Introduction

During early embryonic development, organizers play an important role in patterning and directing the differentiation of surrounding cells into specific tissues and organs. The embryonic organizer establishes the developmental polarity in vertebrates; and similarly, in Dictyostelium, the mound/slug tip acts as an organizer (Rubin and Robertson, 1975) playing a pivotal role in guiding collective cell migration and patterning. Despite numerous investigations on Dictyostelium tip formation, the processes by which the tip establishes and maintains the primary developmental axis remains elusive. Gaining insights into the mechanisms regulating tip organizer function will offer valuable insights on orchestrated cell movements and processes underlying development.

Dictyostelium transitions from a unicellular amoeba to a multicellular organism in response to starvation. The cells aggregate into a mound, which gives rise to a migrating slug and ultimately a fruiting body composed of spores and a dead stalk (Raper, 1940; Kessin, 2001). This process is regulated by diffusible signals including cAMP, adenosine, ammonia, and a chlorinated hexaphenone, ‘differentiation-inducing factor’ (DIF) (Bloom and Kay, 1988; Williams, 1988; Gross, 1994; Mahadeo and Parent, 2006; Shu et al., 2006). Importantly, the cell cycle phase soon after starvation is known to strongly influence the cell fate either as prestalk (pst) or prespore (psp) cells (Weeks and Weijer, 1994; Jang and Gomer, 2011).

Tip development in Dictyostelium depends on cAMP signaling in turn regulating protein kinase A (PKA) activity, adenosine signaling, and morphogenetic cell movements (Schaap and Wang, 1986; Mann and Firtel, 1993; Siegert and Weijer, 1995). Adenosine, a by-product of cAMP hydrolysis, acts as an inhibitory morphogen suppressing additional tip formation (Schaap and Wang, 1986).

Adenosine deaminases (ADA) catalyse the breakdown of adenosine to generate inosine and ammonia, and are conserved among bacteria, invertebrates, vertebrates including mammals (Cristalli et al., 2001). In humans, two isoforms of ADA are known including ADA1 and ADA2, and the Dictyostelium homolog of ADA2 is adenosine deaminase-related growth factor (ADGF). Unlike ADA that is intracellular, ADGF is extracellular and also has a growth factor activity (Li and Aksoy, 2000; Iijima et al., 2008). Loss-of-function mutations in ada2 are linked to lymphopenia, severe combined immunodeficiency (SCID) (Gaspar, 2010), and vascular inflammation due to accumulation of toxic metabolites like dATP (Zhou et al., 2014; Notarangelo, 2016). In mice, ada2 disruption leads to perinatal mortality (Wakamiya et al., 1995), and overexpression of ada2 results in aberrant heart and kidney development (Riazi et al., 2005). In frogs, mutations in ada2 manifest in reduced body size and altered polarity (Iijima et al., 2008). In Drosophila, certain isoforms of ADGF are known to play a pivotal role in cell proliferation by depleting extracellular adenosine (Zurovec et al., 2002), and loss of function of adgfA promotes melanotic tumour formation and larval death (Dolezal et al., 2005). Adenosine deaminases are known to interact with dipeptidyl peptidase IV (DPP), cluster of differentiation (CD26) expressed on T-cells and the adenosine receptor, A2AR (expressed on dendritic cells) to facilitate cell-cell signaling (Moreno et al., 2018). Thus, adgf plays a pivotal role in the regulation of cell proliferation and development in several organisms.

Four isoforms of ADA are annotated in the Dictyostelium discoideum genome (Eichinger et al., 2005) including adenosine deaminase (ada; DDB_G0287371), adenosine deaminase acting on tRNA-1 (DDB_G0278943), adenosine deaminase tRNA-specific (DDB_G0288099) and adenosine deaminase-related growth factor (adgf; DDB_G0275179), and their role in growth and development is not known. In this study, we demonstrate that ammonia generated by ADGF plays a direct role in establishing the tip organizer, highlighting a novel link between nucleoside metabolism and developmental polarity in Dictyostelium.

Results

Differential regulation of adgf expression during growth and development

Dictyostelium cell lines carrying mutations in the gene adgf were obtained from the genome wide Dictyostelium insertion (GWDI) bank, and were subjected to further analysis to know the role of adgf during Dictyostelium development. To verify the insertion of blasticidin (bsr) resistance cassette in adgf mutant, a diagnostic PCR was carried out and the integration was validated (Figures 1A and 1B), and qRT-PCR analysis confirmed the absence of adgf expression (Figure 1C). In vertebrates, adenosine deaminases are expressed in a tissue specific manner to control growth and development. To determine whether adgf expression is differentially regulated during Dictyostelium development, qRT-PCR was performed using RNA isolated at 0 h, 8 h, 12 h, 16 h, 20 h and 24 h post starvation. adgf expression peaks at 16 h (Figure 1D) implying an important role for adgf later in development. At this time point, the expression of the other three isoforms of ADA were not significantly different from wild-type (WT), suggesting that the loss of adgf (data not shown) function is not compensated by the other isoforms.

Figure 1.

The predicted structures of Dictyostelium ADGF and human ADA2 share a strong structural similarity

The D. discoideum adgf gene is predicted to encode a protein of 543 amino acids, and belongs to the metallo-transferases superfamily (Figure S1A), having an ADA and an N-terminal domain similar to human ADA2 (Figure S1B). The ADGF sequence from the protein family database (https://www.ebi.ac.uk/interpro/) was analysed using the online tool SMART (Simple Modular Architecture Research Tool: http://smart.embl-heidelberg.de), to know the presence of structurally similar domains. The different domains of Dictyostelium ADGF (DdADGF) show a high degree of similarity with human ADA2. DdADGF shares 37.5% identity with human ADA2 and shares a sequence similarity of 59.5% with D. fasciculatum, 61.2% with D. pupureum, 53.6% with D. lacteum and 62.1% with Polysphondylium pallidum (www.uniprot.org). Multiple sequence alignment reveals the presence of an N-terminal signal sequence characteristic of extracellular proteins (Figure S1C).and conserved histidine and glutamine residues in the active site of both D. discoideum ADGF and human ADA2 (Figure S1D).

The phylogenetic relation of ADGF to the classic ADA subfamily has been reported previously (Maier et al., 2005). To determine the evolutionary relationship of DdADGF with that of other organisms, a phylogenetic analysis was carried out, and the amino acid alignment was created using MEGAX software (Kumar et al., 2018). The evolutionary history of ADGF was inferred using a Maximum Likelihood approach with Bootstrap analysis (100 iterations) as described by Felsenstein (1985). The resulting phylogenetic tree indicated that DdADGF is closely related to the ADGF proteins of other Dictyostelids, including D. purpureum, Heterostelium pallidum, and Cavenderia fasciculata. DdADGF forms a distinct clade, likely representing a distant relative of its vertebrate homolog (Figure S1E). Using the crystal structure of human ADA2, Cat eye syndrome critical region protein 1 (CECR1) as a template (PDB-3LGD), the structure of DdADGF was predicted by homology modeling (https://alphafold.ebi.ac.uk/) (Figures S1 F-H). Alignment of DdADGF with human ADA2 yielded a root mean square deviation (RMSD) value of 1 Å, indicating strong structural similarity (Eidhammer et al., 2000; Koehl, 2001).

adgf controls aggregate size in Dictyostelium

To understand the role of adgf during D. discoideum development, adgf mutants were plated at a density of 5×10⁵ cells/cm² on non-nutrient phosphate buffered agar plates and monitored thereafter. In comparison to WT, the aggregates of adgf ⁻ were larger, and thus the number of aggregates were fewer (Figure 2A) than the WT. To determine the pathways impairing the tissue size in the mutant, RNA expression of countin (ctn) and small aggregates (smlA) were examined, and their levels were reduced significantly compared to controls (Figure 2B). Countin factor (CF) regulates group size by reducing the expression of cell-cell adhesion proteins cadherin (cadA) and contact site A (csaA) (Siu et al., 1985; Coates and Harwood, 2001). Compared to WT, both cell-to-cell adhesion and cadA, csaA expression were higher in the adgf mutant (Figures 2C and 2D) suggesting that adgf regulates the overall size of the aggregates.

