Chemoattractant-Induced Intracellular Calcium Responses Using a Calcium Biosensor GCaMP3 in Dictyostelium discoideum

Calcium (Ca²⁺) is a critical second messenger in eukaryotic chemotaxis and development, yet the molecular coordination of its spatiotemporal dynamics across different chemoattractant systems remains incompletely understood. Using the genetically encoded calcium indicator GCaMP3, we characterized real-time intracellular calcium ([Ca²⁺]i) dynamics in Dictyostelium discoideum under two developmental stages. Both cAMP-stimulated developed cells and folate-stimulated vegetative cells exhibited a rapid, transient [Ca²⁺]i elevation peaking at approximately 12 sec, strictly dependent on extracellular Ca²⁺ influx. Genetic and pharmacological analyses using signaling mutants (Gβ-, GCA/sGC-, IplA-, PlaA-null) revealed a universal requirement for IplA and for SERCA-dependent ER calcium sequestration, consistent with a store-operated calcium entry (SOCE)-like mechanism. Strikingly, folate-stimulated vegetative Gβ-null cells displayed an accelerated [Ca²⁺]i transient peaking at 8-12 sec, suggesting a G-protein-independent Ca²⁺ entry mechanism. cGMP and calmodulin act as downstream kinetic modulators rather than upstream gating factors. Together, these findings advance our understanding of the molecular architecture of chemoattractant-induced calcium signaling in Dictyostelium.