Ca 2+ influx is an essential component of the positive-feedback loop that maintains leading-edge structure and activity in macrophages

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Abstract
In migrating eukaryotic cells, phosphatidylinositol 3-kinase (PI3K), filamentous actin (F-actin), and monomeric Rho GTPases are key components of a complex positive-feedback system that maintains and amplifies a phosphatidylinositol-3,4,5-trisphosphate signal at the leading edge of the cell. This lipid signal is required for cell polarization and movement. In leukocytes and Dictyostelium, activation or inhibition of any one of these components leads to the activation or inhibition, respectively, of the others via undefined feedback interactions. The role of Ca(2+) signals in migrating leukocytes is controversial, and there has been no indication that Ca(2+) participates in positive feedback. Here, we demonstrate that an extracellular Ca(2+) influx is required for positive feedback at the leading edge of spontaneously polarized macrophages. Inhibition of extracellular Ca(2+) influx leads to loss of leading-edge PI3K activity, disassembly of F-actin, cessation of ruffling, and decay of chemoattractant signals. Conversely, increasing cytosolic Ca(2+) enhances membrane ruffling, PI3K activity, and F-actin accumulation. Overall, these findings demonstrate that an extracellular Ca(2+) influx is an essential component, together with PI3K and F-actin, of the positive-feedback cycle that maintains leading-edge structure and ruffling activity and that supports the chemoattractant response. Strikingly, the Ca(2+)-sensitive enzyme protein kinase Calpha (PKCalpha) is enriched at the leading edge, and its enrichment is sensitive to blockade of Ca(2+) influx, to inhibition of PI3K activity, and to F-actin depolymerization. These findings support the working hypothesis that a local, leading-edge Ca(2+) signal recruits PKCalpha as a central player in the positive-feedback loop.