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The Histidine KinasedhkCRegulates the Choice between Migrating Slugs and Terminal Differentiation inDictyostelium discoideum
Abstract
An early decision that a newly formed aggregate of Dictyostelium cells must make is whether to form a migrating slug or to proceed through culmination, the process of forming the mature fruiting body. The choice between these alternative morphological pathways is influenced by external and internal cues. dhkC was identified as a potential hybrid sensor kinase possessing domains homologous to the histidine kinase and receiver motifs of two-component signaling systems. Null strains of dhkC show a rapidly developing phenotype for aggregation through finger formation, and culmination commences immediately thereafter and proceeds at a normal rate to generate typical fruiting bodies. Ammonia, an endogenous regulator of the slug versus culmination choice, results in a prolonged slug stage for wild-type strains while the dhkC- strain bypasses the slug stage in the presence or absence of ammonia. Conversely, expression in wild-type cells of a modified DHKC protein composed of only the histidine kinase domain results in normal timing through early aggregation, but subsequent development is significantly delayed. The resulting fingers, once formed, readily convert to slugs that do not undergo culmination but instead migrate until their energy sources are depleted. The slugger phenotype is dependent on the presence of a functional response regulator REGA, and it is rescued by exogenously supplied cAMP. Together, the results indicate that DHKC contributes to the integration of environmental and cellular signals so that the appropriate choice is made between slug formation and culmination. We suggest that DHKC may function as a sensor for ammonia, and that it is the initial component of a phosphorelay signaling system that may modulate the activity of cAMP-dependent protein kinase to either inhibit or promote culmination. Additionally, dhkC- spores were found to be defective in germination, indicating a role for the DHKC signaling pathway in activating spore germination.
... Dictyostelium discoideum stands out among these eukaryotes because of the relatively large number of histidine kinases in its genome [2]. Several of the 15 histidine kinases of Dictyostelium have been characterized, and they function in a number of different processes during the multicellular developmental program, including spore encapsulation, spore dormancy, osmoregulation, prespore to spore differentiation, and the slug to culmination transition [3][4][5][6][7][8][9][10][11]. ...
... When a slug-like structure was observed, little to no migration of that entity was apparent as development progressed. In contrast, under standard conditions of development the parental Ax4 cells always showed a transient period of 1 to 2 hours of slug formation with the slugs migrating during this brief period prior to initiating culmination [6]. ...
... The dhkD-pollywog phenotype may reflect a disruption of the normal patterns of cell movement in the mounds, with the atypical movement of cells resulting in a horizontally mobile mound as opposed to tip formation and vertical extension of the mound. The histidine kinases in Dictyostelium that have been characterized all seem to function through the single response regulator, RegA [3,6,10,43,44]. Given that the pollywog phenotype in dhkD null cells was dependent upon a functional RegA, it is possible that DhkD also functions by modulating RegA activity. We postulate that DhkD modulates cAMP production in response to an as yet unidentified extracellular signal. ...
Background:
Histidine kinases are receptors for sensing cellular and environmental signals, and in response to the appropriate cue they initiate phosphorelays that regulate the activity of response regulators. The Dictyostelium discoideum genome encodes 15 histidine kinases that function to regulate several processes during the multicellular developmental program, including the slug to culmination transition, osmoregulation, and spore differentiation. While there are many histidine kinases, there is only a single response regulator, RegA. Not surprisingly given the ubiquitous involvement of cAMP in numerous processes of development in Dictyostelium, RegA is a cAMP phosphodiesterase that is activated upon receiving phosphates through a phosphorelay. Hence, all of the histidine kinases characterized to date regulate developmental processes through modulating cAMP production. Here we investigate the function of the histidine kinase DhkD.
Principal findings:
The dhkD gene was disrupted, and the resulting cells when developed gave a novel phenotype. Upon aggregation, which occurred without streaming, the mounds were motile, a phenotype termed the pollywog stage. The pollywog phenotype was dependent on a functional RegA. After a period of random migration, the pollywogs attempted to form fingers but mostly generated aberrant structures with no tips. While prestalk and prespore cell differentiation occurred with normal timing, proper patterning did not occur. In contrast, wild type mounds are not motile, and the cAMP chemotactic movement of cells within the mound facilitates proper prestalk and prespore patterning, tip formation, and the vertical elongation of the mound into a finger.
Conclusions:
We postulate that DhkD functions to ensure the proper cAMP distribution within mounds that in turn results in patterning, tip formation and the transition of mounds to fingers. In the absence of DhkD, aberrant cell movements in response to an altered cAMP distribution result in mound migration, a lack of proper patterning, and an inability to generate normal finger morphology.
... The catabolite ammonia, which is produced by protein degradation through autophagy in the starving cells, is a negative regulator of stalk cell differentiation in Ddis [31,32]. Ammonia activates the histidine kinase DhkC, which by phosphorylating the cAMP phosphodiesterase RegA, lowers cAMP levels, preventing activation of PKA [33]. The ammonia transporter AmtC, which is expressed at the sorogen tip and in prespore cells, facilitates local loss of ammonia, allowing the Ddis stalk and thereby the fruiting body to form [34]. ...
... One such factor is NH 3 , an inhibitor of stalk cell differentiation, which is abundantly produced by protein degradation in the starving cells. NH 3 acts on the sensor histidine kinase DhkC to activate the intracellular cAMP phosphodiesterase RegA, thereby preventing PKA activation by ACA [33]. The position of the aerially exposed tip cells, combined with the action of the ammonia transporter AmtC, locally decreases NH 3 at the tip allowing PKA to be activated and stalk cells to differentiate. ...
Background
Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid social amoebas acquired diguanylate cyclase ( dgcA ) by horizontal gene transfer. Dictyostelium discoideum ( Ddis ) in taxon group 4 uses c-di-GMP as a secreted signal to induce differentiation of stalk cells, the ancestral somatic cell type that supports the propagating spores. We here investigated how this role for c-di-GMP evolved in Dictyostelia by exploring dgcA function in the group 2 species Polysphondylium pallidum ( Ppal ) and in Polysphondylium violaceum ( Pvio ), which resides in a small sister clade to group 4.
Results
Similar to Ddis , dgcA is upregulated after aggregation in Ppal and Pvio and predominantly expressed in the anterior region and stalks of emerging fruiting bodies. DgcA null mutants in Ppal and Pvio made fruiting bodies with very long and thin stalks and only few spores and showed delayed aggregation and larger aggregates, respectively. Ddis dgcAˉ cells cannot form stalks at all, but showed no aggregation defects. The long, thin stalks of Ppal and Pvio dgcAˉ mutants were also observed in acaAˉ mutants in these species. AcaA encodes adenylate cyclase A, which mediates the effects of c-di-GMP on stalk induction in Ddis. Other factors that promote stalk formation in Ddis are DIF-1, produced by the polyketide synthase StlB, low ammonia, facilitated by the ammonia transporter AmtC, and high oxygen, detected by the oxygen sensor PhyA (prolyl 4-hydroxylase). We deleted the single stlB , amtC and phyA genes in Pvio wild-type and dgcAˉ cells. Neither of these interventions affected stalk formation in Pvio wild-type and not or very mildly exacerbated the long thin stalk phenotype of Pvio dgcAˉ cells.
Conclusions
The study reveals a novel role for c-di-GMP in aggregation, while the reduced spore number in Pvio and Ppal dgcAˉ is likely an indirect effect, due to depletion of the cell pool by the extended stalk formation. The results indicate that in addition to c-di-GMP, Dictyostelia ancestrally used an as yet unknown factor for induction of stalk formation. The activation of AcaA by c-di-GMP is likely conserved throughout Dictyostelia.
... Precocious development has been observed in a number of Dictyostelium knockout mutants. Specifically, early tipped mound formation has been reported in strains overexpressing cyclin C, cyclin-dependent kinase 8, or the G-protein alpha 5 subunit, and in knockout mutants of histidine kinase C, a metabotropic glutamate receptor-like protein, protein inhibitor of STAT, and SCAR/WAVE [70][71][72][73][74][75]. Several knockout mutants that display increased slug migration have been described, including mutants for genes important for oxysterol binding, the assembly of mitochondrial complex I, and the targeting of proteins for degradation via proteasomes [76][77][78]. ...
... Several knockout mutants that display increased slug migration have been described, including mutants for genes important for oxysterol binding, the assembly of mitochondrial complex I, and the targeting of proteins for degradation via proteasomes [76][77][78]. This phenotype has also been observed in cells overexpressing histidine kinase C or in cells where calcium-binding protein 3 expression has been knocked down by RNAi [72,79]. The diversity of functions associated with these proteins as well as those discussed above for the other developmental phenotypes in cln3 2 cells, highlight the importance of elucidating the signal transduction pathways underlying the function of Cln3 during Dictyostelium development. ...
... Precocious development has been observed in a number of Dictyostelium knockout mutants. Specifically, early tipped mound formation has been reported in strains overexpressing cyclin C, cyclin-dependent kinase 8, or the G-protein alpha 5 subunit, and in knockout mutants of histidine kinase C, a metabotropic glutamate receptor-like protein, protein inhibitor of STAT, and SCAR/WAVE [70][71][72][73][74][75]. Several knockout mutants that display increased slug migration have been described, including mutants for genes important for oxysterol binding, the assembly of mitochondrial complex I, and the targeting of proteins for degradation via proteasomes [76][77][78]. ...
... Several knockout mutants that display increased slug migration have been described, including mutants for genes important for oxysterol binding, the assembly of mitochondrial complex I, and the targeting of proteins for degradation via proteasomes [76][77][78]. This phenotype has also been observed in cells overexpressing histidine kinase C or in cells where calcium-binding protein 3 expression has been knocked down by RNAi [72,79]. The diversity of functions associated with these proteins as well as those discussed above for the other developmental phenotypes in cln3 2 cells, highlight the importance of elucidating the signal transduction pathways underlying the function of Cln3 during Dictyostelium development. ...
The neuronal ceroid lipofuscinoses (NCL) are a group of inherited, severe neurodegenerative disorders also known as Batten disease. Juvenile NCL (JNCL) is caused by recessive loss-of-function mutations in CLN3, which encodes a transmembrane protein that regulates endocytic pathway trafficking, though its primary function is not yet known. The social amoeba Dictyostelium discoideum is increasingly utilized for neurological disease research and is particularly suited for investigation of protein function in trafficking. Therefore, here we establish new overexpression and knockout Dictyostelium cell lines for JNCL research. Dictyostelium Cln3 fused to GFP localized to the contractile vacuole system and to compartments of the endocytic pathway. cln3- cells displayed increased rates of proliferation and an associated reduction in the extracellular levels and cleavage of the autocrine proliferation repressor, AprA. Mid- and late development of cln3- cells was precocious and cln3- slugs displayed increased migration. Expression of either Dictyostelium Cln3 or human CLN3 in cln3- cells suppressed the precocious development and aberrant slug migration, which were also suppressed by calcium chelation. Taken together, our results show that Cln3 is a pleiotropic protein that negatively regulates proliferation and development in Dictyostelium. This new model system, which allows for the study of Cln3 function in both single cells and a multicellular organism, together with the observation that expression of human CLN3 restores abnormalities in Dictyostelium cln3- cells, strongly supports the use of this new model for JNCL research.
... The phosphodiesterase (PDE) activity of RegA is controlled by phosphorylation of its intrinsic response regulator domain by sensor histidine kinases/phosphatases. There are 15 sensor histidine kinases/phosphatases in the Ddis genome and at least four of these are receptors for signals that control the timely formation and germination of spores in an intricate network of communication between the maturing spore and stalk cells [24][25][26][27][28][29][30][31][32]. ...
... While PKA activity requires cAMP synthesis by several adenylate cyclases, it is actually RegA that controls PKA activity by integrating the external stimuli that regulate its cAMP hydrolytic activity. These stimuli, such as solute stress, NH 3 , spore-inducing peptides and cytokinins are detected by sensor histidine kinases/phosphatases [27][28][29]50], which target the phosphoryl accepting aspartate residue in the response regulator of RegA [51]. ...
Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts pathogenic amoebas resist antibiotic treatment, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation.
... We did not investigate in more detail other possible minor developmental abnormalities of DH1 and JH10 DhkM mutants. Developmental ab-normalities, sometimes with a similar "slugger" phenotype, were observed in mutants of other histidine kinase receptor genes in Dictyostelium (Schuster et al., 1996;Singleton et al., 1998;Zinda and Singleton, 1998;Wang et al., 1999;Thomason et al., 2006). ...
... More specifically, the molecules involved in the ins and del mutations may be in spatial proximity. discadenine (DhkB), ammonia (DhkC), and osmolarity (DokA) (Schuster et al., 1996;Singleton et al., 1998;Zinda and Singleton, 1998;Wang et al., 1999). It is believed that several of these histidine kinases control cAMP levels by signaling to the response regulator phosphodiesterase RegA, probably coordinated with cAMP-controlling G protein-dependent pathways (Wolanin et al., 2002). ...
Dictyostelium constitutes a genetically tractable model for the analysis of autophagic cell death (ACD). During ACD, Dictyostelium cells first transform into paddle cells and then become round, synthesize cellulose, vacuolize, and die. Through random insertional mutagenesis, we identified the receptor histidine kinase DhkM as being essential for ACD. Surprisingly, different DhkM mutants showed distinct nonvacuolizing ACD phenotypes. One class of mutants arrested ACD at the paddle cell stage, perhaps through a dominant-negative effect. Other mutants, however, progressed further in the ACD program. They underwent rounding and cellulose synthesis but stopped before vacuolization. Moreover, they underwent clonogenic but not morphological cell death. Exogenous 8-bromo-cAMP restored vacuolization and death. A role for a membrane receptor at a late stage of the ACD pathway is puzzling, raising questions as to which ligand it is a receptor for and which moieties it phosphorylates. Together, DhkM is the most downstream-known molecule required for this model ACD, and its distinct mutants genetically separate previously undissociated late cell death events.
