Inferring feedback regulation from static snapshots of a single signal.

Direct measurement of feedback regulation remains difficult because it usually relies on pathway perturbations and prior knowledge of circuit topology. We introduce AoF (Assay of Feedback), a single-cell imaging framework that combines fixed-cell measurements of a target (together with DNA and Geminin as cell-cycle ordering markers) with ergodic rate analysis to reconstruct target dynamics in steady-state proliferating populations and quantify how a target's inferred rate of change depends on its own level. AoF therefore estimates feedback sign, magnitude, and state dependence from snapshot data without resolving surrounding circuitry. As a proof of concept, we applied AoF to map Akt phosphorylation across the cell cycle, unmasking a highly localized negative feedback loop restricted to the G1/S transition. Biochemical experiments point to an mTORC1/S6K1-dependent inhibitory phosphorylation of IRS1, which would stabilize Akt activation during early S phase, as the likeliest source of this snapshot-derived signature, but they do not establish that edge uniquely. AoF provides a rapid, scalable, and perturbation-free methodology to map context-specific feedback control.