Figure 2.

cAMP chemotaxis significantly influences cell-cell adhesion (Konijn et al., 1967), and to determine if chemotaxis is impaired in the adgf ^- lines, an under-agarose chemotaxis assay was carried out. The chemotactic activity was not significantly different between the two cell types (Figure 2E), suggesting that the increased mound size in the mutant is not due to altered chemotaxis.

adgf mutants form large, tipless mounds

Subsequent to plating on KK2 agar, WT cells formed mounds by 8-9 h, and culminate to form fruiting bodies by the end of 24 h. In contrast, the adgf ⁻ lines were blocked as rotating mounds with no tips till 30 h (Figure 2F; Supplementary videos S1 and S2). Such a mound arrest phenotype could be mimicked by adding the ADA specific inhibitor deoxycoformycin (DCF) to WT cells (Figure 2G). After 36 h, a fraction of adgf ⁻ mounds (34.77 ± 4%) formed fruiting bodies with bulkier spore sac (79.6 ± 6.3 mm²) and stalk (Figures 2H and 2I). This late recovery from the mound arrest may be due to the expression of other ADA isoforms after 36 h.

Reduced ADA activity leads to high adenosine levels in the adgf mutant

If adgf function is compromised, ADA enzyme activity is expected to be low. To verify this, total ADA activity in WT and adgf ⁻ mounds was measured using a commercial assay kit. This assay relies on the conversion of inosine (derived from ADA activity) to uric acid, and was measured spectrophotometrically. Although ADA activity dropped significantly in the mutant (Figure 3A), it was not completely abolished, and possibly, other isoforms may have a basal activity both at 12 h and 16 h.

Figure 3.

ADGF quenches extracellular adenosine, and if blocked, as expected in adgf ⁻, the mutants will have increased extracellular adenosine. Using a commercial kit, the total adenosine levels were measured, and both at 12 h and 16 h, the levels were elevated (Figure 3B), and this difference was highly significant at 16 h. adgf expression at this time point was also high in WT cells. The elevated adenosine levels at 16 h may result from reduced ADA activity and increased expression of genes such as 5’ nucleotidase (5’nt) and phosphodiesterases (pdsA and regA), all involved in adenosine formation (Headrick and Willis, 1989; Godecke, 2008). Hence, their expression levels were examined. Although the expression of both 5’nt and regA were enhanced at 8 h, pdsA levels remained unaltered. Interestingly, the expression of all three genes trended lower at 12 h but were significantly upregulated at 16 h (Figure 3C), suggesting an important role of these genes at a specific time in development.

Addition of ADA or overexpression of adgf cDNA restored tip development in adgf ⁻

adgf mutants carry significantly high levels of adenosine. By administering ADA enzyme on top of the adgf ⁻ mounds (Figure 4A), excess adenosine can be quenched resulting in ammonia formation, possibly rescuing the phenotype. Indeed, addition of 10 U ADA onto mutant mounds restored tip development. Besides, overexpression of WT adgf cDNA (driven by actin15 promoter) complemented the developmental defects of adgf mutants (Figures 4B and 4C), confirming that the developmental block is the result of adgf gene disruption alone. However, adgf cDNA overexpression in WT cells does not result in any observable defects (Figure 4D). Taken together, these findings support an important function of adgf in tip development.

Figure 4.

WT or its conditioned media (CM) restored tip formation in adgf ⁻ mounds

To determine whether adgf is necessary for tip formation in a cell-autonomous manner, WT and the mutant cells were mixed in different proportions and plated for development. In a mix of 50% WT: 50% mutant, the mound defects of adgf ⁻ were rescued, but with 20% WT and 80% adgf ⁻, the rescue was partial (31 ± 4 %) (Figure 4E) suggesting that the mound arrest phenotype of the mutant is due to the absence of some secreted factor(s).

Further, when developed in the presence of WT CM, adgf ⁻ cells formed tipped mounds and eventually fruiting bodies (Figure 4F). Conversely, in the presence of adgf ⁻ CM, WT cells developed as large mounds with no tips (Figure 4G) and the size was comparable to the mounds formed by adgf ⁻. These findings imply that the developmental phenotype of adgf ⁻ is not due to a cell autonomous defect but due to faulty secreted factor signaling. While this finding is consistent with the involvement of a secreted factor, it is also possible that a membrane-bound extracellular factor may have a role in complementing the mutant phenotype.

Volatile ammonia rescued the mound arrest phenotype of adgf ⁻

If ADGF enzyme activity is compromised, ammonia levels are expected to be low. Hence, the concentration of ammonia from WT and adgf ⁻ mounds was measured using a commercial kit. Ammonia levels were significantly reduced in adgf ⁻ lines (Figure 5A). By mixing sodium hydroxide and ammonium chloride (Thadani et al., 1977), ammonia could be generated, and in such conditions, tip formation was restored in adgf ⁻ mounds (Figure 5B). All the rescue experiments involving ammonia were carried for a 3.5 h time period.

Figure 5.

Physically separated WT restored tip development in the mutant

WT mounds are expected to release different set of volatiles including ammonia, possibly rescuing the mound arrest phenotype of the mutants nearby. To verify if this is correct, adgf ⁻ cells were developed in one half of a compartmentalized Petri dish, and WT cells on the other side. Interestingly, the mound arrest of adgf ⁻ was fully rescued (Figure 5C) in the presence of WT, although they were physically separated from each other. This suggests that volatiles, likely to be ammonia released from WT is sufficient enough to rescue the mutant phenotype.

The adgf ⁻ CM is expected to have high adenosine levels, possibly generating low or no ammonia. Addition of ADA to adgf ⁻ CM in one compartment of the partitioned dish led to partial rescue (57 % ± 2) of adgf ⁻ kept on the other side of the dish (data not shown) implying that the ammonia generated in such conditions may not be enough for a full rescue.

Adenosine deamination alone drives the rescue of adgf ⁻

To know if adenosine deamination is exclusively responsible for the rescue of the mutant, 10 ml cold KK2 buffer mixed with different concentrations of adenosine (10 µM, 0.1 mM, 1 mM) was added in one half of the compartmentalized dish, and the other side of the dish had the mutant on phosphate buffered agar arrested at the mound stage. Addition of ADA enzyme (10 U) to the buffered solution containing adenosine led to a full rescue after 3.5 h (Figure 5D), strongly indicating that volatile ammonia generated from adenosine deamination alone is rescuing the defect. Protein/RNA catabolism also generates ammonia in Dictyostelium (White and Sussman, 1961; Hames and Ashworth, 1974; Schindler and Sussman, 1977; Walsh and Wright, 1978), but their levels were not significantly different between WT and adgf mutants (Figure S2A-B) suggesting that ammonia released from adenosine deamination plays a direct role in tip formation.

Caffeine, not ammonia rescued the mound size of adgf ^-

Given that adenosine levels were elevated in the mutants, we attempted to rescue the large mound size observed in the adgf mutants by treating them with the adenosine receptor antagonist, caffeine (Costenla et al., 2010; Ribeiro and Sebastiao, 2010). Caffeine rescued this early developmental defect in a dose-dependent manner (Figure 5E), suggesting that adgf may be one of the regulators of group size in Dictyostelium. Since ammonia levels were reduced in the mutant, we tested whether exposure to ammonia could rescue the large mound size of the adgf mutant. Exposing the mutants to 0.01 M ammonia soon after plating or six hours after plating, had no effect on the mound size of the mutants (Figure 5F), suggesting that while some early effects may be mediated through adenosine receptors, the later effects appear to be independent and are likely to be influenced by ammonia.