... The presence of c-di-GMP activates AcaA in tip cells and cAMP synthesized by AcaA in turn activates PKA to induce the differentiation of prestalk cells into stalk (Chen et al., 2017). Conversely, ammonia acts on RegA via DhkC, which suppresses cAMP signaling, thereby inhibiting stalk differentiation (Schindler and Sussman, 1977;Singleton et al., 1998). Our observation of c-di-GMP accumulation in the tip during stalk formation (Figures 2, 3) is consistent with the localization of AcaA (Verkerke-van Wijk et al., 2001), supporting the previous spatiotemporal model (Chen et al., 2017). ...
The bacterial signaling molecule cyclic diguanosine monophosphate (c-di-GMP) is only synthesized and utilized by the cellular slime mold Dictyostelium discoideum among eukaryotes. Dictyostelium cells undergo a transition from a unicellular to a multicellular state, ultimately forming a stalk and spores. While Dictyostelium is known to employ c-di-GMP to induce differentiation into stalk cells, there have been no reports of direct observation of c-di-GMP using fluorescent probes. In this study, we used a fluorescent probe used in bacteria to visualize its localization within Dictyostelium multicellular bodies. Cytosolic c-di-GMP concentrations were significantly higher at the tip of the multicellular body during stalk formation.
... Here, we showed that TH-DIF-1 and DIF-1A(+1) as well as DIF-2 negatively modulate chemotaxis, at least in part, via the DhkC-RdeA-RegA pathway ( Figure 6D). DhkC is a receptortype hybrid sensor kinase that carries domains homologous to the histidine kinase and receiver motifs of two-component phospho-relay signaling systems ( Figure 2BII), and ammonia is thought to be an activator (ligand?) of DhkC in vivo [25,43,44]. However, because the receptors for cytokinins (plant hormones that are also small compounds) are histidine kinases and components of a two-component phospho-relay signaling system in plants [45,46] and also because the cytokinin discadenine is proposed to suppress spore formation through another Dictyostelium histidine kinase, DhkB, in D. discoideum [47,48], we suspect that DIF-2 is a ligand for DhkC, i.e., that DhkC might be DR-2C. ...
Differentiation-inducing factors 1 and 2 (DIF-1 and DIF-2) are small lipophilic signal molecules that induce stalk cell differentiation but differentially modulate chemotaxis toward cAMP in the cellular slime mold Dictyostelium discoideum; DIF-1 suppresses chemotactic cell movement in shallow cAMP gradients, whereas DIF-2 promotes it. The receptor(s) for DIF-1 and DIF-2 have not yet been identified. We examined the effects of nine derivatives of DIF-1 on chemotactic cell movement toward cAMP and compared their chemotaxis-modulating activity and stalk cell differentiation–inducing activity in wild-type and mutant strains. The DIF derivatives differentially affected chemotaxis and stalk cell differentiation; for example, TM-DIF-1 suppressed chemotaxis and showed poor stalk-inducing activity, DIF-1(3M) suppressed chemotaxis and showed strong stalk-inducing activity, and TH-DIF-1 promoted chemotaxis. These results suggest that DIF-1 and DIF-2 have at least three receptors: one for stalk cell induction and two for chemotaxis modulation. In addition, our results show that the DIF derivatives can be used to analyze the DIF-signaling pathways in D. discoideum.
... An alternative interpretation of the mode of action of ammonia has been proposed to explain its well-known inhibitory effect on cAMP levels (Singleton et al., 1998), in which ammonia transporters act as ammonia sensors activating or inhibiting the hybrid histidine kinase DhkC in response to the local ammonia level; the level of DhkC activation would then be relayed to the RegA cAMP phosphodiesterase which would in turn be activated or inhibited. The authors discount a role for vacuolar pH in this process. ...
mTORC1 and AMPK are mutually antagonistic sensors of nutrient and energy status that have been implicated in many human diseases including cancer, Alzheimer’s disease, obesity and type 2 diabetes. Starved cells of the social amoeba Dictyostelium discoideum aggregate and eventually form fruiting bodies consisting of stalk cells and spores. We focus on how this bifurcation of cell fate is achieved. During growth mTORC1 is highly active and AMPK relatively inactive. Upon starvation, AMPK is activated and mTORC1 inhibited; cell division is arrested and autophagy induced. After aggregation, a minority of the cells (prestalk cells) continue to express much the same set of developmental genes as during aggregation, but the majority (prespore cells) switch to the prespore program. We describe evidence suggesting that overexpressing AMPK increases the proportion of prestalk cells, as does inhibiting mTORC1. Furthermore, stimulating the acidification of intracellular acidic compartments likewise increases the proportion of prestalk cells, while inhibiting acidification favors the spore pathway. We conclude that the choice between the prestalk and the prespore pathways of cell differentiation may depend on the relative strength of the activities of AMPK and mTORC1, and that these may be controlled by the acidity of intracellular acidic compartments/lysosomes (pHv), cells with low pHv compartments having high AMPK activity/low mTORC1 activity, and those with high pHv compartments having high mTORC1/low AMPK activity. Increased insight into the regulation and downstream consequences of this switch should increase our understanding of its potential role in human diseases, and indicate possible therapeutic interventions.
... L'ammonium agirait via une cascade de phosphorylation qui inhiberait la PKA, dont la kinase Dhkc serait un des relais (Singleton et al, 1998). Si l'ammonium agit de la même façon sur l'activation de la PKA dans les muscles de larve, une élévation de la concentration intracellulaire en ammonium pourrait inactiver la PKA, qui, par conséquent, ne serait pas en mesure d'inactiver les sous-unités A des récepteurs du glutamate. ...
L’ammonium (couple NH4+/NH3) est impliqué dans de nombreux processus biologiques comme le maintien de l’équilibre acido-basique par le rein et la synthèse et le recyclage des neurotransmetteurs. Des transporteurs spécifiques rendent les membranes cellulaires perméables à l’ammonium, en particulier les glycoprotéines Rhésus (Rh50). Décrites notamment dans les globules rouges, le foie et le rein, les protéines Rh50 ont à la fois une fonction de transport de NH3 et un rôle dans le maintien de l’architecture cellulaire. La drosophile (Drosophila melanogaster) possède un unique gène Rh50, exprimé dans le système nerveux central (SNC) et neuromusculaire. De ce fait, elle est un modèle de choix pour l’étude fonctionnelle in vivo de ces protéines dans le système nerveux.Dans ce travail de thèse, nous montrons que Rh50 est exprimée fortement dans les muscles et enrichie à la jonction neuromusculaire (JNM) de la larve de drosophile, où elle colocalise avec des protéines des densités postsynaptiques, dont Discs-large. Une inactivation du gène Rh50 induit une atrophie musculaire, des défauts locomoteurs, la surexpression des récepteurs glutamatergiques postsynaptiques, et une létalité pupale. Des enregistrements électrophysiologiques à la JNM larvaire montrent que la fréquence des potentiels postsynaptiques excitateurs spontanés augmente lorsque le gène Rh50 est inactivé dans les muscles. Les larves déficientes pour Rh50 ont également une plus grande sensibilité à l’exposition à de fortes concentrations d’ammonium. Dans le SNC, Rh50 est exprimée dans des cellules neuroendocrines et nos résultats suggèrent qu’il contrôle l’expression des FMRFamides, neuropeptides impliqués dans la régulation de l’activité à la JNM.En conclusion, nos résultats montrent que Rh50 est une nouvelle protéine essentielle chez la drosophile, qui intervient dans le développement et la physiologie synaptique, et régule les taux d’ammonium et l’homéostasie acido-basique dans le système neuromusculaire.
... It should be noted that in both high oxygen and low temperature, depth of the weak-base-rich outer zone is increased. This change in ecmB expression is consistent with the idea that a decrease in ammonia plays a part in switching from the slug stage to the fruiting body formation (Singleton et al., 1998). ...
Recently it was demonstrated that a rapidly forming, self-organizing pattern that emerges within two-dimensional Dictyostelium discoideumcell cultures could later give rise to stripes of distinct zones, each comprising different cell types. Here we report physiological aspects of the initial rapid patterning and its relationship to cell differentiation. We found that as the temperature is lowered the characteristic length of the pattern increases. From this we estimated the activation energy of the patterning kinetics. Fluorescence of fluorescein-conjugated dextran revealed that the cytosolic pH of cells in the inside zone becomes lower than that in the outer zone facing the air. The patterning could be inhibited by addition of the plasma-membrane proton pump inhibitors diethystilbestrol (DES) or miconazole. Preincubation of cells with weak acid delayed the timing of the patterning, whereas weak base hastened it. A pH-indicating dye revealed localized accumulation of ammonia in the extracellular space. These results suggest that gradients of secreted metabolites may be directly responsible for the rapid patterning and its consequence on cell differentiation in a confined geometrical situation. Possible diffusible candidate molecules and a reaction scheme coupled to the imposed oxygen gradient are discussed.
... The transition from slug migration to fruiting body formation is inhibited by ammonia, which is produced by autophagy in the starving cells [75]. Ammonia activates the SHK DhkC to phosphorylate and, thereby, activate RegA to hydrolyse intracellular cAMP and, thereby, block further PKA activation [76]. Aerial projection of the slug tip in response to incident light allows dissipation of ammonia away from the tip, thus relieving the block. ...
Multicellularity evolved repeatedly in the history of life, but how it unfolded varies greatly between different lineages. Dictyostelid social amoebas offer a good system to study the evolution of multicellular complexity, with a well-resolved phylogeny and molecular genetic tools being available. We compare the life cycles of the Dictyostelids with closely related amoebozoans to show that complex life cycles were already present in the unicellular common ancestor of Dictyostelids. We propose frost resistance as an early driver of multicellular evolution in Dictyostelids and show that the cell signalling pathways for differentiating spore and stalk cells evolved from that for encystation. The stalk cell differentiation program was further modified, possibly through gene duplication, to evolve a new cell type, cup cells, in Group 4 Dictyostelids. Studies in various multicellular organisms, including Dictyostelids, volvocine algae, and metazoans, suggest as a common principle in the evolution of multicellular complexity that unicellular regulatory programs for adapting to environmental change serve as “proto-cell types” for subsequent evolution of multicellular organisms. Later, new cell types could further evolve by duplicating and diversifying the “proto-cell type” gene regulatory networks.
... The transition from slug migration to fruiting body formation is inhibited by ammonia, which is produced by autophagy in the starving cells. Ammonia activates the SHK DhkC to phosphorylate and thereby activate RegA to hydrolyse intracellular cAMP and thereby block further PKA activation [70]. Aerial projection of the slug tip in response to incident light allows dissipation of ammonia away from the tip, thus relieving the block. ...
Multicellularity evolved repeatedly in the history of life, but how it unfolded varies greatly between different lineages. Dictyostelid social amoebas offer a good system to study the evolution of multicellular complexity, with a well-resolved phylogeny and molecular genetic tools being available. We compare the life cycles of the Dictyostelids with closely related amoebozoans to show that complex life cycles were already present in the unicellular common ancestor of Dictyostelids. We propose frost resistance as an early driver of multicellular evolution in Dictyostelids and show that the cell signalling pathways for differentiating spore and stalk cells evolved from that for encystation. The stalk cell differentiation program was further modified, possibly through gene duplication, to evolve a new cell type, cup cells, in Group 4 Dictyostelids. Studies in various multicellular organisms including Dictyostelids, volvocine algae, and metazoans suggest as a common principle in the evolution of multicellular complexity that unicellular regulatory programs for adapting to environmental change serve as “proto-cell types” for subsequent evolution of multicellular organisms. Later, new cell types could further evolve by duplicating and diversifying the “proto-cell type” gene regulatory networks.
... DhkC is a sensor for NH 3 [38], which is produced by autophagy in the starving cells. The migrating slug is partially submerged in its own NH 3 , but the aerial projection of the slug tip in response to incident light enables dissipation of NH 3 gas. ...
Dictyostelid social amoebas respond to starvation by self-organizing into multicellular slugs that migrate towards light to construct spore-bearing structures. These behaviours depend on excitable networks that enable amoebas to produce propagating waves of the chemoattractant cAMP, and to respond by directional movement. cAMP additionally regulates cell differentiation throughout development, with differentiation and cell movement being coordinated by interaction of the stalk inducer c-di-GMP with the adenylate cyclase that generates cAMP oscillations. Evolutionary studies indicate how the manifold roles of cAMP in multicellular development evolved from a role as intermediate for starvation-induced encystation in the unicellular ancestor. A merger of this stress response with the chemotaxis excitable networks yielded the developmental complexity and cognitive capabilities of extant Dictyostelia.
This article is part of the theme issue ‘Basal cognition: conceptual tools and the view from the single cell’.
... In addition to Erk1, the role of other protein kinases in RegA regulation has been analysed. Histidine kinase and phosphorelay proteins facilitate the phosphorylation of RegA at residue D212 and this regulation impacts spore formation later in development [30,35,58]. It has not been determined whether RegA can be simultaneously phosphorylated at multiple sites or what possible impact one phosphorylation event might have on another. ...
