Cell-cell adhesion in Dictyostelium discoideum

Three separate mechanisms of cell-cell adhesion have been shown to appear at different stages of development in Dictyostelium discoideum. During the first few hours of development, the cells synthesize and accumulate a glycoprotein of 24,000 daltons (gp24) that is positioned in the membrane. The time of appearance of gp24 correlates exactly with the time of appearance of cell-cell adhesion in two strains in which temporal control varies by several hours. Antibodies specific to gp24 are able to block cell-cell adhesion during the first few hours of development but not during later development. By 8 hr of development, another glycoprotein, gp80, that is not recognized by antibodies to gp24 accumulates on the surface of cells. This membrane protein mediates an independent adhesion mechanism during the aggregation stage that is resistant to 10 mM EDTA. Antibodies specific to gp80 can block EDTA-resistant adhesion during this stage. During subsequent development, gp80 is removed from the cell surface and replaced by another adhesion mechanism that is insensitive to antibodies to either gp24 or gp80. A lambda gt11 expression vector carrying a Dictyostelium cDNA insert was isolated that directs the synthesis of a fusion protein recognized by antibodies specific to gp24. This cDNA was used to probe a genomic library. A clone carrying a 1.4-kb insert of genomic DNA was recognized by the cDNA and shown to hybridize to a 0.7-kb mRNA that accumulates early in development. This unusually small RNA could code for the small protein, gp24. Southern analysis of restriction fragments generated by various enzymes on Dictyostelium DNA with both the cDNA and genomic clones indicated the presence of two tandem copies of the gene. This may account for the failure to recover mutations resulting in the lack of gp24. Mutations have been recovered that result in the lack of accumulation of gp80, and cells carrying these mutations have been shown to be missing the second adhesion mechanism. These mutant strains are able to complete development because the other adhesion mechanisms are not impaired. Sequential addition of adhesion mechanisms provides a means for the formation of multicellular organisms from previously solitary cells.     

Cell-cell adhesion molecules in Dictyostelium

Multicellularity in the cellular slime mold Dictyostelium discoideum is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive adhesion sites are expressed very early in the development cycle and a surface glycoprotein of 24,000 Da is known to be responsible for these sites. The EDTA-resistant contact sites begin to accumulate on the cell surface at the aggregation stage of development. Several glycoproteins have been implicated in the EDTA-resistant type of cell-cell binding and the best characterized one has an Mr of 80,000 (gp80). gp80 mediates cell-cell binding via homophilic interaction and its cell binding site has been mapped to an octapeptide sequence. The mechanism by which gp80 mediates cell-cell adhesion will be discussed.     

Cell-cell contact mediates cAMP secretion in Dictyostelium discoideum.

Cyclic adenosine 3':5' monophosphate (cAMP) and cell-cell contact regulate developmental gene expression in Dictyostelium discoideum. Developing D. discoideum amoebae synthesize and secrete cAMP following the binding of cAMP to their surface cAMP receptor, a response called cAMP signaling. We have demonstrated two responses of developing D. discoideum amoebae to cell-cell contact. Cell-cell contact elicits cAMP secretion and alters the amount of cAMP secreted in a subsequent cAMP signaling response. Depending upon experimental conditions, bacterial-amoebal contact and amoebal-amoebal contact can enhance or diminish the amount of cAMP secreted during a subsequent cAMP signaling response. We have hypothesized that cell-cell contact regulates D. discoideum development by altering cellular and extracellular levels of cAMP. To begin testing this hypothesis, these responses were further characterized. The two responses to cell-cell contact are independent, i.e., they can each occur in the absence of the other. The responses to cell-cell contact also have unique temperature dependences when compared to each other, cAMP signaling, and phagocytosis. This suggests that these four responses have unique steps in their transduction mechanisms. The secretion of cAMP in response to cell-cell contact appears to be a non-specific response; contact between D. discoideum amoebae and Enterobacter aerogenes, latex beads, or other amoebae elicits cAMP secretion. Despite the apparent similarities of the effects of bacterial-amoebal and amoebal-amoebal contact on the cAMP signaling response, this contact-induced response appears to be specific. Latex beads addition does not alter the magnitude of a subsequent cAMP signaling response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modelling Dictyostelium discoideum morphogenesis: the culmination

