Dictyostelium (Ax2) cells were grown axenically and development was performed as described (Gosain et al., 2012). Log phase cultures were inoculated into fresh media at a density of 5 × 10⁵ cells/mL and cell proliferation was monitored over 5 days using hemocytometer. For development, log phase cultures were harvested, washed and developed at a density of 5 × 10⁷ cells/mL, followed by synchronization at 4°C for 4–6 h and further incubated at 22°C to proceed in development. The timings and pattern of development was noted and photographed under Nikon SMZ-1500 stereomicroscope. Analyses were performed using NIS-ELEMENTS AR v. 4.0.

Tctp overexpressing (tctp^OE) strain was made as described earlier (Kumar et al., 2017). The knockout of tctp gene was created by disrupting the tctp gene with BSR cassette, followed by homologous recombination in the genome of D. discoideum. The 5’ and 3’ homology regions were PCR amplified using gene-specific primer pairs (Supplementary Tables S1, S2) and cloned at the flanking sides of BSR cassette, followed by linearization of the construct (Supplementary Figure S1). The linearized product (5’ homology region + BSR cassette + 3’ homology region) was transformed into Ax2 cells by electroporation and transformants were selected at 10 μg/mL Blasticidin-S (Invitrogen).

RNA isolation and cDNA synthesis were performed as described (Gosain et al., 2012). Briefly, Dictyostelium cells were developed and samples were collected at various time points, washed and lysed in TRI reagent (Sigma-Aldrich, United States). cDNA was synthesized and PCR amplification was performed. The gene-specific primer pairs used for RT-PCR reactions were mentioned in Supplementary Table S2.

Pinocytosis assay was performed as described (Maurya et al., 2020). Briefly, log phase cultures of Ax2 and tctp^– cells were harvested, re-suspended in fresh media at a density of 3 × 10⁶ cells/mL and kept under shaken conditions for 20 min. Furthermore, FITC dextran (2 mg/mL; Mr = 70 kDa; Sigma-Aldrich) was added and kept in dark under shaken conditions. 1 mL cell suspension was taken at different time points, washed with 1xKK₂ + 5 mM sodium azide, and further by 1xKK₂. Cells were lysed and fluorescence was measured at excitation 470 nm and emission at 520 nm. Samples were normalized with protein content to exclude any difference due to cell size.

Cell dry weight measurement was done according to Maurya et al. (2017). Log phase cultures were harvested, washed and pellet having 5 × 10⁷ cells were vacuum dried at 55°C for 1 h and cell weight was scored by subtracting empty tube weight.

Cell size analyses were performed as described (Maurya et al., 2017). Briefly, approximately 1 × 10⁷ cells were harvested, washed with 1xKK₂ buffer, resuspended in 1.5 mL buffer (0.9% NaCl, 2% sucrose, 5 mM EDTA in KK₂ buffer) followed by fixation. Just prior to analysis 1 × 10⁶ cells were washed, treated with RNaseA and incubated at 37°C for 30 min followed by addition of propidium iodide (Sigma-Aldrich, United States) and incubated at RT. Sample was loaded on BD FACS Calibur flow cytometer and cell size analysis was performed using Cell Quest software.

Spore viability assay was performed according to the procedure as described (Myre et al., 2011). Approximately, 1 × 10⁸ cells were harvested, washed and developed on 1.5% non-nutrient agar. Spores from mature fruiting bodies were collected, washed and aliquots of 100 spores were mixed with the suspension of Klebsiella aerogenes (bacteria) and allowed to grow for 5 days under moist conditions.

Cells were developed at a density of 5 × 10⁷ cells/mL on nitrocellulose membrane, already placed on agar till the formation of fruiting bodies, followed by treatment with calcofluor white (Sigma) at 0.01% wt/vol for 10 min. The staining solution was removed and images were captured using fluorescence microscope (Nikon Eclipse TiE).

RFP marked Ax2 and GFP marked tctp^– cells were mixed in various proportions (10–75%) and developed as described above. The development of chimeras was monitored under fluorescence microscope (Nikon SMZ-1500) and photographed.

For scoring contribution of Ax2 and tctp^– cells in chimeric spore formation, individual spore-heads were picked on a glass-slide and photographed, both under brightfield and fluorescence using Nikon Eclipse 80i microscope. Red and green fluorescent spores were counted and graph was plotted. A minimum of 10–15 fruiting bodies developed from each ratio mixture per individual experiment were counted.

