Shweta Saran

cAMP production by adenylyl cyclase G induces prespore differentiation in Dictyostelium slugs

Encystation and sporulation are crucial developmental transitions for solitary and social amoebae, respectively. Whereas little is known of encystation, sporulation requires both extra- and intracellular cAMP. After aggregation of social amoebae, extracellular cAMP binding to surface receptors and intracellular cAMP binding to cAMP-dependent protein kinase (PKA) act together to induce prespore differentiation. Later, a second episode of PKA activation triggers spore maturation. Adenylyl cyclase B (ACB) produces cAMP for maturation, but the cAMP source for prespore induction is unknown. We show that adenylyl cyclase G (ACG) protein is upregulated in prespore tissue after aggregation. acg null mutants show reduced prespore differentiation, which becomes very severe when ACB is also deleted. ACB is normally expressed in prestalk cells, but is upregulated in the prespore region of acg null structures. These data show that ACG induces prespore differentiation in wild-type cells, with ACB capable of partially taking over this function in its absence.

Dissecting the Functional Role of Polyketide Synthases in Dictyostelium discoideum BIOSYNTHESIS OF THE DIFFERENTIATION REGULATING FACTOR 4-METHYL-5-PENTYLBENZENE-1,3-DIOL

Dictyostelium discoideum exhibits the largest repository of polyketide synthase (PKS) proteins of all known genomes. However, the functional relevance of these proteins in the biology of this organism remains largely obscure. On the basis of computational, biochemical, and gene expression studies, we propose that the multifunctional Dictyostelium PKS (DiPKS) protein DiPKS1 could be involved in the biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol (MPBD). Our cell-free reconstitution studies of a novel acyl carrier protein Type III PKS didomain from DiPKS1 revealed a crucial role of protein-protein interactions in determining the final biosynthetic product. Whereas the Type III PKS domain by itself primarily produces acyl pyrones, the presence of the interacting acyl carrier protein domain modulates the catalytic activity to produce the alkyl resorcinol scaffold of MPBD. Furthermore, we have characterized an O-methyltransferase (OMT12) from Dictyostelium with the capability to modify this resorcinol ring to synthesize a variant of MPBD. We propose that such a modification in vivo could in fact provide subtle variations in biological function and specificity. In addition, we have performed systematic computational analysis of 45 multidomain PKSs, which revealed several unique features in DiPKS proteins. Our studies provide a new perspective in understanding mechanisms by which metabolic diversity could be generated by combining existing functional scaffolds.

Dictyostelium discoideum Sir2D modulates cell-type specific gene expression and is involved in autophagy

Sirtuins (SIRTs) belong to class III histone deacetylases and require NAD+ for their activity. Their activity is associated with the nutritional status of the cell and they directly link cellular metabolic signalling to the state of protein post-translational modifications. Sirtuins play an important role in healthy aging, longevity and age-related diseases, as well as in cell survival mechanisms, such as autophagy. Here, we investigate the functions of Dictyostelium discoideum Sir2D which shows similarity to human SIRT1. This gene is expressed throughout growth and development. Overexpression of sir2D promotes cell proliferation and the corresponding fusion protein shows nuclear localization. To facilitate the study of the function of Sir2D, we created a sir2D knockout by gene disruption. This mutant exhibits inhibited cell proliferation and developmental defects, including smaller aggregates and multi-tipped structures. When developed as chimeras with wild-type cells, the sir2D- cells show a reduced ability to form spores. Prespore and prestalk differentiation was also impaired in the mutant strain. Sir2D regulates the expression of several autophagic genes (Atgs) and the sir2D deficient strain shows reduced autophagic flux. In conclusion, Sir2D plays a role in cell differentiation, modulates the expression of both prespore and prestalk genes and participates in the process of autophagy.

Overexpression of S-adenosylmethionine decarboxylase impacts polyamine homeostasis during development of Dictyostelium discoideum

The polyamines putrescine, spermidine and spermine are essential polycations involved in the regulation of cellular proliferation. They exert dynamic effects on nucleic acids and macromolecular synthesis in vitro but their specific functions in vivo are poorly understood. Here, we have modulated the spermidine levels either by overexpressing the S-adenosylmethionine decarboxylase (samdc) gene or treating the cells with methylglyoxal-bis (guanylhydrazone) (MGBG), an inhibitor of SAMDC. In Dictyostelium, overexpression of SAMDC slowed cell proliferation, delayed development and arrested cells in the S-phase of the cell cycle. Treatment with MGBG reduced cell proliferation and stimulated development, but in samdcOE cells, it increased cell proliferation suggesting critical levels of spermidine to be important. In samdcOE cells, spermidine levels remained high throughout development but only small changes in the spermine levels were observed. Initial putrescine levels did increase but reverted to wild-type levels after the mound stage. As tight regulation of polyamine homeostasis is required, we identified genes that could be involved in its maintenance. In conclusion, we characterised samdcOE cells and observed the maintenance of polyamine homeostasis during the development of Dictyostelium cells.

Deletion of etoposide-induced 2.4 kb transcript (ei24) reduced cell proliferation and aggregate-size in Dictyostelium discoideum

The etoposide-induced 2.4 kb transcript (ei24) gene is induced both by p53 and etoposide, an anti-cancer tumour drug. There is no p53 gene present in Dictyostelium discoideum. Thus, the functions of ei24 in the absence of p53 were analysed. Both overexpressor (ei24OE) and knockout (ei24-) mutants were made to study its role during growth, development and differentiation. Additionally, cell cycle and its response to DNA-damage were also analysed. We identified, characterized and elucidated the functions of the ei24 gene in Dictyostelium. In silico analyses demonstrated the conservation across eukaryotes and in situ hybridization showed it to be prestalk-specific. ei24- cells showed reduced cell proliferation and cell-cohesive properties, ultimately forming small-sized aggregates that developed into miniature and stalky fruiting bodies. The ei24OE cells formed fruiting bodies with engorged or double-decker type sori with short stalks. The ei24- cells showed reduced cAMP signalling with lower intracellular cAMP levels resulting in diminished migration of cells along cAMP gradients. Deletion of ei24 resulted in mis-expression of prestalk-specific markers. Cell cycle analysis revealed an increased bias towards the stalk-pathway by ei24- cells and vice-versa for ei24OE cells. EI24 in Dictyostelium functions even in the absence of p53 and is induced in response to both UV-radiation and etoposide treatments. ei24OE cells showed enhanced DNA-damage repair mechanisms. Also, etoposide treatment and overexpression of ei24 caused G2/M arrest in the cell cycle. Our results indicate that EI24 is important for the growth, development and differentiation of Dictyostelium apart from being a DNA-damage response gene.