Μελέτη του ρόλου των φωσφοϊνοσιτιδίων στο μηχανισμό της φαγοκύτωσης

Abstract

Phosphoinositides, the phosphorylated derivatives of PtdIns, are important regulators of eukaryotic cell functions including phagocytosis. In higher organisms, phagocytosis is attributed to specific cells, named professional phagocytes, to which macrophages and neutrophils belong. In lower organisms, as protozoa and slime molds, phagocytosis is a process utilized primarily for food uptake. Phosphoinositides contribute to the regulation of phagocytosis, as the product of PI3K I, PtdIns3,4,5P3, which is restricted precisely to the phagosomal cup, is essential for pseudopod extension. In addition, during the initial phase of phagocytosis, the levels of PtdIns4,5P2 are increased followed by its disappearence as the phagosome seals. Loss of PtdIns4,5P2 has been correlated with mobilization of PLCγ1 in macrophages, as a localized formation of DAG occurs. After phagosome formation, PtdIns3P accumulates onto the phagosomal membranes and participates in the regulation of maturation. The unicellular ciliate T.thermophila has been well characterized in terms of intracellular trafficking pathways, including phagocytosis. Ιn addition, studies from our group have shown that 3- and 4-phosphoinositides are present in Tetrahymena, where they serve as regulators of lysosomal enzyme secretion and PI-PLC-dependent cell signalling. In this thesis, we studied whether phosphoinositides are implicated in the regulation of phagocytosis in this organism. Phagocytosis was studied using two different types of particles, indian ink and iron-dextran particles, as well as fluorescent E.coli cells using flow cytometry analysis. Analysis of total T.thermophila phosphoinositides, using in vivo [3H]inositol labelling and TLC, showed that PtdInsP2 levels are increased 2-fold during the first minute of phagocytosis and then they gradually decreased over a 10min period; the rapid increase of PtdInsP2 is attributed to both PtdIn3,5P2 and PtdIns4,5P2, as revealed by HPLC analysis. In agreement with this result, in vitro assay of PI-PLC showed a 65% decrease of this activity within the first minute of phagocytosis. During the HPLC analysis, a putative PtdIns5P, the third PtdInsP isomer, was identified in T.thermophila. PtdIns5P levels constitute approximately 2% of total PtdInsP and they remained stable during phagocytosis. Treatment of cells with wortmannin, a specific inhibitor of mammalian PI3K, which was found to inhibit PtdIns3P and PtdIns3,5P2 synthesis in Tetrahymena, or U73122, which inhibits PI-PLC activity in Tetrahymena, was used to test whether PI3K and PI-PLC are required for the regulation of phagocytosis. Wortmannin caused an inhibition of phagosome formation and an arrest in the maturation and defecation of dextran-iron-containing phagosomes, while U73122 caused a delay in phagosome formation. These results suggest the involvement of 3-phosphoinositides in more than one different stages of phagocytosis in T.thermophila and furthermore, they indicate that there is a need for proper regulation of PtdIns4,5P2 levels during the initiation of phagocytosis. The above results were further supported by the study of a mutant T.thermophila strain (A2), blocked in the biogenesis of phagosomes. This strain was found to possess reduced amounts of total protein and phospholipids, including PtdCho and PtdEth, probably due to the complete absence of phagosomes. T.thermophila A2 cells contained also reduced amounts of PtdIns, however, PtdInsP2 levels were found to be increased by 60% compared to the wild type cells. In addition, PtdIns3P levels were decreased by 40%, while wortmannin-induced depletion of 3-phosphoinositides was similar between wild type and phagocytosis-deficient cells. Interestingly, PI-PLC activity was increased in T.thermophila A2 homogenate and microsomal fractions by 2-fold compared to the wild type cells. These results suggest that phosphoinositide metabolism, and particularly PI-PLC activity, is deregulated in phagocytosis-deficient T.thermophila cells. In order to study the composition and possible changes of phosphoinositides on phagosomal membranes during the initiation and maturation process, dextran-iron-containing phagosomes were isolated and analyzed. Isolation was carried out by magnetic separation from a T.thermophila strain, which was blocked in mucocyst secretion, since phagosome isolation is impaired by mucocyst secretion. The distribution of phosphoinositides in isolated 30min phagosomes was found to be different compared to the cell homogenate, but further analysis of phagosomal phosphoinositides during maturation was hindered due to the very low amounts of [3H]inositol incorporated. Instead, fatty acid profile of phagosomal lipids was analyzed in phagosomes by establishing a pulse-chase protocol. Maturation was accompanied by distinct changes in the fatty acid profile. Saturared fatty acids were increased in the first 10min of maturation while, linolenic acid was decreased and 18:1 and 18:2 fatty acids were increased during the whole maturation process. Moreover, fatty acid profile of the homogenate lipids did not change during phagocytosis. In conclusion, in T.thermophila like in mammalian cells, different phosphoinositides and phosphoinositide metabolism enzymes play a regulatory role in the different stages of phagocytosis.