Mutant validation.

A) Blasticidin resistance cassette (bsr) insertion in the adgf gene. B) PCR analyses using P1 and P4 primers. 1.4 kb shift in the adgf mutant. PCR using P1 and P2 primers showed an amplicon from the mutant (M) and not from the wild type (WT). PCR using P3 and P4 primers showed an amplicon with adgf mutant while WT did not show any amplicon. C) Semi- quantitative RT-PCR of internal control, rnlA and adgf -. adgf expression during development in Dictyostelium. D) Total RNA was isolated from Dictyostelium during vegetative growth and development using TRIzol method. To quantify adgf expression, qRT-PCR was carried out with rnlA as a control and the fold change was calculated accordingly. Time points are shown in hours (bottom). Error bars represent the mean and SEM (n = 3). E) RT-PCR analysis was performed to check the expression of the isoforms of ADA during 16 h in the mutant.

Bioinformatic analyses of ADGF.

A) BLAST analysis of ADGF. ADGF belongs to the metallo dependent hydrolases superfamily.

B) SMART analysis of different ADA domains within ADGF. ADGF protein has an adenosine deaminase-ADA domain and a N-terminal deaminase domain which is similar to the human ADA2. Multiple sequence alignment of Dictyostelium ADGF with ADGF from other organisms. C) The shaded region depicts the N-terminal signal sequence characteristic of extracellular proteins. D) The active site residue is highlighted in red. Active site residue is conserved between D.discoideum and human ada2. E) Phylogenetic analysis of ADGF across different organisms. Maximum likelihood method was used for constructing the tree using MEGAX (Molecular Evolutionary Genetic Analysis X). Structural Comparison of human ADA2 and Dictyostelium ADGF. Identical tertiary structures of human ADA2 and Dictyostelium ADGF. F) ADA2 (CECR1) Homo sapiens and G) ADGF Dictyostelium discoideum have many structural similarities. H) Alignment of Dictyostelium ADGF with Human ADA2 (CECR1).

Aggregates formed by adgf− mutants are larger in size.

A) The graph shows the number of aggregates formed by WT and adgf - and their respective mound size. The values represent mean ± S.E; n=3. Significance level is indicated as *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. (Student’s t-test). B) Expression levels of genes countin (ctn) and small aggregates (smlA) during aggregation in adgf - compared to WT. rnlA was used as the internal control in the realtime PCR. C) WT and adgf - cells were developed on KK2 agar, and after 16 h, the multicellular mounds/slugs were dissociated by vigorous vortexing in KK2 buffer. Individual cells were counted using a hemocytometer and resuspended in a phosphate buffer. Non-adherent single cells were counted 45 min after incubation. The percent cell-cell adhesion was plotted by normalizing the values to the non-adherent WT count to 100%. Error bars represent the mean ± SEM (n=3). Cell-cell adhesion, but not cAMP chemotaxis, is significantly impaired in adgf -. D) qRT-PCR analysis of cadherin (cadA) and contact site (csA) during aggregation. The fold-change in RNA transcript levels is relative to WT at the indicated time points. rnlA was used as the internal control (n=3). ns = not significant. E) Under agarose chemotaxis assay: The average cell speed in response to 10 μM cAMP was recorded. The graph represents the mean ± SEM (n=3). Developmental phenotype of adgf -. F) WT and adgf - cells were washed, plated on 1% KK2 agar plates at a density of 5x105 cells/cm2, incubated in a dark, moist chamber and images were taken at different time intervals. G) WT cells treated with 100 nM of DCF mimicked the mound arrest phenotype of the mutant. The time points are indicated in hours at the top of the figure. Scale bar: 2 mm; (n=3). H) WT and adgf - cells after 36 h of development. Scale bar: 0.5 mm; (n=3). I) Fruiting bodies of WT and adgf -. Scale bar: 0.5 mm; (n=3).

Reduced ADA activity and high adenosine levels in adgf -.

A) ADA activity in Dictyostelium cell extracts harvested at 16 h. The enzymatic assay for ADA was performed in adgf - with the corresponding WT control. Error bars represent the mean and SEM (n=3). Significance level is indicated as *p< 0.05, **p< 0. 01. Adenosine quantification and expression profile of genes involved in adenosine formation. B) Quantification of adenosine levels. Level of significance is indicated as *p< 0.05, **p< 0.01, ***p< 0.001; (n=3). C) Expression profile of the genes, 5’ nucleotidase (5’nt) and phosphodiesterases (regA, pdsA) involved in cAMP-to- adenosine conversion. The fold-change in RNA transcript levels is relative to WT at the indicated time points. rnlA was used as an internal control. Error bars represent the mean and SEM (n=3).

Overexpression of adgf rescued the mound arrest phenotype.

A) adgf - mounds were treated with 5 U and 10 U ADA enzyme, plated for development and imaged at 16 h. Scale bar: 2 mm; (n=3). B) The full-length adgf gene was cloned in the vector pDXA-GFP2. The overexpression construct w a s v e r i f i e d b y r e s t r i c t i o n d i g e s t i o n w i t h H i n d I I I a n d K p n 1 enzymes.C) Overexpression of adgf in the mutant rescued the mound arrest. D) Overexpression of adgf in the WT background. Scale bar: 2 mm; (n=3). The time points in hours are shown at the top. WT cells reconstituted with adgf - rescued the adgf mutant phenotype. E) Reconstitution of WT with adgf - in a 1:4 ratio showing a partial rescue and a full rescue of the adgf - mound arrest phenotype in a 1:1 ratio with WT. F) Development of adgf mutants in the presence of adgf - CM and WT CM on KK2 agar plates. WT CM rescued the mound arrest. G) Development of WT in the presence of WT CM and adgf - CM on KK2 agar plates. adgf - CM induced mound arrest in WT cells. Scale bar: 2 mm; (n=3).