Faulty expression of cAMP relay genes in adgf ⁻

cAMP signaling is crucial for tip development and determining cell fate in Dictyostelium (Schaap and Wang, 1986; Saxe et al., 1993; Firtel, 1996; Singer et al., 2019). Hence, the expression of genes involved in cAMP relay were measured in WT and adgf ⁻ cells by qRT-PCR. Total cAMP levels and acaA gene expression were both low in the adgf ⁻ lines (Figures 6A and 6B). Treating the cells with the pkA activator 8-Br-cAMP or the activator of adenyl cyclase, cyclic-di-GMP (Wang and Schaap, 1985; Chen and Schaap, 2012), reversed the adgf ⁻ mound arrest phenotype (Figures 6C and D), and increasing the cyclic-di-GMP dose from 0.5 mM to 1 mM resulted in multi tipped phenotype (Figure 6E). These observations reinforce that the mound arrest phenotype of the adgf ⁻ lines is due to faulty cAMP signaling. A transient increase in cAMP levels can be achieved by blocking the phosphodiesterase (pde4) activity. When treated with a known PDE4 inhibitor, 3-Isobutyl-1-methylxanthine (IBMX) (Bader et al., 2007; Siegert and Weijer, 1989), there was no effect on tip formation in adgf ⁻ mounds (Figure 6F), although caffeine restored tip formation (Figure 6G).

Figure 6.

Circular instead of spiral cAMP waves in adgf ⁻ mounds

Low acaA expression, reduced cAMP levels and enhanced cell-cell contacts in adgf ⁻ is likely to impair cAMP wave propagation, and to ascertain if this is true, the cAMP signal propagation in WT and adgf ⁻ mounds were compared using dark field optics. The cAMP wave propagation was spiral in WT, and in contrast, the waves were circular in the adgf mutant, suggesting that the cAMP relay is impaired (Figure 6H; Supplementary videos S3 and S4). While this strongly suggests a defect in the mutant’s ability to sustain spiral wave formation, the possibility of short-lived or transient spiral waves in the mutant cannot be excluded.

Ammonia restores tip formation by regulating acaA/pde4 expression

To find if adgf expression is regulated by the substrate or the product, WT cells were treated with different concentrations of adenosine (100 nM, 1 µM, 0.5 mM), plated on KK2 agar plates and harvested at 16 h for RNA isolation. Independently, WT mounds on KK2 agar plates were exposed to different concentrations of volatile ammonia (0.1 mM, 1 mM, 10 mM) for 3 h and thereafter, the mRNA expression levels of adgf, acaA and pde4 were examined. With 100 nM adenosine, the expression of both adgf and acaA decreased, but at higher adenosine concentrations (1 µM, 0.5 mM), the expression levels of these two genes were comparable to controls (Figure 7A). Interestingly, pde4 expression decreased gradually with increasing adenosine concentrations (100 nM to 0.5 mM), but increased steadily with increasing ammonia levels.

Figure 7.

Exposure of WT mounds to 0.1 mM ammonia, led to decreased adgf expression but exposure to 1 mM and 10 mM ammonia, respectively caused a 3 and 2.3-fold upregulation of acaA expression. These observations suggest that ammonia may be rescuing the mound arrest phenotype by enhancing acaA expression and thus cAMP levels. In similar conditions, a considerable increase in pde4 expression was observed (Figure 7B), which may be necessary for controlling cAMP levels in response to ammonia treatment. In conclusion, the expression of adgf is influenced both by the substrate adenosine, as well as the product ammonia in a dose dependent manner. To ascertain if ammonia exposure indeed increases the cAMP levels in the mutant thereby restoring the phenotype, cAMP levels were measured in the adgf mutant mounds following the rescue with ammonia. Indeed, the cAMP levels were higher in the rescued mounds compared to untreated adgf controls (Figure 7C), supporting the idea that ammonia promotes tip development by restoring the cAMP signaling.

adgf ^- cells sort to prestalk in a chimera with WT

To determine if adgf favours the differentiation of one cell type over the other, the expression of pst and psp markers were examined in the mutant by realtime PCR. While the expression of pst markers, ecmA and ecmB were significantly upregulated, the psp-specific pspA gene expression was reduced in the mutant (Figure 7D), suggesting that WT adgf favours psp expression.

To know the cell type preference of the adgf mutants in a chimera with WT, a fraction (20%) of any one cell type was labelled with a cell tracker stain 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine Perchlorate (DIL), and the development was tracked thereafter. In the slugs that formed after mixing labelled WT or adgf ^OE cells with 80% unlabelled adgf ⁻ cells, the fluorescence was largely confined to the psp region (Figures 8B-C). Conversely, when adgf ⁻ cells stained with DIL were mixed with unlabelled WT or adgf ^OE, the fluorescence was restricted to the pst part of the slug (Figure 8A). In the chimeric slugs, consistently, labelled WT or adgf ^OE cells occupy the psp, whereas the adgf ⁻ cells end up in the pst region. Thus, the distribution of cell types in the mound/slug is significantly influenced by adgf, as the mutant cells sort out in a mixture with WT cells and differentiate to pst cells.

Figure 8.

Further, when adgf ⁻ slugs were stained with the pst marker, neutral red (Yamamoto and Takeuchi, 1983), the staining was intense in the pst region and anterior-like cells (ALCs), and such an intense coloration was not apparent in WT especially in the ALCs (Figure S3A). To further investigate if reduced ADA activity affects cell type specific marker expression or their pattern in slugs, pst (ecmA-GFP, ecmO-GFP) or psp (pspA-RFP) lines were treated with the ADA inhibitor DCF. While there was no change in pst/psp patterns in the fraction of mounds and slugs that developed after a long delay, the ecmA-GFP fluorescence was intense when compared to WT (Figures S3B). Similar to NR staining pattern in adgf mutant slugs, a prominent expression of ecmA-GFP was observed in the ALC region also. Visually, there was no change in ecmO or pspA marker expression in DCF treated slugs (Figures S3C and S3D) suggesting that impaired ADGF activity affects tip specific expression.

To know if adgf expression is differentially regulated between the two major cell types, psp-GFP tagged WT slugs were disaggregated, and using the fluorescence activated cell sorter (FACS), GFP-negative prestalk (pst-GFP⁻) and GFP-positive prespore (psp-GFP⁺) populations were obtained (Figures 9A-E), and RNA was isolated from both cell populations. Real time PCR analysis reveals that adgf expression is 4.95-fold higher in the psp-GFP⁺ than pst-GFP⁻ cells (Figure 9F). The preferential sorting of WT cells to psp region and the adgf mutants (with reduced psp expression) to the pst part of the slug also reinforces the differential expression of adgf. Thus, cells with higher adgf expression preferentially sorted to the psp region and the absence of adgf in mutant cells may hinder their ability to adopt a prespore fate, leading to their preferential sorting in the pst region within the chimera.

Figure 9.

adgf acts downstream of the histidine kinase, dhkD

In an effort to identify the pathway by which adgf acts during tip development, we selected a number of mutant lines with similar mound arrest phenotype such as cAMP receptor B (carB ^-), LIM domain containing protein (limB ^-), mound mutant (mndA ^-) and histidine kinase (dhkD ^-) (Saxe et al., 1993; Carrin et al., 1996; Chien et al. 2000; Singleton and Xiong, 2013), and treated the mounds with ADA enzyme. Treatment with 10 U ADA or exposure to ammonia restored tip formation in dhkD ⁻ mounds and not others tested (Figures S4A and S4C). An increase in ADA concentration (20 U), resulted in the development of multiple tips in dhkD⁻ (Figures 10A-C). Further, in the dhkD mutant mounds that were rescued by ammonia, cAMP levels were significantly elevated compared to untreated dhkD ^- controls (Figure S4D). These findings imply that ammonia induced tip formation increased cAMP levels, thereby restoring the phenotype, and adgf acts downstream of dhkD in controlling tip development. Similarly, we tested few mutants with multi-tipped phenotype, such as tipped mutant (tipA ^-), culinB (culB ^-), autophagy mutants (atg7 ^-, atg8^-, and atg9^-) (Stege et al., 1999; Wang and Kuspa, 2002; Otto et al., 2003; Otto et al., 2004, Tung et al., 2010) by adding the ADA inhibitor, DCF (1 mM) to the cell suspension /agar plates and if rescued, those mutants are likely to be in the same pathway as that of adgf in controlling tip development. However, DCF treatment had no impact on the phenotype of the mutants with multiple tips (Figure S4B).

