R Baskar

Thyroxine deiodinases, the enzymes that regulate thyroxine metabolism, are essential for vertebrate growth and development. In the genome of Dictyostelium discoideum, a single intronless gene (dio3) encoding type III thyroxine 5' deiodinase is present. The amino acid sequence of D. discoideum Dio3 shares 37% identity with human T4 deiodinase and is a member of the thioredoxin reductase superfamily. dio3 is expressed throughout growth and development and by generating a knockout of dio3, we have examined the role of thyroxine 5' deiodinase in D. discoideum. dio3- had multiple defects that affected growth, timing of development, aggregate size, cell streaming, and cell-type differentiation. A prominent phenotype of dio3- was the breaking of late aggregates into small signaling centers, each forming a fruiting body of its own. cAMP levels, its relay, photo- and chemo-taxis were also defective in dio3-. Quantitative RT-PCR analyses suggested that expression levels of genes encoding adenylyl cyclase A (acaA), cAMP-receptor A (carA) and cAMP-phosphodiesterases were reduced. There was a significant reduction in the expression of CadA and CsaA, which are involved in cell-cell adhesion. The dio3- slugs had prestalk identity, with pronounced prestalk marker ecmA expression. Thus, Dio3 seems to have roles in mediating cAMP synthesis/relay, cell-cell adhesion and slug patterning. The phenotype of dio3- suggests that Dio3 may prevent the formation of multiple signaling centers during D. discoideum development. This is the first report of a gene involved in thyroxine metabolism that is also involved in growth and development in a lower eukaryote.

A number of organisms possess several isoforms of protein kinase C but little is known about the significance of any specific isoform during embryogenesis and development. To address this we characterized a PKC ortholog (PkcA; DDB_G0288147) in Dictyostelium discoideum. pkcA expression switches from prestalk in mound to prespore in slug, indicating a dynamic expression pattern. Mutants lacking the catalytic domain of PkcA (pkcA-) did not exhibit tip dominance. A striking phenotype of pkcA[U+02C9] was the formation of an aggregate with a central hollow, and aggregates later fragmented to form small mounds, each becoming a fruiting body. Optical density wave patterns of cAMP in the late aggregates showed several cAMP wave generation centers. We attribute these defects in pkcA- to impaired cAMP signaling, altered cell motility and decreased expression of the cell adhesion molecules - CadA and CsaA. pkcA- slugs showed ectopic expression of ecmA in the prespore region. Further, the use of a PKC-specific inhibitor, GF109203X that inhibits the activity of catalytic domain phenocopied pkcA-.

Multicellular development in Dictyostelium discoideum involves tightly regulated signaling events controlling the entry into development, initiation of aggregation and chemotaxis, and cellular differentiation. Here we show that PkcA, a Dictyostelium discoideum Protein Kinase C-orthologue, is involved in quorum sensing and the initiation of development, as well as cAMP sensing during chemotaxis. Additionally, by epistasis analysis we provide evidence that PkcA and PldB (a Phospholipase D-orthologue) functionally interact to regulate aggregation, differentiation, and cell-cell adhesion during development. Finally, we show that PkcA acts as a positive regulator of intracellular PLD-activity during development. Taken together, our results suggest that PkcA act through PldB, by regulating PLD-activity, in order to control events during development.