Phosphoinositides (PtdInss) play key roles in cell polarization and motility. With a series of biosensors based on Fö rster resonance energy transfer, we examined the distribution and metabolism of PtdInss and diacylglycerol (DAG) in stochastically migrating Madin-Darby canine kidney (MDCK) cells. The concentrations of phosphatidylinositol (4,5)-bisphosphate, phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), phosphatidylinositol (3,4)-bisphosphate, and DAG were higher at the plasma membrane in the front of the cell than at the plasma membrane of the rear of the cell. The difference in the concentrations of PtdInss was estimated to be less than twofold between the front and rear of the migrating MDCK cells. To decode the spatial activities of PtdIns metabolic enzymes from the obtained concentration maps of PtdInss, we developed a one-dimensional reaction diffusion model of PtdIns metabolism. In this model, the activities of phosphatidylinositol monophosphate 5-kinase, phosphatidylinositol 3-kinase, phospholipase C, and PIP 3 5-phosphatases were higher at the plasma membrane of the front than at the plasma membrane of the rear of the cell. This result suggests that, although the difference in the steady-state level of PtdInss is less than twofold, PtdInss were more rapidly turned over at the front than the rear of the migrating MDCK cells. INTRODUCTIONCell migration is an important event during early development, inflammatory responses to infection, and wound healing, and it is an important pathological event during tumor invasion and metastasis. Despite morphological and functional differences, different migratory cells share a conserved set of polarity signals. Kinases for phosphoinositides (PtdInss), Rho GTPases, and the actin and microtubule cytoskeletons play key roles in signaling polarity in cells ranging from Dictyostelium discoideum (Charest and Firtel, 2006) to neurons (Luo, 2000;Aoki et al., 2007) and neutrophils (Van Keymeulen et al., 2006;Wong et al., 2006).PtdInss are a family of phospholipids containing myoinositol as their head group (reviewed in Takenawa and Itoh, 2001). Despite a relatively low abundance in biological membranes, PtdInss have been reported to regulate a myriad of cellular processes. Among them, phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P 2 ] is the major PtdInss at the inner leaflet of the plasma membrane. On growth factor stimulation, intracellular second messengers such as inositol (1,4,5)-trisphosphate (IP 3 ), diacylglycerol (DAG), and phosphatidylinositol (3,4,5)-triphosphate (PIP 3 ) are generated from PI(4,5)P 2 .The discovery that the pleckstrin homology (PH) domain in the signaling proteins recognizes specific phosphoinositides revealed that stimulated proteins translocate to specific regions of the membrane to participate in signaling events via interaction with these lipids (reviewed in Di Paolo and De Camilli, 2006). A series of experiments indicated that the PH domains from different proteins recognize different PtdInss and led to the development of a novel t...