Figure 10.

Impaired expression of other deaminases also results in aberrant tip formation

Several pathways control ammonia levels during development, and knockouts in other deaminases (2-aminomuconate deaminase: DDB_G0275081, adenosine monophosphate deaminase: DDB_G0292266, dCTP deaminase: DDB_G0293580, threonine deaminase: DDB_G0277245, N-acetyl glucosamine deaminase: DDB_G0286195, glucosamine-6-phosphate deaminase: DDB_G0278873) show a partial mound arrest phenotype (Figures S5A-F) although the expression of some of these candidates is stronger during development, suggesting a prominent and unique role of adenosine deamination in tip development.

High adenosine levels and other related purines do not block tip development

ADA catalyses the conversion of adenosine/deoxy adenosine respectively to inosine/ deoxy inosine. As adgf mutants have high adenosine levels, we investigated if the mound arrest could be mimicked in WT by treating with adenosine. However, addition of adenosine does not lead to mound arrest in WT (Figure S6A). It is possible that ADA/ADGF converted adenosine analogues to inosine analogues, which in some manner affected development. Hence, WT cells were treated with adenosine analogue (2’-deoxyadenosine) or guanosine (10 µM each), and they do not cause a mound arrest phenotype either. Similarly, treating adgf ⁻ mounds with inosine does not restore tip formation (Figures S6B and S6C) suggesting that the blocked tip development is due to faulty adenosine deamination alone.

Cross kingdom rescue of the adgf ^- mound arrest phenotype

Just like WT Dictyostelum rescuing the mound arrest phenotype of the adgf mutant, we examined if bacteria physically separated from the mutants would rescue the phenotype. To check this, Klebsiella pneumoniae and adgf mutants were incubated adjacent to each other within a compartmentalized KK2 agar plate. After 12 h incubation period, tip formation was restored in the mutants while in the same time frame, the mounds in controls failed to form tips. Possibly, Klebsiella on KK2 plates with no nutrients would die releasing ammonia thereby restoring tip development in Dictyostelium (Figure S7).

Discussion

Dictyostelium ADGF is likely to be a secreted growth factor

Multiple sequence alignments, experiments with conditioned media and cell mixing with WT suggests that ADGF is secreted. ADGF is also known to interact with 5′-adenosine monophosphate (AMP) and deoxyadenosine. AMP deaminase, previously characterized in the mollusk Helix pomatia, has been identified as a member of ADGF family (Tzertzinis et al., 2023). Studies on the characterization and expression of ADGF in Pacific abalone has shown that it is a secreted protein critical for embryonic and larval development (Hanif et al., 2022). The crystal structure of human ADA2 reveals the presence of a catalytic- and two unique ADA2-specific domains with novel folds, responsible for protein dimerization and interaction with cell surface receptors. Furthermore, the presence of a number of N-glycosylation sites, conserved disulfide bond, and a signal peptide indicate that ADA2 is specifically adapted to function in the extracellular environment (Zavialov et al., 2010).

Possible reasons for increased mound size in the adgf mutant

Enhanced cell adhesion influences cohesion and can impact the mound size (Roisin-Bouffay et al., 2000). The expression of cadA and csaA genes were upregulated in the adgf mutant, possibly leading to increased adhesion and larger mound formation. While over-secretion of ctn leads to stream breaking and small aggregate formation (Brock and Gomer, 1999), disruption of the ctn gene prevents this process, inducing large aggregate formation. ctn in turn regulates smlA (Brock et al., 2003), impacting the overall aggregate size. Thus, adgf mutants that have reduced ctn and smlA expression manifest in large mound formation. Indeed, cells treated with adenosine are known to form large aggregates (Schaap and Wang, 1986; Jaiswal et al., 2012), and the first genetic evidence supporting the previous work is from adgf mutants, which carry excess extracellular adenosine and form large aggregation streams.

Pathways generating ammonia in Dictyostelium

Proteolysis during starvation is believed to be the main source of volatile ammonia in Dictyostelium (Hames and Ashworth, 1974; Schindler and Sussman, 1977; White and Sussman, 1961), while RNA degradation is also attributed to yield ammonia during starvation (Walsh and Wright, 1978). During development, amino acids, total protein and RNA levels are reported to reduce with a significant increase in ammonia levels, thus equivocating the source of volatile ammonia (Hames and Ashworth, 1974). The highly acidic, autophagic vesicles in pst cells (Gross, 2009) are believed to catalyze the breakdown of proteins and RNA, also generating ammonia.

Furthermore, several deaminases or enzymatic reactions in Dictyostelium may also generate ammonia (Supplementary Table S1). The annotated D. discoideum genome contains five evolutionarily conserved family of ammonium transporter/methylammonium permease/rhesus protein (Amt/Mep/Rh) encoding genes (Eichinger et al., 2005), amtA, amtB, amtC, rhgA, and rhgB, which help in regulating ammonia levels during growth and development. amtA and amtC antagonistically control developmental processes and are involved in ammonium sensing or transport (Follstaedt et al., 2003; Kirsten et al., 2005; Singleton et al., 2006).

Role of ammonia during Dictyostelium development

Ammonia is known to inhibit aggregation (Schindler and Sussman, 1979; Williams et al., 1984), affect aggregate territory size (Thadani et al., 1977), aggregate density (Feit, 1988), cell fate (Gross et al., 1988), culmination (Davies et al., 1993) and fruiting body size in Dictyostelium (Lonski, 1976). Ammonia promotes psp over pst differentiation (Newell et al., 1969; Sternfeld and David, 1979; Gross et al. 1983; Oyama and Blumberg, 1986), and favors prolonged slug migration called ‘slugging’ over culmination by suppressing DIF biosynthesis (Neave et al., 1983). Ammonia plays a crucial role in orienting cell masses, accelerating the movement of aggregating cells (Bonner et al., 1986), and prevents the developmental transition of slugs to fruiting bodies (Bradbury and Gross, 1989; Wang and Schaap, 1989). Enzymatic removal of ammonia causes the quick transition from slug to fruiting thus controlling the morphogenetic pathways (Schindler and Sussman, 1977).

In slugger mutants, ammonia is known to inhibit tip formation (Gee et al., 1994), but not in WT NC4. Yoshino et al., (2007) have also reported ammonia inhibiting both aggregation and tip formation in WT cells. However, ammonium chloride was used as a source of ammonia, and the potential interference from chloride ions cannot be ruled out. A mutant impaired in mitochondrial function, midA⁻, is reported to accumulate high levels of ammonia that inhibited development (Torija et al., 2006). Notably, these cells were developed with overhead light, a condition that may influence ammonia sensitivity and the developmental phenotype. However, ammonia exerts no effect on tip formation in AX4 (data not shown). After the tips are established, the slugs/ fruiting bodies move away from ammonia, reflecting a process by which fruiting bodies position themselves from other structures to increase the possibility of spore dispersal (Kosugi and Inouye, 1989).

Cells carrying high ATP, its derivatives cAMP (Bagorda et al., 2009; Singer et al., 2019) and adenosine (Schaap and Wang, 1986), end up in the tip (Hiraoka et al., 2022). Further, 5’-nucleotidase promoter activity is high in pstAB cells (Ubeidat et al., 2002) that significantly covers the tip region suggesting high adenosine levels in the tip. However, extracellular 5’-AMP can be derived from multiple sources such as hydrolysis of cAMP, ATP and RNA (Carpousis et al., 1999).

