3' phosphoinositide dynamics during phagosome formation and maturation in macrophages.
Kamen, Lynn A.
2007
Abstract
In macrophages, enzymes that synthesize or hydrolyze phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P<sub>3</sub>) regulate Fcgamma receptor-mediated phagocytosis. Inhibition of phosphatidylinositol 3-kinase (PI3K) or overexpression of the lipid phosphatases PTEN and SHIP-1, which hydrolyse PI(3,4,5)P<sub> 3</sub> to PI(4,5)P<sub>2</sub> and PI(3,4)P<sub>2</sub>, respectively, inhibit phagocytosis. To examine how these enzymes regulate phagosome formation, the distributions of YFP chimeras of enzymes and PH domains specific for their substrates and products were analyzed quantitatively. PTEN-YFP did not localize to phagosomes, suggesting that PTEN regulates phagocytosis globally within macrophages. SHIP1-YFP and p85-YFP, a regulatory subunit of PI3K, were recruited to forming phagosomes. SHIP1-YFP sequestered to the leading edge and dissociated from phagocytic cups earlier than did p85-CFP, indicating that SHIP-1 inhibitory activities are restricted to the early stages of phagocytosis. These results support a model in which phagosomal PI3K generates PI(3,4,5)P<sub>3</sub> necessary for later stages of phagocytosis, PTEN determines if those late stages can occur and SHIP-1 regulates when and where they occur by transiently suppressing PI(3,4,5)P<sub>3</sub>-dependent activities necessary for completion of phagocytosis. To examine how SHIP-1 activity contributes to phagocytosis, we manipulated SHIP-1 expression to measure its effects on phospholipids and membrane trafficking during FcR-mediated phagocytosis. SHIP-1<super>-/-</super> macrophages demonstrated differences in 3' phosphoinositide synthesis on phagosomal membranes. Surprisingly, we found that SHIP-1 deficiency decreased the early oxidative burst. Therefore, the early and transient association of SHIP-1 with forming phagosomes increased the early oxidative burst, perhaps through PI(3,4)P<sub>2</sub>-dependent enhancement of NADPH-oxidase activity. The dynamics of actin and phosphoinositides were examined on fully-formed <italic> Candida albicans</italic> phagosomes. Phagosomes that exhibited increased levels of actin also had increased levels of PI(3,4,5)P<sub>3</sub>, PI(3,4)P<sub> 2</sub>, PI(3)P and PI(4,5)P<sub>2</sub>. These membrane markers are usually associated with the early stages of FcR-mediated phagocytosis, so it is possible that <italic>C. albicans</italic> disrupted conventional phagosome maturation to evade the antimicrobial activities of the host macrophage. Overall, these studies indicate that progression through several stages of phagocytosis entails transitions in the species of phosphoinositides of the phagosomal membranes, and that these transitions are mediated by lipid kinases and phosphatases. As such, the phosphatase activities of SHIP-1 can be viewed as morphogenetic signals, rather than a simple inhibitory brake on the process.Subjects
Dynamics Macrophages Maturation Phagosome Formation Phagosomeformation Phosphoinositide
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