β-galactosidase staining was performed according to the method described by Escalante and Sastre (2006) with minor modifications (Mishra et al., 2017). Log phase cells were developed at a cell density of 5 × 10⁷ cells/mL on dialysis membrane placed on non-nutrient agar plates. Various developmental structures were collected, fixed with methanol for 30 min followed by washing and permeabilization with NP40 (0.1% in Z buffer) for 1 h at room temperature. Further, structures were washed 2–3 times and staining with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) was performed. The reaction was stopped when evident staining appeared by washing with Z buffer. Images were captured under the Nikon AZ100 microscope.

Image processing and mound size analyses were performed using NIS ELEMENTS ARv.4.0. The statistical analyses were performed with mean, standard deviation, and standard error and values were plotted in graph using GraphPad Prism (GraphPad Software, CA, United States, www.graphpad.com). Student t-test was used where p-values; ^∗∗∗p < 0.001, ^∗∗p < 0.01 and ^∗p < 0.05 were established as significant.

To investigate the similarity between DdTCTP and TCTPs from other species, we performed multiple sequence alignment of protein sequences through MUSCLE program (Edgar, 2004). The protein sequence of DdTCTP is ∼171 amino acids (source from dictybase server; Fey et al., 2009). Amino acid sequence alignment of TCTP from social amoeba (D. discoideum) and other species (including Arabidopsis thaliana, Drosophila melanogaster, Schizosaccharomyces pombe, Mus musculus, and Homo sapiens) was performed (Figure 1A). We aligned the TCTP protein sequences from seven species that cover lower to higher animals, plant and fungi. Conserved residues showed identity between social amoeba, plants, fungi and lower as well as higher animals. A constructed phylogenetic tree suggested that DdTCTP was closely related to the other members of dictyostelids and protozoa such as Theileria annulata, Dictyostelium fasciculatum, and Plasmodium falciparum with high bootstrap values (Figure 1B). DdTCTP was also closer to fungi (Saccharomyces cerevisiae, Schizosaccharomyces japonicas, and Schizosacchromyces pombe). The homology between DdTCTP and S. pombe was 46% while with human it showed 42%.

The expression level of tctp mRNA during growth and development of D. discoideum was checked using semi-quantitative RT-PCR (Figure 1C). tctp mRNA was expressed throughout development, predominantly expressed at initial time points (0–6 h) then showed reduced expression between 8 and 20 h followed by increased expression till 24 h.

To explore the functions of TCTP, its deletion mutant was generated by disrupting the tctp gene with BSR cassette through homologous recombination method in the genome of D. discoideum (Figure 2A and Supplementary Figure S1). Approximately, 400 independent clones were screened for positional integration, confirmed by PCR and one out of the two positive clones was used for further experiments (Figures 2B,C).

For measuring cell proliferation, log phase cultures were identically diluted into fresh HL5 media and observed over 5 days (Figure 3A). Wild type and tctp^OE cells divides at every 12.3 ± 0.2 and 18 ± 0.6 h and attained stationary phase at ∼10.8 × 10⁶ cells/mL and ∼7.2 × 10⁶ cell/mL, respectively, while cell proliferation of tctp^– cells was very slow with a longer lag phase and reached the stationary phase and at a much lower density of 4.7 × 10⁶ cell/mL (Figure 3A). The doubling time for tctp^– cells was 26 ± 2.5 h. Growth of Dictyostelium was accomplished with either increase in cell proliferation or increase in size of the cells. Cell growth and proliferation are regulated independently in the organism. Cell proliferation rate is increase in number while cell growth is increase in size of cell due to the increased molecular mass in the cell. To determine the role of DdTCTP in regulating growth, we estimated the cell mass and observed that Ax2, tctp^OE, and tctp^– cells have an average cell mass of 9 ± 0.08, 8.1 ± 0.2, and 11.4 ± 0.5 mg, respectively (Figure 3B). Increased cell mass of tctp^– cells was attributed to the enhanced cell size revealed by FACS analysis (Figure 3C). The size of the tctp^– cells was also observed by microscopy and found significantly more than wild type cells (Figure 3D and Supplementary Figure S2). Furthermore, the reason behind increased cell size was checked via nuclear staining using DAPI and found that there was increase in number of nuclei per cell in case of tctp^– cells as compared to wild type cells (Figure 3E). Together, we conclude that tctp^– cells display reduced proliferation but enhanced cell growth due to increased number of nuclei. Next, we wanted to check the effect of TCTP knockout on pinocytosis (rate of liquid uptake; Figure 3F). Pinocytosis also known as macropinocytosis is the uptake of liquid media by axenic strain (Ax2) of Dictyostelium cells grown in laboratory. Pinocytosis is a crucial process in the growth of Dictyostelium Ax2 cells, as it is the only mode of taking the nutrients (Bloomfield and Kay, 2016). Vegetative stage of Ax2 cells grow and divide by actively pinocytosing the external nutrients. It can be measured through the rate of uptake of fluid phase marker, fluorescent dextran (Maurya et al., 2020). The pinocytosis rate of tctp^– cells was significantly reduced at all time points as measured by decrease in intracellular FITC content in the tctp^– cells (Figure 3D). Therefore, decreased proliferation could be attributed to defect in nutrient uptake as well.