As a gas, ammonia can diffuse generating a gradient. Our results (Figure 6A) also show that the amount of ammonia released from adenosine is in the same order of magnitude as that from other sources (Yoshino et al., 2007). It is interesting that a mutation in adgf manifests in arrested tip development, although adgf expression was found to be higher in psp than pst cells. If a threshold concentration of ammonia is not present, collective cell movement influencing tip formation may be blocked at the mound stage. Increased adgf expression in psp than pst cell types suggest that low extracellular adenosine (Weijer and Durston, 1985; Schaap and Wang, 1986) and high levels of ammonia influence psp cell fate. The slime sheath at the back of the slug is believed to prevent the diffusion of ammonia (Gross, 2009), and ammonia escaping through the front of the slug neutralizes the acidic vesicles (Bonner, 1952), causing collective cell movement thereby restoring tip development. The decision between the formation of the tip (pst cells) and the ALC is controlled by the tip’s production of ammonia, which prevents the migration of ALCs towards the tip (Sternfeld and David, 1982; Feit et al., 1990).

In adgf mutants, ammonia levels may not be sufficient enough to neutralize the acidic vesicles and hence, NR staining is intense in the pst region and the ALCs (Bonner, 1952). Ammonia has been shown to differentially suppress cAMP chemotaxis in ALC and pst cells in Dictyostelium (Feit et al., 2001).

Ammonia’s effect on cAMP signaling in Dictyostelium

Exposing the cells to ammonia is known to increase intracellular cAMP levels in D. discoideum (Riley and Barclay, 1990; Feit et al., 2001), but Schindler and Sussman, (1977) and Williams et al., (1984) reported that high ammonia levels inhibit the synthesis and release of cAMP. However, these experiments use ammonium carbonate (Schindler and Sussman, 1977), or ammonium chloride (Williams et al., 1984) as a source of ammonia, and the possibility of carbonate and chloride ions interfering with development cannot be ruled out. Ammonia is reported to show no effect on cAMP levels in D. discoideum slugs (Schaap et al., 1995), but in D. mucoroides, high ammonia levels are known to block the production of extracellular cAMP. The effect of ammonia on aggregation seems to be species specific notably in P. violaceum, P. pallidum, and D. mucoroides resulting in wider aggregation territories, while it was not observed in other species such as D. discoideum and D. purpureum (Bonner and Hoffman, 1963).

Adenylate cyclase (AC) activity is regulated by various factors (Steer, 1975). At physiological levels, ammonium ions increase AC activity in the rat brain by 40% (Yeung et al., 1989), but lowered its activity in the liver by about 30% (Wiechetek et al., 1979). Ammonia has been shown to increase the activity of AC-G in Dictyostelium sori (Cotter et al., 1999). High ammonia levels by altering the pH, can affect the activity of numerous enzymes whose activity is pH dependent and thus the activity of AC can be impacted by pH variations. adgf mutants with low ammonia levels have reduced cAMP levels, and increase in ammonia causes a significant increase in acaA expression. In rat brain, ammonia is known to interact with manganese (Rivera-Mancía et al., 2012; Lu et al., 2020), that is known to increase acaA expression in Dictyostelium (Loomis et al., 1979; Khachatrian et al., 1987). Possibly, ammonia interacts with metal ions like manganese to form ‘ammine complexes’ (Lipkowski and Galus, 1973), particularly in aqueous environments or under specific conditions where the appropriate ligands are available, and enhances cAMP levels, thus rescuing the mound defects of the mutant.

Tip organizer development in Dictyostelium depends on the differentiation and appropriate sorting of pst and psp cells (Kay et al., 1978; Williams et al., 1989; Saxe et al., 1993; Williams, 2006), a process that relies on signaling of different morphogens including cAMP, adenosine, DIF and ammonia (Bloom and Kay, 1988; Williams, 1988; Riley and Barclay, 1990; Gross et al., 1994). These morphogens modify one another’s effects, and determine the choice of the differentiation pathway as well as the spatial arrangement of cells (Bloom and Kay, 1988). Thus, the rescue of the adgf mutant upon exposure to ammonia is likely due to cAMP signaling and cell-cell contact.

Possible reasons for reduced cAMP levels in the adgf mutant

Higher pde4 and reduced acaA expression may result in low cAMP levels in the mutant. ADA significantly regulates adenosine levels, thus reducing the activation of adenosine-mediated receptors (Van Haastert, 1983). Two classes of adenosine receptors including adenosine alpha- and beta-receptors are known to be expressed in Dictyostelium (Theibert and Devreotes, 1984). When adenosine concentrations are high, beta-receptors bound with adenosine inhibit the binding of cAMP to its receptors, thereby inhibiting cAMP signaling (Newell, 1982; Van Haastert, 1983; Theibert and Devreotes, 1984). High adenosine levels in the adgf mutant may also reduce cAMP levels via a similar mechanism.

The mound/slug tip is believed to carry high adenosine levels restricting additional tip formation (Wang and Schaap, 1985), and our results also suggest that adgf ⁻ cells with high adenosine end up as pst cells.

Distorted cAMP waves in mutant

Collective cell movement within mounds is essential for cell sorting and the progression from mounds to finger structures (Kellerman and McNally, 1999). In WT mounds, cAMP waves propagate as spirals, while in the mutants, the wave propagation was in concentric circles. Initially, the cAMP waves arise as concentric circles, that, upon symmetry breaking, form spiral waves (Siegert and Weijer, 1995). With successive wave propagation, the circular ring distorts, and one end curls towards the pulsatile center, creating a spiral wave around the organizing center. In adgf mutants, however, defective cAMP relay prevents this transition. A surge in phosphodiesterase inhibition is thought to regulate this shift from concentric to spiral wave propagation (Palsson and Cox, 1996), and indeed the phosphodiesterase expression was higher in the adgf mutant possibly distorting the wave propagation.

The mound/slug tip of Dictyostelium generates cAMP pulses (Traynor et al., 1992), and is known to suppress additional tip formation (Farnsworth, 1973). Adenosine, that inhibits additional tip formation represses pde4 expression whereas ammonia, that promotes tip elongation, increases pde4 expression. Thus, the restoration of cAMP signaling and spiral wave propagation possibly leads to the rescue of the adgf ^- mound arrest phenotype upon exposure to ammonia. Surprisingly adenosine exerts little effect on tip development in WT cells and studies of Inouye (1989) also reinforce our observations, showing that adenosine does not significantly influence the conversion of pst to psp cells in shaking culture conditions.

Adenosine deamination drives tip organizer development

A crucial evidence supporting that adenosine deamination is singularly responsible for the rescue of adgf ^- mound arrest comes from experiments where partitioned dish with buffered solution containing adenosine on one side, and the mutants on the other half of the dish. Addition of ADA to the buffer containing adenosine resulted in adgf mutants forming tipped mound. Although ammonia’s role in Dictyostelium development is well established, this report is the first to show a novel role of ammonia in tip development. If natural variants of Dictyostelium that fail to form tips exist in soil ecosystems, it is conceivable that these strains could still form fruiting bodies when in proximity to WT strains. This interaction would likely occur through volatile signals such as ammonia diffusing through the shared environment. These diffusible signals may help rescue or coordinate developmental processes among neighbouring cells. Notably, the development of an organism in its natural environment is strongly influenced by volatile compounds present in its surroundings (Schulz-Bohm et al., 2017).

