Imaging and quantitative analysis of phosphoinositide 3-kinase signaling in living fibroblasts

During wound healing, fibroblasts in connective tissue are directed to migrate to sites of clotting by gradients of platelet-derived growth factor (PDGF). Mammalian cells detect chemical gradients by spatial sensing, in which the cell can differentiate signaling through receptors at its front and rear. The phosphoinositide 3-kinase enzyme generates specific phospholipid second messenger products, 3´ phosphoinositides (3´ PIs), in the cell membrane, a signal transduction event required for directed migration. Significantly, this activity is polarized by gradients of PDGF and other chemoattractants, indicating a role in spatial sensing. The pattern of 3´ PIs in the plasma membrane is governed by generation, lateral diffusion, and turnover. Using a realtime fluorescence imaging technique in conjunction with a generalized reaction-diffusion model, we have devised a methodology for the accurate determination of 3´ PI generation rate, diffusion coefficient, and effective turnover rate constant in individual cells under uniform stimulation conditions. We report the dependence of these parameters on PDGF concentration, relationships that allow us to evaluate current models of the spatial sensing process. In concert with future experiments analyzing cells exposed to PDGF gradients, these relationships are expected to yield an integrated, quantitative model of directed migration at the level of intracellular processes.