To delineate the role of TCTP in multicellular developmental program of Dictyostelium, wild type, tctp^OE, and tctp^– cells were plated at a density of 5 × 10⁷ cells/mL on non-nutrient agar and allowed to develop at 22°C following synchronization (Figure 4). Ax2 cells completed its developmental life cycle by 24 h forming aggregation streams by 6 h, aggregates (mounds) by 12 h, slugs by 16 h, and fruiting bodies by 24 h whereas the formation of aggregates was delayed by 4 h in tctp^OE and tctp^– strains. The tctp^OE and tctp^– cells formed mound at 16 h, slugs at 20 h and fruiting body at 28 h (Figure 4). Additionally, the number of aggregates were significantly reduced in tctp^OE and tctp^– strains as compared to Ax2 (Figures 5A,B) but the size of aggregates was 3-fold more in case of tctp^OE whereas tctp^– showed comparatively smaller aggregates than Ax2. tctp^OE cells formed long and slender slugs as compared to wild type whereas tctp^– slugs was similar to wild type slugs. Then, we analyzed the fruiting body morphology of wild type, tctp^OE and tctp^– strains and found that tctp^OE cells formed fruiting body with long, thin stalk and small sorus while tctp^– cells developed into fruiting body with a small and thick stalk and a large sorus as compared to wild type fruiting bodies (Figure 5C). Moreover, in case of tctp^OE and tctp^– fruiting bodies stalk cells are arranged in a septate pattern as compared to the polyhedral manner in wild type cells (Figure 5D). Further, cellulose deposition in fruiting bodies of these strains were checked by calcofluor staining and found less cellulose deposition in the septum of tctp^– stalk (Figure 5D and Supplementary Figure S3A). The spores formed by tctp^OE cells were elliptical similar to Ax2 but tctp^– spores were round in shape (Figure 5D and Supplementary Figure S3B). cotA (spore COaT protein, SP96) and cotB (spore COaT protein, SP70) codes for major spore coat proteins, SP96 and SP70, respectively, that are necessary for the integrity of the spore coat (Srinivasan et al., 2000). Altered levels of any of these coat proteins are responsible for defect in shape and size of spores formed. Since the shape of tctp^– spore was largely altered, thus we checked the expression of spore coat genes cotA and cotB at the mRNA level by semi-quantitative RT-PCR in different stages of development (Figure 5F). We observed the expression of cotA remained unaffected, while the transcript of cotB was down regulated in tctp^– strain as compared to wild type (Figure 5F). Collectively, the above results suggest that TCTP is required for timely development and appropriate formation of aggregates and fruiting bodies.

Aberrant fruiting body formation by mutant cells led us to ask whether spores formed by mutant cells were viable or not therefore, spore viability assay was performed. Wild type and tctp^OE cells produced spores with average spore viability of 85.1 ± 1.7 and 65.3 ± 5.5%, respectively, whereas tctp^– cells produced 27.7 ± 1.2% viable spores (Figure 5E). Therefore, TCTP is essential for maintaining the viability of Dictyostelium spores.