Mitogen-activated protein kinase (MAPK) regulation of cAMP-specific phosphodiesterase function has been demonstrated in mammalian cells and suspected to occur in other eukaryotes. Epistasis analysis in the soil amoeba Dictyostelium discoideum suggests the atypical MAPK Erk2 downregulates the function of the cAMP-specific phosphodiesterase RegA to regulate progression of the developmental life cycle. A putative MAPK docking motif located near a predicted MAPK phosphorylation site was characterized for contributions to RegA function and binding to Erk2 because a similar docking motif has been previously characterized in the mammalian PDE4D phosphodiesterase. The overexpression of RegA with alterations to this docking motif (RegAD-) restored RegA function to regA- cells based on developmental phenotypes, but low-level expression of RegAD- from the endogenous regA promoter failed to rescue wild-type morphogenesis. Co-immunoprecipitation analysis indicated that Erk2 associates with both RegA and RegAD-, suggesting the docking motif is not required for this association. Epistasis analysis between regA and the only other Dictyostelium MAPK, erk1, suggests Erk1 and RegA can function in different pathways but that some erk1- phenotypes may require cAMP signalling. These results imply that MAPK downregulation of RegA in Dictyostelium is accomplished through a different mechanism than MAPK regulation of cAMP-specific phosphodiesterases in mammalian cells and that the regulation in Dictyostelium does not require a proximal MAPK docking motif.
... 4). Homologs of D. discoideum DhkB, which regulates spore germination and maintenance of spore dormancy (Zinda and Singleton 1998), and DhkC, which is required for the initiation of fruiting body formation (Singleton et al. 1998) and also affects chemotaxis (Kuwayama and Kubohara 2016), were also found in P. pallidum ( fig. 4). Finally, homologs of D. discoideum DhkF and DhkG, for which no functions are known, were identified in P. pallidum but were absent from the genomes of Physa. ...
Histidine kinases (HKs) are primary sensor proteins that act in cell signaling pathways generically referred to as "two component systems" (TCSs). TCSs are among the most widely distributed transduction systems used by both prokaryotic and eukaryotic organisms to detect and respond to a broad range of environmental cues. The structure and distribution of HK proteins are now well documented in prokaryotes but information is still fragmentary for eukaryotes. Here, we have taken advantage of recent genomic resources to explore the structural diversity and the phylogenetic distribution of HKs in the prominent eukaryotic supergroups. Searches of the genomes of 67 eukaryotic species spread evenly throughout the phylogenetic tree of life identified 748 predicted HK proteins. Independent phylogenetic analyses of predicted HK proteins were carried out for each of the major eukaryotic supergroups. This allowed most of the compiled sequences to be categorised into previously described HK groups. Beyond the phylogenetic analysis of eukaryotic HKs, this study revealed some interesting findings: (i) characterisation of some previously undescribed eukaryotic HK groups with predicted functions putatively related to physiological traits; (ii) discovery of HK groups that were previously believed to be restricted to a single kingdom in additional supergroups and (iii) indications that some evolutionary paths have led to the appearance, transfer, duplication, and loss of HK genes in some phylogenetic lineages. This study provides an unprecedented overview of the structure and distribution of HKs in the Eukaryota and represents a first step towards deciphering the evolution of TCS signaling in living organisms.
... For RegA, phosphorylation of the receiver domain activates the phosphodiesterase activity, decreasing intracellular cAMP levels [94,95]. In D. discoideum, the SHKPs detect signals like the spore-inducing peptide SDF2, ammonia, high osmolarity and the cytokinin, discadenine, that regulate the timely maturation of spores and stalk cells and the maintenance of spore dormancy in the fruiting body [91,96,97]. ...
Not long ago, protists were considered one of four eukaryote kingdoms, but recent gene-based phylogenies show that they contribute to all nine eukaryote subdomains. The former kingdoms of animals, plants and fungi are now relegated to lower ranks within subdomains. Most unicellular protists respond to adverse conditions by differentiating into dormant walled cysts. As cysts, they survive long periods of starvation, drought and other environmental threats, only to re-emerge when conditions improve. For protists pathogens, the resilience of their cysts can prevent successful treatment or eradication of the disease. In this context, effort has been directed towards understanding the molecular mechanisms that control encystation. We here firstly summarize the prevalence of encystation across protists and next focus on Amoebozoa, where most of the health-related issues occur. We review current data on processes and genes involved in encystation of the obligate parasite Entamoeba histolytica and the opportunistic pathogen Acanthamoeba. We show how the cAMP-mediated signalling pathway that controls spore and stalk cell encapsulation in Dictyostelium fruiting bodies could be retraced to a stress-induced pathway controlling encystation in solitary Amoebozoa. We highlight the conservation and prevalence of cAMP signalling genes in Amoebozoan genomes and the suprisingly large and varied repertoire of proteins for sensing and processing environmental signals in individual species.
... The culmination of migrating slugs is regulated by internal (e.g., ammonia and cAMP) and external (e.g., light, temperature, and humidity) factors (Newell et al., http://dx.doi.org/10.1016/j.biocel.2017.07.019 Received 2 April 2017; Received in revised form 13 July 2017; Accepted 27 July 2017 1969; Schindler and Sussman, 1977), which have been studied by mutating slugs to be defective in the culmination phase (Davies et al., 1993;Fukuzawa et al., 1997;Gee et al., 1994;Nelson et al., 2000;Singleton et al., 1998). However, despite the high resistance of D. discoideum to H 2 O 2 , little is known about oxidative stress-induced responses associated with methylglyoxal (MG), its inducing partner, and slug migration (Garcia et al., 2000). ...
... Stalk cell differentiation is inhibited in the slug stage by ammonia, the product of protein degradation. Ammonia activates the SHK DhkC, which activates RegA and thereby inhibits PKA [32]. Ammonia is lost by diffusion from the aerially projecting fruiting body tip of the early fruiting body, thus lifting PKA inhibition. ...
Dictyostelia represent a tractable system to resolve the evolution of cell-type specialization, with some taxa differentiating into spores only, and other taxa with additionally one or up to four somatic cell types. One of the latter forms, _Dictyostelium discoideum_, is a popular model system for cell biology and developmental biology with key signalling pathways controlling cell-specialization being resolved recently. For the most dominant pathways, evolutionary origins were retraced to a stress response in the unicellular ancestor, while modifications in the ancestral pathway were associated with acquisition of multicellular complexity. This review summarizes our current understanding of developmental signalling in _D. discoideum_ and its evolution.
... When ligand activates the histidine phosphatase, phosphorelay runs in reverse and inactivates RegA. Ammonia activates the histidine kinase DhkC and consequently RegA (Singleton et al. 1998), resulting in cAMP degradation and preventing activation of PKA. PKA activation is essential for spore and stalk cell maturation, and ammonia activation of RegA therefore effectively blocks both processes (Harwood et al. 1992;Hopper et al. 1993). ...
Dictyostelid social amoebas represent one of several groups of genetically divergent lineages that display aggregative multicellularity. In this chapter, we describe the evolution of developmental complexity in Dictyostelia and discuss the signalling mechanisms that control the developmental programme of the model organism Dictyostelium discoideum. We also reconstruct the evolutionary history of these developmental control mechanisms from environmental sensing in the unicellular ancestors of Dictyostelia. Finally, we explore the parameters that define the boundary between uni- and multicellularity.
... Stalk cell differentiation is under negative regulation of ammonia, which is produced in large quantities by protein degradation in the starving cells [51]. Ammonia activates the SHK DhkC, thereby activating RegA and inhibiting PKA [52]. Ammonia is lost from the aerially projecting tip of the early fruiting body, thus inactivating RegA and lifting PKA inhibition. ...
Aggregative multicellularity, resulting in formation of a spore-bearing fruiting body, evolved at least six times independently among both eukaryotes and prokaryotes. Among eukaryotes, this form of multicellularity is mainly studied in the social amoeba Dictyostelium discoideum. In this review, we summarize trends in the evolution of cell-type specialization and behavioural complexity in the four major groups of Dictyostelia. We describe the cell-cell communication systems that control the developmental programme of D. discoideum, highlighting the central role of cAMP in the regulation of cell movement and cell differentiation. Comparative genomic studies showed that the proteins involved in cAMP signalling are deeply conserved across Dictyostelia and their unicellular amoebozoan ancestors. Comparative functional analysis revealed that cAMP signalling in D. discoideum originated from a second messenger role in amoebozoan encystation. We highlight some molecular changes in cAMP signalling genes that were responsible for the novel roles of cAMP in multicellular development.
Copyright © 2015. Published by Elsevier Ltd.
... The slug responds to environmental signals that direct its migration and regulate the slug-to-fruit switch– the process of culmination leading to formation of the fruiting body. Signals include light, low NH 3 , low moisture, higher temperature, and high O 2 which, in the native environment of the soil, draw the subterranean slug to above ground where culmination is most pro- ductive [11,12,151617181920. In the laboratory, the process takes place over the course of 24 h after deposition of amoebae on moist agar or filter surfaces wetted with low salt buffers. ...
Background
Oxygen sensing is a near universal signaling modality that, in eukaryotes ranging from protists such as Dictyostelium and Toxoplasma to humans, involves a cytoplasmic prolyl 4-hydroxylase that utilizes oxygen and α-ketoglutarate as potentially rate-limiting substrates. A divergence between the animal and protist mechanisms is the enzymatic target: the animal transcriptional factor subunit hypoxia inducible factor-α whose hydroxylation results in its poly-ubiquitination and proteasomal degradation, and the protist E3SCFubiquitin ligase subunit Skp1 whose hydroxylation might control the stability of other proteins. In Dictyostelium, genetic studies show that hydroxylation of Skp1 by PhyA, and subsequent glycosylation of the hydroxyproline, is required for normal oxygen sensing during multicellular development at an air/water interface. Because it has been difficult to detect an effect of hypoxia on Skp1 hydroxylation itself, the role of Skp1 modification was investigated in a submerged model of Dictyostelium development dependent on atmospheric hyperoxia.
Results
In static isotropic conditions beneath 70-100% atmospheric oxygen, amoebae formed radially symmetrical cyst-like aggregates consisting of a core of spores and undifferentiated cells surrounded by a cortex of stalk cells. Analysis of mutants showed that cyst formation was inhibited by high Skp1 levels via a hydroxylation-dependent mechanism, and spore differentiation required core glycosylation of Skp1 by a mechanism that could be bypassed by excess Skp1. Failure of spores to differentiate at lower oxygen correlated qualitatively with reduced Skp1 hydroxylation.
Conclusion
We propose that, in the physiological range, oxygen or downstream metabolic effectors control the timing of developmental progression via activation of newly synthesized Skp1.
... High concentrations of ammonia keep slugs migrating and block the initiation of culmination (Schindler and Sussman, 1977). The exhaustion of the ammonia supply triggers culmination at least in part by activating protein kinase A through the DhkC signaling pathway (Singleton et al., 1998). In fruiting bodies, extremely high concentration of ammonium phosphate in sori maintain spore dormancy through the activation of the sporulation-specific adenylyl cyclase ACG (Cotter et al., 1999). ...
The signaling molecules NH(3) (unprotonated volatile ammonia), as well as cyclic adenosine monophosphate and differentiation-inducing factor, play important roles in the multicellular development of the slime mould Dictyostelium discoideum. One of the downstream metabolic products catalyzed by allantoicase (allC) is ammonia. We observed the role of allC by RNAi-mediated manipulation of its expression. The allC gene of D. discoideum was silenced by RNAi. We found significant downregulation of allC mRNA and protein expression levels. Recombinant allC RNAi mutant cell lines had a shortened cell cycle, a reduction in cell size relative to wild-type cells and interrupted development. We conclude that the normal functions of allC include retarding cell division until a specific cell size is reached and coordinating the progression of development.
... Homologs of COS1/CANIK1 have been identified in the filamentous fungi Neurospora crassa (NIK-1) (72) and Aspergillus nidulans (ANNIK1) (71). The slime mold Dictyostelium discoideum contains at least 11 HKs (DOKA, DHKA-D, and ESTs) involved in a range of activities that include osmotic response and development (73)(74)(75)(76)(77). Two-component proteins are not limited to eukaryotic microorganisms; they have also been found in plants, such as Arabidopsis thaliana (ETR1, ETR2, ERS, and EIN4) (55,78) and tomato (NR) (79,80), in which they regulate ethylene-mediated fruit ripening. ...
Most prokaryotic signal-transduction systems and a few eukaryotic pathways use phosphotransfer schemes involving two conserved components, a histidine protein kinase and a response regulator protein. The histidine protein kinase, which is regulated by environmental stimuli, autophosphorylates at a histidine residue, creating a high-energy phosphoryl group that is subsequently transferred to an aspartate residue in the response regulator protein. Phosphorylation induces a conformational change in the regulatory domain that results in activation of an associated domain that effects the response. The basic scheme is highly adaptable, and numerous variations have provided optimization within specific signaling systems. The domains of two-component proteins are modular and can be integrated into proteins and pathways in a variety of ways, but the core structures and activities are maintained. Thus detailed analyses of a relatively small number of representative proteins provide a foundation for understanding this large family of signaling proteins.
... There is, for example, a well-established correlation between ammonia hypersensitivity and an increased propensity for slug migration (Gee et al., 1994) and the fbxA strain shows both these properties. Entry into culmination is regulated by cAMP dependent protein kinase (Harwood et al., 1992; Mann et al., 1992) and recently a pathway that links ammonia to production of intracellular cAMP via a sensor histidine kinase has been proposed (Singleton et al., 1998). It will be of interest to see whether the fbxA strain has altered levels of any of the proteins in this pathway. ...
FbxA is a novel member of a family of proteins that contain an F-box and WD40 repeats and that target specific proteins for degradation via proteasomes. In fruiting bodies formed from cells where the fbxA gene is disrupted (fbxA− cells), the spore mass fails to fully ascend the stalk. In addition, fbxA− slugs continue to migrate under environmental conditions where the parental strain immediately forms fruiting bodies. Consistent with this latter behaviour, the development of fbxA− cells is hypersensitive to ammonia, the signaling molecule that regulates the transition from the slug stage to terminal differentiation. The slug comprises an anterior prestalk region and a posterior prespore region and the fbxA mRNA is highly enriched in the prestalk cells. The prestalk zone of the slug is further subdivided into an anterior pstA region and a posterior pstO region. In fbxA− slugs the pstO region is reduced in size and the prespore region is proportionately expanded. Our results indicate that FbxA is part of a regulatory pathway that controls cell fate decisions and spatial patterning via regulated protein degradation.
