Lipids regulate P2X7-receptor-dependent actin assembly by phagosomes via ADP translocation and ATP synthesis in the phagosome lumen

Abstract

Latex bead phagosomes isolated from J774 macrophages polymerize actin. We show here that five lipids – phosphatidylinositol-4-phosphate, phosphatidylinositol-(4,5)-bisphosphate, sphingosine-1-phosphate (S1P), ceramide-1-phosphate and phosphatidic acid – stimulate both actin assembly and transport of ADP across the phagosomal membrane into the lumen. Once there, this ADP is converted to ATP by adenylate kinase activity. High luminal ATP concentrations correlated well with phagosome actin assembly under different conditions. The ATP-binding P2X7 receptor (P2X7R) was detected in phagosomes. Although S1P stimulated actin assembly by phagosomes from P2X7R-containing bone marrow macrophages, S1P-stimulated actin assembly was inhibited in phagosomes from cells lacking P2X7R. We propose that luminal ATP accumulates in response to selected lipids and activates the P2X7R that signals across the phagosomal membrane to trigger actin assembly on the cytoplasmic membrane surface. In the accompanying paper by Kuehnel et al. (doi:10.1242/jcs.034207), more evidence is provided in support of this model from the analysis of actin assembly at the plasma membrane of intact macrophages.

P2X7 Is a Scavenger Receptor for Apoptotic Cells in the Absence of Its Ligand, Extracellular ATP

“…3A, yellow arrows). Phagosome localization of P2X 7 has been shown by others (15). In our study, accumulation of P2X 7 is clearly seen at the point of attachment between a captured HPB cell and the HEK-293 cell (Fig.…”

“…We and others have shown that most cellular P2X 7 receptors are located intracellularly rather than on the cell surface (15,20,39). In this study, an increase of P2X 7 surface expression on phagocytes was observed following phagocytosis of either apoptotic cells or beads, similar to the upregulation of the P2X 4 receptor on THP-1 cells after phagocytosis of beads (40).…”

“…Recent findings from our group and others (14,15) have suggested a direct involvement of the purinergic P2X 7 receptor in phagocytosis of nonopsonized particles and bacteria. We showed that overexpression of the P2X 7 receptor greatly augmented the phagocytosis of nonopsonized beads and heat-killed bacteria by transfected HEK-293 cells, whereas blocking P2X 7 expression by small interfering RNA significantly reduced the phagocytic ability of human monocytic cells.…”

A new role for the P2X7 receptor: a scavenger receptor for bacteria and apoptotic cells in the absence of serum and extracellular ATP

“…However, P2X7 receptors may play a role in linking innate and adaptive immunity after phagocytosis has occurred. Confocal microscopy shows P2X7 in the phagosome membrane surrounding engulfed particles or bacteria [29,30] and recent reports suggest that P2X7 together with pannexin-1 may facilitate the delivery of bacterial products from phagosome lumen into the cytosol, where they can activate inflammasome assembly [31,32]. Moreover P2X7 has a role in facilitating the fusion of phagosome with lysosome by a process involving stimulation of phospholipase D activity presumably on the phagolysosomal membrane [33,34].…”

Intracellular P2X receptors as novel calcium release channels and modulators of osmoregulation inDictyostelium

“…2 In addition to a cell surface residency, some P2XR subtypes are localized to intracellular compartments of mammalian and other eukaryotic cells, including lysosomes [7][8][9] and phagosomes. 10 In 2007 we cloned the first P2XR from a unicellular organism, from the amoeba Dictyostelium. 3 In contrast to the recognized cell surface role of P2XRs, the receptors of Dictyostelium are exclusively intracellular.…”