Initial development of Dictyostelium is mainly regulated by proteins involved in aggregation such as acaA (adenyle cyclase aggregate A), carA (cAMP receptor A), cell adhesion molecules csA (contact site A), and cadA (Calcium-dependent ADhesion). acaA and carA are major components for cAMP signal relay. Since, we observed delay in initiation of streaming we checked the mRNA expression of acaA and carA in Ax2, tctp^OE, and tctp^– strains (Figures 5G,H). The transcript level of acaA remain unaffected while tctp^OE showed reduced expression at 8 h (Figure 5G). The mRNA expression of carA was significantly down regulated in tctp^OE at 8 h and tctp^– at 8, 12, and 16 h. Furthermore, we checked the mRNA expression of cell adhesion molecules csA and cadA that are required for aggregate formation in early development. cadA expression remained unchanged and csA level was significantly reduced in tctp^OE cells resulting in larger aggregate formation. The cadA mRNA was down regulated and csA expression was up regulated at 12 h contributing to decrease cell-cell adhesion and stream break up in tctp^– aggregates (Figure 5H). The above data suggest that reduced acaA and/or carA may be responsible for the delay in development and altered cadA and/or csA expression contribute to altered aggregates formation in tctp^OE and tctp^– strains.

Aberrant fruiting body formation by mutant cells incited us to study its role in cell type differentiation and patterning. The spatial distribution of prespore/prestalk cells in multicellular structures developed by wild type and tctp^– cells was analyzed using lacZ reporter fused to promoter of prestalk/prespore specific marker genes such as ecmAO, ecmB, and pspA (d19). The ecmAO/lacZ staining in wild type slugs was observed in the anterior region of slugs and in ALCs (anterior-like cells) of posterior region while in tctp^– mound it was found in anterior region but absent in ALCs of posterior region of slug. In tctp^– early culminant and culminant, ecmAO/lacZ staining was absent in the lower cup, basal disk, and the stalk tube (Figure 6A). In wild type, the ecmB/lacZ staining was observed in the central core region of prestalk cells of migrating slugs and distributed in the basal disk, upper and lower cups of fruiting bodies formed (Figure 6B). In tctp^– cells, ecmB/lacZ expression is precocious as found at mound stage when compared with the wild type. The ecmB/lacZ staining in tctp^– early and late culminant was found similar to wild type. The pspA/lacZ staining was found in the prespore/spore region of the multicellular structures developed but the pspA/lacZ staining region was reduced in tctp^– slugs, early and late culminant (Figure 6C). Thus, we suggest that TCTP plays an important role in the distribution of cell types in multicellular structures.

To trace the fate of tctp^– cells in a chimera, cell types mixing experiments were performed in which Ax2 (RFP-tagged) and tctp^– (GFP-tagged) mixed in varying ratios of 9:1, 3:1, 1:1, and 1:3 and allowed to co-develop (Figure 7 and Supplementary Figures S4–S6). Chimeras are the mixture of cells from two or more genetically different backgrounds. The differential presence of wild type and/or mutant cells in development and differentiation can be clearly observed by chimera study. Here, we wanted to observe the differential preference of mutant cells in presence of wild type cells. During early development, Ax2 and tctp^– cells contribute equally in formation of aggregation centers of various chimeras (1:9, 1:3, 1:1, and 3:1 of tctp^– and Ax2 cells; Supplementary Figure S4). As the development proceed further, in chimera of 10% tctp^– and 90% wild type cells, we observed that tctp^– cells occupied the prestalk/stalk region while the Ax2 cells occupied the prespore/spore region. During slug formation, tctp^– cells majorly occupied the anterior prestalk region, rear-guard and ALCs (Figure 7 and Supplementary Figure S5). At early culminant stage, tctp^– cells occupied the anterior prestalk region, ALCs, and basal disk. At culminant, it majorly contributed to anterior tip region, upper and lower stalk portion and basal disk (Figure 7 and Supplementary Figure S5). This trend was observed as the percentage of tctp^– cells increased further as it also occupied entire stalk and basal disk region. Next, the contribution of tctp^– cells in spore formation was assessed and it exhibited decreased contribution in the prespore/spore region as compared to wild type cells. The contribution of tctp^– cells in chimeras of varying proportion, i.e., 10, 25, 50, 75, and 90% of tctp^– cells with Ax2 cells resulted in chimeric spore formation in which tctp^– cells contributed only to 1.95 ± 0.35, 11 ± 1, 15 ± 1, 15.5 ± 0.5, and 49 ± 9% (Figure 8). Our results suggested that in chimeras with wild type cells, tctp^– cells differentiated into prestalk/stalk cells preferentially, further suggesting that TCTP is a crucial component controlling the equilibrium between prestalk and prespore cell-types.