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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 Ca 2+ , we uncovered a directional, front-to-rear propagation of cAMP signaling and a biphasic Ca 2+ response coordinated with actin wave dynamics. Grid-based mapping revealed asymmetric cAMP synthesis and decay kinetics, and vesicle localization suggested spatially regulated cAMP secretion. 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. A method to enlarge living Dictyostelium cells by selectively inhibiting cytokinesis while preserving cellular functions enables direct visualization of directional cAMP and Ca 2+ signal propagation in single cells.

The bacterial signaling molecule cyclic diguanosine monophosphate (c-di-GMP) is only synthesized and utilized by the cellular slime mold Dictyostelium discoideum among eukaryotes. Dictyostelium cells undergo a transition from a unicellular to a multicellular state, ultimately forming a stalk and spores. While Dictyostelium is known to employ c-di-GMP to induce differentiation into stalk cells, there have been no reports of direct observation of c-di-GMP using fluorescent probes. In this study, we used a fluorescent probe used in bacteria to visualize its localization within Dictyostelium multicellular bodies. Cytosolic c-di-GMP concentrations were significantly higher at the tip of the multicellular body during stalk formation.

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.