The culmination of the morphogenesis of the cellular slime mould Dictyostelium discoideum involves complex cell movements which transform a mound of cells into a globule of spores on a slender stalk. We show that cyclic AMP signalling and differential adhesion, combined with cell differentiation and slime production, are sufficient to produce the morphogenetic cell movements which lead to culmination. We have simulated the process of culmination using a hybrid cellular automata/partial differential equation model. With our model we have been able to reproduce the main features that occur during culmination, namely the straight downward elongation of the stalk, its anchoring to the substratum and the formation of the long thin stalk topped by the spore head. We conclude that the cyclic AMP signalling system is responsible for the elongation and anchoring of the stalk, but in a roundabout way: pressure waves that are induced by the chemotaxis towards cyclic AMP squeeze the stalk through the cell mass. This mechanism forces the stalk to elongate precisely in the direction opposite to that of the chemotactically moving cells. The process turns out to be ‘guided’ by inactive ‘pathfinder’ cells, which form the tip of the stalk. We show that the entire development is enacted by means of the aforementioned building blocks. This means that no global gradients or different modes of chemotaxis are needed to complete the culmination. MPEG movies of the simulations are available on-line: http://www-binf.bio. uu.nl/stan/bmb.     

The trishanku gene and terminal morphogenesis in Dictyostelium discoideum

SUMMARY Multicellular development in the social amoeba Dictyostelium discoideum is triggered by starvation. It involves a series of morphogenetic movements, among them being the rising of the spore mass to the tip of the stalk. The process requires precise coordination between two distinct cell types—presumptive (pre-) spore cells and presumptive (pre-) stalk cells. Trishanku ( triA ) is a gene expressed in prespore cells that is required for normal morphogenesis. The triA⁻ mutant shows pleiotropic effects that include an inability of the spore mass to go all the way to the top. We have examined the cellular behavior required for the normal ascent of the spore mass. Grafting and mixing experiments carried out with tissue fragments and cells show that the upper cup, a tissue that derives from prestalk cells and anterior-like cells (ALCs), does not develop properly in a triA⁻ background. A mutant upper cup is unable to lift the spore mass to the top of the fruiting body, likely due to defective intercellular adhesion. If wild-type upper cup function is provided by prestalk and ALCs, trishanku spores ascend all the way. Conversely, Ax2 spores fail to do so in chimeras in which the upper cup is largely made up of mutant cells. Besides proving that under these conditions the wild-type phenotype of the upper cup is necessary and sufficient for terminal morphogenesis in D. discoideum , this study provides novel insights into developmental and evolutionary aspects of morphogenesis in general. Genes that are active exclusively in one cell type can elicit behavior in a second cell type that enhances the reproductive fitness of the first cell type, thereby showing that morphogenesis is a cooperative process.     

The cell adhesion molecule DdCAD-1 regulates morphogenesis through differential spatiotemporal expression in Dictyostelium discoideum

During development of Dictyostelium, multiple cell types are formed and undergo a coordinated series of morphogenetic movements guided by their adhesive properties and other cellular factors. DdCAD-1 is a unique homophilic cell adhesion molecule encoded by the cadA gene. It is synthesized in the cytoplasm and transported to the plasma membrane by contractile vacuoles. In chimeras developed on soil plates, DdCAD-1-expressing cells showed greater propensity to develop into spores than did cadA-null cells. When development was performed on non-nutrient agar, wild-type cells sorted from the cadA-null cells and moved to the anterior zone. They differentiated mostly into stalk cells and eventually died, whereas the cadA-null cells survived as spores. To assess the role of DdCAD-1 in this novel behavior of wild-type and mutant cells, cadA-null cells were rescued by the ectopic expression of DdCAD-1-GFP. Morphological studies have revealed major spatiotemporal changes in the subcellular distribution of DdCAD-1 during development. Whereas DdCAD-1 became internalized in most cells in the post-aggregation stages, it was prominent in the contact regions of anterior cells. Cell sorting was also restored in cadA(-) slugs by exogenous recombinant DdCAD-1. Remarkably, DdCAD-1 remained on the surface of anterior cells, whereas it was internalized in the posterior cells. Additionally, DdCAD-1-expressing cells migrated slower than cadA(-) cells and sorted to the anterior region of chimeric slugs. These results show that DdCAD-1 influences the sorting behavior of cells in slugs by its differential distribution on the prestalk and prespore cells.     

