Hayashida, Y.; Gomibuchi, Y.; Oosawa, C.; Yasunaga, T.; Morimoto, Y. V.

Intracellular signaling dynamics are often obscured by the spatial and temporal limitations of cell size. Here, we developed a method to enlarge Dictyostelium discoideum cells by partial cytokinesis inhibition, generating multinucleated yet functional giant cells. These cells retained chemotactic signaling, polarity, and motility, enabling high-resolution live-cell imaging. Using fluorescent probes for cAMP and Ca2+, we uncovered a directional, front-to-rear propagation of cAMP signaling and a biphasic Ca2+ response coordinated with actin wave dynamics. Grid-based mapping revealed asymmetric cAMP synthesis and decay kinetics, and vesicle localization suggested spatially regulated cAMP secretion. Combining giant cells with super-resolution or electron microscopy allowed detailed visualization of intracellular local structures at high resolution. Our findings demonstrate that intracellular signaling involves self-organized, spatially structured propagation events aligned with cellular polarity. The giant cell platform offers a powerful and generalizable strategy for dissecting the spatiotemporal logic of single-cell signaling.