... High concentrations of ammonia lead to a drop in intracellular cAMP concentrations and subsequent inhibition of PKA, while low levels of ammonia lead to cAMP accumulation and subsequent activation of PKA. Ammonia is thought to regulate cAMP levels by triggering a phosphorelay from the histidine kinase DhkC to the protein RdeA and then to RegA, a cAMP phosphodiesterase (Singleton et al. 1998). Several ammonia transporter genes have been discovered in Dictyostelium (Kirsten et al. 2008). ...
Copines are calcium-dependent membrane-binding proteins found in many eukaryotic organisms. We are studying the function of copines using the model organism, Dictyostelium discoideum. When under starvation conditions, Dictyostelium cells aggregate into mounds that become migrating slugs, which can move toward light and heat before culminating into a fruiting body. Previously, we showed that Dictyostelium cells lacking the copine A (cpnA) gene are not able to form fruiting bodies and instead arrest at the slug stage. In this study, we compared the slug behavior of cells lacking the cpnA gene to the slug behavior of wild-type cells. The slugs formed by cpnA- cells were much larger than wild-type slugs and exhibited no phototaxis and negative thermotaxis in the same conditions that wild-type slugs exhibited positive phototaxis and thermotaxis. Mixing as little as 5% wild-type cells with cpnA- cells rescued the phototaxis and thermotaxis defects, suggesting that CpnA plays a specific role in the regulation of the production and/or release of a signaling molecule. Reducing extracellular levels of ammonia also partially rescued the phototaxis and thermotaxis defects of cpnA- slugs, suggesting that CpnA may have a specific role in regulating ammonia signaling. Expressing the lacZ gene under the cpnA promoter in wild-type cells indicated cpnA is preferentially expressed in the prestalk cells found in the anterior part of the slug, which include the cells at the tip of the slug that regulate phototaxis, thermotaxis, and the initiation of culmination into fruiting bodies. Our results suggest that CpnA plays a role in the regulation of the signaling pathways, including ammonia signaling, necessary for sensing and/or orienting toward light and heat in the prestalk cells of the Dictyostelium slug.
... Ammonia is one of several signals that regulate the activity of sensor histidine kinases ⁄ phosphatases, which, upon ligand binding initiate either forward or reverse phosphoryltransfer that ultimately leads to phosphorylation or dephosphorylation of a conserved aspartate in the response regulator of the intracellular cAMP phosphodiesterase RegA (Shaulsky et al. 1998;Thomason et al. 1998). Ammonia activates forward phosphoryl transfer by acting on the histidine kinase DhkC, resulting in RegA activation, hydrolysis of cAMP and inhibition of PKA (Singleton et al. 1998). Loss of ammonia from the tip therefore allows PKA activation and stalk maturation at this position. ...
The Dictyostelid social amoebas represent one of nature's several inventions of multicellularity. Though normally feeding as single cells, nutrient stress triggers the collection of amoebas into colonies that form delicately shaped fruiting structures in which the cells differentiate into spores and up to three cell types to support the spore mass. Cyclic adenosine monophosphate (cAMP) plays a very dominant role in controlling morphogenesis and cell differentiation in the model species Dictyostelium discoideum. As a secreted chemoattractant cAMP coordinates cell movement during aggregation and fruiting body morphogenesis. Secreted cAMP also controls gene expression at different developmental stages, while intracellular cAMP is extensively used to transduce the effect of other stimuli that control the developmental program. In this review, I present an overview of the different roles of cAMP in the model D. discoideum and I summarize studies aimed to resolve how these roles emerged during Dictyostelid evolution.
... The second coding region also possessed the S51A mutation made as described above. pif2␣-25 was digested to release the eIF2␣-bsr DNA, and this was transformed into Ax4 cells using electroporation (33). Genomic DNA was isolated from blasticidin-resistant clones possessing insertions at the endogenous eIF2␣ gene (determined by PCR screening) and used as a template in a PCR to amplify a 1,000-bp fragment spanning the proximal promoter and coding region past the S51 codon. ...
Growing Dictyostelium cells secrete CfaD and AprA, two proteins that have been characterized as chalones. They exist within a high-molecular-weight
complex that reversibly inhibits cell proliferation, but not growth, via cell surface receptors and a signaling pathway that
includes G proteins. How the production of these two proteins is regulated is unknown. Dictyostelium cells possess three GCN2-type eukaryotic initiation factor 2 α subunit (eIF2α) kinases, proteins that phosphorylate the translational
initiation factor eIF2α and possess a tRNA binding domain involved in their regulation. The Dictyostelium kinases have been shown to function during development in regulating several processes. We show here that expression of an
unregulated, activated kinase domain greatly inhibits cell proliferation. The inhibitory effect on proliferation is not due
to a general inhibition of translation. Instead, it is due to enhanced production of a secreted factor(s). Indeed, extracellular
CfaD and AprA proteins, but not their mRNAs, are overproduced in cells expressing the activated kinase domain. The inhibition
of proliferation is not seen when the activated kinase domain is expressed in cells lacking CfaD or AprA or in cells that
contain a nonphosphorylatable eIF2α. We conclude that production of the chalones CfaD and AprA is translationally regulated
by eIF2α phosphorylation. Both proteins are upregulated at the culmination of development, and this enhanced production is
lacking in a strain that possesses a nonphosphorylatable eIF2α.
... The phosphoryl group is then transferred in two steps towards or away from the response regulator domain of RegA, thereby activating or inactivating its cAMP-phosphodiesterase activity, respectively (Thomason et al., 1999). For instance, ammonia, which inhibits the onset of fruiting body formation and stalk maturation, activates the histidine kinase activity of sensor histidine kinase C (DhkC), resulting in RegA activation, hydrolysis of cAMP and inhibition of PKA activity ( Singleton et al., 1998). Spore differentiation factor 2 (SDF-2) activates the histidine phosphatase activity of DhkA (histidine phosphatase A), which results in the inactivation of RegA, the accumulation of cAMP and in PKA activation, which subsequently causes spore maturation ( Wang et al., 1999). ...
Dictyostelium discoideum belongs to a group of multicellular life forms that can also exist for long periods as single cells. This ability to shift between uni- and multicellularity makes the group ideal for studying the genetic changes that occurred at the crossroads between uni- and multicellular life. In this Primer, I discuss the mechanisms that control multicellular development in Dictyostelium discoideum and reconstruct how some of these mechanisms evolved from a stress response in the unicellular ancestor.
Dictyostelids were once grouped with fungi because their fruiting structures are superficially similar. However, either group evolved quite distinct strategies to reach this mode of species propagation. This chapter describes the cell communication systems that cause dictyostelid amoebas to aggregate and regulate cell-type specialisation and cell movement during fruiting body formation. It also highlights how increasingly complex morphologies appeared during dictyostelid evolution and how some of these morphological innovations were caused by modification of specific cell–cell signalling systems.KeywordsAmoebozoaCampCystDictyosteliaFruiting bodyEvolutionMulticellularitySignallingSocial amoebasSpore
Protein kinases are major regulators of cellular processes, but the roles of most kinases remain unresolved. Dictyostelid social amoebas have been useful in identifying functions for 30% of its kinases in cell migration, cytokinesis, vesicle trafficking, gene regulation and other processes but their upstream regulators and downstream effectors are mostly unknown. Comparative genomics can assist to distinguish between genes involved in deeply conserved core processes and those involved in species-specific innovations, while co-expression of genes as evident from comparative transcriptomics can provide cues to the protein complement of regulatory networks. Genomes and developmental and cell-type specific transcriptomes are available for species that span the 0.5 billion years of evolution of Dictyostelia from their unicellular ancestors. In this work we analysed conservation and change in the abundance, functional domain architecture and developmental regulation of protein kinases across the 4 major taxon groups of Dictyostelia. All data are summarized in annotated phylogenetic trees of the kinase subtypes and accompanied by functional information of all kinases that were experimentally studied. We detected 393 different protein kinase domains across the five studied genomes, of which 212 were fully conserved. Conservation was highest (71%) in the previously defined AGC, CAMK, CK1, CMCG, STE and TKL groups and lowest (26%) in the "other" group of typical protein kinases. This was mostly due to species-specific single gene amplification of "other" kinases. Apart from the AFK and α-kinases, the atypical protein kinases, such as the PIKK and histidine kinases were also almost fully conserved. The phylogeny-wide developmental and cell-type specific expression profiles of the protein kinase genes were combined with profiles from the same transcriptomic experiments for the families of G-protein coupled receptors, small GTPases and their GEFs and GAPs, the transcription factors and for all genes that upon lesion generate a developmental defect. This dataset was subjected to hierarchical clustering to identify clusters of co-expressed genes that potentially act together in a signalling network. The work provides a valuable resource that allows researchers to identify protein kinases and other regulatory proteins that are likely to act as intermediates in a network of interest.
Background
The two-component signaling (TCS) system is an important signal transduction machinery in prokaryotes and eukaryotes, excluding animals, that uses a protein phosphorylation mechanism for signal transmission.
Conclusion
Prokaryotes have a primitive type of TCS machinery, which mainly comprises a membrane- bound sensory histidine kinase (HK) and its cognate cytoplasmic response regulator (RR). Hence, it is sometimes referred to as two-step phosphorelay (TSP). Eukaryotes have more sophisticated signaling machinery, with an extra component - a histidine-containing phosphotransfer (HPT) protein that shuttles between HK and RR to communicate signal baggage. As a result, the TSP has evolved from a two-step phosphorelay (His–Asp) in simple prokaryotes to a multi-step phosphorelay (MSP) cascade (His–Asp–His–Asp) in complex eukaryotic organisms, such as plants, to mediate the signaling network. This molecular evolution is also reflected in the form of considerable structural modifications in the domain architecture of the individual components of the TCS system. In this review, we present TCS system's evolutionary journey from the primitive TSP to advanced MSP type across the genera. This information will be highly useful in designing the future strategies of crop improvement based on the individual members of the TCS machinery.
Amoebozoa, such as Entamoeba and Acanthamoeba, survive environmental stress by encystment, but also comprise the Dictyostelia which form spores in multicellular fruiting bodies to survive starvation stress. Sporulation is triggered by cAMP activation of cAMP-dependent protein kinase (PKA), with cAMP levels being controlled by the adenylate cyclases ACG and ACR and the phosphodiesterase RegA. Many Dictyostelia can also alternatively encyst and we showed that environmental stress acts on ACG and ACR to increase cAMP and thereby activate PKA to trigger encystation, with RegA preventing precocious encystation and inducing cyst germination. RegA activity requires phosphorylation of its response regulator domain, which is controlled by Sensor Histidine Kinases/Phosphatases (SHKPs), which in Dictyostelium respond to developmental signals. Comparative genomics showed that RegA, AcrA and PKA and a wealth of SHKPs are deeply conserved in Amoebozoa, where SHKs may sense food and SHPs environmental stress.
Signal transduction systems configured around a core phosphotransfer step between a histidine kinase and a cognate response regulator protein occur in organisms from all domains of life. These systems, termed two-component systems, constitute the majority of multi-component signaling pathways in Bacteria but are less prevalent in Archaea and Eukarya. The core signaling domains are modular, allowing versatility in configuration of components into single-step phosphotransfer and multi-step phosphorelay pathways, the former being predominant in bacteria and the latter in eukaryotes. Two-component systems regulate key cellular regulatory processes that provide adaptive responses to environmental stimuli and are of interest for the development of antimicrobial therapeutics, biotechnology applications, and biosensor engineering. In bacteria, two-component systems have been found to mediate responses to an extremely broad array of extracellular and intracellular chemical and physical stimuli, whereas in archaea and eukaryotes, the use of two-component systems is more limited. This review summarizes recent advances in exploring the repertoire of sensor histidine kinases in the Archaea and Eukarya domains of life.
The well-orchestrated multicellular life cycle of Dictyostelium discoideum has fascinated biologists for over a century. Self-organisation of its amoebas into aggregates, migrating slugs and fruiting structures by pulsatile cAMP signalling and their ability to follow separate differentiation pathways in well-regulated proportions continue to be topics under investigation. A striking aspect of D. discoideum development is the recurrent use of cAMP as chemoattractant, differentiation inducing signal and second messenger for other signals that control the developmental programme. D. discoideum is one of >150 species of Dictyostelia and aggregative life styles similar to those of Dictyostelia evolved many times in eukaryotes. Here we review experimental studies investigating how phenotypic complexity and cAMP signalling co-evolved in Dictyostelia. In addition, we summarize comparative genomic studies of multicellular Dictyostelia and unicellular Amoebozoa aimed to identify evolutionary conservation and change in all genes known to be essential for D. discoideum development.
DIF-1 and DIF-2 are signaling molecules that control chemotaxis in Dictyostelium discoideum. Whereas DIF-1 suppresses chemotaxis in shallow cAMP gradients, DIF-2 enhances chemotaxis under the same conditions via a phosphodiesterase, RegA, which is a part of the DhkC-RdeA-RegA two-component signaling system. In this study, to investigate the mechanism of the chemotaxis regulation by DIF-2, we examined the effects of DIF-2 (and DIF-1) on chemotaxis in rdeA(-) and dhkC(-) mutant strains. In the parental wild-type strains, chemotactic cell movement was suppressed with DIF-1 and enhanced with DIF-2 in shallow cAMP gradients. In contrast, in both rdeA(-) and dhkC(-) strains, chemotaxis was suppressed with DIF-1 but unaffected by DIF-2. The results suggest that DIF-2 modulates chemotaxis via the DhkC-RdeA-RegA signaling system. This article is protected by copyright. All rights reserved.