Adenosine deamination reaction rescues the mound arrest of adgf–.

A) Quantification of ammonia using the ammonia assay kit. WT and adgf - mounds were harvested and lysed using a cell lysis buffer. Cell debris were removed by centrifugation, and the supernatant was used to quantify ammonia.

B) Exposing adgf - mounds to ammonia. Ammonia was generated by mixing 2 ml of NH4Cl and 2 ml of 1N NaOH. The mixture was poured on top of the lid and the KK2 plates with the mounds were inverted and sealed thereafter. Images were taken 3.5 h post treatment. Dose dependent effect of ammonia on the rescue. Scale bar: 2 mm; (n=3). C) WT and adgf− cells on either side of a compartmentalized Petri dish. D) adgf− cells on one side of the plate in the presence of adgf− CM treated with ADA enzyme in the other half of the dish. E) adgf− cells on one side and KK2 buffer containing adenosine and ADA on the other half of the compartmentalized dish, rescued the mound defect. Caffeine rescues the large mound size of adgf mutant. F) adgf - cells were treated with different concentrations of caffeine (100 nM, 1 µM) while plating and images were taken 3.5 h post chemical treatment. Scale bar: 2 mm; n=3. G) Exposure to ammonia does not rescue the aggregate size of adgf mutant. adgf - mounds were exposed to 0.01 M ammonia and images were taken 3.5 h post chemical treatment. Scale bar: 2 mm; (n=3).

Impaired cAMP signaling in adgf -.

A) Total cAMP levels in WT and adgf - mounds were quantified using cAMP- XP assay kit (Cell signaling, USA). Level of significance is indicated as *p< 0.05, **p< 0.01; (n=3). B) acaA expression was quantified using qRT-PCR. The error bars represent the mean ± SEM (n=3). C) adgf - mounds. D) adgf - mounds were treated with 2 mM pkA activator 8-Br-cAMP and imaged after 3.5 h. Scale bar: 2 mm; (n=3). Treatment with cyclic di-GMP and caffeine rescues the mound arrest phenotype. E) Addition of cyclic-di-GMP restored tip formation in adgf - 4 h after the treatment. Scale bar: 1 mm; (n=3). F) PDE inhibitor (IBMX) treatment failed to rescue the adgf - mound arrest. Scale bar 1 mm; (n=3). G) adgf - mounds treated with caffeine formed tips 3.5 h post treatment. Scale bar: 2 mm; (n=3). Altered cAMP wave pattern in adgf−. H) Optical density wave images depicting cAMP wave generating centers in WT and adgf -. WT shows spiral wave pattern and adgf - exhibits circular wave propagation.

Expression levels of adgf, acaA and pde4 in response to adenosine and ammonia treatment.

A) Expression levels of adgf, acaA and pde4 in response to adenosine treatment (100 nM, 500 nM, 1 µM). B) Effect of different concentrations of ammonia (0.1 mM, 1 mM, 10 mM) on the expression levels of adgf, acaA and pde4. Levels of s ignificance i s indicated as * p< 0 . 05 , ** p< 0 . 01 , and *** p< 0 . 001 ; (n=3). Expression levels of prestalk, ecmA, ecmB and prespore, pspA cell type markers in adgf -. The expression profiles of C) prestalk (ecmA, ecmB) and prespore (pspA) specific markers in WT and adgf - were quantified using qRT-PCR. Level of significance is indicated as *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001; (n=3). Three independent biological replicates were performed and the error bars represent the mean and SEM. The fold-change in RNA transcript levels is relative to WT at the indicated time points. rnlA was used as the internal control (n=3). ns = not significant

Reconstitution of WT cells with adgf - following DIL staining.

A-C) DIL labelled cells were reconstituted with unlabeled cells and plated for development. Images were captured during the migrating slug stage. The left panel shows bright field, and the right panel shows the corresponding fluorescence images. Scale bar: 0.5 mm; (n=3). adgf acts downstream of histidine kinase dhkD. D) dhkD -mutants on KK2 agar plates. E-F) 5 U, 10 U or 20 U ADA was added on top of the mounds. Scale bar: 1 mm; (n=3). Images were taken 3.5 h post treatment. Addition of the ADA enzyme rescued the mound arrest phenotype in a dose dependent manner. Scale bar: 2 mm; (n=3).

Model illustrating the role of adgf in development adgf supresses the expression of genes involved in cell adhesion, cadA and csaA.

adgf regulates aggregate size and tip development by directly acting on adenosine, ammonia levels and cAMP signaling. Line ending in an arrow implies that the previous gene/factor either directly or indirectly raises the activity or levels of the second; line ending in a cross-bar indicates inhibition. Dotted lines indicate ADGF interacting with APRA, and the cytokinins zeatin, dihydro zeatin and isopentenyl adenine.