ADA in organizer development and gastrulation in vertebrates

ada/adgf expression is found to be high during gastrulation stages (Figure S8) in several vertebrates (Pijuan-Sala et al., 2019; Tyser et al., 2021), and collective cell migration during gastrulation and collective cell movement within the Dictyostelium mound are remarkably similar (Weijer, 2009), suggesting an overlooked role of ammonia in organizer development. The human homologue of adgf, CECR1, is a potential gene for the genetic disorder, Cat-eye syndrome and thus, Dictyostelium may also serve as a model to study this condition. Ammonia is crucial in regulating differentiation, metabolism, and gene expression (Stein et al., 2013; Liu et al., 2022). Ammonium has been shown to induce aberrant blastocyst differentiation, disrupt normal metabolic processes, alter pH regulation, and subsequently affect fetal development in mice (Lane and Gardner, 2003). Newt Triturus exposed to ammonia shows dorsalization of the ventral marginal zone highlighting the capacity of ammonia to alter embryonic axis formation (Yamada, 1950). Furthermore, research on avian embryos has revealed significant ammonia content in developing eggs, suggesting a potential role for ammonia in the energy metabolism during ontogenesis (Needham, 1926).

dhkD functions upstream of adgf

The histidine kinase dhkD, a member of the two-component family of histidine kinases, appears to function upstream of adgf, adding to our understanding of the signaling cascade in Dictyostelium. Histidine kinases play essential roles in regulating developmental transitions, with dhkC, for example, initiating the late developmental program that ultimately leads to fruiting body formation (Singleton et al., 1998). This function of dhkC is regulated in part by ammonia levels, as low ammonia concentrations have been shown to inhibit the dhkC phospho-relay, thereby influencing the developmental outcomes (Kirsten et al., 2005). In Dictyostelium, several histidine kinases, including dhkA, dhkB, dhkC, and dokA, coordinate cAMP signaling to modulate the activity of PKA, a cAMP-dependent protein kinase that is essential for proper development (Anjard and Loomis, 2003). These findings suggest the interdependencies within the signaling network in regulating multicellular development in Dictyostelium.

Model of ADGF action

Ammonia generated from multiple sources is likely to be quenched rapidly by the intracellular acidic vesicles of pst cells, increasing its pH and favoring collective cell movement driving mound/ slug tip development. However, the slime sheath at the back of the slug may prevent the diffusion of ammonia, and thus the major route of ammonia emission will be from the slug front (Farnsworth and Loomis, 1974). Although ammonia is likely to be formed from several sources in Dictyostelium, a critical threshold concentration of ammonia may be necessary for tip development. The failure of even one pathway may result in a drop-in ammonia levels, exerting an effect on development (Figure 11). Thus, ammonia is likely to be important for the maturation of tip cells.

Figure 11.

Our study shows that adgf acts downstream of dhkD and in silico studies have shown that dhkD interacts with adgf. Therefore, it is likely that in the presence of ammonia, dhkD activates the phosphorelay, in turn increasing intracellular cAMP levels, leading to tip formation. Histidine kinase dhkC is known to phosphorylate regA in Dictyostelium (Thomason et al.,1999). However, based on docking we found no direct interaction of regA with ADGF (data not shown). Thus, the integration of histidine kinase signaling ensures coordinated multicellular organization in Dictyostelium (Aoki et al., 2020). Previous work in other systems have found ADA to be interacting with CD 26 (dpp IV) (Tanaka et al., 1993), and docking DdADGF with AprA (dpp8) also supports this observation. However, adding ADA inhibitor to aprA mutant (dpp8), showed no effect on the phenotype (data not shown).

Supplementary Results

Genes involved in autophagy and tipped mound formation do not show altered expression in the adgf mutant

Autophagy helps in maintaining cellular homeostasis by promoting the breakdown of damaged proteins and organelles (Mizushima et al., 2008), and ammonia is recognised to be a diffusible regulator of autophagy in human cells (Eng et al., 2010). To investigate if autophagy is impaired in the mutant, the expression of autophagy markers atg8 and atg18 were examined and there was no discernible change in the expression levels when compared to WT. The tipless to tipped mound transition as well as late developmental gene expression is regulated by gbfA, a G-box binding factor (GBF). However, in the adgf mutant the gbfA expression levels were comparable with no significant difference between the WT at 16 h (data not shown). Without the assay for autophagy as well as assay for GBF activity, these expression studies alone are not conclusive to rule out the role of these factors in tip formation.

ADA does not deaminate cytokinin

ADA is known to interact with 5’AMP (Hanif et al., 2022) and in Dictyostelium, 5′-AMP serves as a direct precursor of cytokinin, playing a significant role in cellular signaling and development (Taya et al., 1978). Although docking studies suggest low to moderate binding of ADGF with cytokinins (zeatin, dihydrozeatin, isopentenyl adenine), we carried a functional assay by mixing cytokinin and ADA with KK2 buffer in one side of the Petridish and found no rescue of the mutant in such conditions (data not shown), suggesting no functional activity between ADA and the versions of cytokinin tested.

Supplementary discussion

Plausible routes of caffeine action rescuing the mound arrest phenotype

The adgf mound arrest could be rescued by the addition of the adenosine antagonist, caffeine but treatment with the pde4 inhibitor, IBMX failed to rescue the mound arrest. Caffeine is known to reduce adenosine levels in blood plasma (Conlay et al., 1988) and also increase ammonium levels in urine samples of rabbits (Bernheim and Bernheim, 1945). Thus, the mound rescue upon caffeine treatment may be a result of reduced adenosine and increased ammonia levels. With respect to caffeine action on cAMP levels, the reports are contradictory. Caffeine has been reported to increase adenylate cyclase expression thereby increasing cAMP levels (Hagmann, 1986) whereas Alvarez-Curto et al., (2007) found that caffeine reduced intracellular cAMP levels in Dictyostelium. Although, caffeine is moderately potent in inhibiting PDE enzyme activity, the in vivo concentrations are likely to be low to be associated with effective PDE inhibition (Burg and Werner, 1975; Daly, 1993).

Material and Methods

Bioinformatic analyses of ADGF

The genomic sequence and the protein sequence of ADGF were obtained either from dictybase (http://dictybase.org) or NCBI database (https://www.ncbi.nlm.nih.gov/). The ADA domain within the DdADGF was identified by SMART and BLAST (http://smart.embl-heidelberg.de/) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1990; Letunic and Bork, 2018) analyses. A phylogenetic tree was generated by aligning multiple amino acid sequences of ADGF from several taxa. Neighbour Joining approach and the MUSCLE alignment tool of the MEGAX programme were used for constructing the tree (Saitou and Nei, 1987). The tertiary structure of ADGF was obtained using the online programme alphafold (https://alphafold.ebi.ac.uk/) (Jumper et al., 2021), and PyMol was used for viewing the images. The expression profiles of ada and ada2 during mouse and human gastrula development were retrieved from the marionilab.cruk.cam.ac.uk/MouseGastrulation2018/ and human-gastrula.net/ databases (Pijuan-Sala et al., 2019; Tyser et al., 2021), respectively. Protein-protein docking was carried out using High Ambiguity Driven Protein-Protein Docking (HADOCK) version 2.4.

Culture and development of Dictyostelium discoideum

D. discoideum (WT-AX4) cells or the mutant adgf derived from AX4, (DBS0237637) were cultured in modified maltose-HL5 medium (Formedium, UK) with 100,000 U/L penicillin and 0.1 g/L streptomycin. Three independent mutants (GWDI_17_D_7, GWDI_47_C_1, GWDI_132_H_3; insertion in exon 2 in all three mutants) of the adgf gene (DDB_G0275179) were obtained from the GWDI bank (https://remi-seq.org), in Dictyostelium stock centre, North Western University, USA (Gruenheit et al., 2021). The mound defects were identical in all three and strain GWDI_47_C_1 alone was characterised further. The culture was raised as a monolayer in Petri plates or grown in an Erlenmeyer flask in shaking conditions at 150 rpm and 22 °C, to a cell density of 2-4×10⁶ cells/ml. The cells were also grown on SM/5 agar plates supplemented with Klebsiella pneumoniae at 22 °C (2 g/L glucose, 2 g/L protease peptone, 0.4 g/L yeast extract, 1 g/L MgSO₄.H₂O, 0.66 g/L K₂HPO₄, 2.225 g/L KH₂PO₄, 1% Bactoagar, pH 6.4). For developmental assays, freshly starved cells were washed twice with ice cold KK2 buffer (2.25 g KH₂PO₄ and 0.67 g K₂HPO₄ per litre, pH 6.4), and plated on 1% non-nutrient KK2 agar plates at a density of 5×10⁵ cells/cm² (Nassir et al., 2019). Thereafter, the plates were incubated in dark conditions at 22 °C for development.