Cell-cell signaling and adhesion in phagocytosis and early development of Dictyostelium

Cell-cell signaling and adhesion regulate transition from the unicellular to the multicellular stage of development in the cellular slime mold Dictyostelium. Essential gene networks involved in these processes have been identified and their interplay dissected. Heterotrimeric G protein-linked signal transduction plays a key role in regulating expression of genes mediating chemotaxis or cell adhesion, as well as coordinating actin-based cell motility during phagocytosis and chemotaxis. Two classes of cell adhesion molecules, one cadherin-like and the second belonging to the IgG superfamily, contribute to the strength of adhesion in Dictyostelium aggregates. The developmental role of genes involved in motility and adhesion, and their degree of redundancy, have been re-assessed by using novel developmental assay conditions which are closer to development in nature.     

Monoclonal antibodies block cell-cell adhesion in Dictyostelium discoideum

Of 39 monoclonal antibodies that bind the cell surface of aggregating Dictyostelium discoideum, 4 block 76-98% of cell-cell adhesion measured in an in vitro assay. The active antibodies all bind in the range of 10(6) antigenic sites/cell surface and react with more than one material on nitrocellulose blots prepared after polyacrylamide gel electrophoresis of whole aggregating cells in sodium dodecyl sulfate. Active antibodies can by grouped into two classes, each with two very similar members. Class I binds several molecules that are prominent in aggregating cells but scarce or undetectable in vegetative cells, blocks cell adhesion only in the presence of EDTA, and has no detectable effect on cell morphology. Class II binds a wide range of molecules present in both vegetative and aggregating cells, inhibits adhesion as well in the absence as in the presence of EDTA, and reversibly alters cell shape.     

Two distinct adhesion systems are responsible for EDTA-sensitive adhesion in Dictyostelium discoideum

Abstract. Early in their developmental program, Dictyostelium discoideum exhibit EDTA-sensitive and EDTA-resistant adhesion. The molecules which mediate the adhesions have been called contact sites, with contact sites A mediating EDTA-resistant adhesion and contact sites B mediating EDTA-sensitive adhesion. The studies described here have revealed that prior to aggregation, a second EDTA-sensitive adhesion system emerges. In keeping with previously established nomenclature, the molecules mediating the newly discovered adhesion system have been called contact sites C. Unlike contact sites B, contact sites C are unaffected by a contact sites B-blocking peptide. Contact sites C-mediated adhesion is also distinct from contact sites B-mediated adhesion in that contact sites C-mediated adhesion is EGTA-resistant and in the presence of EDTA it can be rescued by the addition of Mg²⁺. Thus Mg²⁺ may be the cation present under physiological conditions that is essential for contact sites C activity. Unlike contact sites B-mediated adhesion, contact sites C-mediated adhesion is not observed in growing amoebae. Contact sites C-mediated adhesion first becomes apparent within hours after the initiation of development and its strength appears to increase throughout the first 10 h of the developmental program. A mutant lacking the EDTA-resistant contact sites A exhibits normal contact sites B- and C-mediated adhesion, demonstrating that both EDTA-sensitive adhesion systems are independent of contact sites A. Thus aggregating D. discoideum amoebae possess three distinct adhesion systems, one of them is EDTA-resistant and the other two are EDTA-sensitive.     

Cell-cell interactions in Dictyostelium development

The development of an organism requires extensive cell-cell communication; however, little is known about the signals transmitted among differentiating cells. Observations of Dictyostelium amoebae reveal that transmembrane signaling systems have been highly conserved in evolution. The signals that cause these cells to differentiate are processed by mechanisms similar to those that process sensory and hormonal stimuli in higher animals.     

