adgf mutant validation

A) Schematic representation of adgf locus showing the relative positions of the primers (P1–P4) and the blasticidin resistance cassette (bsr). Primer P1 (adgf fwd) is at the start codon of adgf, and primer P4 (adgf rev) is 264 bp upstream of the stop codon, flanking the insertion site. Primers P2 (pGWD2) and P3 (pGWD1) are located within the bsr cassette. bsr insertion is in exon 2 of adgf. B) PCR analyses using P1 and P4 primers. A 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 the adgf mutant while WT did not show any amplicon. C) Semi-quantitative RT-PCR of the 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).

Aggregates formed by adgf mutants were larger in size

A) The graph shows the mound size and the number of aggregates formed by WT and adgf −. The values represent mean ± S.E; n=3. B) Expression levels of the genes, countin (ctn) and small aggregates (smlA) during aggregation in adgf − compared to WT. rnlA was used as the internal control. 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). 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. (n=3). ns = not significant. Significance level is indicated as *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. (Student’s t-test). E) Under agarose chemotaxis assay. The average cell speed in response t o 10 μ M c AMP was r ecorded. The graph r eprese n t s t h e m e a n ± SE M (n=3). Developmental phenotype of adgf −. F) WT and adgf − cells were washed, plated on 1% KK2 agar plates at a density of 5×105 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: 2 mm; (n=3).

adgf mounds have reduced ADA activity and high adenosine levels.

A) ADA activity in WT and adgf − harvested at 12 h and 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< 0.01. B) Quantification of adenosine levels in WT and adgf mutants at 12 h and 16 h. Level of significance is indicated as *p< 0.05, **p< 0.01, ***p< 0.001; (n=3). C) Expression profile of 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, 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 was verified by restriction digestion with HindIII and KpnI enzymes. C) adgf overexpression in the mutant rescued the mound arrest. D) Overexpression of adgf in WT background. Scale bar: 2 mm; (n=3). The time points in hours are shown at the top. WT cells mixed with adgf − rescued the adgf mutant phenotype. E) Mixing of WT with adgf − in a 1:4 ratio showed 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 was removed by centrifugation, and the supernatant was used to quantify ammonia. B) Treatment of adgf − mounds with 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 led to tip fornation in adgf −. D) adgf − cells on one side and KK 2 buffer containing adenosine and ADA on the other s ide of the compartmentalized dish, rescued the mound defect. Caffeine rescues the large mound size of adgf mutant. E) adgf − cells were treated with different concentrations of caffeine (100 nM, 1 µM) while plating, and images were taken during mound stage. Scale bar: 2 mm; n=3. F) Exposure to ammonia does not rescue the mound size of adgf mutant. adgf − mounds were exposed to 0.01 M ammonia and images were captured 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) Time course of adgf − mounds treated with 8-Br-cAMP and imaged at different intervals. 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 − 3.5 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 mutants. H) Optical density waves depicting cAMP wave generating centers in WT and adgf −. WT shows spiral 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) And ammonia treatment (0.1 mM, 1 mM, 10 mM). Level of significance is indicated as *p<0.05, **p<0.01, and ***p<0.001; (n=3). C) cAMP levels in adgf mutants rescued with ammonia. Level of significance is indicated as **p< 0.01; (n=3). Expression levels of prestalk (pst), ecmA, ecmB and prespore (psp), pspA cell type markers in adgf −. The expression profile of D) pst (ecmA, ecmB) and psp (pspA) specific markers in WT and adgf − were quantified using qRT-PCR. Level of significance is indicated as **p<0.01 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.

Mixing of WT cells with adgf − following DIL staining

A-C) DIL labelled cells were mixed with unlabelled cells and plated on KK2 agar. 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).

Sorting of pst-GFP− and psp-GFP⁺ Dictyostelium cells by fluorescence activated cell sorter

A) Forward scatter (FSC) vs. side scatter (SSC) plot used to gate total cells. B) Singlets were gated based on FSC-H vs. FSC-A to exclude doublets and aggregates. C) GFP fluorescence profile of gated single cells reveals two populations: pst-GFP⁻ and psp-GFP⁺, corresponding to pst and psp cells, respectively. D) GFP⁻ (pst) and E) GFP⁺ (psp) fractions. F) adgf expression in FACS sorted samples was quantified by qRT-PCR. Level of significance is indicated as **p<0.01 (n=3).

adgf acts downstream of the histidine kinase dhkD

A) dhkD mutants on KK2 agar plates. B) 5 U, 10 U ADA rescued the mound arrest phenotype in a dose dependent manner. Scale bar: 1 mm; (n=3). Images were taken 3.5 h post treatment. C) Addition of 20 U ADA led to formation of multiple tips. Scale bar: 2 mm; (n=3).

Model illustrating the role of adgf in development

adgf suppresses the expression of genes involved in cell adhesion, cadA and csaA, and regulates the mound size and tip development by directly acting on adenosine, ammonia levels and cAMP signaling. Line ending in an arrow indicates 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.