Dictyostelium has become an important model system to study the molecular details of the signalling pathways controlling gradient sensing and cell polarisation that control localised activation of the actin-myosin cytoskeleton responsible for evolutionary highly conserved mechanisms of chemotactic cell movement up chemoattractant gradients. 3′-5′ cyclic AMP is the chemoattractant that controls the chemotactic cell movements that result in aggregation of up to several hundred thousand cells, slug formation, migration and fruiting body formation. The coordination of these complex cell movements require long-range cAMP mediated cell-cell signalling based on periodic initiation of cAMP signals in the aggregation centre and slug tip and relay by surrounding cells, resulting in highly dynamic patterns of cAMP wave propagation. Model calculations have shown that the dynamic feedbacks between autocatalytic cell-cell cAMP signalling and cAMP-mediated collective chemotactic cell movement result in emergent properties that readily explain multicellular morphogenesis. cAMP signalling not only controls cell movement but also acts as a key morphogen to control cell differentiation, which in turn affects cell type specific cell-cell signalling and cell movement, adding an additional layer of feedback. To fully understand the multicellular morphogenesis of this organism at the level of cell behaviours, it will be needed to integrate the detailed cell type proportioning mechanisms in models describing cell-cell signalling and movement. Dictyostelium is likely to be the first eukaryotic organism where it will be possible to quantitatively understand how multicellular development and morphogenesis arise as emergent properties from a few relatively simple collective cell behaviours. © 2013 Springer-Verlag Berlin Heidelberg. All rights are reserved.
Phosphorelay from histidine kinases to response regulators controls a myriad of processes in bacteria but appears to be more specialized in eukaryotes. At least 15 members of the histidine kinase family can be recognized in the genomic sequences of Dictyostelium discoideum. The predicted products of each of these genes have well-conserved catalytic and receiver domains. One member of the family, DhkD, is a double histidine kinase with two catalytic and two receiver domains. Alignment of this large family with the Sln1 histidine kinase of yeast extends the sequence profile that characterizes eukaryotic histidine kinases. DhkA is a transmembrane receptor kinase that autophosphorylates and relays the phosphate to the receiver aspartate when dimerized. Genetic studies on dhkA, dhkB, dhkC, and dokA have indicated their roles in cellular and developmental processes. It is likely that DhkC relays phosphate to the N-terminal receiver domain of the cAMP phosphodiesterase RegA through the H2 domain of RdeA. DhkA and DhkB appear to inhibit RegA activity by indirectly activating the MAP kinase ERK2. When the carboxy-terminal region of RegA is phosphorylated, phosphodiesterase activity is inhibited. Histidine kinases may also activate the late adenylyl kinase, ACR. Thus, these histidine kinases seem to be focused on regulating cAMP to modulate the activity of the cAMP-dependent protein kinase, PKA.
A suite of organisms from different taxonomical and ecological positions are needed to assess environmentally relevant soil toxicity. We herein present a new bioassay based on Dictyostelium that is aimed at integrating slime moulds into such a testing framework. Toxicity tests on elutriates and the solid phase developmental cycle (DDDC) assay were successfully applied to a soil spiked with a mixture of Zn, Cd and diesel fuel freshly prepared (RC) and after 2 yr ageing (AC). The elutriates of both soils provoked toxic effects but toxicity was markedly lower in the aged soil. In the DDDC assay both soils affected amoeba viability and aggregation, with fewer multicellular units, smaller fruiting bodies and, overall, inhibition of fruiting body formation. This assay is quick and requires small amounts of test soil, which might facilitate its incorporation into a multispecies multiple-endpoint toxicity bioassay battery suitable for environmental risk assessment in soils. This article is protected by copyright. All rights reserved.
Cyclic-di-GMP is currently recognized as the most widely used intracellular signal molecule in prokaryotes, but roles in eukaryotes were only recently discovered. In the social amoeba Dictyostelium discoideum, c-di-GMP, produced by a prokaryote-type diguanylate cyclase, induces the differentiation of stalk cells, thereby enabling the formation of spore- bearing fruiting bodies. In this review, we summarize the currently known mechanisms that control the major life cycle transitions of Dictyostelium and focus particularly on the role of c-di-GMP in stalk formation. Stalk cell differentiation has characteristics of autophagic cell death, a process that also occurs in higher eukaryotes. We discuss the respective roles of c-di-GMP and of another signal molecule, DIF, in autophagic cell death in vitro and in stalk formation in vivo.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
The social amoeba Dictyostelium discoideum has been proposed for assessing stress responses to pollutants in soil and it has already been successfully employed in the aquatic environment. Presently, we developed the DDDC assay (D. discoideum developmental cycle assay) for both soil toxicity assessment and soil health screening. The DDDC assay is primarily aimed at determining the capacity of D. discoideum to undergo its developmental programme forming a fruiting body, measured in terms of fruiting body formation inhibition and fruiting body size factor, which may be considered an indication of its ecological fitness (potential for spore dispersal). A second objective of the solid phase DDDC assay is to identify potential mechanisms of toxic action on the developmental cycle, for which three checkpoints are examined: (a) aggregation arrest, (b) migration arrest, and (c) culmination arrest. Presently, conditions for the DDDC assay such as soil texture, soil water content, soil pH, food availability and incubation time were investigated and optimized. In addition, both solid and liquid phase variants of the DDDC assay were applied to assess the toxicity of Hg, at regulatory concentrations. The developmental cycle and ecological fitness were affected from the exposure to 0.3mgHg/kg dry-wt soil onwards. The DDDC assay has been shown to be a high sensitivity test.
Multicellular development of the social amoeba Dictyostelium discoideum results from the chemotactic aggregation of single cells to form a fruiting body consisting of a stalk supporting a spore mass. The interplay of two extracellular signalling molecules, cAMP (cyclic adenosine monophosphate) and the chlorinated hexaphenone DIF‐1 (differentiation‐inducing factor 1), directs cellular differentiation, and cAMP is also the chemo‐attractant that orchestrates all of morphogenesis. Although this is a relatively unusual mode of pattern formation, most of the fundamental processes that typify development in higher organism are on display, and the genetic accessibility of the organism makes it a very powerful and relevant model system. It has proven particularly valuable for understanding the basic mechanism of chemotaxis and the role that chemotaxis can play in the morphogenesis of a multicellular structure.
Key concepts
Dictyostelium cells aggregate by chemotaxis in response to propagating cAMP waves.
cAMP waves propagate through cAMP relay, the ability of the cells to detect a small cAMP signal, synthesis and secrete cAMP in response and thus pass the cAMP signal on to their neighbours.
Adaptation of the relay response ensures unidirectional wave propagation away from the centre.
Chemotactic cell movement involves detection of a cAMP gradient across the length of the cell, resulting in a polarization of actin–myosin cytoskeletal dynamics and movement up the gradient.
Multicellular morphogenesis, the arrangement of tissue in space and time, is controlled by the interplay of cAMP wave propagation and chemotaxis during all stages of development.
Dictyostelium development is separated from cell division but a cell's fate can be biased by its cell cycle position when development starts.
Prespore cells are formed in response to cAMP signalling.
Differentiation of prestalk cells is induced by a small organic molecule, DIF, which is secreted by the prespore cells. This induction forms part of a feedback loop that controls the prestalk–prespore ratio.
The tip‐organizer cells are a subset of the prestalk cells that differentiation in response to a high local cAMP concentation.
Dictyostelium pattern formation occurs by the sorting out of differentiated cell types.
Dictyostelids were once grouped with fungi because their fruiting structures are superficially similar. However both protists
evolved quite distinct strategies to reach this mode of species propagation. This chapter describes the cell communication
systems that cause Dictyostelid amoebas to aggregate and that regulate cell-type specialization and cell movement during fruiting
body formation. It also highlights how increasingly complex morphologies appeared during dictyostelid evolution and how some
of these morphological innovations were caused by modification of specific cell–cell signalling systems.
Multicellular organisms have evolved several times from unicellular protists giving rise to the familiar forms of animals,
plants and fungi. An important question in biology is how such transitions occurred. Multicellular life is typically dependent
on complex communication between cells, whereas unicellular organisms respond mainly to environmental signals. Social amoebae
are eminently suited to study the evolution of multicellularity, since they still combine a unicellular feeding stage with
a stage where thousands of cells aggregate to form motile slugs and fruiting structures. In this chapter we summarize the
signalling mechanisms that coordinate multicellular development in social amoebae and we discuss how these signalling mechanisms
evolved from a response to environmental stress in solitary amoebae.
One of the central aims of the study of development is to understand how distinct cellular behaviours, for example, division,
differentiation, apoptosis, and movement, are coordinated in space and in time to result in reproducible pattern formation
and morphogenesis. Coordination of these cellular behaviours requires extensive communication between cells of different types
and between the cells and their environment. The social amoeba Dictyostelium discoideum, a simple genetically tractable organism situated at the threshold of single and multicellular organisms in the evolutionary
tree of life, is well suited for the study of these interactions because its genome has been sequenced and it is amenable
to experimental manipulation through targeted gene disruption and replacement (Kreppel, Fey et al. 2004; Eichinger, Pachebat
et al. 2005). Dictyostelium cells normally live as single cells in the leaf litter of the soil where they feed on bacteria and divide by binary fission.
Under starvation conditions, up to several hundred thousand cells aggregate chemotactically to form a multicellular structure
the slug, that directed by light and temperature gradients migrates to the surface of the soil to form a fruiting body. The
fruiting body is composed of a stalk supporting a mass of spores. The spores are a dormant stage which after dispersal may
germinate to release amoebae, thus closing the life cycle (Fig. 1). We describe here key aspects of the signalling mechanisms
coordinating cellular behaviours responsible for pattern formation and morphogenesis.
In Dictyostelium amoebae, cell-type differentiation, spatial patterning, and morphogenesis are controlled by a combination of cell-autonomous mechanisms and intercellular signaling. A chemotactic aggregation of approximately 10(5) cells leads to the formation of a multicellular organism. Cell-type differentiation and cell sorting result in a small number of defined cell types organized along an anteroposterior axis. Finally, a mature fruiting body is created by the terminal differentiation of stalk and spore cells. Analysis of the regulatory program demonstrates a role for several molecules, including GSK-3, signal transducers and activators of transcription (STAT) factors, and cAMP-dependent protein kinase (PKA), that control spatial patterning in metazoans. Unexpectedly, two component systems containing histidine kinases and response regulators also play essential roles in controlling Dictyostelium development. This review focuses on the role of cAMP, which functions intracellularly to mediate the activity of PKA, an essential component in aggregation, cell-type specification, and terminal differentiation. Cytoplasmic cAMP levels are controlled through both the regulated activation of adenylyl cyclases and the degradation by a phosphodiesterase containing a two-component system response regulator. Extracellular cAMP regulates G-protein-dependent and -independent pathways to control aggregation as well as the activity of GSK-3 and the transcription factors GBF and STATa during multicellular development. The integration of these pathways with others regulated by the morphogen DIF-1 to control cell fate decisions are discussed.
Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of kinases. The protein kinase B-related kinase (PKBR1)
has been studied with emphasis on its role in chemotaxis, but its roles in late development remained obscure. The pkbR1 null mutant stays in the first finger stage for about 16 h or longer. Only a few aggregates continue to the migrating slug
stage; however, the slugs immediately go back probably to the previous first finger stage and stay there for approximately
37 h. Finally, the mutant fingers diversify into various multicellular bodies. The expression of the pkbR1 finger protein probably is required for development to the slug stage and to express ecmB, which is first observed in migrating slugs. The mutant also showed no ST-lacZ expression, which is of the earliest step in differentiation to one of the stalk cell subtypes. The pkbR1 null mutant forms a small number of aberrant fruiting bodies, but in the presence of 10% of wild-type amoebae the mutant
preferentially forms viable spores, driving the wild type to form nonviable stalk cells. These results suggest that the mutant
has defects in a system that changes the physiological dynamics in the prestalk cell region of a finger. We suggest that the
arrest of its development is due to the loss of the second wave of expression of a protein kinase A catalytic subunit gene
(pkaC) only in the prestalk region of the pkbR1 null mutant.
Large vacuoles are characteristic of plant and fungal cells, and their origin has long attracted interest. The cellular slime mould provides a unique opportunity to study the de novo formation of vacuoles because, in its life cycle, a subset of the highly motile animal-like cells (prestalk cells) rapidly develops a single large vacuole and cellulosic cell wall to become plant-like cells (stalk cells). Here we describe the origin and process of vacuole formation using live-imaging of Dictyostelium cells expressing GFP-tagged ammonium transporter A (AmtA-GFP), which was found to reside on the membrane of stalk-cell vacuoles. We show that stalk-cell vacuoles originate from acidic vesicles and autophagosomes, which fuse to form autolysosomes. Their repeated fusion and expansion accompanied by concomitant cell wall formation enable the stalk cells to rapidly develop turgor pressure necessary to make the rigid stalk to hold the spores aloft. Contractile vacuoles, which are rich in H(+)-ATPase as in plant vacuoles, remained separate from these vacuoles. We further argue that AmtA may play an important role in the control of stalk-cell differentiation by modulating the pH of autolysosomes.
Identifying candidate genes in genetic networks is important for understanding regulation and biological function. Large gene expression datasets contain relevant information about genetic networks, but mining the data is not a trivial task. Algorithms that infer Bayesian networks from expression data are powerful tools for learning complex genetic networks, since they can incorporate prior knowledge and uncover higher-order dependencies among genes. However, these algorithms are computationally demanding, so novel techniques that allow targeted exploration for discovering new members of known pathways are essential.
