Cell spreading and migration involve surface attachment and cytoskeletal reorganization, leading to membrane extension and ultimately cell polarization, processes that are stimulated and coordinated through intracellular signal transduction pathways. In fibroblasts and many other cells, activation of the phosphoinositide 3-kinase (PI3K) pathway is required for motility. PI3Ks generate specific 3' PI lipid products, which act as membrane second messengers, and the spatial pattern of 3' PI density in the membrane is thought to control the directionality of membrane protrusion and cell migration. Using total internal reflection fluorescence microscopy in conjunction with a fluorescent 3' PI probe, we have observed spontaneous activation of PI3K signaling during spreading of mouse fibroblasts that is independent of integrin-mediated adhesion (fibronectin vs. poly-lysine) and is frequently localized to areas of active, membrane protrusion. Inhibition of PI3K blocks active cell spreading, whereas inhibition of actin polymerization halts spreading but not PI3K signaling. Inhibition of microtubule polymerization gives rise to a dynamic motility phenotype that is also sensitive to PI3K inhibition. Inhibiting protein kinase C (PKC), isoforms of which are known mediators of PI3K signaling, prevented cell spreading and, surprisingly, lead to a significant decrease in PI3K activity that could not be rescued by stimulation with platelet-derived growth factor. The kinetics of these effects suggests roles of PKC both upstream and downstream of PI3K. Taken together, we conclude that PI3K and PKC are spontaneously and dynamically activated during cell spreading, in a manner that it is independent from known receptor-mediated stimulation or feedback from F-actin/microtubules, and PI3K/PKC signaling is required for actin-based protrusion.

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