Trishanku, a novel regulator of cell-type stability and morphogenesis in Dictyostelium discoideum

We have identified a novel gene, trishanku (triA), by random insertional mutagenesis of Dictyostelium discoideum. TriA is a Broad complex Tramtrack bric-a-brac domain-containing protein that is expressed strongly during the late G2 phase of cell cycle and in presumptive spore (prespore (psp)) cells. Disrupting triA destabilizes cell fate and reduces aggregate size; the fruiting body has a thick stalk, a lowered spore: stalk ratio, a sub-terminal spore mass and small, rounded spores. These changes revert when the wild-type triA gene is re-expressed under a constitutive or a psp-specific promoter. By using short- and long-lived reporter proteins, we show that in triA(-) slugs the prestalk (pst)/psp proportion is normal, but that there is inappropriate transdifferentiation between the two cell types. During culmination, regardless of their current fate, all cells with a history of pst gene expression contribute to the stalk, which could account for the altered cell-type proportion in the mutant.     

Altered cyclic-AMP receptor activity and morphogenesis in a chemosensory mutant of Dictyostelium discoideum

The functional properties of the cell-surface cyclic-AMP receptor that controls chemotaxis were found to be altered in an aggregation mutant of Dictyostelium discoideum. The mutant aggregated without stream formation and had a tenfold increased cell-density requirement for the initiation of aggregation. After aggregation, mounds formed multiple tips and subsequently subdivided to give multiple fruits that were small and abnormally proportioned. Cyclic-AMP-induced light-scattering changes in cell suspensions indicated that the mutant had a diminished response to external cyclic-AMP signals. Associated with these altered functional responses was a physical change in the cyclic-AMP sensory system. Cyclic-AMP-binding studies showed that the parent had two classes of cyclic-AMP binding sites, i.e., Kd = 32 and 110 nM. In contrast, the mutant had two- to threefold or more high-affinity sites (Kd = 25 nM) and altered low-affinity sites (Kd less than 3 microM). These results indicate that both affinity classes of binding site are independently mutable. This observation suggests that the two affinity classes can be interconverted by mutation, or the mutation alters a single molecular species and its equilibrium between binding sites with different affinities for cyclic AMP, as postulated in receptor cycling models.     

Altered cyclic-AMP receptor activity and morphogenesis in a chemosensory mutant of Dictyostelium discoideum

Abstract. The functional properties of the cell-surface cyclic-AMP receptor that controls chemotaxis were found to be altered in an aggregation mutant of Dictyostelium discoideum. The mutant aggregated without stream formation and had a tenfold increased cell-density requirement for the initiation of aggregation. After aggregation, mounds formed multiple tips and subsequently subdivided to give multiple fruits that were small and abnormally proportioned. Cyclic-AMP-induced light-scattering changes in cell suspensions indicated that the mutant had a diminished response to external cyclic-AMP signals. Associated with these altered functional responses was a physical change in the cyclic-AMP sensory system. Cyclic-AMP-binding studies showed that the parent had two classes of cyclic-AMP binding sites, i.e., Kd = 32 and 110 nM. In contrast, the mutant had two- to threefold or more high-affinity sites (Kd = 25n M ) and altered low-affinity sites (Kd < 3μ M ). These results indicate that both affinity classes of binding site are independently mutable. This observation suggests that the two affinity classes can be interconverted by mutation, or the mutation alters a single molecular species and its equilibrium between binding sites with different affinities for cyclic AMP, as postulated in receptor cycling models.     

Changes in morphogenesis and developmental enzyme levels in Dictyostelium discoideum after gamma irradiation.