Here we describe a Bayesian network approach that addresses a specific network within a large dataset to discover new components. Our algorithm draws individual genes from a large gene-expression repository, and ranks them as potential members of a known pathway. We apply this method to discover new components of the cAMP-dependent protein kinase (PKA) pathway, a central regulator of Dictyostelium discoideum development. The PKA network is well studied in D. discoideum but the transcriptional networks that regulate PKA activity and the transcriptional outcomes of PKA function are largely unknown. Most of the genes highly ranked by our method encode either known components of the PKA pathway or are good candidates. We tested 5 uncharacterized highly ranked genes by creating mutant strains and identified a candidate cAMP-response element-binding protein, yet undiscovered in D. discoideum, and a histidine kinase, a candidate upstream regulator of PKA activity.
The single-gene expansion method is useful in identifying new components of known pathways. The method takes advantage of the Bayesian framework to incorporate prior biological knowledge and discovers higher-order dependencies among genes while greatly reducing the computational resources required to process high-throughput datasets.
1. In the life history of Dictyostelium discoideum there is a period of migration in which the aggregated cell mass wanders about in the form of a sausage prior to the formation of a final fruiting body.2. The period of migration was known to be variable, and now a limiting factor has been demonstrated. It was possible to show that in the absence of solutes migration will occur for long periods of time, and that the duration of migration decreased approximately exponentially with the concentration of added solute. The effect cannot be accounted for entirely as an osmotic phenomenon because different substances have different degrees of effectiveness, electrolytes being more effective than non-electrolytes.
Reductions in the levels and activities of enzymes that utilize thiamine diphosphate (ThDP) as a cofactor are thought to be responsible for the tissue damage suffered during thiamine deficiency. Although loss of cofactor can account in part for loss of enzyme activity, thiamine and its phosphorylated derivatives may also regulate the expression of the genes encoding these proteins. To examine this possibility, steady-state mRNA levels for three ThDP-dependent enzymes were measured in human fibroblasts, lymphoblasts and neuroblastoma cells cultured under conditions of thiamine sufficiency and deficiency. In all three cell types, the mRNA levels of transketolase and the E1beta subunit of pyruvate dehydrogenase complex were lower in thiamine-deficient cultures. In contrast, mRNA levels for a ThDP-binding subunit of alpha-ketoglutarate dehydrogenase, the E1 subunit did not differ. These results indicate that thiamine or a thiamine metabolite regulates the expression in humans of some, but not all, genes encoding ThDP-utilizing enzymes.
We have previously reported the analysis of DdPK3, a developmentally regulated putative serine/threonine kinase that shares approximately 50% amino acid sequence identity with metazoan cAMP-dependent protein kinase A (PKA) and protein kinase C, within their catalytic domains. Cells in which the DdPK3 gene has been disrupted do not aggregate but they are able to induce aggregation-stage genes in response to cAMP pulses and the prestalk-specific ras gene DdrasD in response to high continuous levels of cAMP but will not induce prespore gene expression. In this report, we present conclusive evidence that DdPK3 encodes the catalytic subunit of the Dictyostelium PKA. DdPK3 null cells lack kinase activity that phosphorylates a PKA-specific substrate and is specifically inhibitable by recombinant cAMP-dependent protein kinase inhibitor. DdPK3 expressed in Escherichia coli has PKA activity that is inhibitable by protein kinase inhibitor. When Ddpk3 null cells are complemented with DdPK3 expressed from an actin promoter on an extrachromosomal vector (low copy number), PKA activity is restored and the cells proceed to the slug stage but will not culminate, suggesting that properly regulated PKA activity is essential for culmination. Moreover, overexpressing DdPK3 in wild-type cells on integrating vectors (high copy number) from either an actin or prespore-specific promoter results in accelerated development and the ability to form mature spores in monolayer culture in the presence of high cAMP, a developmental potential lacking in wild-type cells.
The Dictyostelium discoideum genome contains an estimated 17 to 20 actin genes. We report the identification of a new member
of this multigene family, actin 15, and its complete nucleotide sequence and transcription initiation sites. We constructed
transformation vectors carrying either the actin 15 promoter fused to the neomycin phosphotransferase gene from transposon
Tn903 or the actin 6 promoter fused to the neomycin phosphotransferase gene from Tn5. Cells transformed with the actin 15
vector carried less than five copies of vector DNA, while cells transformed with the actin 6 vector carried more than 200
copies. In both cases, the vector appeared to be integrated into the chromosome as a tandem array. Gene fusion RNAs transcribed
from the actin 15 and actin 6 vectors were regulated like endogenous actin genes during D. discoideum development. DNA sequences
required for temporal and cell type-specific regulation of these genes were contained within 2.8 kilobases of 5' noncoding
DNA for actin 15 and 0.7 kilobases of 5' noncoding DNA for actin 6.
A 2.8-kb fragment of 5' non-coding DNA from the Dictyostelium actin 15 gene has previously been shown to contain all of the cis-acting DNA sequence elements required for normal developmentally-regulated transcription of actin gene fusion RNAs when reintroduced into the genome by DNA-mediated transformation. Deletion analysis of this promoter fragment indicates that all of the necessary information is contained within a 270-bp fragment of actin 15 5' non-coding DNA. This fragment contains four short G/C-rich repeated sequences that are also found in other co-regulated Dictyostelium actin genes. A 12-bp consensus sequence, AAAAATGGGG/ATT, is present in the regions essential for expression of two different Dictyostelium actin genes, actin 6 and actin 15, but is absent from an actin gene showing a different temporal pattern of developmental regulation. Deletion analysis and DNase I footprinting implicate this sequence as a functional cis-acting element required for transcription of the actin 15 gene.
We have established a rapid and sensitive transient expression system for Dictyostelium discoideum. We constructed a gene fusion containing the promoter from the Dictyostelium Actin 15 gene fused to the firefly luciferase gene. The enzymatic activity of this gene fusion, expressed at very high levels
in stable transformants, was measured to determine optimum conditions for transient expression using electroporation to introduce
the DNA into cells. With these conditions, we show that a luciferase gene fusion driven by a prestalk, cell-type specific
promoter from the pst-cathepsin gene expresses luciferase at the appropriate developmental stage. In addition, we present
results suggesting that the system will be useful for expressing genes in non-axenic cell lines. Finally, we observe that
electroporation is more efficient for obtaining stable transformations than the standard calcium phosphate procedure using
extrachromosomally replicating shuttle vectors but less efficient for vectors that integrate into the Dictyostelium chromosomes.
In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate nonmuscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium discoideum phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed in RMLC null Dictyostelium cells (mLCR-) from an actin promoter on an integrating vector. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wild-type RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions.
The activity of cAMP-dependent protein kinase (PKA) is required for proper development at several stages during the Dictyostelium life cycle. We present evidence that activation of PKA is rate-limiting for the differentiation of prespore cells to spores and that PKA activation may be the developmental trigger for sporulation. Strains that overexpress the gene encoding the catalytic subunit of PKA (PKAcat) or lack a functional regulatory subunit (rdeC strains) undergo rapid, heterochronic development. We show that overexpression of PKAcat in prespore cell is sufficient to directly induce expression of the spore maturation marker spiA and differentiation to spores, in a cell-autonomous manner. Moreover, overexpression of PKAcat in prespore cells can bypass a mutation that blocks an earlier developmental step to induce spiA expression. Our results suggest that the regulatory pathway in prespore cells between the activation of PKA and spiA induction/spore maturation is quite short and that PKAcat expression in prespore cells may mediate spore differentiation at the level of transcription. This induction of sporulation requires the prior activation of the prespore cell pathway. In addition, we show that beta-galactosidase activity expressed from a PKAcat promoter/lacZ reporter construct is highly enriched in the anterior prestalk A region during the tipped aggregate, slug, and early culminant stages and that this pattern switches abruptly to a prespore pattern at the time of spore maturation, supporting the proposed role of PKA in this process.
Expression of a dominant inhibitor of the Dictyostelium cAMP-dependent protein kinase in prespore cells blocks their differentiation into spore cells. The resultant structures comprise a normal stalk supporting a bolus of cells that fail to express a sporulation-specific gene and that show greatly reduced levels of expression of several prespore-specific genes. The latter result suggests that in addition to activating spore formation, the cAMP-dependent protein kinase may play a role in initial prespore cell differentiation. Development of the strain expressing the dominant inhibitor is hypersensitive to the inhibitory effects of ammonia, the molecule that is believed to repress entry into culmination during normal development. This result supports a model whereby a decrease in ambient ammonia concentration at culmination acts to elevate intracellular cAMP and hence induce terminal differentiation.
We have used PCR to identify a Dictyostelium homolog of the bacterial two-component system. The gene dokA codes for a member of the hybrid histidine kinase family which is defined by the presence of conserved amino acid sequence motifs corresponding to an N-terminal receptor domain, a central kinase and a C-terminal response regulator moiety. Potential function of the regulator domain was demonstrated by phosphorylation in vitro. dokA mutants are deficient in the osmoregulatory pathway, resulting in premature cell death under high osmotic stress. Under less stringent osmotic conditions, cells grow at a normal rate, but development at the multicellular stage is altered. dokA is a member of a family of histidine kinase-like genes that play regulatory roles in eukaryotic cell function.
Dictyostelium cells express a G-protein-coupled adenylyl cyclase, ACA, during aggregation and an atypical adenylyl cyclase, ACG, in mature
spores. The ACG gene was disrupted by homologous recombination. acg− cells developed into normal fruiting bodies with viable spores, but spore germination was no longer inhibited by high osmolarity,
a fairly universal constraint for spore and seed germination. ACG activity, measured in aca−/ACG cells, was strongly stimulated by high osmolarity with optimal stimulation occurring at 200 milliosmolar. RdeC mutants, which display unrestrained protein kinase A (PKA) activity and a cell line, which overexpresses PKA under a prespore
specific promoter, germinate very poorly, both at high and low osmolarity. These data indicate that ACG is an osmosensor controlling
spore germination through activation of protein kinase A.
A 2.8‐kb fragment of 5′ non‐coding DNA from the Dictyostelium actin 15 gene has previously been shown to contain all of the cis‐acting DNA sequence elements required for normal developmentally‐regulated transcription of actin gene fusion RNAs when reintroduced into the genome by DNA‐mediated transformation. Deletion analysis of this promoter fragment indicates that all of the necessary information is contained within a 270‐bp fragment of actin 15 5′ non‐coding DNA. This fragment contains four short G/C‐rich repeated sequences that are also found in other co‐regulated Dictyostelium actin genes. A 12‐bp consensus sequence, AAAAATGGGG/ATT, is present in the regions essential for expression of two different Dictyostelium actin genes, actin 6 and actin 15, but is absent from an actin gene showing a different temporal pattern of developmental regulation. Deletion analysis and DNase I footprinting implicate this sequence as a functional cis‐acting element required for transcription of the actin 15 gene.
This chapter discusses some genetic approaches for studying signaling pathways and for elucidating the molecular mechanisms of information processing by modular signaling proteins. Bacteria live in precarious environments. Nutrient and toxin levels, acidity, temperature, osmolarity, humidity, and many other conditions can change rapidly and unexpectedly. Bacterial signaling systems are amenable to detailed genetic and biochemical analyses. In this chapter, some general strategies for using genetic methods to study sensory pathways and signaling proteins are discussed. Many signaling proteins, from both gram-positive and gram-negative bacteria, contain characteristic "transmitters" and "receivers" domains that promote information transfer within and between proteins. Transmitters and receivers are ideally suited as circuit elements for assembling signaling pathways. The only demonstrated mechanisms of transmitter-receiver communication involve phosphorylation and dephosphorylation reactions. With caution, the logic of epistatic analysis can be extended to more elaborate signaling pathways that have branches, feedback loops, and so on. Genetic approaches can provide considerable insight into the operation of signaling pathways and proteins. Even though actual signaling circuits are unlikely to be as simple as the two-component examples in the chapter, the same basic principles should apply.
During the process of fruiting body construction in the cellular slime mould Dictyostelium discoideum, prestalk cells become mature stalk cells in a well-controlled manner. To identify the natural inducer of stalk cell maturation, substances known to induce stalk cell differentiation under in vitro conditions, and some other related compounds, were examined for their effects in vivo on migrating slugs, the precursor structures of the fruiting bodies.
Among these substances, addition of weak acids such as CO2, and addition followed by removal of weak bases such as NH3, strikingly induced the maturation of prestalk cells in situ in slugs. On the other hand, inhibitors of the plasma membrane proton pump did not efficiently induce the maturation of prestalk cells in intact slugs. Differentiation inducing factor (DEF), an endogenous inducer of prestalk differentiation, seemed to be an even poorer inducer of stalk cell maturation when applied to intact slugs.
The activities of these substances in inducing stalk cell maturation showed a good correlation with their effects on the cytoplasmic pH (pH|) of prestalk cells; the larger the pH| drop, the stronger the induction of stalk cell maturation, suggesting a requirement for a pHi decrease for the maturation of prestalk cells. Based on these results, it was proposed that stalk cell differentiation, which is induced by DIF, is blocked halfway during normal development by (an) agent(s) that prevent(s) the decrease in pH,.
Spore maturation occurs during normal development in Dictyostelium when environmental influences induce a migrating slug to transform into a fruiting body. As the amoeboid prespore cells turn into refractile spores there is a burst of enzyme accumulation, including UDP-galactose epimerase, and at a later stage the exocytosis of preformed components of the spore coat. Evidence is presented here that this process is triggered by an elevated intracellular cAMP concentration.
First, a number of rapidly developing (rde) mutants, whose cAMP metabolism had been investigated previously, are shown to be able to form spores in submerged monolayers, whereas wild-type strains are not. The phenotypes of these mutants are best explained by a derepression of the signal transduction pathway utilizing intracellular cAMP.
