Adenylate cyclase A acting on PKA mediates induction of stalk formation by cyclic diguanylate at the Dictyostelium organizer

Abstract

Coordination of cell movement with cell differentiation is a major feat of embryonic development. The Dictyostelium stalk always forms at the organizing tip, by a mechanism that is not understood. We previously reported that cyclic diguanylate (c-di-GMP), synthesized by diguanylate cyclase A (DgcA), induces stalk formation. Here we used transcriptional profiling of dgca− structures to identify target genes for c-di-GMP, and used these genes to investigate the c-di-GMP signal transduction pathway. We found that knockdown of cAMP-dependent protein kinase (PKA) activity in prestalk cells reduced stalk gene induction by c-di-GMP, whereas PKA activation bypassed the c-di-GMP requirement for stalk gene expression. c-di-GMP caused a persistent increase in cAMP, which still occurred in mutants lacking the adenylate cyclases ACG or ACR, or the cAMP phosphodiesterase RegA. However, both inhibition of adenylate cyclase A (ACA) with SQ22536 and incubation of a temperature-sensitive ACA mutant at the restrictive temperature prevented c-di-GMP–induced cAMP synthesis as well as c-di-GMP–induced stalk gene transcription. ACA produces the cAMP pulses that coordinate Dictyostelium morphogenetic cell movement and is highly expressed at the organizing tip. The stalk-less dgca− mutant regained its stalk by expression of a light-activated adenylate cyclase from the ACA promoter and exposure to light, indicating that cAMP is also the intermediate for c-di-GMP in vivo. Our data show that the more widely expressed DgcA activates tip-expressed ACA, which then acts on PKA to induce stalk genes. These results explain why stalk formation in Dictyostelia always initiates at the site of the morphogenetic organizer.

Cell-type specific RNA-Seq reveals novel roles and regulatory programs for terminally differentiated Dictyostelium cells

“…8a , b ), but not in the basal disc. Expression of these genes occurs during and after maturation of the spores, and is similar to the expression pattern of the two cup genes DDB_G0276063 ( beiA ) and DDB_G0278537 (beiB) [ 14 ] that instigated the current study. The third cup gene tgrR1, encoding a transmembrane protein, was expressed in cells scattered in mounds and throughout slugs with more pronounced expression at the slug anterior and rear-guard.…”

“…The earlier finding that many cup genes are only expressed late in the spore head [ 14 ] and that a number of prespore genes are highly expressed in cup cells (Fig. 6b ) suggests that prespore cells transdifferentiate into cup cells during late culmination and that late cup cells are a different population from cup cells derived from ALCs.…”

“…A recent RNA-Seq study of a null mutant in diguanylate cyclase, which synthesizes the stalk inducing factor c-di-GMP, identified several cup-specific genes [ 14 ], which were first expressed very late in fruiting body formation, when the spore mass has already ascended the stalk. This suggested that the cup cells also have other roles in the fruiting body and that the late cup cells are distinct from the early ALC or pstB derived cup cells, which were found to end up mostly in the stalk [ 39 ] and basal disc [ 40 ].…”

“…A recent study comparing the transcriptomes of wild-type cells and null mutants in diguanylate cyclase, which synthesizes the stalk-inducing factor c-di-GMP [ 13 ] revealed both novel stalk genes and a number of genes which are expressed exclusively in the cup cells [ 14 ]. Unlike previously recognized cup-expressed genes, most of these genes are not expressed earlier in anterior-like cells, but only very late when spores are maturing in the elevated spore head.…”

Evolution of developmental signalling in Dictyostelid social amoebas

“…c-di-GMP strongly activates AcaA, which in slugs is predominantly expressed at the utmost tip to coordinate morphogenetic cell movement. Increased intracellular cAMP then acts on PKA to activate stalk gene expression [25]. …”