Changes in the levels of specific activity of two enzymes believed to be involved in developmental regulation were observed after irradiating differentiating cells of Dictyostelium discoideum. Stimulation of the levels of specific activity of alkaline phosphatase occured after irradiation at the beginning of development and at the end of the aggregation period, but not after irradiation at the beginning of aggregation. A stimulation in UDP-glucose pyrophosphorylase specific activity was also observed, but to a lesser extent and only after irradiation at the end of aggregation. Dose-dependent delays in the appearance of peaks of specific activity were noted. The delay per unit dose was less when irradiation took place at the beginning of development as opposed to the beginning or end of the aggregation period. Radiation-induced delays in progression through visible developmental stages were almost identical to delays in enzyme appearance. Other radiation effects on morphogenesis included the induction of a migratory slug phase.     

Cell-cell adhesion in the cnidaria: insights into the evolution of tissue morphogenesis

Cell adhesion is a major aspect of cell biology and one of the fundamental processes involved in the development of a multicellular animal. Adhesive mechanisms, both cell-cell and between cell and extracellular matrix, are intimately involved in assembling cells into the three-dimensional structures of tissues and organs. The modulation of adhesive complexes could therefore be seen as a central component in the molecular control of morphogenesis, translating information encoded within the genome into organismal form. The availability of whole genomes from early-branching metazoa such as cnidarians is providing important insights into the evolution of adhesive processes by allowing for the easy identification of the genes involved in adhesion in these organisms. Discovery of the molecular nature of cell adhesion in the early-branching groups, coupled with comparisons across the metazoa, is revealing the ways evolution has tinkered with this vital cellular process in the generation of the myriad forms seen across the animal kingdom.     

Cell-cell contact and cAMP regulate the expression of a UDP glucose pyrophosphorylase gene of Dictyostelium discoideum

UDP glucose pyrophosphorylase (UDPGP) (EC.2.7.7.9) is a developmentally regulated enzyme of Dictyostelium discoideum. Two polypeptides of UDPGP are translated from Dictyostelium mRNA. Recently we isolated a cDNA clone which encodes one of the UDPGP polypeptides (B. R. Fishel, J. A. Ragheb, A. Rajkovic, B. Haribabu, C. W. Schweinfest, and R. P. Dottin (1985). Dev. Biol. 110, 369-381). By hybridization with the cDNA and by in vitro translation and immunoprecipitation, we examined the effect of cell-cell contact and cAMP on the regulation of UDPGP expression. Disaggregation of slugs resulted in a rapid loss of UDPGP mRNA. Addition of cAMP to these cells resulted in increased levels of UDPGP mRNA, though not to the same extent as seen during normal development. The two UDPGP polypeptides observed in vitro are coordinately regulated. Unaggregated cells, starved and shaken rapidly in suspension, did not show UDPGP mRNA accumulation. However, addition of cAMP to these cells caused UDPGP induction, suggesting that the requirement for cell-cell contact could be bypassed in part by cAMP addition.     

Cell adhesion during the migratory slug stage of Dictyostelium discoideum

Prespore-specific Antigen (PsA) is selectively expressed on the surface of prespore cells at the multicellular migratory slug stage of Dictyostelium discoideum development. It is a developmentally regulated glycoprotein that is anchored to the cell membrane through a glycosyl phosphatidylinositol (GPI) anchor. We present the results of an in vitro immunological investigation of the hypothesis that PsA functions as a cell adhesion molecule (CAM), and of a ligand-binding assay indicating that PsA has cell membrane binding partner(s). This is the first evidence to implicate a direct role for a putative CAM in cell-cell adhesion during the multicellular migratory slug stage of D. discoideum development. Cell-cell adhesion assays were carried out in the presence or absence of the monoclonal antibody (mAb) MUD1 that has a single antigenic determinant: a peptide epitope on PsA. These assays showed specific inhibition of cell-cell adhesion by MUD1. Further, it was found that a purified recombinant form of PsA (rPsA), can neutralize the inhibitory effect of MUD1; the inhibitory effect on cell-cell adhesion is primarily due to the blocking of PsA by the mAb. The resistance of aggregates to dissociation in the presence of 10 mM EDTA (ethylenediamintetraacetic acid) indicates that PsA mediates EDTA-stable cell-cell contacts, and that PsA-mediated cell adhesion is likely to be independent of divalent cations such as Ca(2+) or Mg(2+).