Second and more direct, it is shown that the permeant cAMP analogues 8-Br-cAMP and 8-chlorophenylthio-cAMP, but not cAMP itself, can rapidly induce spore differentiation in wild-type amoebae incubated in submerged monolayers. These analogues also stimulate accumulation of UDP-galactose epimerase in slug cells transferred to shaken suspension.
The ability to induce spore differentiation with Br-cAMP in wild-type strains provides a new technique that can be exploited in various ways. For instance, spore differentiation in strain V12M2 is induced by 8-Br-cAMP at very low cell densities, suggesting that neither cell contact nor additional soluble inducers are necessary in these conditions. In contrast NC4 cells may require an additional inducer. Spore differentiation is inhibited by the stalk-specific inducer DIF-1 suggesting that DIF-1 inhibits a target downstream of intracellular cAMP in the signal transduction pathway inducing spore differentiation.
In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate nonmuscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium discoideum phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed in RMLC null Dictyostelium cells (mLCR-) from an actin promoter on an integrating vector. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wild-type RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions.
1. Equipment and procedures incidental to determining the nitrogen metabolism of the vegetative amoebae, whole pseudoplasmodia, and fragments of pseudoplasmodia of the slime mold Dictyostelium discoideum during growth and morphogenesis have been described.2. During the transition from the vegetative amoebae to the migrating pseudoplasmodia no statistically significant changes were found in any of the nitrogenous components under investigation.3. During the transition from the migrating pseudoplasmodia to the mature sorocarps statistically significant decreases were found in certain nitrogenous components.4. By analyzing spores and stalks separately it was possible to attribute nitrogen changes occurring in the intact mature sorocarps to particular regions.5. The excretion of ammonia during the transition from the migrating pseudoplasmodia to the mature sorocarps was demonstrated.6. The relationship between nitrogen metabolism and the synthesis of cellulose was discussed.
During the course of development, cells of Dictyostelium discoideum are able to produce a multicellular body (a ‘slug’) which is capable of movement over the substratum. This phase, which is induced by production of ammonia by the starving cells, may last for hours or days depending on environmental stimuli. In order to probe the regulatory system controlling formation and duration of the slug phase, mutants were isolated that remained in the slug phase for an extended period. Thirty-two such 'slugger’ mutants were analysed by parasexual genetic techniques and placed into 10 complementation groups (slgA-slgJ). The linkage groups bearing representatives of these complementation groups were determined by segregation of diploids formed between mutants and tester strains. Phenotypic studies of mutants indicated that members of slgD, slgE and slgG were over-sensitive to the ammonia slug-inducing stimulus.
We showed some years ago that the rising cell masses of cellular slime moulds repel one another, and that this is achieved by a gas given off from the cell masses1. It is now clear that this gas is ammonia. We also have evidence that NH3 tends to speed up the movement of cells in aggregating streams which would account for its ability to repel; more NH3 on one side of a cell mass would cause a speed-up of the cells on that side, thus making the mass veer to one side.
Two compounds, ammonia (NH 3 ) and 3′5′ cyclic AMP (cAMP) act as specific morphogens in regulating the development of Dictyostelium discoideum [1–11]. A previous study [12] demonstrated that NH 3 at concentrations that affect the course of morphogenesis completely inhibits the extracellular release of cAMP by aggregation competent cells incubated in shaken suspension. The present study extends this finding in two respects: Exposure of aggregation competent cells to NH 3 (supplied as ammonium carbonate) is followed within a few minutes by the complete disappearance of intracellular cAMP. Subsequent removal of NH 3 is followed by a rapid, complete restoration of the level. Neither the disappearance nor the reappearance is affected by the presence of cycloheximide, an inhibitor of protein synthesis.
In a mutant strain of D discoideum, greatly increased sensitivity to NH 3 as a regulator of morphogenesis is coupled with a correspondingly increased sensitivity to NH 3 as an inhibitor of cAMP accumulation.
These results are consistent with a recently proposed [13, 14] model of morphogenetic regulation that is based on the supposition that NH 3 , by inhibiting cAMP production, restricts cAMP accumulation to specified constrained areas within the developing multicellular aggregate and thereby dictates the course of morphogenesis and cytodifferentiation.
Recent monumental advances in microbial genetics and in the chemistry of macromolecular biosynthesis have been applied to problems with developmental import. The program's appearance and macromolecules' disappearance plays crucial roles in developmental sequences. As a consequence, a new technology has arisen that must be adapted to the peculiarities of each kind of biological material. This chapter describes genetic, biochemical, and immunochemical techniques that have been successfully applied in the laboratory to the study of cellular slime mold development. The cellular slime molds are designated as an order within the phylum Myxomycophyta. Several genera and many species have been described. However, the bulk of the current investigation has been carried out with Dictyostelium discoideum. This species has a particularly interesting morphogenetic sequence and constructs fruiting bodies rapidly and well under a wide variety of environmental conditions. Many mutant strains have been isolated that display a wealth of developmental aberrations.
A model of morphogenetic regulation in Dictyostelium discoideum is presented which is based on the assumption that NH3 inhibits the synthesis and/or release of extracellular 3′,5′-cyclic-AMP and by topographical restriction of c-AMP production to specified zones within the cell aggregate, NH3 is presumed to set up the conditions for apical dominance and directed morphogenetic movements.
Stalk cell differentiation in Dictyostelium can be induced by the differentiation-inducing factor, DIF, or by conditions that decrease intracellular pH (pHi). We have investigated whether cytoplasmic acidification acts directly to induce expression of pDd56 and pDd63, two DIF-regulated genes, specifically expressed in prestalk cells. The weak base methylamine, which increases pHi, inhibits DIF-induced transcription. The weak acid 5,5-dimethyl-2,4-oxazolidinedione (DMO), which decreases pHi, stimulates DIF-induction of the two prestalk genes. After relatively long incubation periods, DMO also induces a low level of prestalk gene expression in the absence of added DIF. However, unlike DIF-mediated induction, the apparent DMO-mediated induction decreases to undetectable levels when the cell density is reduced from 107 to 105 cells/ml. This indicates that DMO does not itself induce gene expression, but acts to enhance the effects of an autonomously secreted stalk-inducing factor, presumably DIF. These results suggest that the effects of DIF on gene expression are regulated by intracellular pH, but do not support a role for protons as direct intermediates in the DIF signal transduction pathway.
There are cells acattered in the rear, prespore region of the Dictyostelium slug that share many of the properties of the prestalk cells and that are therefore called anterior-like cells (ALCs). By placing the gene encoding a cell surface protein under the control of an ALC-specific promoter and immunologically labeling the living cells, we analyze the movement of ALCs within the slug. There is a posterior to anterior cellular flow, and the ALCs change their movement pattern as they enter the prestalk zone. Prestalk cells are periodically shed from the migrating slug. They must be replaced if the correct ratio of prestalk to prespore cells is to be maintained, and we present evidence for the trans-differentiation of prespore into prestalk cells, with ALCs functioning as intermediates in the transition. The slug has, therefore, a surprisingly dynamic structure, both with respect to cellular differentiation and cell movement.
Previous work (Newell et al., 1969) had indicated that the transformation of a newly formed multicellular aggregate into a migrating slug rather than a fruiting body depended on the local accumulation of a diffusible metabolite excreted before and during aggregation. The subsequent shift back to the fruiting mode could be accounted for by the disappearance or removal of the metabolite. All these morphogenetic transformations were associated with profound and consistent changes in the patterns of enzyme accumulation and disappearance (Sussman et al., 1975). In the present study, evidence is presented to support the conclusion that the metabolite in question is NH3. Thus: 1.(a) Cell populations accumulated and excreted significant amounts of NH3 + NH4+ before and during cell aggregation under the conditions employed.2.(b) Excretory products collected from such cells induced newly formed aggregates to develop into migrating slugs under conditions which otherwise would permit them to construct fruiting bodies directly. The inducing activity of such preparations was precisely correlated with the NH3 + NH4+ concentration and was destroyed by incorporation of the NH3 into glutamic acid in the presence of glutamate dehydrogenase.3.(c) The slug-inducing activities of the extracellular metabolite preparations and purified fractions thereof were mimicked by equimolar solutions of ammonium carbonate at identical pH values.4.(d) Isolated migrating slugs treated with 0.5-μ1 samples of a reaction mixture containing glutamate dehydrogenase, α-ketoglutarate and NADH immediately stopped migrating and constructed fruiting bodies. Samples lacking a single component or containing boiled enzyme were ineffective.5.(e) By several criteria, the biological and biochemical performances of autoinduced slugs and of those induced by the metabolite preparations or by ammonium carbonate solutions were indistinguishable.
Two representatives of a newly recognized class of D. discoideum mutants termed “sluggers” are described. They display the following properties: 1.1. The newly formed mutant aggregates invariably transform into migrating slugs even under conditions which lead their wild-type counterparts to eschew the slug mode completely and to construct fruiting bodies directly in place. Depending on environmental conditions, the mutant aggregates may remain indefinitely as migrating slugs or may, after a lapse of 10–12 h, re-enter the fruiting mode and construct normal fruiting bodies.2.2. The patterns of accumulation and disappearance of three enzymes previously shown to be under morphogenetic feedback control in the wild type [3] were found to be profoundly disturbed in the mutants but in ways at least partly comparable to the performance of wild-type aggregates when the latter are caused to vacillate between the alternative morphogenetic pathways of slug migration and fruit construction.3.3. Both morphogenetic lesions can be completely or partially suppressed by synergistic development in mixed aggregates with wild-type cells. A small proportion of wild-type cells is required. In the case of JC-2, as little as 2.5–10% was effective. Moreover, the presence of the wild type in the mixtures was not needed prior to the actual time at which the morphogenetic lesion is expressed in order to have suppressed it.4.4. In neither case does the initiation of the slug stage or its duration appear to be due to the over-production of NH3 or enhanced sensitivity to it.5.5. The capacity of the mutant slugs to abandon that mode and initiate fruit construction is lost by removal of a diffusible metabolite(s) released into the extracellular milieu during prior morphogenesis and the capacity can be restored by its replacement. The metabolite appears to trigger fruit construction and is not required for its completion.6.6. The inducer accumulates during morphogenesis of both mutants and the wild type apparently in comparable amounts. It appears to be a small anionic species, dialysable and volatile at acid but not alkaline pH. Of many known compounds tested for inductive activity only sodium propionate and 2-methyl propionate were active but the concentrations required rule them out as the metabolite in question.7.7. Slight but significant differences in their morphogenetic sequences, patterns of enzyme accumulation and disappearance, differing sensitivities to synergistic interaction with wild type and the fruit-inducing metabolite suggest that JC-2 and JC-4, though phenotypically similar, may represent independent mutational events. However, synergistic complementation was not observed thereby raising the possibility that the morphogenetic lesions stem from the same metabolic defect.
After activation, wild-type Dictyostelium discoideum spores germinate rapidly and synchronously in phosphate buffer as well as in complex medium. Mutants defective in spore germination were isolated and characterized. These mutants (called grm) did not germinate normally in buffer but did germinate in complex medium in the presence of bacteria. One mutant (grm B) swelled normally, but amoebae were not formed. Another mutant (grm F) swelled and germinated poorly in buffer. The members of the third group of mutants (A, C, D, and E) did not swell or give rise to amoebae in buffer.
We placed a specific inhibitor of cyclic AMP-dependent protein kinase (PKA) under the control of a prestalk-specific promoter. Cells containing this construct form normally patterned slugs, but under environmental conditions that normally trigger immediate culmination, the slugs undergo prolonged migration. Slugs that eventually enter culmination do so normally but arrest as elongated, hairlike structures that contain neither stalk nor spore cells. Mutant cells do not migrate to the stalk entrance when codeveloped with wild-type cells and show greatly reduced inducibility by DIF, the stalk cell morphogen. These results suggest that the activity of PKA is necessary for the altered pattern of movement of prestalk cells at culmination and their differentiation into stalk cells. We propose a model whereby a protein repressor, under the control of PKA, inhibits precocious induction of stalk cell differentiation by DIF and so regulates the choice between slug migration and culmination.
In Dictyostelium there are multiple prestalk cell types that have a complex pattern of directed cell movement during slug formation and culmination. Three extracellular signals, cyclic AMP, DIF and ammonia, control cell type differentiation. Recently there has been considerable progress in understanding their modes of action and interaction.
The ecmA (pDd63) and ecmB (pDd56) genes encode extracellular matrix proteins of the slime sheath and stalk tube of Dictyostelium discoideum. Using fusion genes containing the promoter of one or other gene coupled to an immunologically detectable reporter, we previously identified two classes of prestalk cells in the tip of the migrating slug; a central core of pstB cells, which express the ecmB gene, surrounded by pstA cells, which express the ecmA gene. PstB cells lie at the position where stalk tube formation is initiated at culmination and we show that they act as its founders. As culmination proceeds, pstA cells transform into pstB cells by activating the ecmB gene as they enter the stalk tube. The prespore region of the slug contains a population of cells, termed anterior-like cells (ALC), which have the characteristics of prestalk cells. We show that the ecmA and ecmB genes are expressed at a low level in ALC during slug migration and that their expression in these cells is greatly elevated during culmination. Previous observations have shown that ALC sort to surround the prespore cells during culmination (Sternfeld and David, 1982 Devl Biol. 93, 111-118) and we find just such a distribution for pstB cells. We believe that the ecmB protein plays a structural role in the stalk tube and its presence, as a cradle around the spore head, suggests that it may play a further function, perhaps in ensuring integrity of the spore mass during elevation. If this interpretation is correct, then a primary role of anterior-like cells may be to form these structures at culmination. We previously identified a third class of prestalk cells, pstO cells, which lie behind pstA cells in the slug anterior and which appeared to express neither the ecmA nor the ecmB gene. Using B-galactosidase fusion constructs, which give more sensitive detection of gene expression, we now find that these cells express the ecmA gene but at a much lower level than pstA cells. We also show that expression of the ecmA gene becomes uniformly high throughout the prestalk zone when slugs are allowed to migrate in the light. Overhead light favours culmination and it may be that increased expression of the ecmA gene in the pst 'O' region is a preparatory step in the process.
