Adenylate cyclase A amplification and functional diversification during Polyspondylium pallidum development

Area of Science:

• Cellular and Molecular Biology
• Developmental Biology
• Biochemistry

Background:

• Dictyostelium discoideum (Ddis) uses adenylate cyclase A (ACA) for cAMP oscillations coordinating aggregation and morphogenesis.
• Unlike Ddis, other species like Polyspondylium pallidum (Ppal) may not rely on extracellular cAMP for aggregation, but cAMP receptors (cARs) and phosphodiesterase (PdsA) are essential for fruiting body formation.
• Ppal's acaA gene has duplicated, prompting investigation into the roles of these three Ppal ACA genes and Ppal's capacity for transient cAMP-induced cAMP accumulation.

Purpose of the Study:

• To investigate the specific roles of the three Ppal ACA genes (aca1, aca2, aca3) in Ppal development.
• To determine if Ppal exhibits transient cAMP-induced cAMP accumulation, a key feature of oscillatory cAMP signaling.
• To understand the evolutionary conservation and divergence of cAMP signaling pathways in cellular slime molds.

Main Methods:

• Gene deletion experiments were performed to create single and multiple Ppal aca mutants (aca1-, aca2-, aca3-, aca1-aca2-, aca1-aca3-, aca2-aca3-, aca1-aca3-aca2-).
• Phenotypic analysis of mutants included observations of aggregation, stalk formation, and fruiting body development.
• Cells were stimulated with 2'H-cAMP (a cAR agonist) to assess cAMP production, and aggregation was rescued using 8Br-cAMP (a PKA activator).

Main Results:

• Ppal cells acquired the ability to produce a cAMP pulse upon stimulation only after aggregation, unlike Ddis.
• Single aca gene deletions resulted in distinct defects: aca1- (thin stalks), aca2- (delayed secondary sorogen formation), and aca3- (fewer aggregation centers).
• Multiple aca mutants exhibited combined defects and significant delays in aggregation; aggregation could be rescued by 8Br-cAMP, indicating PKA activation is critical. Ddis aca-/ACG cells also produced cAMP pulses, suggesting ACA is not solely responsible for oscillations.

Conclusions:

• The three Ppal ACA genes have specialized functions in stalk morphogenesis, secondary branch formation, and aggregation, collectively enabling development via PKA activation.
• Even in the absence of ACA-induced oscillations, Ppal can form fruiting bodies, suggesting alternative signaling mechanisms.
• Adenylate cyclase G (ACG) also mediates transient cAMP-induced cAMP accumulation, supporting the model that cAMP oscillations, potentially dominated by cAR-regulated cAMP hydrolysis, organize post-aggregative morphogenesis in Ppal.