Atypical PI3Ks coordinate chemotaxis, signaling dynamics, and multicellular development in Dictyostelium.

Phosphoinositide 3-kinase (PI3K) signaling regulates protrusion, polarity, membrane uptake, and multicellular development in Dictyostelium discoideum, but these functions have been interpreted largely through canonical Class I PI3Ks and PI(3,4,5)P₃ production. This framework does not fully explain how PI3K-dependent pathways restrain Ras activity, organize relay signaling, or coordinate the transition from single-cell migration to multicellular aggregation. Here, we show that three atypical PI3K-family enzymes, PikF, PikG, and PikH, define genetically separable functions within this broader PI3K signaling network. PikF attenuates Ras-phosphoinositide-actin signaling, limiting protrusive activity so that chemotactic responses remain spatially and temporally constrained. PikG is required for aggregation and supports ACA-dependent cAMP relay, coupling cellular polarity to the collective signaling needed for streaming and multicellular development. PikH separates uptake from these chemotactic and developmental functions by supporting efficient phagocytosis with little effect on acute cAMP-stimulated signaling. Together, these findings expand the Dictyostelium PI3K framework beyond a Class I PI(3,4,5)P₃-centered pathway and identify atypical PI3Ks as specialized regulators of signal attenuation, cAMP relay organization, and membrane uptake.