Dictyostelium genomic DNA isolation

WT and adgf ^- cells grown axenically were harvested, and the pellet was resuspended in 1 ml of lysis buffer (50 mM Tris-Cl, pH 8; 10 mM EDTA; 0.8% SDS). To this mixture, 200 µl of Nonidet P-40 (NP40), a non-ionic detergent, was added, vortexed and centrifuged at 12,000 g for 15 min at room temperature (RT). The resultant pellet was gently vortexed, resuspended in 500 µl of lysis solution containing 200 µg/ml Proteinase K, and incubated at 65 °C for 30 min. 300 µl of phenol: chloroform was added to this suspension, centrifuged at 18,000 g for 10 min at RT, and the resultant aqueous phase was extracted carefully. An equal amount of chloroform was added and centrifuged at 18,000 g for 10 min at RT. Following this, 750 µl of pure ethanol was added to the suspension, which was then centrifuged at 12,000 g for 15 min at 4 °C. Genomic DNA was precipitated by adding twice the volume of absolute ethanol and 1/10^th the volume of 3 M sodium acetate. After a 10 min centrifugation at 15,000 g, the pellet was cleaned using 70% ethanol and stored at 4 °C. Electrophoresis was conducted in TAE buffer at 50 V using a Medox power pack system (India), and the integrity of DNA was confirmed on a 1% agarose gel.

Validation of the adgf mutant

To validate the blasticidin (bsr) resistance cassette insertion in the adgf mutant, WT and adgf ⁻ genomic DNA were isolated, and a diagnostic PCR was performed using the gene and bsr insert specific primers in accordance with the guidelines provided in the GWDI website. A qRT-PCR was carried out to confirm the absence of adgf expression in the mutant cells. The primers used for mutant validation are listed in Supplementary Table S2.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from WT and adgf ⁻ cells using Trizol reagent (Favorgen, USA) at specified intervals (every four hours from 0 to 24 h). cDNA was synthesized from the total RNA using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Japan). Random primers from the manufacturer were used to generate the cDNA from a template of 1 µg total RNA. qRT-PCR was performed using SYBR Green Master Mix (Thermo Scientific, USA) and 1 µl of cDNA. The expression levels of adgf, acaA, cAMP receptor A (carA), phosphodiesterases (pdsA, regA), 5’ nucleotidase (5’nt), extracellular matrix A (ecmA), extracellular matrix B (ecmB), prespore A (pspA), countin (ctn), and small aggregate (smlA) were quantified with a QuantStudio Flex 7 (Applied Biosystems, USA). The mitochondrial large RNA subunit (rnlA) served as an internal control. qRT-PCR data analysis was conducted according to the method described by Schmittgen and Livak (2008). The primer sequences used for qRT-PCR are provided in Supplementary Table S4.

Generation of adgf over expression construct

The full-length 1.7 kb adgf sequence was PCR-amplified using ExTaq polymerase (Takara, Japan) with WT genomic DNA as the template. The amplified product was then ligated into the pDXA-GFP2 vector at the HindIII and KpnI restriction sites. Both adgf ^- and WT cells were electroporated with this vector, and G418-resistant clones (10 μg/ml) were isolated for further analysis. The expression of adgf was confirmed by semi-quantitative PCR. The corresponding primer sequences are listed in Supplementary Table S3.

Transformation of Dictyostelium discoideum

WT and adgf ^- cells grown axenically were harvested, washed twice with ice-cold EP buffer (10 mM KH₂PO₄, 10 mM K₂HPO₄, 50 mM sucrose, pH 6.2), and resuspended in 100 µl of EP++ solution (10 K₂HPO₄ mM, 10 mM KH₂PO₄, 50 mM sucrose, 1 mM MgSO₄, 1 mM NaHCO₃, 1 mM ATP, 1 µM CaCl₂) containing 10 µg of plasmid vector (Nassir et al., 2019) in pre-cooled cuvettes (BioRad, USA). The cells were electroporated using a BTX ECM830 electroporator (Harvard Apparatus, USA) at 300 V with 2 ms pulses and five square wave pulses at 5-second intervals. The cells were then transferred to a Petri dish with 10 ml of HL5 medium and incubated at 22 °C. After 24 hours, G418 (10 µg/ml) was added to the medium, and resistant colonies were selected for further analysis.

Preparation of conditioned media (CM)

WT and adgf ⁻ cells grown in HL5 medium were collected at the mid-log (ML) phase, resuspended in KK2 buffer at a density of 1×10⁷ cells/ml, and incubated at 22 °C with shaking for 20 hours. The clarified supernatant obtained after centrifugation was used for the experiments.

Assay for ADA activity

Total ADA activity from Dictyostelium was determined as per the protocol of the manufacturer (Abcam, USA; Cat No: ab204695). For sample preparation, 5×10⁵ cells/cm² were seeded onto KK2 agar plates and at the mound stage, ice cold ADA assay buffer was flooded, then vigorously pipetted to disrupt the mound integrity. The cell homogenate was agitated on a rotary shaker at 4 °C for 15 min and then centrifuged at 12,000 rpm for 10 min in a cold microfuge tube. The supernatant was subjected to the ADA assay. This ADA test relies on adenosine to inosine formation. The intermediate formed combines with the probe to produce uric acid, which is quantified at 293 nm. BCA (Bicinchonic acid) assay kit (Thermoscientific, USA) was used for determining the protein concentration. One unit of ADA activity is defined as the amount of enzyme that hydrolyses adenosine to yield 1 µmol of inosine per min under the assay conditions.

Adenosine quantification

Total adenosine levels from Dictyostelium mounds were measured according to the manufacturer’s protocol, using the adenosine assay kit (Abcam, USA; Cat No: ab211094). Cells grown in HL5 were collected, washed and plated on KK2 agar plates. The cell lysis buffer was added to the plates with mounds and mixed thoroughly. 50 μl of the lysate was mixed with 2 U of ADA and was subjected to incubation for 15 min at RT. Adenosine quantification involves the use of ADA and after a series of enzymatic reactions, an intermediate is formed which reacts with the adenosine probe generating a fluorescent product. Using a spectrofluorometer (Perkin Elmer, USA; λEx = 544 nm/λEm = 590), the fluorescence intensity was measured, which is proportional to the concentration of adenosine. The adenosine levels were quantified using the adenosine standard curve.

Quantification of ammonia

Ammonia assay kit (Sigma-Aldrich, USA; Cat No: AA0100) was used for estimating the total ammonia levels. WT and adgf ^- cells developed on KK2 agar plates were sealed with parafilm and incubated at 22 °C. The mounds were collected using lysis solution, and the debris was removed by centrifugation at 10,000 g for 10 min. The supernatant was used for further analysis. For the ammonia assay, 1 ml of assay reagent was mixed thoroughly with either 100 µl of samples or standards, incubated for 5 min at RT, and the absorbance was measured at 340 nm. Then, 10 µl of L-glutamate dehydrogenase (GDH) solution was added to each cuvette, and after a 5-min incubation at 25 °C, the absorbance was measured again at 340 nm using a spectrophotometer (Eppendorf, Germany). In the presence of GDH, ammonia combines with α-ketoglutaric acid and reduced NADPH to produce L-glutamate and oxidised NADP⁺. The decrease in absorbance at 340 nm is proportional to the ammonia concentration. The ammonia standard curve was used to calculate the ammonia levels.

Volatile ammonia generation

To generate ammonia, 1 ml of 1 N NaOH and 1 ml of NH₄Cl (concentrations used 0.1 mM, 1 mM, 10 mM) were mixed thoroughly (Thadani et al., 1977; Feit et al., 1990) and from this mix, 2 ml was aliquoted in the upper half of the Petri dish. The other half of the plate with the adgf ⁻ mounds on KK2 agar were inverted, sealed and incubated at 22 °C. To determine whether WT mounds, physically separated from the mutants could rescue the mound arrest, WT (1×10⁶ cells/cm²) and adgf ⁻ (5×10⁵ cells/cm²) cells were developed on KK2 agar plates on either side of compartmentalised and sealed Petri plates. adgf ⁻ cells developed on either side of the dish served as controls.