Ammonia appears to be an important regulatory signal for several aspects of the Dictyostelium life cycle. The postulated role of ammonia in the determination of the prespore pathway in cells of the slug stage has led us to examine the effect of ammonia on the prestalk/prespore ratio of migrating slugs. In the presence of 10(-3) M ammonium chloride, the volume of the prestalk region decreases by 40.8%. The kinetics of the process make it unlikely that this is due to a shift in the differentiation pathway. A test of the hypothesis that the decrease in volume of the prestalk region is due to the conversion of prestalk cells to anterior-like cells shows that the percent of anterior-like cells in the posterior region increases by the amount predicted by the hypothesis. This suggests that ammonia may be the molecular signal, produced by the tip, that prevents anterior-like cells from chemotactically migrating to the tip and thereby becoming anterior cells. The effect of enzymatic removal of ammonia from vitally stained migrating slugs is the appearance of a series of dark stripes beginning at the posterior end and progressing forward. We interpret this as a result of progressive removal of anterior-like cells from tip dominance and essentially as the formation of new potential tips. Indeed, in a few cases one or even two of the stripes separate from the posterior of the cell mass and form small fruiting bodies. We consider the phenomenon of stripe formation further evidence that the tip acts on anterior-like cells through ammonia.
The cellular slime mold Dictyostelium discoideum undergoes a transition from single-celled amoebae to a multicellular organism
as a natural part of its life cycle. A method of cell-cell signaling that controls chemotaxis, morphogenesis, and gene expression
has developed in this organism, and a detailed understanding of this signaling system provides clues to mechanisms of intercellular
communication in the development of metazoans.
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We have isolated cDNA clones derived from three mRNA sequences which are inducible by DIF, the putative stalk-specific morphogen of Dictyostelium. The three mRNA sequences are selectively expressed in cells on the stalk cell pathway of differentiation and we have compared them with previously characterized prestalk-enriched mRNA sequences. We find these latter sequences are expressed without a dependence on DIF, are much less highly enriched in prestalk over prespore cells and are expressed earlier during development than the DIF-inducible mRNA sequences. We propose two distinct mechanisms whereby a mRNA may become enriched in prestalk cells. An apparently small number of genes, represented by those we have isolated, is inducible by DIF and accumulates only in prestalk cells. We suggest that a second class of prestalk-enriched mRNA sequences are induced by cAMP to accumulate in all cells during aggregation and then become enriched in prestalk cells by selective loss from prespore cells.
We have identified and begun characterizations of the differential expression of 15 genes whose corresponding mRNA levels decrease during the preaggregative period of the developmental program of Dictyostelium discoideum. Upon the onset of development, the mRNAs decrease from 5- to 1000-fold over the first 8 hr. The rates of loss of each mRNA were similar to one another but distinct, and the decreases were dependent on progress through the developmental program. One exception to this dependency was observed, and the decrease in this mRNA was dependent on the absolute time after initiation of development instead of progress through development. With two exceptions, the decreases in mRNA levels were dependent on developmental conditions and were not seen when cells were shaken in starvation buffer. When the polysomal distributions of each species were examined, three classes were found: most showed no significant shifts off of polysomes upon initiation of development, two were characterized by a 20% shift to nonpolysomal RNA fractions upon development, and two gave a 40-50% shift. Collectively, these characterizations reveal differences in behavior which suggest that deactivation of genes upon initiation of development in Dictyostelium involves more than one regulatory pathway.
The cellular slime mold Dictyostelium discoideum grows in the soil as a population of independent, uninucleate amoebae. Upon entrance to the stationary phase, the amoebae collect in multicellular aggregates to form organized fruiting bodies composed of spores and stalk cells. Depending upon environmental conditions, the developing aggregate either constructs the fruiting body at the site of aggregation or transforms into a structure that can migrate to a more favorable location. Environmental conditions that favor migration are (i) the accumulation of metabolite(s) produced by the aggregate and (ii) a low ionic strength in the substratum. Conditions that prevent migration or that stop a migrating slug are (i) the presence of buffer and (ii) illumination by overhead light.
Rapidly developing (rde) mutants of Dictyostelium discoideum, in which cells precociously differentiated into stalk and spore cells without normal morphogenesis, were investigated genetically and biochemically. Genetic complementation tests demonstrated that the 16 rde mutants isolated could be classified into at least two groups (groups A and C) and that the first described rde mutant FR17 (D. R. Sonneborn, G. J. White, and M. Sussman, 1963, Dev. Biol. 7, 79-93) belongs to group A. Morphological studies revealed several differences in development and final morphology between group A and group C mutants. In group A mutants, the time required for cell differentiation from vegetative cells to aggregation competent cells is reduced, whereas the time required for spore and stalk cell differentiation following the completion of aggregation is shortened in group C mutants. This suggests that group C mutants represent a new class of rde mutants and that there exist at least two mechanisms involved in regulating the timing of development in D. discoideum. Measurements of cell-associated and extracellular phosphodiesterase activities, and intracellular and total cAMP levels revealed that cAMP metabolism in both groups is significantly altered during development. Group A mutants showed precocious and excessive production of phosphodiesterase and cAMP during the entire course of development; intracellular cAMP levels in group C mutants were extremely low, and spore and stalk cell differentiation occurred without an apparent increase in these levels. Thus, while cAMP metabolism is abnormal in all the rde mutants studied, there exist several distinct types of derangement, not necessarily involving the overproduction of cAMP.
We have identified genes that are expressed preferentially in either prestalk or prespore cells in Dictyostelium. The prestalk mRNAs are detectable at 7.5 hr prior to the completion of cell aggregation, while the prespore mRNAs are not detectable until approximately 15 hr of development. Exogenous cAMP in the absence of sustained cell contact is sufficient to induce prestalk-specific gene expression, while multicellularity is required for the induction of prespore-specific genes. A gene expressed equally in both cell types, which has the same developmental kinetics as the prestalk genes, is induced in shaking culture in the absence of either cAMP or stable cell associations. Dissociation of aggregates results in the rapid loss of prespore- and prestalk-specific mRNAs, and these can be induced to reaccumulate with the addition of cAMP. We conclude that there are substantial differences in the timing and requirements for tissue-specific gene expression in Dictyostelium.
spiA, a marker for sporulation, is expressed during the culmination stage of Dictyostelium development, when the mass of prespore cells has moved partly up the newly formed stalk. Strains containing a full-length spiA promoter/lacZ fusion were stained for beta-galactosidase activity at intervals during development. The results indicate that expression of spiA initiates in prespore cells at the prestalk/prespore boundary (near the apex) and extends downward into the prespore mass as culmination continues. A spatial gradient of staining expands from the top of the prespore mass and intensifies until the front of activation reaches the bottom, whereupon the entire region stains darkly. The spiA promoter can be deleted to within 301 bp of the transcriptional start site with no effect on the relative strength, timing or spatial localization of expression. Further 5' deletions from -301 to -175 reduce promoter strength incrementally, although timing and spatial expression are not affected. Deletions to -159 and beyond result in inactive promoters. Treatment of early developmental structures with 8-Br-cAMP in situ activates the intracellular cAMP-dependent protein kinase (PKA) and precociously induces spiA expression and sporulation. The absence of an apparent gradient of staining in these structures suggest that PKA is equivalently activatable throughout the prespore region and that all prespore cells are competent to express spiA. Thus, we postulate that the pattern of expression of spiA reveals the progression of an inductive signal for sporulation and suggest that this signal may originate from the prestalk cells at the apex.
The cotA, cotB, and cotC genes encode the major spore coat proteins of Dictyostelium. All three cot genes are coordinately expressed as aggregation is nearing completion. Induction and maintenance of their expression is dependent upon the presence of extracellular cAMP. We show that expression of a dominant inhibitor of the cAMP dependent protein kinase (PKA) in prespore cells greatly reduces the transcription rates of the cotB and cotC genes. All three cot genes contain, in their upstream regulatory regions, short sequence elements that have a high content of cytosine and adenosine residues. These CA-rich sequences are essential for optimal cot gene transcription. We show that expression of the dominant PKA inhibitor results in a greatly reduced level of the binding activity that recognizes the CA-rich sequences upstream of the cotB gene. Thus PKA acts, either directly or indirectly, to control expression of the cot genes and it may do so by modulating the activity of a DNA binding protein. However, we find that mutant cells where PKA is constitutively active still require exogenous cAMP for optimal cot gene expression in dissociated cells, suggesting that a separate, PKA-independent, signalling pathway is also involved in the regulation of cot gene expression by extracellular cAMP.
During the past several years, it has become apparent that prokaryotic organisms process much of their sensory information through families of similar proteins that make up two-component signal transducing systems. Biochemical and genetic analyses have clarified our understanding of how these proteins mediate the signaling circuitry necessary to allow an appropriate response. Recent advances in this field suggest that 'primitive' signaling systems may involve molecular strategies and mechanisms that are conserved in more complex organisms.
Analysis of the expression patterns of two genes encoding extracellular matrix proteins shows there to be an unexpectedly complex pattern of prestalk cell differentiation and movement during the morphogenesis of Dictyostelium. The organism employs both cell sorting and positional differentiation to generate a patterned structure but these two mechanisms are used at different times during development. During slug formation prestalk cells arise at scattered positions within the aggregate and then move to its apex to form the tip. In contrast, during culmination, stalk cell differentiation occurs in a positionally localized manner at the entrance to the stalk tube. Two interacting signalling pathways regulate the differentiation of prestalk and stalk cells. Prestalk cell differentiation is induced by DIF, a chlorinated hexaphenone, and a repression mechanism prevents DIF acting to induce premature stalk cell differentiation during slug migration. At culmination intracellular cAMP levels rise, the cAMP dependent protein kinase (PKA) is activated and the block to stalk cell differentiation is lifted. Activation of PKA is also necessary in order that prestalk cells move to the entrance of the stalk tube at culmination. Thus, in Dictyostelium, PKA plays a role both in the regulation of cellular differentiation and in morphogenetic cell movement.
A new selectable marker for transformation of Dictyostelium discoideum cells was constructed by using the bsr gene from Bacillus cereus, which confers resistance to Blasticidin S. The bsr gene was driven by Dictyostelium actin 15 promoter and Dictyostelium actin 8 terminator for expression in Dictyostelium cells. To demonstrate the feasibility of using the bsr marker, we constructed an extrachromosomal replication vector by replacing the Neor gene of pnDeI (B. Leiting and A. Noegel (1988) Plasmid 20, 241-248) with the bsr gene cassette. A mutant Dictyostelium actin 15 gene was constructed and inserted into the vector. Dictyostelium cells were transformed with the resulting vector and then transformants were selected with Blasticidin S. The selected cells showed high level expression of the mutant actin, indicating an efficient selection of transformed cells with the bsr marker.
The weak base ammonia is implicated in a number of key processes in Dictyostelium development, notably aggregation and culmination. To determine its intracellular site of action, we compared its biological potency with that of other weak bases. All the bases inhibited these developmental processes effectively, but differed manyfold in potency in accordance with their activity in dissipating pH gradients, as measured by in vivo 31P NMR with pH-sensitive phosphonate probes. These results indicate that weak bases influence development by raising the pH of an intracellular acidic compartment.
Prespore and prestalk cells can be distinguished within aggregates of Dictyostelium by the expression of well-characterized cell type-specific genes. Fusion of the tagB regulatory region to Escherichia coli beta-galactosidase revealed that this prestalk specific gene marks the differentiation of the initial prestalk cell population, PST-1. The reporter gene was expressed normally in tagB- mutant cells despite the fact that they do not accumulate measurable levels of DIF-I, a morphogen that was previously implicated in prestalk differentiation. In an independent experimental system, wild-type cells respond to the addition of DIF-I by induction of the prestalk marker ecmA and repression of the prespore marker cotB. We found that DIF-1 did not affect the expression of the tagB or carB genes, both of which are prestalk specific and essential for PST-A cell differentiation. We conclude that the initiation of prestalk development is not dependent on DIF-1 and suggest that the morphogen participates mainly at later stages.
A mutant which failed to complete development was isolated from a population of cells that had been subjected to insertional mutagenesis using restriction enzyme-mediated integration. The disrupted gene, dhkA, encodes the conserved motifs of a histidine kinase as well as the response regulator domain. It is likely that the histidine in DhkA is autophosphorylated and the phosphate passed to one or more response regulators. Such two-component systems function in a variety of bacterial signal transduction pathways and have been characterized recently in yeast and Arabidopsis. In Dictyostelium, we found that DhkA functions both in the regulation of prestalk gene expression and in the control of the terminal differentiation of prespore cells.
We have found conditions for saturation mutagenesis by restriction enzyme mediated integration that result in plasmid tagging of disrupted genes. Using this method we selected for mutations in genes that act at checkpoints downstream of the intercellular signalling system that controls encapsulation in Dictyostelium discoideum. One of these genes, mkcA, is a member of the mitogen-activating protein kinase cascade family while the other, regA is a novel bipartite gene homologous to response regulators in one part and to cyclic nucleotide phosphodiesterases in the other part. Disruption of either of these genes results in partial suppression of the block to spore formation resulting from the loss of the prestalk genes, tagB and tagC. The products of the tag genes have conserved domains of serine protease attached to ATP-driven transporters, suggesting that they process and export peptide signals. Together, these genes outline an intercellular communication system that coordinates organismal shape with cellular differentiation during development.