter-chromosomal mixing, the dynamic plasticity of DNA foci within individual CTs allows continual transformation of CT structure so that different domains of chromatin mixing evolve over time. Notably, chromatin mixing at the boundaries of adjacent CTs had little impact on the innate structural properties of DNA foci. However, when TSA was used to alter the extent of histone acetylation changes in chromatin correlated with increased chromatin mixing. We propose that DNA foci maintain a structural integrity that restricts widespread mixing of DNA and discuss how the potential to dynamically remodel genome organization might alter during cell differentiation.     

Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule, N-cadherin.

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2 mM Ca++ or 0.2 mM Ca++, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca++, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca++ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca++ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca++ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca++ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca++, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca++. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca++ levels had similar effects as lowering the Ca++ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.