Quantification of cAMP

Using the cAMP-XP test kit and following the manufacturer’s instructions, total cAMP levels were determined from both the WT and the mutant (Cell signaling, USA). WT and adgf ⁻ mounds were disrupted and collected in 1 ml ice cold KK2 buffer. After centrifuging the pellet, 100 μl of 1X lysis solution was added, and the mixture was incubated in ice for 10 min. Subsequently, 50 μl of lysate and 50 μl of HRP-conjugated cAMP solution were added to the test plates, which were then shaken horizontally at RT. After 3 h incubation, the wells were emptied and washed three times with 200 μl of 1X wash buffer. 100 μl of stop solution was added to halt the reaction, and the absorbance was measured at 450 nm using a spectrophotometer (Biorad, USA). The cAMP standard curve was used to determine the cAMP levels.

Visualisation of cAMP waves using dark field optics

5×10⁵ cells/cm² were developed on KK2 agar plates under moist, dark conditions to monitor the propagation of cAMP waves. Using a Nikon DS-5MC camera mounted on a Nikon SMZ-1000 stereo zoom and Nikon Eclipse TE2000 inverted microscope, a time-lapse video of the aggregation was captured in real-time (Nikon, Japan). cAMP optical density waves were displayed by subtracting the image pairs (Siegert and Weijer, 1995) after processing the images with the NIS-Elements D program or ImageJ (NIH, USA).

Under agarose cAMP chemotaxis assay

The under-agarose cAMP chemotaxis assay (Woznica and Knecht, 2006; Singh and Insall, 2022) was carried out with cells obtained from WT and adgf ⁻ mounds. Briefly, the cells were starved in KK2 buffer at 1×10⁷ cells/ml density. Three parallel troughs, each measuring 2 mm in width, were set up on a Petri dish containing 1% agarose. In the outer two troughs, 100 µl of WT and adgf ⁻ cell suspension was aliquoted respectively and 10 μM cAMP was added to the central trough. Cell migration toward cAMP was recorded every 30 s over a total duration of 20 min using the NIS-Elements D software and an inverted Nikon Eclipse TE2000 microscope (Nikon, Japan). 35 cells were tracked each time and subsequently analysed using ImageJ (NIH, USA).

Mixing WT with mutant cells

WT and adgf ⁻ cells cultured in HL5 medium, were harvested at the mid log (ML) phase by centrifugation, rinsed twice with ice-cold KK2 buffer. The cells mixed in different ratios (1:9, 2:8 and 1:1) were adjusted to a final density of 5×10⁵ cells/cm², plated on 1% KK2 agar plates, and incubated at 22 °C for development.

Tracking cell fate after mixing

Dictyostelium cells harvested from fresh HL5 media were resuspended at a density of 1×10⁶ cells/ml in KK2 buffer, followed by incubation at 22 °C for 1.5 h in shaking conditions with 0.2 µM DIL (Invitrogen, USA). DIL has been used extensively as a cell tracker in C. elegans, Xenopus and mice (Schultz and Gumienny, 2012; Xu et al., 2020; Erdogan et al., 2016). WT or adgf ⁻ cells labelled with DIL were mixed with the unlabelled cells in a ratio of 1:9, 2:8 and 1:1 and plated for development. In the ratios mentioned, the smaller fraction represents the stained cells. DIL is a lipophilic dye that selectively stains the plasma membranes of living cells, allowing visualization and analysis of cell morphology.

Neutral red staining of mounds and slugs

WT and adgf ⁻ cells harvested from HL5 cultures were resuspended at a density of 1×10⁷ cells per ml in KK2 buffer and treated with 0.005% neutral red (NR) solution for 15 min at RT in shaking conditions (Bonner, 1952). The stained cells were washed twice with KK2 buffer, plated on buffered agar plates and thereafter, NR stained slugs were observed using an upright microscope.

Cell-type specific expression analysis of adgf

To determine the differential expression of adgf, if any, between the two major cell types, psp-GFP cells in AX4 background were developed on KK2 agar plates at a density of 5 × 10⁵ cells/cm² until they reached the slug stage, as described by Ratner and Borth (1983). Fluorescence was initially monitored using microscopy to confirm GFP expression. Slugs were then dissociated in 20 mM phosphate buffer containing 40 mM EDTA, pH 7.0 (Nadin et al., 2000), filtered through a 10-μm nylon mesh, chilled on ice, and using FACS (FACS Discover S8 Image Sorter (BD Biosciences, USA)) the two cell types were sorted. Sorting was performed at a rate of 10,000 cells/s with a 488 nm laser. GFP-positive prespore (psp) cells and GFP-negative prestalk (pst) cells were collected separately on ice over several hours. Total RNA was extracted from both cell populations, and the expression of adgf was analyzed using RT-PCR.

Cell-cell adhesion assay

After 14 h of development on phosphate buffered agar plates, WT and adgf ⁻ mounds were disaggregated by repeated pipetting and vortexing using ice-cold KK2 buffer. Dissociated cells were resuspended in KK2 buffer and incubated at 150 rpm for 45 min. Single and non-adherent cells were counted using a Neubauer chamber (Lam et al., 1981).

Treatment of Dictyostelium cells with different compounds

A highly specific ADA inhibitor, DCF that inhibits both extra and intra-cellular ADAs (Cha et al., 1975) was mixed with the WT cell suspension and plated on 1% non-nutrient KK2 agar plates at a density of 5×10⁵ cells/cm². Of the different DCF concentrations tested (10 nM, 50 nM, 100 nM and 1 µM), 100 nM showed complete tip inhibition.

For the enzymatic rescue assay, 10 U of bovine ADA dissolved in KK2 buffer (Sigma-Aldrich, USA) was added onto mutant mounds. The concentrations of 8-Br-cAMP (2 mM), cAMP (0.1 mM, 0.5 mM), c-di-GMP (0.1 mM, 0.5 mM), adenosine (1 µM, 10 µM, 100 µM, 1 mM), caffeine (10 nM, 100 nM, 1 µM) and IBMX (0.5 mM) were based on previous publications (Chen et al., 2017; Chen and Schaap, 2012; Nassir et al., 2019; Siegert and Weijer, 1989) and these compounds were added independently either on top of the mounds or supplemented while plating. All the fine chemicals were from Sigma-Aldrich, USA except when mentioned.

Microscopy

Microscopy was performed using a Nikon SMZ-1000 stereo zoom microscope with epifluorescence optics, Nikon 80i Eclipse upright microscope, or a Nikon Eclipse TE2000 inverted microscope, connected with a digital sight DS-5MC camera (Nikon). Image processing was carried out using NIS-Elements D (Nikon) or Image J.

Statistical tools

Data analyses were carried out using Microsoft Excel (2016). Statistical significance was determined by paired or unpaired, two-tailed Student’s t-test and ANOVA analysis (GraphPad Prism, version 7).

Acknowledgements

We gratefully acknowledge the help of NBRP Nenkin, Japan and Dictyostelium Stock Center (DSC), Northwestern University, USA for providing various strains and plasmids used in this study. We thank Prof. Richard Gomer, Texas A&M, USA for providing aprA mutant strain. We thank Prof. Kei Inouye, Kyoto University, Japan, Prof. Kees Weijer, University of Dundee, UK and Prof. Gad Shaulsky, Baylor College of Medicine USA for their comments on an earlier version of the manuscript/ thesis. We acknowledge Dr. Srividhya, Facility-in-Charge, Flow Cytometry facility, Bio-SAIF, IIT Madras, for assistance with FACS and technical support. We gratefully acknowledge the help from Indian Council of Medical Research (ICMR), New Delhi for their support in this project. We thank Steve Hawthorne from Scholarly memory for his feedback and suggestions on an earlier version of the manuscript.

Additional information

Author contributions

P.H. and R.B. conceptualized the work. P.H. performed all experiments. P.H. and R.B. analysed the data and wrote the manuscript.

Funding

Indian Council of Medical Research (2012-0467)

Additional files

Supplementary videos.