Abstract
Phagocytosis and chemotaxis are receptor-mediated processes that require extensive rearrangements of the actin cytoskeleton, and are controlled by lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. We used a panel of pleckstrin homology (PH) domains with distinct binding specificities for PtdIns(3,4,5)P3 and PtdIns(3,4)P2 to study the spatiotemporal dynamics of these phosphoinositides in vivo. During phagocytosis and macropinocytosis PtdIns(3,4,5)P3 levels transiently increased at sites of engulfment, followed by a rapid PtdIns(3,4)P2 production round the phagosome/macropinosome upon its internalisation, suggesting that PtdIns(3,4,5)P3 is degraded to PtdIns(3,4)P2. PTEN null mutants, which are defective in phagocytosis, showed normal rates of PtdIns(3,4,5)P3 degradation, but unexpectedly an accelerated PtdIns(3,4)P2 degradation. During chemotaxis to cAMP only PtdIns(3,4,5)P3 was formed in the plasma membrane, and no PtdIns(3,4)P2 was detectable, showing that all PtdIns(3,4,5)P3 was degraded by PTEN to PtdIns(4,5)P2. Furthermore, we showed that different PtdIns(3,4,5)P3 binding PH domains gave distinct spatial and temporal readouts of the same underlying PtdIns(3,4,5)P3 signal, enabling distinct biological responses to one signal.
Highlights
In recent years phosphorylated inositol lipids have emerged as important second messenger molecules
We used the PtdIns(4)P-specific pleckstrin homology (PH) domain of FAPP1 to detect possible phosphoinositide intermediates in the generation and/or degradation especially of PtdIns(3,4)P2 (Dowler et al, 2000). We studied both macropinocytosis, which has previously been shown to require PI3-kinase activity, and phagocytosis during the vegetative stage of the Dictyostelium life cycle when cells are growing and dividing, as well as cells during the different developmental stages when they become responsive to the chemoattractant cAMP and aggregate to form multicellular structures (Weijer, 1999)
Previous studies in Dictyostelium suggested that PI3-kinase signalling is only required for macropinocytosis, not phagocytosis (Cardelli, 2001)
Summary
In recent years phosphorylated inositol lipids (phosphoinositides) have emerged as important second messenger molecules. Multiple isoforms of phosphoinositide 3-kinases (PI3kinases) phosphorylate phosphatidylinositol (PtdIns), phosphatidylinositol (4)-phosphate [PtdIns(4)P] and phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] at the 3-OH position of the inositol ring, generating PtdIns(3)P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3, respectively (Vanhaesebroeck et al, 2001). These 3-phosphoinositides, in turn, are recognised by a variety of lipid binding domains including PH, PX and FYFE domains (Lemmon, 2003), enabling the recruitment of specific proteins to the membrane sites where these lipids were generated and regulating downstream signalling events.
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