In vivo analysis of 3-phosphoinositide dynamics during Dictyostelium in phagocytosis and chemotaxis

Phagocytosis and chemotaxis are receptor-mediated processes that require extensive rearrangements of the actin cytoskeleton, and are conetrolled by lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P-3] and phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P-2]. We used a panel of pleckstrin homology (PH) domains with distinct binding specificities for PtdIns(3,4,5)P-3 and PtdIns(3,4)P-2 to study the spatiotemporal dynamics of these phosphoinositides in vivo. During phagocytosis and macropinocytosis PtdIns(3,4,5)P-3 levels transiently increased at sites of engulfment, followed by a rapid PtdIns(3,4)P-2 production round the phagosome/macrophiosome upon its internalisation, suggesting that PtdIns(3,4,5)P-3 is degraded to PtdIns(3,4)P-2. PTEN null mutants, which are defective in phagocytosis, showed normal rates of PtdIns(3,4,5)P-3 degradation, but unexpectedly an accelerated PtdIns(3,4)P-2 degradation. During chemotaxis to cAMP only PtdIns(3,4,5)P-3 was formed in the plasma membrane, and no PtdIns(3,4)P-2 was detectable, showing that all PtdIns(3,4,5)P-3 was degraded by PTEN to PtdIns(4,5)P-2. Furthermore, we showed that different PtdIns(3,4,5)P-3 binding PH domains gave distinct spatial and temporal readouts of the same underlying PtdIns(3,4,5)P-3 signal, enabling distinct biological responses to one signal.