Ricci, M. M. C.; Patel, H.; Montoya, M. J.; Jayasuriya, K. L.; Rectenwald, A.; Motter, M. A. K.; Wills, R. C.; Hammond, G. R.

Class I phosphoinositide 3-kinases (PI3Ks) generate the lipid second messengers PIP3 and PI(3,4)P2 to control diverse cellular processes including growth, metabolism, and survival. Although these signals are classically thought to arise at the plasma membrane, several recent studies have proposed that PI3K signaling is propagated from intracellular membranes along the endocytic pathway. Here, we combined genomic tagging of endogenous PI3K pathway enzymes with single-molecule imaging and sensitive lipid biosensors to define the spatial organization of PI3K signaling in living cells. We find that PI3K catalytic subunits are recruited to the plasma membrane but do not undergo detectable endosomal translocation during receptor activation. Consistently, PIP3 and PI(3,4)P2 accumulation is restricted to the plasma membrane, despite enrichment of lipid phosphatases along the endocytic pathway. Functional perturbation experiments further show that degradation of PI(3,4)P2 occurs predominantly at the plasma membrane, indicating that both synthesis and termination of proximal lipid signals are spatially confined to this compartment. Together, these results resolve the subcellular localization of proximal PI3K signaling and support a model in which lipid second messenger production is restricted to the plasma membrane, with diversification of downstream pathway outputs occurring through redistribution of activated effector proteins rather than intracellular propagation of lipid signals.