Regulation of dynamic cyclic nucleotide signalling in social amoebas

Over the past years, much information has been gathered about the importance of the individual key components that control cAMP signalling in eukaryotes and prokaryotes. In mammalians, several different classes of ACs produce cAMP and much is known about their mechanism of stimulation and function. In contrast, much less is known about the enzymes that control degradation, the cAMP-PDEs and their regulatory mechanism. Nonetheless, it has become evident that cAMP is degraded by many isoforms of PDEs and that PDEs play a crucial role in controlling dynamic cAMP signalling. Throughout my PhD I have been interested in several aspects of cAMP signalling in the model organism Dictyostelium discoideum with particular emphasis on the roles of the phosphodiesterases in dynamic cAMP signalling. PdsA is the most critical enzyme in the control of aggregation and post-aggregative morphogenesis. It is controlled by three promoters; the vegetative, aggregative and the late promoter, which direct expression during growth, aggregation and late development, respectively. Despite its known crucial role in multicellular development not much is known about the regulation of PdsA or its expression after aggregation. In chapter 2, I present studies on the patterns of the aggregative and late promoter activity throughout development, the signals that regulate promoter activity and the promoter sequences that mediate regulation by these signals. D. discoideum and related species use extracellular cAMP as chemoattractant for aggregation, but most other species use other chemoattractants than cAMP. In chapter 3, I identified orthologues of the PdsA gene throughout the Dictyostelid phylogeny and studied their expression during development. Additionally, I show that one of them encodes a fully functional PDE. Cyclic AMP waves propagate throughout the Dictyostelium slug. This is somewhat enigmatic since ACA, the enzyme considered to be responsible for pulsatile cAMP signalling, is only expressed at the slug anterior. ACG is expressed at the posterior region of the slug, where it was shown to be responsible for induction of prespore differentiation. In chapter 4, I describe experiments showing that similar to ACA, ACG is transiently activated by stimulation of cAMP receptors. This could explain why cAMP waves can propagate in slug posteriors. In Dictyostelium, cGMP is associated with chemotaxis and plays a major role in myosin II regulation during cytoskeletal reorganisation. In the chapter 5, I describe experiments showing that modulation of the expression of the PdeD gene, a cGMPstimulated cGMP-PDE, has a pronounced effect on the expression of early genes, necessitating re-evaluation of the role of cGMP in Dictyostelium chemotaxis