There is a dearth of approaches to experimentally direct cell migration by continuously varying signal input to a single cell, evoking all possible migratory responses and quantitatively monitoring the cellular and molecular response dynamics. Here we used a visual blue opsin to recruit the endogenous G-protein network that mediates immune cell migration. Specific optical inputs to this optical trigger of signaling helped steer migration in all possible directions with precision. Spectrally selective imaging was used to monitor cellwide phosphatidylinositol (3,4,5)-triphosphate (PIP3), cytoskeletal, and cellular dynamics. A switch-like PIP3 increase at the cell front and a decrease at the back were identified, underlying the decisive migratory response. Migration was initiated at the rapidly increasing switch stage of PIP3 dynamics. This result explains how a migratory cell filters background fluctuations in the intensity of an extracellular signal but responds by initiating directionally sensitive migration to a persistent signal gradient across the cell. A twocompartment computational model incorporating a localized activator that is antagonistic to a diffusible inhibitor was able to simulate the switch-like PIP3 response. It was also able simulate the slow dissipation of PIP3 on signal termination. The ability to independently apply similar signaling inputs to single cells detected two cell populations with distinct thresholds for migration initiation. Overall the optical approach here can be applied to understand G-proteincoupled receptor network control of other cell behaviors.optogenetics | ultrasensitivity A variety of cells sense gradients of chemoattractants and respond by migrating toward increasing concentrations. Migration is central to immune cell function, morphogenesis, cancer cell metastasis, and the life cycle of the social amoeba, Dictyostelium discoideum (1). Cell migration is made up of a characteristic sequence of identifiable cellular events that are governed by G-protein-coupled receptor (GPCR)-driven signaling networks. Although considerable information exists about molecules involved in migration, the challenge is in translating a static map of these molecules into a spatially and temporally dynamic network that orchestrates migratory behavior. Effective methods to probe the basis of network control of migration need to be able to faithfully evoke migratory behavior experimentally and quantitatively monitor response dynamics at the cellular and molecular level. Microfluidic devices and electrical fields have been used to regulate migration and provide insights into the process (2-6). However, there are limitations at present in the ability to direct a series of signaling inputs to a single cell in spatially and temporally complex patterns. Such inputs are essential to continually choreograph the events that constitute the migratory response: initiation, translocation, directional changes, and adaptation. A light-sensitive domain of a plant protein has been inserted into Rac1, a downstrea...