In vivo analysis of 3-phosphoinositide dynamics during Dictyostelium phagocytosis and chemotaxis.
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.
Dormann
Dirk
D
Division of Cell and Developmental Biology, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK.
Weijer
Gerti
G
Dowler
Simon
S
Weijer
Cornelis J
CJ
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002633:Chemotaxis; D000242:Cyclic AMP / Q000378:metabolism; D004023:Dictyostelium / Q000502:physiology*; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D050356:Lipid Metabolism; D051059:PTEN Phosphohydrolase; D010587:Phagocytosis; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010873:Pinocytosis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C060980:phosphatidylinositol 3,4-diphosphate; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase